単回投与毒性試験代替法

目次
評価会議報告書 ------------------------------------------------------------------------------

1

第三者評価報告書 ---------------------------------------------------------------------------

7

Background Review Document, NIH Publication No: 07-4518
In Vitro Cytotoxicity Test Methods for Estimating Acute Oral Systemic Toxicity---------------------------------------------------------------------------------------------------- 55

単回投与毒性試験代替法の評価会議報告書

JaCVAM 評価会議

平成 23 年（2011 年）4 月 20 日

1

JaCVAM 評価会議
西川秋佳（国立医薬品食品衛生研究所 安全性生物試験研究センター）
田中憲穂（食品薬品安全センター 秦野研究所）
吉田武美（昭和大学薬学部）
横関博雄（東京医科歯科大学）
吉村 功（東京理科大学）
渡部一人（日本製薬工業協会）
岡本裕子（日本化粧品工業連合会）
大島健幸（日本化学工業協会）
小野寺博志（医薬品医療機器総合機構）
小笠原弘道（医薬品医療機器総合機構）
吉田 緑 （国立医薬品食品衛生研究所 安全性生物試験研究センター 病理部）
五十嵐良明（国立医薬品食品衛生研究所 生活衛生化学部）
長谷川隆一（独立行政法人 製品評価技術基盤機構）
浅野哲秀（元日東電工株式会社）
任期：平成 22 年 4 月 1 日～平成 24 年 3 月 31 日

オブザーバー：JaCVAM 運営委員
大野泰雄（国立医薬品食品衛生研究所）
関野祐子（国立医薬品食品衛生研究所 安全性生物試験研究センター 薬理部）
増田光輝（国立医薬品食品衛生研究所 安全性生物試験研究センター 薬理部）
小島 肇（国立医薬品食品衛生研究所 安全性生物試験研究センター 薬理部）
秋田正治（日本動物実験代替法学会）
柴辻正喜（厚生労働省 医薬食品局 審査管理課 化学物質安全対策室）
実国慎一（経済産業省 製造産業局 化学物質安全対策室）
任期：平成 22 年 4 月 1 日～平成 23 年 4 月 30 日
以上

2

単回毒性試験代替法について、第三者評価委員会からの報告を受け１）、以下の 9 項目について審議した。本 2～8 項目は OECD ガイダンス文書 No．34 に示された検討項目である２）。なお、本動物実
験代替法の利用にあたっては、適用範囲を十分に配慮した上で使用されるべきである。
＜審議内容＞
１．

検討対象の試験法は、日本のどの法規制やガイドラインに関係しているか。
毒物に関しては、毒物及び劇物取締法についての通知 ＊で、「毒物又は劇物の指定等を判断するための試験法として、OECD 化学物質試験ガイドライン 401 に替わり、OECD 化学物質試験ガイドライン 420(固定用量法)、同 423(急性毒性等級法)、同 425(上げ下げ法)を推奨する」こととしている。これに関係している。
＊

平成 14 年 12 月 9 日、医薬化発第 1209001 号：毒物及び劇物取締法における毒物又は劇物の指定等を判断するために必要とされる試験法について
新規化学物質に関しては、化学物質の審査及び製造等の規制に関する法律（化審法）における試験法に関する通知＊で、急性毒性試験を反復投与毒性試験の予備試験として実施する際には、OECD Test Guideline の試験法を参考にするのが望ましい、としている。これが関連している。
＊平成 15 年 11 月 21 日 薬食発第 1121002 号、
平成 15･11･13 製局第 2 号、環保企発 第 031121002
号、「新規化学物質等に係る試験の方法について」（最終改正：平成 18 年 11 月 20 日）
農薬に関しては、通知＊「農薬の登録申請時に提出される試験成績の作成に係る指針の付表、単回毒性試験」で、「固定用量法」と「急性毒性等級法」が推奨されている。これが関係している。
＊農林水産省農産園芸局長通知、平成 12 年 11 月 24 日、１２農産第８１４７号：農薬の登録申請に係る試験成績について
医薬品に関しては、薬事法の施行規則で「医薬品の製造（輸入）承認申請に際して添付すべき資料」を指定している。その中に単回投与毒性試験の資料が含まれており、これが一般に急性毒性試験と呼ばれている試験法で得られる資料である。これについては通知＊で、単回投与毒性試
験のやり方が定められている。そこでは「概略の致死量」が求められているだけで、LD50 を求めることは必須とされていない。
＊平成 5 年 8 月 10 日、薬新薬第 88 号：単回及び反復投与毒性試験ガイドラインの改正について
医療機器に関しては、急性毒性を示すような抽出物が存在しないことが求められている。しかし、そこでは、生理食塩液と植物油の２種類の抽出媒体で調整した抽出液で試験することが求められているだけで、LD50 等を求める必要がないので、当該試験法とは関連がない。
＊平成 15 年 2 月 13 日、医薬審発第 0213001 号「医療用具の製造（輸入）承認申請に必要な生物学的安全性試験の基本的考え方について」、平成 15 年 3 月 19 日：事務連絡 医療機器審査 No. 36「生物学的安全性評価の基本的考え方に関する参考資料について」3

輸液用ゴム栓に関しては、「第十五改正日本薬局方 7.03」 で急性毒性試験が求められている。
しかしこれは、一定用量での試験であり、当該試験法とは関連がない。
２．検討対象の試験法とその妥当性を示すデータは、透明で独立な評価を受けているか。

当該試験法の妥当性は、日本国内では実験的に評価されていない。
しかし国外では、NICEATM 及び ECVAM が、2002 年から 2005 年にかけて、72 種類の化学物質を用いて妥当性を検証している。
その評価状況は、Background Review Document (BRD) ３）として公開されている。
３．当該試験法で得られるデータは、対象毒性を十分に評価あるいは予測できるものであるか。デー
タは、当該試験法と従来の試験法の、代替法としての繋がりを示しているか。あるいは（同時に）そのデータは、当該試験法と、対象としているあるいはモデルとしている動物種についての影響との繋がりを示しているか。
当該試験法は、従来の試験法の全体を代替するのではなく、従来の試験法での初回投与量の設定法を代替するものである。
４．当該試験法は、ハザードあるいはリスク、あるいはその両方を評価するのに有用であるか。
当該試験法は、前項の問に対する回答と同じ理由で、ハザード評価に直接資するものではないが、間接的には有用である。
５．当該試験法とその妥当性を示すデータは、その試験法で安全性を保証しようとする、行政上のプログラムあるいは関係官庁が対象としている化学物質や製品を、十分広く対象としたものとなっているか。当該試験法が適用できる条件及び適用できない条件が明確であるか。
当該試験法は、化学物質の安全性を保証するためのものではない。次の場合に適用できない
ことは明確である。
•

代謝により活性化されて毒性を発現する場合•

神経毒性、心臓毒性等特異的な作用機序により毒性を発現する場合。

•

細胞培養液に不溶性の物質、揮発性の物質、ライソゾームへの特異的影響を与える物質，
neutral red の吸光度と重なる有色の物質。

６．当該試験法は、プロトコルの微細な変更に対して十分頑健で、適切な訓練経験を持つ担当者と適切な設備のある施設において、技術習得が容易なものであるか。
当該試験法のプロトコルの頑健性について、実験データ上の根拠はない。
しかし、当該試験法の実験手順は、適切な訓練経験を持つ担当者と適切な設備のある施設において 技術習得が容易なものである。
７．当該試験法は、時間的経費的に有用性があり、行政上で用いられやすいものであるか。
当該試験法の時間的経費的有用性を示す決定的定量的データは、シミュレーション試算以外に存在しない。従って、定量的にどのような有用性があるかは確かでない。
定量的にどのような有用性があるかは確かでないので、行政上で用いられやすいものとは言え4

ない。
８．当該試験法は、従来の試験法と比べて、科学的・倫理的・経済的に、新しい試験法あるいは改訂
試験法であることが正当化されているか。
従来法が評価対象としている個体の死にはいくつかのメカニズムが存在している。これに対して当該試験法は、細胞死を指標として評価しているので、科学的妥当性は限定的である。
しかし、初回投与量を当該試験法で定めることは、従来法と比べて、決定的な悪影響を与える
ものでないことから、限定した条件の下では、科学的妥当性が認められる。
倫理的には新しい提案であるが、経済的有用性は明らかではない。
9.安全性評価のための行政的資料として、 受け入れ可能な試験法であるか。
本試験法は，急性毒性試験における初回投与量を設定する手法となる。
以上の審議の結果、JaCVAM 評価会議は、単回毒性試験代替法について以下のように結論した。
当該試験法の有用性はそれほど大きなものでない。
しかし、これを採用することの利点は存在し、しかも欠点は致命的なものでない。
強制力を持たせないで、これを行政的に提案し、推奨することは、３Ｒ原則にそったものである。
参考文献
１．急性毒性試験代替法の第三者評価報告書
２．OECD (2005) OECD

Series on testing and assessment Number 34, Guidance document on thevalidation and international acceptance of new or updated test methods for hazard
assessment, ENJ/JM/MONO(2005) 14
３．Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM),

National Toxicology Program (NTP), et al. Background Review Document:“in vitro
Cytotoxicity Test Methods for Estimating Acute Oral systemic Toxicity”, NIH
Publication No: 07-4518

5急性毒性試験代替法の第三者評価報告書

評価対象試験： in vitro 細胞毒性試験による急性経口毒性試験の
初回投与量設定試験

In vitro Cytotoxicity Test Methods for Estimating Starting Doses
for Acute Oral Systemic Toxicity Test

平成 22 年 5 月 31 日 草案
平成 22 年 10 月 18 日 改訂
平成 22 年 11 月 11 日 改訂
平成 23 年 1 月 14 日 改訂

急性毒性試験代替法評価委員会

委員長

髙橋 祐次

（国立医薬品食品衛生研究所）

委員

笠原 利彦

（富士フイルム株式会社）

若栗 忍（（財）食品薬品安全センター秦野研究所）

小平 輝朋（旭化成ファーマ株式会社）

岩井 久和（株式会社三和化学研究所）

萩野 滋延（株式会社資生堂）

河野 茂生（ブリストル・マイヤーズ株式会社）

岩瀬 裕美子 （田辺三菱製薬株式会社）

JaCVAM 急性毒性試験評価委員会

目次
略語 ...................................................................................................................................... 2
要旨 ...................................................................................................................................... 5
1．試験法の科学的、規制の上での妥当性 ........................................................................... 7
2．試験法の妥当性 ............................................................................................................. 14
3．バリデーションに用いられた物質の分類と妥当性 ....................................................... 21
4．試験法の正確性を評価するために用いられた参照化合物の in vivo 参照データ ........... 25
5．試験法のデータと結果の利用性 .................................................................................... 27
6．試験方法の正確性 ......................................................................................................... 28
7．試験方法の信頼性 ......................................................................................................... 32
8．試験方法のデータの質 .................................................................................................. 35
9．その他の試験方法の科学的な報告 ................................................................................ 37
10．3Rs への関与 .............................................................................................................. 39
11．試験方法の有用性と限界............................................................................................. 41
12．結論 ............................................................................................................................. 43
13．参考文献...................................................................................................................... 45

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JaCVAM 急性毒性試験評価委員会

略語
ADME

Absorption, distribution, metabolism, and elimination

ANOVA

Analysis of varianceATC

Acute Toxic Class method

ATWG

Acute Toxicity Working GroupBRD

Background Review Document
本報告書では In Vitro Cytotoxicity Test Methods for Estimating Acute Oral
Systemic Toxicity（NIH Publication No. 07-4518）を示す。

CNS

Central nervous systemCV

Coefficient of variation

DMEM

Dulbecco’s Modification of Eagle’s Medium

DMSO

Dimethyl sulfoxide

D-PBS

Dulbecco’s phosphate buffered saline

EC50

Concentration of a substance that produces 50% of the maximum
possible response for that substance

ECBC

U.S. Army Edgewood Chemical Biological Center

ECVAM

European Centre for the Validation of Alternative Methods

ETOH

Ethanol (Ethyl alcohol)

FAL

FRAME Alternatives Laboratory

FDP

Fixed Dose Procedure

FRAME

Fund for the Replacement of Animals in Medical Experiments

GHS

Globally Harmonized System (of Classification and Labeling of Chemicals)

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JaCVAM 急性毒性試験評価委員会

GLP

Good Laboratory Practices

HBSS

Hanks’ balanced salt solution

IC50

Concentration producing 50% inhibition of the endpoint measured

ICCVAM

Interagency Coordinating Committee for the Validation of Alternative Methods

IIVS

Institute for In Vitro Sciencesi.p.

Intraperitoneali.v.

Intravenous

KBM

Keratinocyte basal medium

Kow

Octanol-water partition coefficient

LD50

Dose that produces lethality in 50% of test animals

MTT

3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide

N

Number (of substances)NHK

Normal human epidermal keratinocytes

NICEATM

National Toxicology Program Center for the Evaluation of Alternative
Toxicological Methods

NR

Neutral red

NRU

Neutral red uptake

OD

Optical density

OD540

Optical density (absorbance) at a wavelength of 540 nm

PBS

Phosphate buffered salinePC

Positive control

ｐH

Power of hydrogen
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JaCVAM 急性毒性試験評価委員会

QA

Quality assurance

QC

Quality controlｒ

Pearson correlation coefficient

R2

Coefficient of determinationrs

Spearman correlation coefficient

RC

Registry of Cytotoxicity

RI

Radioisotope

RTECS ®

Registry of Toxic Effects of Chemical SubstancesSD

Standard deviation

SLS

Sodium lauryl sulfate

SMT

Study management team

3T3

BALB/c mouse fibroblasts, clone A31 (ATCC # CCL-163)

UDP

Up-and-Down Procedure

VC

Vehicle control

ZEBET

Zentralstelle zur Erfassung und Bewertung von Ersatz-und
Ergänzungsmethoden zum Tierversuch (German Center forDocumentation and Evaluation of Alternative Methods to Animal
Experiments)

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JaCVAM 急性毒性試験評価委員会

要旨
本報告書は、「in vitro 細胞毒性試験による急性毒性試験の初回投与量設定試験」のバリデーショ
ン研究を ICCVAM が第三者評価し、その情報をまとめた Background Review Document (BRD)を
もとに JaCVAM 急性毒性試験代替法評価委員会が第三者評価を実施したものである。
化学物質のハザードを評価する急性毒性試験はげっ歯類を使用して実施されている。急性毒性
試験によって得られる LD50 値（Dose that produces lethality in 50% of test animals、半数致死量）
は、GHS （Globally Harmonized System of Classification and Labeling of Chemicals)における化合物の分類及びラベリング、日本においては毒物及び劇物取締法の判定基準に利用されている。その一方で、動物の死亡をエンドポイントとする試験方法に対する批判があり、また、ヒトが化合物を過剰摂取した場合における急性毒性試験データの有用性に関して議論がある。細胞毒性試験を用いた代替法が検討されてきたが、規制当局が受け入れ可能な信頼性、妥当性、有用性及び適応範囲を評価した試験は存在していない。現在、OECD 毒性試験法ガイドラインの急性経口毒性
試験は、Fixed Dose Procedure (OECD 420 : FDP)、Acute Toxic Class method (OECD 423: ATC)及び Up-and-Down Procedure （OECD 425：UDP）が採択されている。これらのガイドラインは、あらかじめ設定された 4 または 8 段階の用量の一つを選択して動物に投与し、死亡した場合には低用量、生存した場合には高用量を逐次投与することで化合物の LD50 が求められるようにデザインされている。従って、適切な初回投与量の選択が使用動物数削減の鍵となる。
本試験方法は、使用動物数の削減を目的として、細胞毒性試験から急性毒性試験の初回投与用
量を推測する in vitro アプローチである。具体的には、Neutral Red Uptake（NRU） 法による細胞毒性試験で IC50 値（mM）を求め、RTECS®のデータを基にした LD50 値（mmol/kg）と IC50 値の回帰式から急性毒性試験の初回投与用量を推測する。本試験方法のバリデーションは、GHS 急性経口
毒性の区分全体に分布するように選択した 72 種類の参照化合物の細胞毒性試験を、ヒト細胞株（ Normal human epidermal keratinocytes; NHK ） 及 び げ っ 歯 類 の 細 胞 株 （ BALB/c mouse
fibroblasts； 3T3）を用いて実施した。
LD50 値と IC50 値の相関性から急性毒性試験の初回投与用量を推測する回帰式が得られているが、動物の死と細胞の死が類似するメカニズムの説明が不十分であると考える。相関性は必ずしも因果関係を説明するものではない。また、溶解性、沈殿物及び揮発性を有する化合物、代謝活性により毒性を発現する化合物、肝、中枢神経、腎、心臓、肺及び造血器に対する特異的な毒性を有する化合物は細胞毒性試験では評価することができないため、試験対象から除外すべきである。

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JaCVAM 急性毒性試験評価委員会

細胞毒性試験方法は妥当であると判断するが、用量設定、媒体、被験物質の調製に使用する試
験管、被験物質の添加方法は試験精度が担保できる範囲で自由度を持たせるべきである。
本試験法によって予測された LD50 を GHS 区分全体に渡って評価すると 3T3 では 31％（21/67）、NHK 細胞では 29％（20/68）であり、特に強毒性を示す化合物の予測性は低かった。毒性メカニズ
ムの面からは、中枢神経系と心臓への作用を示す化合物ではずれ値が認められた。この成績からも、臓器特異的な毒性を示す化合物の予測性は低く評価に適さない。
バリデーション試験は 3 施設において、GLP または GLP の精神で実施された。データの質については問題がないと判断した。
使用動物の削減数については、コンピュータによるシミュレーションにより評価している。一試験あたり、UDP 法では平均 0.49 匹～0.66 匹、ATC 法では平均 0.51 匹～1.09 匹の動物が削減される
ことが示された。特に弱毒性物質の場合（LD50 値：>2000 mg/kg 又は>5000 mg/kg）、UDP 法では1.28 匹～1.65 匹、ATC 法では 2.03 匹～3.33 匹の動物が削減されることが示された。使用動物の削減には本当に繋がる試験であるかは、実際に使用した動物数が記載されている化合物の試験
情報と、この試験で予測された初回投与量から予測される動物数を比較して検証することが必要である。また本試験法をガイドラインに導入した場合には、試験情報を集計して動物数の削減が実現できているかについて検証する必要がある。また、強毒性の化学物質の予測性が低いことは動物へ与える苦痛の低減にはつながらない。
以上のことから、ICCVAM で実施された細胞毒性試験による急性毒性試験の初回投与量設定試
験の第三者評価は、バリデーションに必要な項目、プロセス及びデータが検討されており、ICCVAM のバリデーション結果を受け入れることに問題はないと判断した。本試験方法は低毒性の化合物については予測性があり、動物数の削減できる可能性が示されていることから、急性毒性試験の初回投与量決定の情報として必要に応じて活用可能であると判断する。しかしながら、強毒性に分類される化合物の予測性は低く、動物へ与える苦痛の低減、使用動物数削減に寄与は低い。臓器特異的な毒性を有する化合物の評価には適しておらず、揮発性を有する物質、溶解度が低い物質の試験は実施が困難である。したがって、急性毒性試験の実施に際して、一律に本試
験法を用いて初回投与量を決定することは合理的ではなく、化合物の物性、類縁化合物の情報と同様の位置づけとして利用することが望ましい。

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JaCVAM 急性毒性試験評価委員会

1．試験法の科学的、規制の上での妥当性
化学物質のハザードを評価する急性毒性試験はげっ歯類を使用して実施されている。急性毒性
試験によって得られる LD50 値（Dose that produces lethality in 50% of test animals、半数致死量）
は、GHS （Globally Harmonized System of Classification and Labeling of Chemicals)における化合物の分類及びラベリング、日本においては毒物及び劇物取締法の判定基準に利用されている。その一方で、動物の死亡をエンドポイントとする試験方法に対する批判があり、また、ヒトが化合物を過剰摂取した場合における急性毒性試験データの有用性に関して議論がある。
動物愛護の観点から、急性毒性試験の代替法が検討されてきた。
1983 年にはスカンジナビアの Society for Cell Toxicology は The Multicentre Evaluation of in vitro
Cytotoxicity (MEIC) プログラムを立ち上げ in vitro 試験とヒトの経口摂取致死量における血中濃度を比較検討した。
1992-1993 年には、The Fund for the Replacement of Animals in Medical Experiments (FRAME)がげっ歯類の急性致死性を複数の in vitro 試験（MTT reduction、LDH release、cell function）で予測する事を検討した。げっ歯類における化合物の急性致死性の予測には in vitro 試験のバッテリー（①細胞死、②肝細胞毒性、③細胞毒性が現れない濃度域における細胞膜電位への干渉）として評価することが推奨された。中でも、細胞死を指標とした試験では、細胞株（V79、3T3-L1 またはBALB/c 3T3）、暴露時間（24-72h）及びエンドポイント（MTT または NRU）によって評価に大きな差異が認められないことが示された。
1998 年及び 2003 年、Dr. Willi Halle は RTECS®から分子量が既知である化合物のげっ歯類における LD50 値と細胞株及びエンドポイントが多様な細胞毒性試験の IC50 値を比較したデータベース
である Registry of Cytotoxicity (RC) を報告した。RC では細胞毒性試験で得られた IC50 値のモル濃度（mM）とげっ歯類の LD50 値を mmol/kg に変換した数値の相関が以下の回帰式（RC millimole
regression）で示された。

log LD50 (mmol/kg) = 0.435 x log IC50 (mM) + 0.625
この回帰式には、参照した化合物の 73% (252/347)の化合物が含まれる。

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JaCVAM 急性毒性試験評価委員会

RC millimole regression（BRD より抜粋）

1994 年 ECVAM は in vitro 試験で化合物のハザードを分類する事を目的としたワークショップを組織した。1996 年ワークショップの参加者によって、in vitro 試験結果を用いて経口投与急性毒性試
験の初回投与量を決定することで使用動物の削減する構想が議論された。同時期には OECD 急
性毒性ガイドライン（420: Acute Oral Toxicity - Fixed Dose Procedure、423: Acute Oral toxicity Acute Toxic Class Method、425: Acute Oral Toxicity: Up-and-Down Procedure）のドラフトが提案されていた。これらのガイドラインは、あらかじめ設定された 4 または 8 段階の用量から一つを選択して動物に投与し、死亡した場合には低用量、生存した場合には高用量を逐次選択することで化合物の LD50 が求められるようにデザインされている。従って、適切な初回投与量の選択が使用動物数の削減の鍵となる。

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JaCVAM 急性毒性試験評価委員会

Acute Oral toxicity - Acute Toxic Class Method の LD50 決定までの流れ図（OECD ガイ
ドライン 423 より抜粋）

300 mg/kg を初回投与量とした場合の流れ図を示す。各試験段階では 3 例の動物を使用する。評価する化合物の動物実験における LD50 値が＞300-2000 mg/kg（Category 4）の場合には、最少で 3 段階、最多で４段階の試験を実施して LD50 値が求められる。動物実験におけるLD50 値が＞5-50 mg/kg（Category 2）の場合には、最少で 4 段階、最多で 6 段階の試験を実施して LD50 値が求められる。

1999 年、The German Center for Documentation and Evaluation of Alternative Methods to Animal
Experiments (ZEBET) は RC millimole regression を用いたシミュレーションで急性毒性試験の初回投与量を決めることにより、UDP 法のドラフトガイドラインにおける使用動物が 25-40% 削減できることを示した。
2000 年、NIEHS、NTP 及び EPA は協力して International Workshop on in vitro Methods forAssessing Acute Systemic Toxicity (Workshop 2000)を開いた。このワークショップでは、①ZEBETの RC millimole regression によって急性毒性試験の初回投与量を見積もる手法、②ECVAM から

9JaCVAM 急性毒性試験評価委員会

提案された試験方法の構想、③動物数削減を目的とした①及び②以外の in vitro 細胞毒性試験の構想について議論された。その結果、急性毒性試験の代替法として試みられた in vitro 細胞毒性試験には、試験方法の信頼性、妥当性、有用性及び試験の適応範囲を適切に評価された試験方法はなく、規制当局が受け入れ可能な in vitro 試験または試験バッテリーは存在しないと結論された。ZEBET が提案した in vitro 細胞毒性の IC50 値と急性毒性試験の LD50 値の millimole
regression を用いて急性毒性試験の初回投与用量を推測する in vitro アプローチを最優先課題とすることが決定された。げっ歯類の急性毒性試験からヒトの致死性予測の試みとしてヒト細胞株（Normal human epidermal keratinocytes; NHK）を用いた試験、また、げっ歯類の急性毒性をより正確に予測できる可能性を考慮して、げっ歯類の細胞株（BALB/c mouse fibroblasts; 3T3）を用いNRU 法を一つの代表例として検討した。
ICCVAM の提案に応じて、NICEATM および ECVAM は、2002 年の 8 月から 2005 年 1 月の間、げっ歯類を使った急性毒性試験法の初回投与用量の予測に使用される in vitro 細胞毒性試験の有用性と限界について調べるために、72 種類の化合物に対して、3T3 細胞あるいは NHK 細胞を用いた NRU テスト（Neutral Red Uptake）のバリデーション試験を複数の施設で実施した。
バリデーション試験は、以下に示すように 4 段階で実施された。

Phase Ia: Laboratory Evaluation
Development of a positive control database for each laboratory
Phase Ib: Laboratory Evaluation
Limited substance testing to demonstrate the reliability of the protocol
Phase II: Laboratory Qualification
Evaluation of protocol refinements
Phase III: Laboratory Testing Phase
Test of optimized protocols
現在、各極における急性毒性試験による化合物分類を BRD から抜粋して記載した（Table 1-2）。
BRD に記載されているように、現在のところ in vivo 急性毒性試験に置き換わる in vitro 試験はない。
OECD ガイドラインでは、in vitro 細胞毒性試験は類縁化合物、構造活性相関などとならんで、invivo 試験の初回投与量を決める毒性情報として利用できることが記載されている。初回投与量を決めるための参考情報が入手できない場合、ATC 法では 300 mg/kg、UDP 法では 175 mg/kg である。FDP 法では main study を実施する前に sighting study を行って初回投与量を決める。sighting
study の初回投与量も試験計画の時点で得られている情報を基にするが、情報が入手できない場
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JaCVAM 急性毒性試験評価委員会

合には 300 mg/kg を選択し、各用量で 1 匹の動物を使用して main study と同様の手順を踏んで初回投与量を決定する。最多で 4 匹の動物を sighting study で使用することになる。
現行の急性毒性ガイドラインにおいても、強制力はないが初回投与量の決定に in vitro 細胞毒性試験は利用されていると考えられる。

経口投与急性毒性試験による化合物の分類（BRD より抜粋）

ニュートラル・レッド（NR）は水溶性の弱陽イオン超生体染色色素である。正常な細胞では、NR は細胞形質膜を透過して陰イオン性のライソゾームマトリクスに結合して濃縮されるが、化合物によって細胞傷害が生じた場合には、細胞に取り込まれる NR が減少する。3T3 細胞を使用した NRU 試
験は Borenfreund and Puerner (1985)が最初に報告した試験である。細胞毒性をエンドポイントとした試験方法は幾つか報告されている。NICETM/ECVAM のバリデーション試験で NRU 法を選択した理由としては、3T3 細胞及び NHK 細胞を用いた NRU 試験は、以前に実施したバリデーション試験において再現可能な試験であること (ICCVAM 2001b)、両細胞の入手は容易であり、LD50 値を予測する RC millimole regression が既に得られていること、加えて、この試験は自動化が可能であり、RI を使用せず、危険性のある試薬を使用しないという実施上の利点があること、である。NRU 試

11JaCVAM 急性毒性試験評価委員会

験法で評価可能な物質は、細胞培養液と反応せずに溶解する限りすべての物質が評価可能である。混合物でも評価可能と考えられるが、このバリデーション試験では使用されていない。
NRU 試験のエンドポイントは細胞死である。一方、in vivo 急性毒性試験のエンドポイントは動物の病的状態あるいは死であり、一見大きく異なる。しかし、細胞障害や細胞死が広範囲の組織に生じると、主要臓器の機能不全が起こり、個体死に繋がる。細胞のもつ基本的な機能として Ekwall(1983) はミトコンドリア活性、形質膜の統合性への影響を提案している。多くの化合物によって誘発される毒性は、これらの基本的な細胞機能への非特異的な影響の結果であって、細胞膜及び細胞骨格の統合性、代謝、合成、分解または細胞構成生物の分解、イオン調節及び細胞分裂に障害が生じ細胞死を招く。また、組織障害は恒常性維持のシグナル干渉も引き起こし、化合物が暴露されていない臓器にも影響する。したがって、細胞死と個体死には、同様のメカニズムが働いていると考えられる。

in vivo 試験において化合物の毒性発現がヒトとげっ歯類で異なるように、in vitro 細胞毒性試験においてもヒト由来の細胞とげっ歯類由来の細胞では反応性が異なり、またヒト由来の細胞であったとしても細胞の種類によってその感受性が異なる(Clemedson et al. 1998a、b)。げっ歯類の致死性を予測するには、げっ歯類由来の細胞が適していると考えられる。
細胞培養系と動物個体では、化合物の暴露の形態が異なる。経口投与された化合物は、消化管で吸収、血漿タンパク結合及び代謝を受け、体外へ排泄される。これらの過程を経ることで化合物の生物学的利用率は低下する。したがって、個体内では投与された化合物の一部のみが標的臓器に到達するに過ぎず、暴露される時間も限定的なものである。一方、細胞培養系では、吸収、分布、代謝及び排泄を有しておらず、化合物は直接細胞に作用するため、細胞培養系は動物の体内の細胞よりも被験物質に長時間暴露される。
化合物の毒性発現機序が細胞培養系と動物個体で異なる場合、細胞毒性試験での評価は適当ではない。例えば、ある種の神経受容体を介して毒性を発現す場合には、その受容体を発現していない 3T3 細胞または NHK 細胞では同様の毒性を捉えることを期待できない。仮に細胞毒性を示したとしても、それは in vivo とは異なったメカニズム、異なった濃度における毒性発現である。培
養神経細胞であっても in vivo と同じ機能を保持していなため、in vivo と同じ反応を期待できない。
神経や心臓に特異的な毒性を発現する物質については、3T3 細胞あるは NHK 細胞を用いた NRU
試験では過小評価されると考えられる。

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JaCVAM 急性毒性試験評価委員会

化合物の物性として、細胞培養液に不溶性の物質、揮発性の物質、ライソゾームへ特異的な影響を与える物質、細胞に残留する性質を有した赤色あるは NR の吸光度と重なる有色の物質の評価は適当ではないかもしれない。3T3 細胞を用いた NRU 試験では、5%の血清を培養液に含むため、
血清タンパクへの結合性が高い物質では、その毒性を過小評価するかもしれない（NHK 細胞の培
地は血清を含まないため、この懸念はない）。
JaCVAM 急性毒性代替法評価委員会の意見としては、72 種類の化合物から得られた NRU 法のIC50 値と急性毒性試験の LD50 値の相関性を根拠とした回帰式から算出しているが、動物の死と細胞の死が類似するメカニズムの説明が不十分であると評価した。相関性は必ずしも因果関係を説明するものではない。神経系、循環器、呼吸器などの生命維持に重要な影響を及ぼす器官に作用する化合物、体内で代謝されることによって毒性を発現する化合物は正しい LD50 値の予測はできないと判断した。また、NRU 法での評価に適当ではない物性（揮発性、難溶解性及び有色の化合物など）を有する化合物は、本実験では正確な評価をすることが困難であるため、試験対象から除外すべきであると判断した。

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JaCVAM 急性毒性試験評価委員会

2．試験法の妥当性
96well マイクロプレートで培養した株化細胞に被験物質を 48 時間暴露させる。その後、ニュートラ
ルレッド（NR）を培地に添加し、一定時間インキュベーションした後、細胞内に取り込まれた NR を抽出してプレートリーダーで測定し、コントロール細胞の吸光度値に対する割合を細胞生存率の指標とする。試験は、溶解性試験、用量設定試験及び本試験から構成される。用量設定試験では、広い範囲の用量をカバーする必要があるため、大きな希釈率で実施する。本試験では IC50 値を用量
段階の中央に設定し希釈率は用量設定試験より小さくする。

細胞の種類と培養液
1）3T3 細胞（BALB/c 3T3 マウス線維芽細胞）培養液：10％新生児ウシ血清（非働化せず）含有 DMEM 培地
2）NHK 細胞（ヒト正常表皮角化細胞）
培養液：KBM 培地（0.0001 ng/mL ヒトリコンビナント EGF、5 μg/mL インスリン、0.5 μg/mL
ハイドロコルチゾン、30 μg/mL ゲンタマイシン、15 ng/mL アンホテリシン B、0.1 mM カル
シウム、30 μg/mL ウシ下垂体抽出物含有）
3T3 細胞を用いた成績は NHK 細胞に比較して、実験結果の再現性は劣るが、動物数の削減に関してはわずかながら効果的であり、GHS の急性毒性のハザード分類もより正確に予測することができる。
NHK 細胞は無血清培地で培養することが可能であることから動物資源への負担が少ない。3T3 細胞を用いた試験は NHK 細胞に比較して費用が安価なことから、一般的に 3T3 細胞の使用が推奨されている。以下のように BRD では、２つの細胞を培養に必要な試薬費用が記載されている。
3T3 細胞:細胞（$200）＋培地 500 mL($20)； 約$220
NHK 細胞:細胞（$380）＋培地 500 mL($100)； 約$480

溶解性試験
溶解性試験は、被験物質が溶解するまで溶媒添加量を段階的に増加させることを基本として実施する。使用する溶媒は、細胞培養液、DMSO、ETOH の順で選択する。溶液を顕微鏡で観察し、溶液が透明で濁りや沈殿物が全く観察されない場合に溶解しているものとみなす。
攪拌方法は、以下のように実施する。
（1）1-2 分、室温でゆっくりボルテックスをかける。
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JaCVAM 急性毒性試験評価委員会（2）被験物質が溶解しなかったら、5 分間超音波処理を実施する。
（3）超音波処理でも溶解しない場合は、ウォーターバスあるいは CO2 インキュベーターで 5-60 分
間加温する。

1）溶解性試験の手順
各 Phase によって手順が変遷しているが、以下に基本となった初期のプロトコルを記載する。
(1） 段階 1：100 mg の被験物質をガラス試験管に秤取する。0.5 mL の培養液を添加して200 mg/mL に調製する。溶解しない場合は段階 2 を実施する。
(2） 段階 2：10 mg の被験物質をガラス試験管に秤取り、0.5 mL の培養液を添加して 20
mg/mL に調製する。溶解しない場合は段階 3 を実施する。
(3） 段階 3：段階 2 の被験物質溶液に、さらに 4.5mL の培養液を添加して全体を 5 mL とし 2 mg/mL に調製する。溶解しない場合は DMSO で溶解する。新しいガラス試験管に被験物質を 100 mg 秤量し、0.5 mL の DMSO を添加して 200 mg/mL に調製する。
DMSO で溶解しない場合は ETOH で溶解する。新しいガラス試験管に被験物質を100 mg 秤量し、0.5 mL の ETOH を添加して 200 mg/mL に調製する。溶解しない場
合は段階 4 を実施する。
(4） 段階 4：被験物質が、培養液、DMDSO、ETOH のいずれにも溶解しない場合は、段階 3 で溶解しなかった培養液（2 mg/mL）、DMSO (200 mg/mL)及び ETOH (200mg/mL)の被験物質溶液をそれぞれの溶媒を添加して 10 倍希釈して、培養液では0.2 mg/mL、DMSO 及び ETOH は 20 mg/mL に調製する。溶解しない場合は、段階5 を実施する。
(5） 段階 5：段階 4 で溶解しなかった DMSO(20 mg/mL)及び ETOH(20 mg/mL)の被験物質溶液に、それぞれの溶媒を添加して 10 倍希釈し 2 mg/mL を調製する。
(6） 段階 6：さらに必要ならば、被験物質を 10 mg ずつ秤量し 50 mL の DMSO またはETOH を添加して 0.2 mg/mL の溶液を調製する。
Phase III においては、20 mg/ｍL を培養液中の最高濃度（終濃度）とし実施することを要求した。
注）実際のバリデーション試験においては ETOH を使用した試験は実施されていない。

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JaCVAM 急性毒性試験評価委員会

溶解性試験のフローチャート（BRD より抜粋）

2）被験物質溶液の希釈
被験物質溶液は、透明且つ沈殿が認められない条件で使用する。被験物質を有機溶媒に溶解した場合は、培養液中の有機溶媒含量は 0.5%以下とする。
被験物質を培養液に溶解した場合は、溶解性試験で溶解した最高濃度の半分、有機溶媒で溶解した場合は、溶解性試験で溶解した最高濃度の 1/200 の濃度を用量設定試験の最高濃度となる。
用量設定試験での上限濃度は、被験物質を培養液で溶解した場合には 10 mg/mL、有機溶媒を使用した場合には 1 mg/mL である。
用量設定試験では公比 10 とし、本試験では希釈段階をｎとして 10 のｎ乗根として計算する。例えば、3 段階希釈では 2.15(3√10)、6 段階希釈では 1.47(6√10)、12 段階希釈では 1.21(12√10)を用いる。
バリデーション試験では、用量設定試験の上限濃度で毒性が認められない場合、培養液で溶解する場合は 100 mg/mL(2 倍のストック液を使用)、DMSO を使用した場合には 2.5 mg/mL を上限濃度として再度用量設定試験を実施した。

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JaCVAM 急性毒性試験評価委員会

用量設定試験及び本試験
1）陽性対照物質（PC）
ラウリル硫酸ナトリウム（SLS）を陽性対照物質として、8 用量で濃度反応曲線を描く。多数の被験物質の試験を実施する際に陽性対照物質のプレートは単独で実施して良い。
2）溶媒対照物質（VC）
被験物質の溶解に有機溶媒を使用した場合には、VC にも被験物質と同じ濃度（0.5%）の有機溶媒を添加する。
3）細胞の播種および前培養
3T3 細胞は、96-well マイクロプレートに 2-3×103 個／100μL／well 播種後 24 時間培養し、NHK細胞は、1.6-2×103 個／125μL／well 播種後 48-72 時間培養する。
4）被験物質の添加
規定時間前培養し（3T3 細胞：24±2 時間、NHK 細胞：48－72 時間）、プレートを注意深くひっくり
返し培養液を除去後、滅菌したペーパータオルに押し付けて well に残った培養液を除去する。すぐに、37±1℃に温めた培養液を 3T3 細胞には 50 μL ずつ、NHK 細胞には 125μL ずつ添加する。それぞれの well に 2 倍濃度の被験物質が入った培養液を 3T3 細胞には 50 μL ずつ、NHK細胞には 125 μL ずつ添加する。添加後 48±0.5 時間培養する。1 用量当たりの well 数は N=6 とする。
5）測定
被験物質の暴露終了後、位相差顕微鏡で細胞を観察して、被験物質の毒性によって生じた細胞の形態学変化、細胞の播種エラーおよび細胞増殖の程度を記録する。（この記録は、細胞毒性の評価には使用しない）。その後、well の培養液を除去し、Dulbecco’s phosphate buffered saline（D-PBS）で洗浄後、NR 染色液(NR dye； 3T3: 25 μg/mL、 NHK: 33 μg/mL)を 250 μL 添加して 37℃、5% CO2 で 3 時間培養する。染色液を除去して D-PBS で洗浄後 100 μL の用時調製した NR 抽出液（水：エタノール：氷酢酸＝49：50：1）を添加しプレートシェイカーで 20－45 分間振盪
して NR を抽出する（BRD にはこの操作における温度の記載がない。室温における操作で十分であると判断した）。振盪後、プレートは少なくとも 5 分間放置する。測定は、NR 抽出液を添加してから 60 分以内に実施する。泡を取り除き、プレートリーダーで 540 nm±10 nm（OD 540nm）の吸光度をする（BRD には泡を取り除く方法について具体的な方法の記載がない。プレートを遠心し取り除く
方法が一般的と考える）。
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JaCVAM 急性毒性試験評価委員会

6）試験成立基準
試験成立基準（Test acceptance criteria）は各 Phase で変遷があるが、以下に Phase III で用いた試
験成立基準を記載する。
(1） PC として使用する SLS の IC50 値は、各研究室で得られたヒストリカルデータの平均値の 2.5 標準偏差（SD）の範囲に入っていること。
(2） VC は 96-well の 2 列目と 11 列目に設定するが、それぞれの列の OD 平均値の差が、全ての VC から算出した平均値から 15%以内であること。
(3） 細胞毒性率が 0%以上かつ生存率 50％未満のものが少なくとも一つ、細胞毒性率が50％以上かつ 10％未満のものが少なくても一つは存在すべきである。
(4） PC の用量相関のＲ2 値が Hill 式のモデルフィットに 0.85 以上の相関があること。
7）データ解析
生物学/科学的な判断により、評価に適していない well はデータ解析から除外可能である。
ブランクの OD540 値を差し引いた後、細胞生存率を VC の平均値に対する割合として算出する。計算には表計算ソフト（例：Microsoft EXCEL®）を使って計算してもよい。
IC50 値を計算するために統計学的ソフト（例：GraphPad Software PRISM®）を用いて、Hill 式の解析を行う。
8）初回投与量の決定
IC50 値（mM）を次の回帰式に代入して logLD50 値（mg/kg）を算出する。
LogLD50(mmol/kg) = 0.439 logIC50(mM) + 0.621(ICCVAM、2006a)

LogLD50 値を LD50 値に変換し、化合物の分子量を乗じて mg/kg 単位に変換する。
UDP 法の用量段階は、2000 mg/kg を上限とする試験では 5、50、300 及び 2000 mg/kg の 4 段階、5000 mg/kg を上限とする試験では、1.75、 5.5、 17.5、 55、 175、 550、1750 及び 5000 mg/kgの 8 段階である。ATC 法の用量段階は、2000mg/kg を上限とする試験では 5、50、300 及び 2000
mg/kg の 4 段階、5000 mg/kg を上限とする試験では 5、 50、 300、2000 及び 5000 mg/kg の 5段階である。動物に投与する用量は、上記回帰式で得られた LD50 値が含まれる用量段階より１段
階低い用量を開始用量とする。
分子量不明の化合物については、μg/mL で算出した IC50 値からは、以下の回帰式で LD50 値（mg/kg）を推測することができる。
LogLD50(mg/kg) = 0.372 logIC50(μg/mL) + 2.024

18（ICCVAM、2006a)

JaCVAM 急性毒性試験評価委員会

既知の適用限界ICCVAM のピアレビューパネルは、in vitro 試験の適用限界について以下のようにコメントしている。

１）溶解性、沈殿物及び揮発性を有する化合物
培養液、また有機溶媒を用いても溶解性が低く、50％の細胞毒性発現濃度が得ることができな
い化合物は in vitro 試験で評価することはできない。
培養液に添加後しばらくしてから結晶が析出する化合物では、正確な IC50 値を求めることはできない。
揮発性の化合物では、VC のｗell にコンタミが認められた。コンタミを防ぐために、well をフィルム状の Plate sealer で密封した試験も実施したが、有機溶媒では sealer に反応してしまうことから試験の実施が困難であった。
2）生物動力学測定（Biokinetic determination）
生体内に投与された化合物は、吸収、分布、代謝、排泄（ADME）の過程において生物学的影響を発現するが、in vitro の試験系では、これらが欠如している（ICCVAM 2001a）。したがって、invitro の試験結果を in vivo に外挿するには、ADME も考慮すべきである。
3）臓器特異的毒性
3T3 及び NHK 細胞を使用した NRU 試験では、肝、中枢神経、腎、心臓、肺及び造血器に対する特異的な毒性を評価することはできない。

JaCVAM 急性毒性代替法評価委員会では、本試験方法に関して以下のように意見をまとめた。
被験物質の用量段階について規定することはなく、ある程度自由度を持たすべきである。公比 3 程度とすることで、1 回の試験でも適切な IC50 値を求めることができるケースも多いはずである。このような場合は、本試験を実施する必要はない。
被験物質の溶媒は、培養液あるいは有機溶媒が推奨されているが、溶媒は被験物質の性質に応じて選択されるべきである。水溶系の溶媒には培養液１種類だけでなく、一般的に in vitro 試験で使用される水や生理食塩液の使用も可能とし、自由度をもたすべきである。
DMSO や ETOH などの有機溶媒に溶解した被験物質が、培養液に添加後に析出した場合の IC50値の取扱いについて特記する必要がある。
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JaCVAM 急性毒性試験評価委員会

被験物質の調製にガラス製試験管を使用することが記載されているが、試験管の材質は被験物質の物性に応じて選択すべきである。従って、被験物質の調製に使用する試験管はガラス製に限定するのではなく自由度をもたすべきである。
被験物質添加の際に、まず細胞へ培養液を添加し、次に 2 倍濃度の被験物質含有培養液を添加することになっているが、1 倍濃度の被験物質含有培養液の直接添加も認めるべきである。
NRU 法の陽性対照物質にラウリル硫酸ナトリウムが指定されているが、陽性対照物質は 1 種類だ
けでなく複数規定し、選択肢を広げるべきである。
陽性対照物質をテストするプレートは、被験物質をテストするプレートと別プレートで実施されているが、試験の成立および信頼性に関わる陽性対照の試験は、できる限り被験物質と同一プレート内で実施することが望ましい。
一つの用量につき６well(N=6)で実施することが指定されている。N 数と結果の信頼性が相関することは理解できるが、N=6 が必要かどうか考慮する必要がある。N=3-4 で十分な評価が可能である。
細胞から NR を抽出し測定するまでの時間が、1 時間以内と記載されている。一般的には 2〜3 時
間後でも問題ないと考える。また、細胞から NR を抽出する時間も 20-45 分と規定している。抽出から測定までの時間や NR 抽出時間などの各処理における時間の規定は、各施設で検討試験を実施し、試験精度が担保できる範囲で自由度をもたせるべきである。
本ガイドライン草案では、LD50 値の算出に Graphpad PRISMⓇが例として挙げられているが、本ガイドライン草案のプロトコルに適したソフトウエアを開発して自由に配布できると利便性が向上し、普及しやすい。

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JaCVAM 急性毒性試験評価委員会

3．バリデーションに用いられた物質の分類と妥当性
バリデーション試験の被験物質として 72 種類の参照化合物を選択した（下表参照）。

1,1,1-Trichloroethane

Diethyl phthalate

Phenobarbital

2-Propanol

DigoxinPhenol5-Aminosalicylic acid

Dimethylformamide

Phenylthiourea

Acetaminophen

Diquat dibromide*

Physostigmine

Acetonitrile

Disulfoton

Potassium cyanide

Acetylsalicylic acid

Endosulfan

Potassium I chloride

Aminopterin

Epinephrine bitartrate

Procainamide**

Amitriptyline HCl

Ethanol

Propanolol HCl

Arsenic III trioxide

Ethylene glycol

Propylparaben

Atropine sulfate*Fenpropathrin

Sodium arsenite

Boric acid

Gibberellic acid

Sodium chloride

BusulfanGlutethimide

Sodium dichromate dihydrate

Cadmium II chloride

GlycerolSodium hypochlorite

Caffeine

Haloperidol

Sodium I fluoride

Carbamazepine

Hexachlorophene

Sodium oxalate

Carbon tetrachloride

Lactic acid

Sodium selenate

Chloral hydrate

LindaneStrychnine

Chloramphenicol

Lithium I carbonate

Thallium I sulfate

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JaCVAM 急性毒性試験評価委員会

Citric acid

Meprobamate

Trichloroacetic acidColchicine

Mercury II chloride

Triethylenemelamine

Cupric sulfate 5H2O

Methanol

Triphenyltin hydroxide

Cycloheximide

Nicotine

Valproic acid

Dibutyl phthalate

Paraquat

Verapamil HCl

Dichlorvos

Parathion

Xylene*試験には一水和物を用いた。
**試験には塩酸塩を用いた。

参照化合物の選択基準は、1）GHS の急性経口毒性の分類（5 区分に加えて LD50 値＞5000 mg/kgの未分類化合物）にげっ歯類 LD50 値が 12 物質ずつ分類できること、2）構造と使用用途が広範囲に渡ること、3）ヒトの毒性データを備えたものであることとした。また、GHS 急性経口毒性の区分全体に分布するように物質を選択した。RC データベースに上げられている物質については ZEBETの RC millimole regression に合うものから選んだ。バリデーション試験で使用した 72 種類の参照化
合物数は十分な数であると ICCVAM ATWG、ICCVAM、ECVAM は判断した。
評価に用いた LD50 値は、OECD ガイドラインでラットを用いた試験が推奨されていること、RC
millimole regression の大部分がラットの LD50 値を使っており、そして大部分の急性経口全身毒性試験ではラットが用いられていることからラットが選ばれた。各物質の LD50 値は RC（LD50 値データの大部分は RTECS® [1983/84]）を優先とし、その他、RTECS®（2001、2002） Hazadous Substances
Data Bank を用いて調査した。選択した 72 種類の物質は RC をはじめとし、各種のデータベースに登録されており、1 物質で複数のデータベースにリストされているものもあった。
選択した参照化合物に含まれる RC 物質（58 物質）については RC millimole regression 全体と比較するとはずれ値の比率が高く、また、過小評価が 17 物質、過大評価が 5 物質と偏りが見られた。
被験物質情報としては分子量、分子構造、化学的分類、代謝活性化/不活性化、作用機序、脳血液関門の透過性などが調べられているが、分子の荷電と界面活性については情報が得られなかった。また、毒性の標的臓器や腐食性に関してもデータを検索した。72 種類の物質のうち 57 物質が有機化合物、15 物質が無機化合物であった。有機化合物にはヘテロサイクリック（14 物質）、カル
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JaCVAM 急性毒性試験評価委員会

ボン酸（14 物質）、アルコール（10 物質）などが多く、その他フェノール、硫黄化合物、アミン、有機
リン化合物などが含まれていた。無機化合物にはナトリウム化合物（6 物質）、塩素化合物（5 物質）などが多く、その他、砒素化合物、金属、カリウム化合物、硫黄化合物などが含まれていた。製品の種類、使用法としては医薬品 27 物質、農薬 17 物質で他に溶媒、食品添加物、殺菌剤/消毒剤などが含まれていた。
72 種類の物質の中で代謝により活性化するもしくは活性化が期待される物質は 22 物質で、代謝により毒性が減少する物質は 5 物質であった。NHK 細胞と 3T3 細胞は活性化能力がほとんどまたは全くないことが報告されている。
試験できなかった、または試験が難しかった物質は主に、毒性が低く IC50 値が得られない、揮発性がある、または溶解性が悪いことが原因であった（試験結果が得られなかった化合物 3T3；
Lithium I carbonate、Methanol、NHK；1,1,1-Trichloroethane、両細胞共通；Carbon tetrachloride、
Xylene）。
試験に使われた被験物質の情報の記載があり、コード化や配付も適切に行われた。
参照化合物に PAHs、触媒、単純なアルデヒド、ケトン、バイオサイド（殺生物剤）、混合物/製剤、植物毒などの天然化合物が含まれていない。ICVAM ピアレビューパネルは、特に、一般的な殺虫剤や家庭用品の評価が必要であるとした。
総じて、in vivo の急性経口毒性で作用様式や機序がはっきりしていないことから、in vitro のバリデーションのために広範囲に亘る標準物質を選択することが戦略的に難しいが、NRU 法は基礎的な細胞毒性を検出するので、選択された物質は reliability と accuracy を評価するには十分である。
JaCVAM 急性毒性代替法評価委員会としては、以下のように意見をまとめた。
バリデーション試験で使用された 72 種類の参照化合物についての選択基準、LD50 値、各種情報（GHS のクラス分け、構造、分類、使用用途等）、情報の由来について十分な記載がされていると判断する。
被験物質数や被験物質名称について文中、表中および Appendix で記載の一部が一致していな
かったが、全体の評価に影響を及ぼす内容ではないと判断した。
急性毒性試験の代替法が早急に求められているのは化粧品業界であるが、NRU 法で使用した 72
種類の化合物に化粧品原料は少ないことから、化粧品原料について本法が有効であるかについ23

JaCVAM 急性毒性試験評価委員会ては、評価ができないと JaCVAM 評価委員会は判断した。なお、化粧品業界は、初回投与量を設定する試験ではなく、動物試験を置換できる完全な代替法を求めている。
ICVAM ピアレヴューパネルが指摘したように、混合物や製品については回帰式から急性毒性の濃度を予測可能であるかについて試験されておらず、データが得られていないことから、これらの物質については開始濃度を予測することができるかどうかの判断はできないと JaCVAM 評価委員会
は判断した。
揮発性を有しているもの、溶解性が低いものなど試験が困難な物質について、試験をどのように実施するかに関する記載は見られなかった。被験物質の物性によって試験が適切に行えないと考えられる場合は、試験を行わない選択についての検討が必要であると JaCVAM 評価委員会は判断した。
参照化合物の選択は RC 全体に物質が亘るように考慮されているが、はずれ値の割合がこの評価方法の基礎となった RC millimole regression よりも多く、また、過小評価となった物質により多く偏りが認められているので、試験結果の評価時に考慮が必要であると JaCVAM 評価委員会は判断した。
使用した細胞は代謝活性化能力をほとんど保有していない。しかしながら、RC millimole regression
で使用されている in vitro データからは代謝活性化系が意図的にはずされており、バリデーション
試験と RC との精度の比較に関しては代謝活性化が含まれていないことは大きな問題とはならないと考える。代謝活性化により活性化または不活化される物質に関して評価が可能であるかどうか、別途考察が必要であると JaCVAM 評価委員会は判断した。

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JaCVAM 急性毒性試験評価委員会

4．試験法の正確性を評価するために用いられた参照化合物の in vivo 参照デー
タ
バリデーション試験の精度を評価するため、げっ歯類急性経口 LD50 値を収集した。LD50 値を求めるために新たな動物実験は行わなかった。各種データベースおよび文献から得られたデータのほとんどが GLP 非適用であり、データの質はよくない。野生のラット、4 週齢未満のラット、麻酔したラット、餌やカプセルで摂取したもの、LD50 値が範囲及びある数値以上として報告されているものを除外し、データの質の改善を行った。試験条件の記載がない場合、ラットは若い生体で一般的な種類、無麻酔、強制経口投与により得たデータとして扱った。
評価に使用した LD50 値（Reference LD50）は、各種クライテリアに合致した LD50 値が複数ある場合には、それらの幾何平均とし、ラットの経口のデータがない 3 物質については、マウスの急性経口
毒性の LD50 値から同様の作業によってデータを得た。Reference LD50 値を用い、各物質について
GHS に基づいて再度クラス分類をしたところ 53 物質が同じ分類、18 物質で LD50 値が高値となり、より毒性の低いクラスへ分類され、1 物質で LD50 値が低くなりより毒性の高いクラスへ分類された。
各物質につき得られた複数の LD50 値について、最大値と最小値の比でばらつきを調べた。72 種類の物質中 2 つ以上の LD50 値が得られたものは 62 物質でその比は平均で 4.3 となり、毒性の強い物質（LD50 値≤ 50 mg/kg）では毒性の弱い物質（LD50 値> 50 mg/kg）よりも比が大きくなる傾向があった。しかしながら、同一のプロトコルで試験した試験施設間でも数倍から 10 倍程度の変動が報告がされており、検索で得られたデータではラット種、性別、観察期間、LD50 値の計算方法などは異なっていることを考慮に入れると文献から得られた結果は評価に使用するのに妥当であると判断した。
RC との共通物質 58 物質について文献より得られた LD50 値と、RC の値を比較したところ、Spearman correlation analysis で対数変換した数値において p<0.0001 で rs = 0.97 と非常に高い相関を示した。
JaCVAM 急性毒性代替法評価委員会としては、以下のように意見をまとめた。
JaCVAM 評価委員会は文献からのデータの取捨選択については十分な記載がされていると判断した。
文中と表の数値、記載が一部一致していないが、全体の評価に影響を及ぼす内容ではないと
JaCVAM 評価委員会は判断した。

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JaCVAM 急性毒性試験評価委員会

全体として、文献から得られた LD50 値は信頼できるものであり、これらの値を用いた評価は可能であると JaCVAM 評価委員会は判断した。
RC、LD50 値を求めた参照化合物の中で、塩の形が異なる物質があったがその同等性に関する記載は認められなかった。したがって、JaCVAM 評価委員会としては、結果への影響が判断できなかった。
文献より得られた LD50 値のばらつきは全体的に LD50 値が 50 mg/kg を境にして低濃度の場合、比が大きい傾向があったが、今回最も比の大きかった物質（25.9 倍）の LD50 値は 329 mg/kg であり、ばらつきが物質固有の物性等に起因する可能性について否定できないと JaCVAM 評価委員会は判断する。
GHS 区分に変更があった物質 19 物質のうち、3 物質はデータがマウスからラットに変更されていたが、動物種を変更したことに対する影響の有無についての記載は認められない。そのため、
JaCVAM 評価委員会では、評価に対する影響についての判断はできなかった。
Reference LD50 値で再評価して GHS クラスが変更された 14 物質における文献値の LD50 値はその化合物が分類される GHS 区分に含まれていた。うち複数物質において LD50 値がほぼ GHS 区分のボーダー上にあり、LD50 値の文献値と GHS 区分に使用された元の数値が必ずしも著しく異なっていたことが原因で GHS クラスが変更された訳ではなかった。GHS 区分のボーダー上で区分変更がされた物質については考察が必要と JaCVAM 評価委員会は判断した。

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JaCVAM 急性毒性試験評価委員会

5．試験法のデータと結果の利用性
バリデーション試験は、2002 年 6 月から、2004 年 1 月に掛けて Bio Reliance Corporation、U.S.
Army Edgewood Chemical Biological Center (ECBC)、Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory (FAL)、Institute for in vitro Sciences (IIVS)の 4 つの施設が参加した。Bio Reliance は、参照化合物の入手、コード化、配布、溶解試験などの作業を行い、細胞毒性試験は ECBC、FAL 及び IIVS で実施した。
Bio Reliance、ECBC および IIVS は技術的な面の解決部分を除き GLP 適用下に実施した。FAL では GLP の精神に従い試験が実施されたが、個別の手順に対する QA のレビューは実施されなかった。各施設での評価と試験方法の改良、改良された方法に基づく評価、の 2 つ Phase を経て、最終化されたことが示されており、改良と最終化は問題ないと判断された。
Positive Control として用いた Sodium lauryl sulfate（SLS）の各施設の IC50 値が各 Phase で示されており、その結果について問題ないと判断した。
3T3 と NHK 細胞との細胞間比較では、種々の化合物において IC50 値に差が認められ、陽性対照の SLS でも 10 倍の差が認められた。バリデーション試験を実施した参照化合物の 85％は、この比が 0.1-10 の間にあった事を示している。
JaCVAM 急性毒性代替法評価委員会は以下のように意見をまとめた。
３T3 細胞と NHK 細胞の各細胞での IC５０値の差は、各化合物で 10 倍以内の差であるが、2 つの細胞系全体では、一定傾向を持つ差ではない。そのため、試験系全体では 100 倍のばらつきがあると判断される。
初回投与量の予測は、それぞれの細胞ごとに得られた化合物の IC50 値と動物の LD50 値からの相関式を用いるが、前述のようなばらつきが存在していても 2 つの細胞系を同様に使用できることの考察が不足していると判断した。
Aminopterin、hexachlorophene および digoxin では両細胞での IC50 値に 100 倍を超える差が認められた。これら極端な差が認められたものについてはそれぞれメカニズムなどについて考察が必要である。
BRD の 7 章では、試験手技の訓練がばらつきを小さくするために必要である事が述べられている。
そこで、陽性対照 SLS の施設間差については、施設間の手技の均一性についても考察する必要がある。
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JaCVAM 急性毒性試験評価委員会

6．試験方法の正確性
3T3 及び NHK NRU 法によりげっ歯類における LD50 値の予測性から正確性が検討された。本試験
方法は置き換えを意図しておらず、LD50 値の予測から初回投与量を決定し、使用動物削減を目的としている。動物へ投与する用量は、回帰直線から得られた LD50 値が含まれる GHS 区分より一段低い用量を用いるとしているため、動物実験で得られる LD50 値に近い予測がされた時、試験に使用する動物の苦痛を軽減することが期待できる（GHS 区分の毒性評価としては保守的となり、強毒性側に偏りのある解釈となる）。
質量換算の相関（mg/kg と mg/mL）から、分子量換算（mM/kg と mM）の相関に変更する事で、vivo
vitro 相関回帰式の相関係数 R2 値が増加し、予測性を向上させたことが示されており、妥当である。
また、質量換算の相関式も分子量不明の化合物、混合物については受け入れる事が出来るとしていることも妥当である。
質量換算データおよび分子量換算データでの正確性とはずれ値について検討されている。2 つの細胞でのそれぞれの IC50 値とラット LD50 値での直線回帰および、予測された LD50 値と、GHS での急性毒性分類の一致性を検討している。その結果、バリデーション参加施設全てのデータを合わせた解析の結果において、いずれの細胞でも類似した直線回帰係数が得られており問題はないと判断する（BRD Table 6-2）。
GHS 区分の予測性は、ラット LD50 値の分子量換算の相関では、3T3 NRU 法と NHK NRU 法でそれぞれ 31％（21/67）、29％（20/68）となった。GHS 区分の幅を±1 まで広げると 3T3 は 69%、NHKが 71%まで予測的中率は増加した。また、各 GHS 区分別に見ると、LD50 値が 300～2000 mg/kg のレンジがもっとも良好であった。一方、毒性の強い化合物、あるいは非常に毒性の弱い化合物の予測性がよくなかった（BRD Table 6-7）。
はずれ値を示した化合物について、物理化学的性質、溶解性、毒性メカニズムなどの分析が実施されている。沸点が 200℃を越えるもの、分子量が 400 を超えるものなどははずれ値を示す可能性が高いことが示された。また、溶解度から見ると、はずれ値を示した化合物において 8/22（3T3）、7/23（NHK）が溶解性の低いものであり、dibutyl phthalate を除き全て under predicted となった。
毒性メカニズムの面から、はずれ値を示した化合物を調査した結果 CNS と心臓への作用を示す化合物ではずれ値が認められた。

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JaCVAM 急性毒性試験評価委員会

バリデーションに参加した各施設の直性回帰（BRD より抜粋）

Table 3 はずれ値を示した化合物のまとめ（BRD より抜粋）
化合物名の後ろに記載されている（＋）は毒性が overpredict されたもの、（-）は毒性がunderpredict されたもの。

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JaCVAM 急性毒性試験評価委員会

はずれ値を示した化合物のまとめ-つづき-（BRD より抜粋）

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JaCVAM 急性毒性試験評価委員会

NRU 法による GHS カテゴリーの予測性（BRD より抜粋）

JaCVAM 急性毒性代替法評価委員会は以下のように意見をまとめた。
毒性の強い化合物の予測性は低く under prediction の傾向がある。結果的に選択された初回投与
量が致死量を超えている可能性が高く、投与した動物の苦痛軽減に寄与できないと判断する。
心臓及び CNS への薬理作用が想定される化合物については本試験による予測性が低いことが示されている。一方、心臓及び CNS 以外の毒性メカニズムについては網羅的に確認されていないことから、他にメカニズム面からはずれ値を示す化合物群が存在するかどうかの検討が不十分であると考える。今後集積されるデータを再度レビューして毒性メカニズムを幅広く考察する事を要望する。
はずれ値の考察のため物理化学的な性質については、分子量、沸点、pH、logKow などが調査されリスト化（BRD AppendixL1）されているが、情報はまだ十分整っていない。物理化学的パラメータによるクラス分けについても今後蓄積されるデータの追加解析により精度を向上させることが出来ると考えるので追加解析を要望する。現状では単独のパラメータを用いた評価にとどまっている事から、今後複合的な解析も必要である。

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JaCVAM 急性毒性試験評価委員会

7．試験方法の信頼性
3T3 および NHK NRU 法の信頼性は、施設間および施設内に関する再現性にて論ぜられた。施設内再現性は、同一の試験計画書にて同じ施設で繰り返し実施された試験結果にて評価された。施
設間再現性は、同一の試験計画書にて同一の参照化合物を用い異なる試験施設にて評価された結果を用いて評価された。再現性の評価には、3 つのすべての研究所において、3T3 細胞で 64 の参照化合物を、NHK 細胞で 68 の参照化合物をそれぞれ試験し、繰り返し IC50 値を算出した結果を用いた。3T3 NRU 法及び NHK NRU 法における IC50 値データの施設内再現性及び施設間再現性について、分散分析（ANOVA）、変動係数（CV）解析、研究所別 IC50 - LD50 回帰の比較及び研究所内平均 IC50 値の最大値／最小値比の比較により評価した。その結果、ばらつきは大きいものの再現性は概して NHK NRU 法の方が良好であった。
今回の試験にて評価された参照化合物は、3 章に記載されているように、GHS 区分全体にわたる
広範囲の領域から選択されている。
施設別 IC50-LD50 回帰による施設内類似性の評価を基とした再現性の解析では、3T3 NRU 法、
NHK NRU 法ともに 95％信頼限界内であった。ANOVA 解析では、3T3 NRU 法の 23 物質において、また NHK NRU 法の 6 物質で施設間に有意な差が認められた。施設内 CV は、3T3 NRU 法に
おいて 1-122％の範囲であり、NHK NRU 法では 1-129％の範囲であった。平均研究所内 CV は、
両 NRU 試験法とも 26％であったが、平均研究所間 CV は NHK ではより低い平均 CV（3T3 の 47％
に対し 28％）であった。施設間の CV は、3T3 試験法において 3-135％の範囲であり、NHK NRU
法では 1-91％の範囲であった。FAL における施設内平均 CV は 3T3 で 33％、NHK で 43％であり、最も高い値であった。再現性は概して NHK NRU 法の方が良好であった。
参照化合物の化学特性と施設間 CV の関係を示す解析を行った結果、化学構造、物理的形状、溶解性や揮発性は CV にほとんど影響を示さなかった。CV の大きさは GHS 急性毒性区分、IC50
値及び沸点に関連すると考えられた。施設間 CV の平均値は、最も毒性の強い GHS 分類の物質では特に 3T3 NRU 法において他の毒性分類の物質より大きかった。3T3 NRU 法において、施設間平均 CV は、LD50 値が 5 mg/kg 以下の区分で 72%、LD50 値が 5 mg/kg 超 50 mg/kg 以下の区
分では 78%であり、全体の施設間平均 CV は 47%であった。NHK NRU 法において、施設間平均
CV は、5 mg/kg 以下の区分で 37%、LD50 値が 5 mg/kg 超 50 mg/kg 以下の区分で 41%であり、全体の施設間 CV の平均値は 28％であった。Spearman の相関解析により IC50 値と施設間 CV との逆相関が、3T3 NRU 法（p=0.0015）及び NHK NRU 法（p=0.014）のいずれにおいても認められた。

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JaCVAM 急性毒性試験評価委員会

沸点と施設間 CV との正の相関（p=0.007）（即ち、沸点が高いほど CV 値も高い）が 3T3 NRU 法でみられたが、NHK NRU 法（p=0.809）では認められなかった。
3T3 NRU 法による陽性対照物質（SLS）の IC50 値の ANOVA 解析では、施設間で有意差（ｐ＝0.006）を示したが、施設内の各試験 Phase における比較では差がみられなかった（ｐ＞0.01）。しかし、施設間の CV は Phase Ia 及び Phase Ib において 6％、Phase II では 10％、PhaseIII では 2%と比較的小さな値であったが、施設内 CV は 5％から 24％であった。
NHK NRU 法の SLS の ANOVA 解析結果、施設間や施設内の各試験 Phase における比較で有意（ｐ＜0.001）であった。Phase Ib 後に FAL では培養法が変更され SLS の IC50 値が経時的に低下傾向にあったが、FAL の施設内 CV は依然として他の施設より高かった。各試験 Phase において、
NHK NRU 法による SLS の IC50 値の施設内 CV は 11％から 51％であるのに対し、3T3 NRU 法による SLS の施設間 CV は Phase III の 8％から PhaseⅠb の 39％であり、3T3 NRU 法に比較して NHK
NRU 法はばらつきが大きかった。
参照化合物の溶解に使用された溶媒は細胞培養液が 38 化合物、DMSO が 34 化合物であった。
参照化合物の溶媒選択の 3 つの施設間の一致性は 76％（55/72）であった。今回の試験の信頼性
保証評価を実施した施設とは別に、溶解性試験のみに参加した BioReliance は他の 3 施設に比較して高い溶解性を得ていた（溶解試験の方法は 2 章記載）。全ての施設で同様のプロトコルで溶解試験を実施しているが、常に同じ結果が得られてはいない。施設によっては、いくつかの参照化合物で IC50 値を算出することができていなかった。IC50 値が得られなかった化合物は、その大部分が有機溶媒であった（Lithium I carbonate、Methanol、1,1,1-Trichloroethane、Carbon tetrachloride、
Xylene）。施設内で行われる手技にばらつきがあるために、施設内および施設間の再現性に差異が生じるという問題点は、試験中にも取り上げられた。GLP を遵守し実施した二つの施設のデータは極めてよく一致していたことが示され、さらにその他の施設の成績に比べ、ばらつきやエラー発
生率がより低い傾向にあったことも示された。すべての施設に対し、共通のトレーニングを行った後では、施設間のバラツキは減少した。このことから、基本的手技訓練と、プロトコル遵守の必要性が示された。試験を実施する研究者は、細胞や培養法さらには適切な科学的方法について、十分にトレーニングを受けるべきである。
JaCVAM 急性毒性代替法評価委員会では以下のように意見をまとめた。
3T3 および NHK NRU 法による試験の信頼性の検討が、3 研究所にて実施された再現性の検討結果をもとに論じられた。試験結果には施設間差がみられており、施設間で生じるバラツキの理由に
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JaCVAM 急性毒性試験評価委員会

ついて考察することは、信頼性確保の上からも有益と考える。試験を実施した 3 施設のうち FAL に
おいては、他の施設と同様のトレーニングを実施したにもかかわらず他施設に比べ高い傾向がみられ、この点について更に考察を加えるべきである。
ANOVA 解析による施設内および施設間の再現性の有意差に関する検討では、サンプルサイズや研究施設内でのばらつきにもよるが、有意差が見られるのは、施設間の差が極めて少ない場合、または有意差が見られないのは、施設間に極めて大きなバラツキがある場合もある。この点も十分考慮した評価を行うことを考えると単純な ANOVA 解析のみの結果で評価を行うことには異論がある。
3T3 および NHK NRU 法による試験結果に関し、NHK NRU 法の再現性が良好であったが、この理由について更に考察を加えるべきである。
試験を実施する場合、試験手技の習熟と計画書遵守に関するトレーニングは最低限必要なことである。その上で化合物の IC50 値が算出できなかった理由を考えるとともに、試験に使用する溶媒の選択計画についても十分に協議すべきである。

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JaCVAM 急性毒性試験評価委員会

8．試験方法のデータの質
バリデーションが実施された施設のうち、ECBC と IIVS は GLP 適合施設、FAL は適合していない施設であった。参照化合物の調達や配布のための準備は、GLP 適合施設である BioReliance が実施した。FAL における試験は GLP 精神に従って実施された。FAL に対して、バリデーション試験マ
ネージメントチーム（SMT）は「記録すべき項目」を提示したことに対し、バリデーション試験開始以前から試験操作において記録を実施する指針を所有していた。様々な実験室の活動はワークブッ
ク、日誌に記録されており、その情報を SMT が確認できた。
GLP における逸脱または不履行の影響については、研究マネージメントチームにより究明された。
EXCEL®または PRISM®のテンプレートへのデータの移行に誤りが認められ、すべてのデータシートが再調査され、修正がなされた。そして正確なデータが統計解析に用いられた。逸脱または不履
行の多くは小さなものであり、データの質に影響はなかった。FAL は他の 2 施設に比べて、データ移行における誤りの割合、試験の受け入れ基準不適合の割合、施設内再現性の検討における変動係数が高い値を示した。しかし、GHS 分類での急性経口毒性の分類の予測能は他の施設と同程度であった。

試験法のデータの質に関わるデータ（BRD より抜粋）
項 目

ECBC

IIVS

FAL

GLP への適合

GLP

GLP

GLP の精神に従って実施

データ移行に誤りが認められた49/402

25/419171/513

21 (3T3)

22 (3T3)

30 (3T3)

8 (NHK)

10 (NHK)

32 (NHK)23 (3T3)

21 (3T3)

33 (3T3)

23 (NHK)

14 (NHK)

42 (NHK)

試験の割合#

受け入れ基準に適合しなかった
本試験の割合# (%)

変動係数$ (%)

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JaCVAM 急性毒性試験評価委員会

予測した GHS 区分が一致した割
合$ (%)

30 (3T3)

27 (3T3)

25 (3T3)

31 (NHK)

31 (NHK)

29 (NHK)#:第 3 フェーズにおけるデータ、$：全フェーズにおけるデータ

3 施設で行われたバリデーション試験におけるデータの質について検討した。ECBC と IIVS における GLP からの逸脱または不履行が小さなものであり、試験責任者または研究マネージメントチーム
が適切に対処していることから、これら 2 施設のデータの質について JaCVAM 評価委員会は問題無いと判断した。
FAL については GLP 適合施設でなく、他の 2 施設に比較して、測定やデータ収集の精度に関して劣っていたものの、テータ移行における誤りは修正され、結果のばらつきも GHS 分類での急性
経口毒性の分類の予測に影響を与えるレベルで無かったことから、JaCVAM 評価委員会はデータの質について問題無いと判断した。
GLP 適合施設である ECBC と IIVS の方が、GLP に適合していない FAL よりもバリデーション時のデータの質が高かったため、本試験が GLP 適用下での実施を必須とするか否かについて検討した。その結果、JaCVAM 評価委員会は、急性毒性試験における初回投与量の予測という目的を考慮し、試験成立条件のクライテリアの適合の確認、その他のクオリティチェックを充分にすれば、必ずしも GLP 適用下で実施する必要はないと判断した。
試験のデータの質を確認するために、ICCVAM の評価ではデータ移行における誤りの割合、試験の受け入れ基準不適合の割合、施設内再現性の検討における変動係数で検討した。JaCVAM 評
価委員会は､本試験を実際に使用する際には、あらかじめ各施設において参照化合物を使用した試験を実施すべきであり、その結果が妥当であるか否かを確認する事が試験のデータの質を確保するうえで必要であると判断し、推奨することとした。

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JaCVAM 急性毒性試験評価委員会

9．その他の試験方法の科学的な報告
種々の細胞を用いた in vitro NRU 細胞毒性試験は、げっ歯類の致死率との相関性が評価されている。
Peloux らと Fautrel らはラット初代培養肝細胞を用いて NRU 測定と腹腔内/静脈内、静脈内による毒性データとの相関性をそれぞれ r=0.877(n=25)、0.88(n=11)とし、良い相関を得ている。Roguet ら
は初代培養肝細胞への 21 時間被験物質暴露後に NRU 測定を行った結果、経口投与の LD50 値との間に有意な直線相関（p<0.001、r=0.80、n=28）を得ている。しかし、一方で、前出の Fautrel らは腹腔内投与による毒性データに対し相関係数は r=0.48(n=14)、経口投与に対し r=0.17(n=15)であり、有意な相関は認められなかったことも報告している。
3T3 および NHK NRU 試験は、急性毒性試験の開始用量の予測以外に、眼刺激性、ヒト致死血中濃度、in vivo の光毒性の予測に関して評価されている。このうち、3T3 NRU 試験は in vivo 光毒性物質を同定する試験として ECVAM によりバリデートされた。2004 年に OECD ガイドライン 432・in

vitro 3T3 NRU 光毒性試験として採択されている。
バリデートされていないが、in vitro 試験法による急性経口毒性の予測を試みた試験は多数報告されており、in vitro 細胞毒性データを利用することによる動物削減が評価されている。急性経口毒性試験の投与開始用量が、動物実験の LD50 値に等しいとした場合、UDP 法における動物削減の理論的な予測値は 25～40%の範囲であった。一方、NICEATM/ECVAM 研究で試験された参照化合物に対し、UDP 法のコンピューターシミュレーションモデルを用いて予測された動物数削減は
5.3～7.8%であった。Halle らは RC の in vitro 細胞毒性データの利用（回帰式を用いて予測した
LD50 値を開始用量として利用）により ATC 法の動物数削減が 32%に達することを見出した。
NICEATM/ECVAM のバリデーション研究で試験された参照化合物は RC millimole regression に対して大部分がはずれ値を取っており、ATC 法の平均動物削減は、コンピューターシミュレーショ
ンモデルで測定した場合、4.8～10.2%であった。
細胞毒性試験を用いるげっ歯類の急性毒性試験の予測に関連した他の報告について検討した。
NRU 試験の再現性については、光毒性のバリデーション研究をはじめ様々な検討が過去に行われており、適切な試験条件を設定することにより再現性を確保できると JaCVAM 評価委員会は推
察した。

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JaCVAM 急性毒性試験評価委員会

急性毒性の予測性については、細胞毒性と LD50 値の間ではずれる物質も多いものの、相関する報告が認められており、急性毒性試験の開始用量を予測し動物数の削減を図る利用方法は妥当なものと JaCVAM 評価委員会は判断した。
細胞毒性試験を用いるげっ歯類の急性毒性試験の予測に関連した他の報告を俯瞰し、JaCVAM
評価委員会は、動物数の削減効果の予測は報告により様々であるが、少なくとも削減を図れる方向であることが予測されており、3Rs の観点から望ましいと判断した。

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JaCVAM 急性毒性試験評価委員会

10．3Rs への関与
3T3 及び NHK NRU 試験は in vivo 急性毒性試験の代替（Replacement）にはなり得ないが、細胞毒性試験の結果に基づいて急性毒性試験の初回投与量を設定する場合、急性毒性試験における 使 用 動 物 数 の 削 減 （ Reduction ） 及 び 死 亡 動 物 の 削 減 や 動 物 へ の 苦 痛 ・ ス ト レ ス の 軽 減（Refinement）に繋がる可能性は考えられる。
使用動物数（Reduction）及び死亡動物数の削減（Refinement）について、コンピューターシミュレー
ションにより次のように検証された。
1)

UDP 法では、NRU 法の IC50 値から推定した初回投与量を用いた場合、固定の初回投与
量（175 mg/kg）を用いた場合と比較して、一試験当たり平均 0.49 匹（6.2%）～0.66 匹（7.0%）の動物数しか削減されないこと（Reduction）が示された。

2)

ATC 法では、NRU 法の IC50 値から推定した初回投与量を用いた場合、固定の初回投与
量（300 mg/kg）を用いた場合と比較して、一試験当たり平均 0.51 匹（4.8%）～1.09 匹（10.2%）の動物が削減されることが（Reduction）示された。

3)

低毒性毒性物質の場合（LD50 値：>2000 mg/kg 又は>5000 mg/kg）、それぞれ UDP 法では一試験当たり 1.28 匹（11.9%）～1.65 匹（16.7%）、ATC 法では一試験当たり 2.03 匹（17.1%）～3.33 匹（27.7%）の動物が削減されることが示され、このクラスの被験物質では比較的多数の使用動物の削減（Reduction）が期待できると判断した。

4)しかし、死亡動物数については、ATC 法で固定の初回投与量を用いるよりも一試験当たり
僅か 0.5～0.6 匹しか削減されず、NRU 法を用いることで死亡動物数の削減及び動物への苦痛やストレスの軽減（Refinement）を明確に示すことは困難である。

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JaCVAM 急性毒性試験評価委員会

コンピュータミュレーションによる使用動物数（BRD より抜粋）

NRU 法は、in vivo 急性毒性試験の Replacement を意図していないため、この点について JaCVAM
評価委員会は判断しなかった。
一定の基準を設けたシミュレーションにより Reduction について検証された結果から、低毒性物質（LD50 値：>2000 mg/kg 又は>5000 mg/kg）の場合では in vitro 試験の導入により一試験当たり 1～3 匹の使用動物数の削減が期待できると判断する。
Refinement に関して、死亡動物数の削減及び動物への苦痛やストレスの軽減を明確に示すことは困難と判断した。強毒性の化合物については初回投与量の予測性が低く、IC50 値に基づいた初回
投与量が動物実験の LD50 値を超えることが予測され、このクラスの化合物では動物への苦痛を与える可能性がある。

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JaCVAM 急性毒性試験評価委員会

11．試験方法の有用性と限界
3T3 及び NHK 細胞を用いた NRU 法は、急性毒性のハザード分類を予測するための代替法ではなく、急性経口投与毒性試験で用いられる UDP 法や ATC 法の初回投与用量を決定するための試験として有用である。
被験物質が低毒性(LD50 値>5000mg/kg)の場合は、NRU 法により使用動物数の削減が可能と考えられる。ATC 法の場合、実験条件によっては、死亡または安楽死に至る動物数の削減も可能と判断する。
3T3 NRU 法は、NHK NRU 法に比べて、実験者の安全面や費用面で優れており、一般的な試験として推奨できる。また再現性の点では劣るものの、動物数削減と正確性の点でわずかに上回っている。
他の同様な細胞毒性試験を利用する場合は、ICCVAM が推奨する 30 種類の参照化合物を用いて評価を行い、3T3 及び NHK NRU 法の精度と信頼性が同等以上であることが必要である。
一方、毒性発現機序（神経毒性や心毒性）によっては、NRU 法による初回投与量の評価は適切ではない。より正確なハザード分類を行うためには、将来的に作用機序や ADME（吸収、分布、代謝、排泄）を評価する in vitro 試験系の利用の可能性を考慮すべきである。今後、さらに混合物の評価も含めて、in vitro 及び in vivo 条件下における高品質のデータベース拡充を図り、in vitro 細胞毒性試験の有用性と限界を特徴づけることが必要と考えられる。今後実施するラット急性経口投与毒性試験では、死亡に至る機序と直接関係のある所見を集めるための標準的な手順を含めるべきであろう。
ただし、in vivo 試験は、データ収集のためだけに実施するべきではない。また、in vivo のデータベ
ースは、他の動物を使用しないアプローチ（構造活性相関のソフトウエア等）の有用性評価にも用いられるべきである。
ICCVAM の評価報告書には、3Rs の検証に用いられたコンピューターシミュレーションのアルゴリズ
ムや計算過程が記載されていなかったことから、シミュレーション方法の妥当性と削減可能な動物数について JaCVAM 評価委員会は判断できなかった。
NRU 法による初回投与量設定試験をガイドラインとして運用する場合には、その後に実施する急性毒性試験での使用動物数や死亡動物数のデータを蓄積して 3Rs について検証することが必要である。
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JaCVAM 急性毒性試験評価委員会

3T3 及び NHK NRU 法は、代謝活性化法が確立されていないため、代謝を介した毒性を評価するには適切ではない。
被験物質が生体内において吸収が低い場合、一般的に in vitro 毒性試験結果から in vivo への外挿は困難である。

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JaCVAM 急性毒性試験評価委員会

12．結論
ICCVAM で実施された細胞毒性試験による急性毒性試験の初回投与量設定試験の第三者評価
は、バリデーションに必要な項目、プロセス及びデータが検討されており、ICCVAM のバリデーショ
ン結果を受け入れることに問題はないと判断した。72 種類の化合物から得られた IC50 値と LD50 値の間に相関が認められ、低毒性の化合物については予測性があると考えられる。したがって、急性毒性試験の実施に際して、NRU 法は化合物の物性、類縁化合物の情報などと並んで、初回投与
量決定の一助になると考えられ、必要に応じて活用可能である。
細胞毒性試験による急性毒性試験の初回投与量設定試験は、72 種類の化合物から得られた IC50
値と LD50 値の相関性を根拠として一般化しているが、動物の死と細胞の死が類似するメカニズム的な根拠が不十分である。相関性は必ずしも因果関係を説明するものではない。急性毒性試験は、個体死またはそれに近い一般状態の変化をエンドポイントにしている。個体死は、呼吸または心臓の停止状態が観察された時点であり、また、苦痛、痙攣、チアノーゼなどの一般状態は安楽死を選択する人道的エンドポイントである。神経系、循環器、呼吸器などコアバッテリーに作用する化合物
は、細胞や組織間のシグナル伝達をかく乱することによって、強力な毒作用が急速に発現して個体死を招くが、細胞死によって発現するものではない。このような化合物の NRU 法による予測性は低く、IC50 値から予測される毒性は過小評価されている。NRU 法を適用することで本来の LD50 値からかけ離れた高用量を投与する可能性があるため、動物へ与える苦痛の低減、使用動物数削減に寄与するとは言い難い。
代謝活性化系が NRU 法の評価系からは除外されているため、活性代謝物が毒性を示す化合物
ついても評価はできないと考えられる。揮発性の化合物、難溶解性の化合物及び有色の化合物は本実験を適用することが困難である。このような物性を有する化合物を NRU 法で評価するのは科学的妥当性に欠ける。したがって、物性情報を基に、本試験の実施の可否を決定するオプションが必要である。
動物試験結果から得られる LD50 値が 4 倍から 14 倍のばらつきがあることを考慮すると IC50 値を正確に測定することは、必ずしも必要とは考えられない。また、複数の、しかも費用のかかる試験法によって IC50 値を正確に特定しても、費用の面から非生産的である。
動物数の削減効果は、コンピューターシミュレーションで確認しているのみであり、実際の試験においては、一般状態の観察から人道的エンドポイントによって安楽殺を選択する場合もありえる。
使用動物の削減には本当に繋がる試験であるかは、実際に使用した動物数が記載されている化合物の試験情報と、この試験で予測された初回投与量から予測される動物数を比較して検証する
43

JaCVAM 急性毒性試験評価委員会

ことが必要である。また、NRU 法をガイドラインに導入した場合には、試験情報を集計して動物数の削減が実現できているかについて検証する必要がある。
データの信頼性保証の面からは、NRU 法の目的は初回投与量を設定するための試験であり、GLP
適用の試験を実施する必要性はない。
その他、JaCVAM 急性毒性代替法評価委員会の議論において少数であるが、以下のような意見が提案された。
(1） In vitro 細胞毒性試験を実施してから急性毒性試験を実施した場合、従来どおり最初から急性毒性試験を実施した場合と比較して、経費、人的リソース、試験期間にどのくらいの違いがあるかを議論すべきである。
(2） 物性情報から生体への吸収が著しく低いと考えられる化合物では、最高用量である 2000
mg/kg の投与のみで試験が成立し、最小限の動物数で化合物の評価ができる場合がある。
従って、急性毒性試験の実施前に必ずしも細胞毒性試験を実施する必要はなく、１つの選択肢として考えるべきあり、定量的構造活性相関(QSAR)等の方法も使用できることを明記すべきである。
(3） 物性が明らかでない被験物質の GLP 試験実施は困難である。
(4） 本当に動物削減に寄与するのか？
(5） 初回投与量を固定して実施した動物を用いた試験に比較すれば、結果が良くなるのは当然ではないか。実際には、一匹の動物に予備的に投与して一般状態を観察した結果から、用量設定をして本試験の用量を決定していることがある。そのような情報を元に、300 mg/kg の固定用量ではない初回用量を設定するはずである。実際には、一匹の動物を使うことで、試験全体での動物数の削減がなされているのではないか。
(6） In vitro の試験を専門とする研究者にとっては、予測性の高い試験として完成させて導入することを望む。
(7） 現在、毒性の強い化合物と毒性の弱い化合物がどれくらいの割合で評価されているか、ということも重要である。この試験では毒性が強いものの予測率が低いので、その割合が高いのであれば、外れることが多い試験と考えられる。
(8） In vitro と in vivo の試験結果が一致しない理由の一つとして、ADME、特に代謝物が毒性に関与する場合が考えられる。CYP 発現細胞を用いた細胞毒性試験でもアフラトキシン等を除くと検出は困難であるが、それを考慮した試験系を構築することが予測性を高めるためには必
要である。
44

JaCVAM 急性毒性試験評価委員会

13．参考文献Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM),
National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM), National Institute of Environmental Health Sciences, National
Institutes of Health, U. S. Public Health Service, Department of Health and Human Services,
BACKGROUND REVIEW DOCUMENT, in vitro Cytotoxicity Test Methods for Estimating Acute
Oral Systemic Toxicity, NIH Publication No: 07-4518
Borenfreund E, Puerner JA. 1985. Toxicity determination in vitro by morphological alterations andneutral red absorption. Toxicol Lett 24:119-124.
Clemedson C, Barile FA, Ekwall Ba, Gon. Toxicol Lett 24:119-124. K, et al. 1998a. MEIC
evaluation of acute systemic toxicity. Part III. In vitro results from 16 additional methods used totest the first 30 reference chemicals and a comparative cytotoxicity analysis. Altern Lab Anim 26(suppl 1):93-129.
Clemedson C, Andersson M, Aoki Y, Barile FA, Bassi AM, Calleja MC, et al. 1998b. MEIC
evaluation of acute systemic toxicity. Part IV. In vitro results from 67 toxicity assays used to test
reference chemicals 31-50 and a comparative cytotoxicity analysis. Altern Lab Anim 26 (suppl
1):131-183.
Ekwall B. 1983. Screening of toxic compounds in mammalian cell cultures. Ann New YorkAcad Sci
407:64-77.
Halle W. 1998. Toxizitätsprüfungen in Zellkulturen für eine Vorhersage der akuten Toxizität (LD50)
zur Einsparung von Tierversuchen. Life Sciences/Lebenswissenschaften, Volume 1, Jülich:
Forschungszentrum Jülich.
Halle W. 2003. The Registry of Cytotoxicity: Toxicity testing in cell cultures to predict acutetoxicity (LD50) and to reduce testing in animals. Altern Lab Anim 31:89-198. (English translation ofHalle 1998)
ICCVAM. 2001a. Report Of The International Workshop On In vitro Methods For Assessing Acute
Systemic Toxicity. NIH Publication No. 01-4499. Research Triangle Park, NC:National Institute45

JaCVAM 急性毒性試験評価委員会

for Environmental Health Sciences. Available: http://iccvam.niehs.nih.gov/ [accessed 01
November 2006].
ICCVAM. 2001b. Guidance Document On Using In vitro Data To Estimate In vivo Starting Doses
For Acute Toxicity. NIH Publication No. 01-4500. Research Triangle Park, NC: National Institutefor Environmental Health Sciences. Available:http://iccvam.niehs.nih.gov/ [accessed 01
November 2006].

ガイドライン
OECD. Guideline for Testing of Chemicals, 425, Acute Oral Toxicity – Up-and-Down Procedure.
OECD. Guideline for Testing of Chemicals, 420, Acute Oral Toxicity – Fixed Dose Method.
OECD. Guideline ｆor Testing of Chemicals, 423, Acute Oral Toxicity – Acute Toxic Class
Method.
OECD Environmental Health and Safety Publications Series on Testing and Assessment No. 19
Guidance Document on The Recognition, Assessment, and Use of Clinical Signs as Humane
Endpoints for Experimental Animals Used in Safety Evaluation

46

NIH Publication No: 07-4518

BACKGROUND REVIEW DOCUMENTIn Vitro Cytotoxicity Test Methods for Estimating
Acute Oral Systemic Toxicity

Volume 1 of 2
Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)
National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)
National Institute of Environmental Health Sciences
National Institutes of Health
U. S. Public Health Service
Department of Health and Human Services

In Vitro Cytotoxicity Test Methods
for Estimating Acute Oral Systemic Toxicity
Background Review Document
Volume 1 of 2
Prepared by
The National Toxicology Program (NTP) Interagency Centerfor the Evaluation of Alternative Toxicological Methods (NICEATM)

National Institute of Environmental Health Sciences (NIEHS)
P.O. Box 12233
Mail Drop: EC-17
Research Triangle Park, NC 27709

November 2006
NIH Publication No. 07-4518
National Institute of Environmental Health Sciences
National Institutes of Health
U.S. Public Health Service
Department of Health and Human Services

This document is available electronically at:
http://iccvam.niehs.nih.gov/methods/invitro.htm

Printed: 11/30/2006

In Vitro Cytotoxicity Test Methods BRD Table of Contents

November 2006

TABLE OF CONTENTSAPPENDICES.................................................................................................................. xiii
LIST OF FIGURES .......................................................................................................... xvi
LIST OF TABLES...........................................................................................................xvii
LIST OF ACRONYMS AND ABBREVIATIONS ........................................................... xx
ACKNOWLEDGMENTS................................................................................................ xxv
PREFACE........................................................................................................................xxix
EXECUTIVE SUMMARY............................................................................................ xxxiii
1.0

INTRODUCTION AND RATIONALE FOR THE USE OF IN VITRO
NEUTRAL RED UPTAKE CYTOTOXICITY TEST METHODS TO
PREDICT STARTING DOSES FOR IN VIVO ACUTE ORALTOXICITY TESTING .........................................................................................1-3
1.1

Historical Background and Rationale for the Use of In Vitro
Cytotoxicity Assays to Predict Starting Doses for Rodent Acute
Oral Toxicity Tests....................................................................................1-4
1.1.1
The Multicentre Evaluation of In Vitro Cytotoxicity (MEIC)
Program........................................................................................1-4
1.1.2
An International Evaluation of Selected In Vitro Toxicity
Test Systems for Predicting Acute Systemic Toxicity ...................1-6
1.1.3
The Registry of Cytotoxicity (RC) ................................................1-7
1.1.4
The ZEBET Initiative to Reduce Animal Use ...............................1-9
1.1.5
The International Workshop on In Vitro Methods forAssessing Acute Systemic Toxicity ...........................................1-10
1.1.6
The NICEATM/ECVAM In Vitro NRU Cytotoxicity Study .......1-11
1.1.6.1
Study Design .............................................................1-12

1.2

Regulatory Rationale and Applicability for the Use of In Vitro
Cytotoxicity Test Methods to Predict Starting Doses for Acute
Oral Toxicity Testing in Rodents............................................................1-14
1.2.1
Current Regulatory Testing Requirements for Acute Oral
Toxicity ......................................................................................1-14
1.2.1.1
Test Methods for Assessing Acute Oral Toxicity ........1-16
1.2.2
Intended Regulatory Uses for In Vitro Cytotoxicity Test
Methods......................................................................................1-16
1.2.3
Similarities and Differences in the Endpoints of In Vitro
Cytotoxicity Test Methods and Rodent Acute Oral Toxicity
Test Methods ..............................................................................1-17
1.2.4
Use of In Vitro Cytotoxicity Test Methods in the Overall
Strategy of Hazard Assessment ...................................................1-18i

In Vitro Cytotoxicity Test Methods BRD Table of Contents

1.3

2.0

November 2006

Scientific Basis for the In Vitro NRU Test Methods .............................. 1-18
1.3.1
Purpose and Mechanistic Basis of In Vitro NRU Test
Methods ..................................................................................... 1-19
1.3.2
Similarities and Differences in the Modes/Mechanisms of
Action for In Vitro NRU Test Methods Compared with theSpecies of Interest ...................................................................... 1-20
1.3.3
Range of Substances Amenable to the In Vitro NRU Test
Methods ..................................................................................... 1-20

TEST METHOD PROTOCOL COMPONENTS OF THE 3T3 AND NHK
IN VITRO NRU TEST METHODS .....................................................................2-3
2.1

Basis for Selection of In Vitro NRU Cytotoxicity Test Methods..............2-4
2.1.1
Guidance Document Rationale for Selection of In Vitro NRU
Cytotoxicity Test Methods............................................................2-4
2.1.2
Guidance Document Rationale for Selection of Cell Types...........2-4

2.2

Overview of the 3T3 and NHK NRU Test Methods ................................2-5
2.2.1
The 3T3 NRU Test Method ..........................................................2-6
2.2.1.1
Initiating and Subculturing 3T3 Cells..........................2-6
2.2.1.2
Preparation of Cells for 96-well Plate Assays .............2-8
2.2.1.3
Reference Substance Application.................................2-8
2.2.2
The NHK NRU Test Method .......................................................2-8
2.2.2.1
Initiating and Subculturing NHK Cells........................2-8
2.2.2.2
Preparation of Cells for 96-well Plate Assays .............2-8
2.2.2.3
Reference Substance Application.................................2-9
2.2.3
Measurement of NRU in the 3T3 and NHK Test Methods ............2-9

2.3

Descriptions and Rationales of the 3T3 and NHK NRU Test
Methods.....................................................................................................2-9
2.3.1
Materials, Equipment, and Supplies..............................................2-9
2.3.1.1
3T3 Cells.....................................................................2-9
2.3.1.2
NHK Cells................................................................. 2-10
2.3.1.3
Tissue Culture Materials and Supplies ...................... 2-10
2.3.1.4
Cell Culture Materials .............................................. 2-10
2.3.1.5
Equipment................................................................. 2-10
2.3.1.6
Culture Medium ........................................................ 2-11
2.3.2
Reference Substance Concentrations/Dose Selection .................. 2-12
2.3.2.1
Range Finder Test..................................................... 2-12
2.3.2.2
Definitive Test ........................................................... 2-12
2.3.3
NRU Endpoints Measured .......................................................... 2-13
2.3.3.1
NRU and Measurement ............................................. 2-13
2.3.3.2
Determination of IC50, IC20, and IC80 Values .............. 2-13
2.3.4
Duration of Reference Substance Exposure ................................ 2-14
2.3.5
Known Limits of Use ................................................................. 2-14
2.3.5.1
Solubility/Precipitation/Volatility.............................. 2-14
2.3.5.2
Biokinetic Determinations ......................................... 2-15
2.3.5.3
Organ-Specific Toxicity............................................. 2-15

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In Vitro Cytotoxicity Test Methods BRD Table of Contents

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2.3.5.4

2.3.6
2.3.7

2.3.8
2.3.9
2.3.102.3.11
2.3.122.3.132.3.14The Role of Cytotoxicity Tests in an In Vitro
Battery Approach for Possible Replacement of In
Vivo Acute Toxicity Testing .......................................2-15
Basis of the Response Assessed ..................................................2-16
Appropriate Positive, Vehicle, and Negative Controls.................2-16
2.3.7.1
Positive Control ........................................................2-16
2.3.7.2
Vehicle Control .........................................................2-16
2.3.7.3
Negative Control .......................................................2-17
Acceptable Ranges of Control Responses ...................................2-17
2.3.8.1
Vehicle Controls as a Quality Control Tool ...............2-18
Nature of Experimental Data Collected.......................................2-18
2.3.9.1
NRU OD Measurements ............................................2-18
2.3.9.2
Information and Data Collected ................................2-18
Data Storage Media ....................................................................2-19
Measures of Variability ..............................................................2-19
Methods for Analyzing NRU Data..............................................2-20
Decision Criteria for Classification of Reference Substances ......2-20
Information and Data Included in the Test Report.......................2-20

2.4

Proprietary Components of the In Vitro NRU Test Methods................2-22

2.5

Basis for the Number of Replicate and Repeat Experiments for the
3T3 and NHK NRU Test Methods..........................................................2-22

2.6

Basis for Modifications to the 3T3 and NHK NRU Test Method
Protocols ..................................................................................................2-22
2.6.1
Phase Ia: Laboratory Evaluation Phase .......................................2-22
2.6.1.1
NR Dye Crystals........................................................2-23
2.6.1.2
3T3 Cell Growth........................................................2-23
2.6.1.3
NHK Cell Growth......................................................2-23
2.6.1.4
Vehicle Control OD Limits ........................................2-24
2.6.1.5
Precipitate Formation ...............................................2-24
2.6.1.6
Dilution Factor .........................................................2-24
2.6.1.7
Test Acceptance Criteria ...........................................2-25
2.6.2
Phase Ib: Laboratory Evaluation Phase .......................................2-25
2.6.2.1
NR Crystal Formation ...............................................2-25
2.6.2.2
Heating of Reference Substance Solutions .................2-26
2.6.2.3
Growth of Untreated Cells.........................................2-27
2.6.2.4
Correction of Reference Substance OD Values ..........2-27
2.6.2.5
Laboratory Error Rates .............................................2-28
2.6.2.6
Resultant Protocol Changes for Phase II ...................2-28
2.6.2.7
Test Acceptance Criteria ...........................................2-29
2.6.3
Phase II: Laboratory Qualification Phase ....................................2-29
2.6.3.1
Testing of Volatile Reference Substances ...................2-29
2.6.3.2
Atypical Concentration-Responses ............................2-31
2.6.3.3
Hill Function.............................................................2-33
2.6.3.4
Insoluble Reference Substances .................................2-33
2.6.3.5
Inadequate Cell Growth in NHK Medium..................2-34
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In Vitro Cytotoxicity Test Methods BRD Table of Contents

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2.6.3.6

Performance Standards for Media to Support
NHK Growth............................................................. 2-34
2.6.3.7
Test Acceptance Criteria for Phase II........................ 2-35
2.6.4 Phase III: Laboratory Testing Phase .............................................. 2-35
2.6.4.1
Required Cytotoxicity Values .................................... 2-35
2.6.4.2
Revisions to Data Analysis Procedures ..................... 2-35

3.0

2.7

Differences Between the 3T3 and NHK NRU Protocols for the
Validation Study and the Guidance Document Standard Protocols...... 2-36

2.8

Overview of the Solubility Protocol........................................................ 2-37

2.9

Basis of the Solubility Protocol............................................................... 2-38
2.9.1
Initial Solubility Protocol Development...................................... 2-38
2.9.2
Basis for Modification of the Phase II Protocol........................... 2-39

2.10

Components of the Solubility Protocol................................................... 2-40
2.10.1 Medium, Supplies, and Equipment Required .............................. 2-40
2.10.1.1 Medium and Chemical Supplies ................................ 2-40
2.10.1.2 Equipment................................................................. 2-40
2.10.1.3 Procedures................................................................ 2-40
2.10.2 Data Collection........................................................................... 2-41
2.10.3 Variability in Solubility Measurements....................................... 2-41
2.10.4 Solubility Issues During the Testing of the Reference
Substances.................................................................................. 2-41
2.10.5 Analysis of Solubility Data......................................................... 2-41

2.11

Summary ................................................................................................. 2-42

REFERENCE SUBSTANCES USED FOR VALIDATION OF THE 3T3
AND NHK NRU TEST METHODS ....................................................................3-3
3.1

Rationale for the 72 Reference Substances Selected for Testing ............3-3
3.1.1
Reference Substance Selection Criteria.........................................3-3
3.1.2
Candidate Reference Substances...................................................3-4
3.1.3
Selection of Reference Substances for Testing..............................3-5

3.2

Characteristics of the Selected Reference Substances .............................3-6
3.2.1
Source Databases Represented by the Selected Reference
Substances....................................................................................3-6
3.2.2
Chemical Classes Represented by the Selected Reference
Substances.................................................................................. 3-28
3.2.3
Product/Use Classes Represented by the Selected Reference
Substances.................................................................................. 3-28
3.2.4
Toxicological Characteristics of the Selected Reference
Substances.................................................................................. 3-28
3.2.4.1
Corrosivity ................................................................ 3-28
3.2.4.2
Toxicity Targets ........................................................ 3-32
3.2.4.3
Metabolism ............................................................... 3-32
3.2.5
Selection of Reference Substances for Testing in Phases Ib
and II.......................................................................................... 3-32
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In Vitro Cytotoxicity Test Methods BRD Table of Contents

3.2.6

4.0

5.0

November 2006

Unsuitable and Challenging Reference Substances .....................3-34

3.3

Reference Substance Procurement, Coding, and Distribution..............3-34
3.3.1
Exceptions..................................................................................3-35

3.4Reference Substances Recommended by the Guidance Document........3-35

3.5

Summary .................................................................................................3-36

RODENT ACUTE ORAL LD50 REFERENCE VALUES USED TO
ASSESS THE ACCURACY OF THE 3T3 AND NHK NRU TESTMETHODS ...........................................................................................................4-3
4.1

Methods Used to Obtain Rodent Acute Oral LD50 Reference
Values ........................................................................................................4-3
4.1.1
Identification of Candidate Rodent Acute Oral LD50
Reference Data .............................................................................4-3
4.1.2
Criteria Used to Select Candidate Rodent Acute Oral Data for
Determination of LD50 Reference Values......................................4-5
4.1.2.1
Final Exclusion Criteria ..............................................4-6
4.1.2.2
Assumptions Regarding Materials, Animals, andMethods ......................................................................4-6
4.1.2.3
Calculation Reference LD50 Values .............................4-7
4.1.2.4
Use of Rat and Mouse Data.........................................4-7

4.2

Final Rodent Acute Oral LD50 Reference Values ....................................4-7

4.3

Relevant Toxicity Information for Humans.............................................4-8

4.4

Accuracy and Reliability of the Rodent Acute Oral LD50 Reference
Values ......................................................................................................4-13
4.4.1
Variability Among the Acceptable LD50 Values..........................4-13
4.4.2
Comparison of Rodent Acute Oral LD50 Reference Values
with the Corresponding RC LD50 Values ...................................4-14
4.4.3
Comparison of the Variability Among Acceptable LD50
Values to Those Obtained in Other Studies .................................4-15

4.5

Summary .................................................................................................4-16

3T3 AND NHK NRU TEST METHOD DATA AND RESULTS .......................5-3
5.1

Study Timeline and Participating Laboratories ......................................5-3
5.1.1
Statements of Work (SOW) and Protocols ....................................5-3
5.1.2
Study Timeline .............................................................................5-4
5.1.3
Participating Laboratories.............................................................5-4

5.2

Coded Reference Substances and GLP Guidelines..................................5-5
5.2.1
Coded Reference Substances ........................................................5-5
5.2.2
Lot-to-Lot Consistency of Reference Substances ..........................5-5
5.2.3
Adherence to GLP Guidelines.......................................................5-5

5.3

3T3 and NHK NRU Test Method Protocols.............................................5-5
5.3.1
Phase Ia: Laboratory Evaluation Phase .........................................5-6v

In Vitro Cytotoxicity Test Methods BRD Table of Contents

5.3.2
5.3.3
5.3.4

November 2006

5.3.1.1
Protocol Changes and the Effect on the Data ..............5-6
Phase Ib: Laboratory Evaluation Phase.........................................5-6
5.3.2.1
Protocol Changes and the Effect on the Data ..............5-7
Phase II: Laboratory Qualification Phase ......................................5-7
5.3.3.1
Protocol Changes and the Effect on the Data ..............5-8
Phase III: Main Validation Phase..................................................5-9
5.3.4.1
Protocol Changes and the Effect on the Data ..............5-9

5.4

Data Used to Evaluate Test Method Accuracy and Reliability ............. 5-10
5.4.1
PC Data...................................................................................... 5-10
5.4.1.1
Phase Ib PC Data Acceptance Limits ........................ 5-12
5.4.1.2
Phase II PC Data Acceptance Limits......................... 5-12
5.4.1.3
Phase III PC Data Acceptance Limits ....................... 5-12
5.4.2
Reference Substance Data .......................................................... 5-12

5.5

Statistical Approaches to the Evaluation of 3T3 and NHK Data.......... 5-26
5.5.1
Statistical Analyses for Phase Ia Data......................................... 5-27
5.5.1.1
Outlier Determination for Replicate Well
Concentration Data................................................... 5-27
5.5.1.2
Curve Fit Criteria ..................................................... 5-27
5.5.1.3
Reproducibility Analyses for PC IC50 Values............. 5-27
5.5.2
Statistical Analyses for Phase Ib Data......................................... 5-28
5.5.2.1
Outlier Determination for Replicate Well
Concentration Data................................................... 5-28
5.5.2.2
Reproducibility Analyses of the Reference
Substance IC50 Values ............................................... 5-28
5.5.3
Statistical Analyses for Phase II Data ......................................... 5-28
5.5.3.1
Outlier Determination for Replicate Well
Concentration Data................................................... 5-28
5.5.3.2
Reproducibility Analyses of the Reference
Substance IC50 Values ............................................... 5-28
5.5.3.3
Comparison of 3T3 and NHK Test Results with
the RC Millimole Regression ..................................... 5-29
5.5.4
Statistical Analyses for Phase III Data ........................................ 5-29
5.5.4.1
Outlier Determination for Replicate Well
Concentration Data................................................... 5-29
5.5.4.2
Reproducibility Analyses of the PC IC50 Data ........... 5-29
5.5.4.3
Reproducibility Analyses of the Reference
Substance
IC50 Values................................................................ 5-30
5.5.4.4
Comparison of 3T3 and NHK Test Results with
the RC Millimole Regression ..................................... 5-31
5.5.5
Summary of the Data Used for Statistical Analyses .................... 5-32

5.6

Summary of NRU Test Results............................................................... 5-32

5.7

Availability of Data ................................................................................. 5-38

5.8

Solubility Test Results ............................................................................ 5-38

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5.8.1
5.8.2
5.9
6.0

November 2006

Solubility Data............................................................................5-43
Solubility and Volatility Effects in the Cytotoxicity Tests...........5-43

Summary .................................................................................................5-44

ACCURACY OF THE 3T3 AND NHK NRU TEST METHODS ......................6-3
6.1

Accuracy of the 3T3 and NHK NRU Test Methods for Predicting
Rodent Acute Oral Toxicity......................................................................6-5
6.1.1
Linear Regression Analyses for the Prediction of Rat Acute
Oral LD50 Values from In Vitro IC50 Values..................................6-5
6.1.2
Comparison of Combined-Laboratory 3T3 and NHK
Regressions to the RC Millimole Regression ................................6-8

6.2

Analysis of Outlier Substances for the RC Millimole Regression ...........6-9
6.2.1
Identification of Outlier Substances ............................................6-10
6.2.2
Evaluation of Outlier Substances ................................................6-12
6.2.2.1
Physical Characteristics............................................6-12
6.2.2.2
Chemical Class .........................................................6-12
6.2.2.3
Solubility...................................................................6-13
6.2.2.4
Metabolism................................................................6-13
6.2.2.5
Mechanism of Toxicity...............................................6-13

6.3

Improving the Prediction of In Vivo Rat Oral LD50 Values from In
Vitro IC50 Data .........................................................................................6-17
6.3.1
The RC Rat-Only Millimole Regression .....................................6-17
6.3.2
The RC Rat-Only Weight Regression .........................................6-17

6.4

Accuracy of the 3T3 and NHK NRU Test Methods for Predicting
GHS Acute Oral Toxicity Categories .....................................................6-18
6.4.1
Prediction of GHS Acute Oral Toxicity Category by the RC
IC50 Values Using the RC Millimole Regression.........................6-20
6.4.2
Prediction of GHS Acute Oral Toxicity Category by the 3T3
and NHK NRU Test Methods Using the RC Rat-Only
Millimole Regression..................................................................6-22
6.4.2.1
In Vitro – In Vivo Concordance Using the RC
Rat-Only Millimole Regression..................................6-22
6.4.2.2
Discordant Substances in the Prediction of GHS
Acute Oral Toxicity Category by the 3T3 andNHK NRU Test Methods and the RC Rat-Only
Millimole Regression.................................................6-24
6.4.3
Prediction of GHS Acute Oral Toxicity Category by the 3T3
and NHK NRU Test Methods Using the RC Rat-Only
Weight Regression......................................................................6-24
6.4.3.1
In Vitro – In Vivo Concordance Using the RC
Rat-Only Weight Regression......................................6-25
6.4.3.2
Discordant Substances in the Prediction of GHS
Acute Oral Toxicity Category by the 3T3 andNHK NRU Test Methods and the RC Rat-Only
Weight Regression.....................................................6-27
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6.4.4

7.0

November 2006

Summary of the Regressions Evaluated ...................................... 6-27

6.5

Correlation of NRU Concentration-Response Slope with Rat
Lethality Dose-Response Slope............................................................... 6-28

6.6

Strengths and Limitations of the Use of In Vitro Cytotoxicity Test
Methods with the IC50–LD50 Regressions for Prediction of Rodent
Acute Oral Toxicity................................................................................. 6-31
6.6.1
In Vitro Cytotoxicity Methods .................................................... 6-31
6.6.2
Use of Mole-Based vs. Weight-Based Regressions for thePrediction of Toxicity for Low and High Molecular Weight
Substances.................................................................................. 6-31

6.7

Salient Issues of Data Interpretation...................................................... 6-34

6.8

Comparison of NRU Test Results to Established PerformanceStandards ................................................................................................ 6-34

6.9

Summary ................................................................................................. 6-40

RELIABILITY OF THE 3T3 AND NHK TEST METHODS ............................7-3
7.1

Reference Substances Used to Determine the Reliability of the 3T3
and NHK Test Methods ............................................................................7-3

7.2

Reproducibility Analyses for the 3T3 and NHK Test Methods ..............7-5
7.2.1
Comparison of Laboratory-Specific IC50-LD50 Linear
Regression Analyses to the Mean Laboratory Regression .............7-5
7.2.2
ANOVA Results for the 3T3 and NHK NRU Test Methods .........7-5
7.2.2.1
Differences Among the IC50 Values inLaboratories Using the 3T3 NRU Test Method............7-5
7.2.2.2
Differences Among the IC50 Values inLaboratories Using the NHK NRU Test Method........ 7-21
7.2.3
CV Results for the 3T3 and NHK NRU Test Methods ................ 7-21
7.2.3.1
Reproducibility of Intralaboratory CV Values ........... 7-21
7.2.3.2
Reproducibility of Interlaboratory CV Values ........... 7-34
7.2.3.3
Variation of CV with Chemical Property ................... 7-34
7.2.3.4
Results of the Intralaboratory CV Analysis................ 7-35
7.2.3.5
Results of the Interlaboratory CV Analysis ................ 7-37
7.2.4
Comparison of Maximum to Minimum IC50 Values Using
Laboratory Means ...................................................................... 7-39
7.2.5
Comparison of Maximum:Minimum IC50 Ratios with the
Maximum:Minimum LD50 Ratios............................................... 7-40
7.2.6
Normalization of Reference Substance IC50 Values Using
SLS IC50 Values ......................................................................... 7-42

7.3

Historical Positive Control (PC) Data ................................................... 7-44
7.3.1
ANOVA and Linear Regression Results for the 3T3 NRU
Test Method .............................................................................. 7-46
7.3.1.1
Variation of the SLS IC50 Values with Time ............... 7-46
7.3.1.2
Comparison of SLS IC50 Values Among theLaboratories ............................................................. 7-46
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7.3.2

8.0

9.0

November 2006

ANOVA and Linear Regression Results for the NHK NRU
Test Method ...............................................................................7-48
7.3.2.1
Variation of SLS IC50 Values with Time.....................7-48
7.3.2.2
Comparison of SLS IC50 Values Among theLaboratories..............................................................7-48

7.4

Laboratory Concordance for Solvent Selection .....................................7-50

7.5

Summary .................................................................................................7-52

3T3 AND NHK NRU TEST METHOD DATA QUALITY ................................8-3
8.1

Compliance with Good Laboratory Practice Regulations.......................8-3
8.1.1
Guidelines Followed for Cytotoxicity Testing...............................8-3
8.1.1.1
Good Laboratory Practices .........................................8-3
8.1.1.2
Spirit of GLP ...............................................................8-3
8.1.1.3
Good Cell Culture Practices (GCCP)..........................8-4
8.1.2
Quality Assurance (QA) for NRU Cytotoxicity Test Data.............8-4
8.1.2.1
Coded Reference Substances .......................................8-4
8.1.2.2
Solubility Testing and Data Review .............................8-5
8.1.2.3
NRU Cytotoxicity Test Tallies......................................8-5
8.1.3
Guidelines Followed for Rodent Acute Oral LD50 Data
Collection .....................................................................................8-6
8.1.3.1
Rodent Acute Oral LD50 Values Used in theRegistry of Cytotoxicity (RC).......................................8-6
8.1.3.2
Rodent Acute Oral LD50 Values Collected by
NICEATM from Other Sources ....................................8-6

8.2

Results of Data Quality Audits .................................................................8-6
8.2.1
QA Statements..............................................................................8-6
8.2.2
QA Statements from the Laboratories ...........................................8-7
8.2.2.1
BioReliance QA Statements .........................................8-7
8.2.2.2
FAL QA Statements .....................................................8-7
8.2.2.3
ECBC QA Statements ..................................................8-7
8.2.2.4
IIVS QA Statements .....................................................8-8
8.2.2.5
Other QA Information .................................................8-8

8.3

Effect of Deviations or Non-compliance with GLPs ................................8-9
8.3.1
Laboratory Error Rates .................................................................8-9
8.3.2
Failure Rates for Definitive and PC Tests ...................................8-10
8.3.3
Intralaboratory Reproducibility...................................................8-11
8.3.4
Prediction of GHS Acute Oral Toxicity Categories .....................8-12

8.4

Availability of Laboratory Notebooks....................................................8-12

8.5

Summary .................................................................................................8-13

OTHER SCIENTIFIC REPORTS AND REVIEWS OF IN VITROCYTOTOXICITY TEST METHODS AND THEIR ABILITY TO
PREDICT IN VIVO ACUTE TOXICITY AND OTHER TOXICEFFECTS..............................................................................................................9-3ix

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10.0

November 2006

9.1

Relevant Studies........................................................................................9-3
9.1.1
Correlation of NRU Cytotoxicity Values with Rodent
Lethality.......................................................................................9-3
9.1.1.1
Peloux et al. (1992).....................................................9-4
9.1.1.2
Fautrel et al. (1993) ....................................................9-5
9.1.1.3
Roguet et al. (1993).....................................................9-5
9.1.1.4
Rasmussen (1999) .......................................................9-6
9.1.1.5
Creppy et al. (2004) ....................................................9-7
9.1.2
Use of In Vitro Cytotoxicity Data to Reduce the Use of
Animals in Acute Oral Toxicity Testing .......................................9-7
9.1.2.1
Halle et al. (1997): Animal Savings with the ATC
Method Using Cytotoxicity Data .................................9-7
9.1.2.2
Spielmann et al. (1999): Animal Savings Using
Cytotoxicity Data with the UDP ..................................9-9
9.1.2.3
EPA (2004): U.S. EPA HPV Challenge Program
Submission ................................................................ 9-10
9.1.3 Other Evaluations of 3T3 or NHK NRU Methods.......................... 9-10
9.1.3.1
Draize Eye Irritation ................................................. 9-11
9.1.3.2
Predicting Human Lethal Blood Concentrations(LC) .......................................................................... 9-13

9.2

Independent Scientific Reviews.............................................................. 9-13
9.2.1
In Vitro Acute Toxicity Testing for the Classification andLabeling of Chemicals................................................................ 9-13
9.2.1.1
Seibert et al. (1996): ECVAM Workshop 16 .............. 9-13
9.2.2
Use of In Vitro Cytotoxicity Data for Estimation of Starting
Doses for Acute Oral Toxicity Testing ....................................... 9-14
9.2.2.1
ICCVAM (2001a): Estimation of Animal Savings
Using Cytotoxicity Data with the ATC Method .......... 9-14
9.2.2.2
ICCVAM (2001a): Estimation of Animal Savings
Using Cytotoxicity Data with the UDP ...................... 9-16
9.2.3
Validation of 3T3 NRU Assay for Phototoxicity......................... 9-17
9.2.3.1
NHK NRU Phototoxicity Assay.................................. 9-17

9.3

Studies Using In Vitro Cytotoxicity Test Methods with Established
Performance Standards .......................................................................... 9-18
9.3.1
Guidance Document (ICCVAM 2001b)....................................... 9-18
9.3.2
King and Jones (2003)................................................................ 9-19
9.3.3
A-Cute-Tox Project: Optimization and Pre-Validation of an
In Vitro Test Strategy for Predicting Human Acute Toxicity(Clemedson 2005) ...................................................................... 9-19

9.4

Summary ................................................................................................. 9-20

ANIMAL WELFARE CONSIDERATIONS (REFINEMENT,REDUCTION, AND REPLACEMENT) ........................................................... 10-3
10.1

Use of 3T3 and NHK NRU Test Methods to Predict Starting Doses
for Rodent Acute Oral Toxicity Assays.................................................. 10-4x

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11.0

November 2006

10.2

Reduction and Refinement of Animal Use for the UDP ........................10-4
10.2.1 In Vivo Testing Using the UDP...................................................10-4
10.2.1.1 Main Test ..................................................................10-4
10.2.1.2 Limit Test ..................................................................10-5
10.2.2 Computer Simulation Modeling of the UDP ...............................10-6
10.2.3 Animal Savings in the UDP When Using 3T3- and NHKBased Starting Doses ..................................................................10-7
10.2.3.1 The Effect of the Dose-Mortality Slope on Animal
Use............................................................................10-7
10.2.3.2 Mean Animal Use for UDP Simulations –Comparison of Regressions and Predictions fromthe 3T3 and NHK NRU Test Methods ........................10-9
10.2.3.3 Animal Savings in the UDP by GHS Acute Oral
Toxicity Category Using 3T3- and NHK-Based
Starting Doses ...........................................................10-9
10.2.4 Refinement of Animal Use for the UDP When Using 3T3and NHK-Based Starting Doses ................................................10-15
10.2.5 Accuracy of the UDP Outcomes Using the IC50-Based
Starting Doses ..........................................................................10-16

10.3

Reduction and Refinement of Animal Use in the ATC Method ..........10-16
10.3.1 In Vivo Testing Using the ATC Method....................................10-16
10.3.1.1 Main Test ................................................................10-17
10.3.1.2 Limit Test ................................................................10-17
10.3.2 Computer Simulation Modeling of the ATC Method ................10-17
10.3.3 Animal Savings for the ATC Method When Using 3T3- andNHK-Based Starting Doses ......................................................10-18
10.3.3.1 The Effect of Dose-Mortality Slope on Animal
Use..........................................................................10-18
10.3.3.2 Mean Animal Use for ATC Simulations –Comparison of Regressions and Predictions fromthe 3T3 and NHK NRU Test Methods ......................10-20
10.3.3.3 Animal Savings in the ATC Method by GHS Acute
Oral Toxicity Category Using the 3T3- and NHKBased Starting Doses...............................................10-22
10.3.4 Refinement of Animal Use in the ATC Method When Using
3T3- and NHK-Based Starting Doses........................................10-26
10.3.5 Accuracy of the ATC Method Outcomes Using the IC50Based Starting Doses ................................................................10-27

10.4

The Impact of Accuracy on Animal Savings........................................10-28

10.5

The Impact of Prevalence on Animal Savings......................................10-30

10.6

Summary ...............................................................................................10-31

PRACTICAL CONSIDERATIONS ..................................................................11-3
11.1

Transferability of the 3T3 and NHK NRU Test Methods .....................11-3
11.1.1 Facilities and Major Fixed Equipment ........................................11-3
xiIn Vitro Cytotoxicity Test Methods BRD Table of Contents

11.1.2

11.1.3

November 2006

11.1.1.1 Facility Requirements ............................................... 11-3
11.1.1.2 Cell Culture Laboratory............................................ 11-4
11.1.1.3 Major Equipment ...................................................... 11-4
Availability of Other Necessary Equipment and Supplies ........... 11-4
11.1.2.1 General Equipment ................................................... 11-4
11.1.2.2 Cell Culture Materials and Supplies.......................... 11-5
11.1.2.3 Cell Cultures............................................................. 11-5
Problems Specific to the NHK NRU Test Method ...................... 11-5

11.2

3T3 and NHK NRU Test Method Training Considerations.................. 11-6
11.2.1 Required Training and Expertise ................................................ 11-6
11.2.1.1 Specific Training and Expertise Needed .................... 11-6
11.2.1.2 General Laboratory Expertise Needed ...................... 11-7
11.2.2 Training Requirements to Demonstrate Proficiency.................... 11-7
11.2.2.1 Proficiency with GLP-Compliance ............................ 11-8
11.2.3 Personnel Needed to Perform the In Vitro NRU Test Methods.... 11-8

11.3

Cost Considerations ................................................................................ 11-8
11.3.1 3T3 and NHK NRU Test Methods.............................................. 11-8
11.3.1.1 Equipment Costs ....................................................... 11-8
11.3.1.2 Costs for Cell Cultures and Supplies ......................... 11-8
11.3.1.3 Commercial Testing .................................................. 11-9
11.3.2 Rodent Acute Oral Toxicity Testing ......................................... 11-11

11.4

Time Considerations for Performing the 3T3 and NHK NRU Tests .. 11-12
11.4.1 The 3T3 NRU Test Method ...................................................... 11-12
11.4.2 The NHK NRU Test Method.................................................... 11-12
11.4.3 Prequalification of NHK Medium............................................. 11-12
11.4.4 In Vivo Testing ......................................................................... 11-12
11.4.5 The Limit Test.......................................................................... 11-13

11.5

Summary ............................................................................................... 11-14

12.0

REFERENCES ................................................................................................... 12-1

13.0

GLOSSARY........................................................................................................ 13-1

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November 2006

APPENDICES
Appendix A NICEATM/ECVAM Validation Study Management.............................A-1
Appendix B Validation Study Test Method Protocols (Phase III) .............................. B-1
B1
Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake (NRU)
Cytotoxicity Test........................................................................................ B-3
B2
Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test .......................................... B-25
B3
Test Method Protocol for Solubility Determination (Phase III)................. B-47
B4
Test Method Procedure for Prequalification of Normal Human
Epidermal Keratinocyte Growth Medium (Phase III)................................ B-59
Appendix C Validation Study Test Method Protocols (Phases Ia, Ib, and II)............C-1
C1
Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase Ia) ....................................................................... C-3
C2
Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test (Phase Ia).......................... C-23
C3
Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase Ib) ..................................................................... C-41
C4
Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test (Phase Ib) ......................... C-63
C5
Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase II)...................................................................... C-85
C6
Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test (Phase II) ........................ C-109
Appendix D SAS Codes ................................................................................................D-1
D1
SAS Code for ANOVA and Contrasts ........................................................D-3
D2
SAS Code for Regression Comparisons......................................................D-7
Appendix E Neutral Red Dye Experiments ................................................................. E-1
E1
Institute for In Vitro Sciences (IIVS) Assessment of Protocol Variablesin the NICEATM/ECVAM Evaluation of Cytotoxicity Assays................... E-5
E2
Neutral Red (NR) Dye Experiments – 3T3 Cells – IIVS........................... E-13
E3
Neutral Red (NR) Dye Experiments – NHK Cells – IIVS......................... E-19
E4
Neutral Red (NR) Dye Experiments – 3T3 Cells – ECBC ........................ E-25
Appendix F Reference Substance Information ........................................................... F-1
F1
NRU Test Information for the 72 Reference Substances ............................. F-3
F2
Chemical, Physical, and Biological Information from the Literature forthe 72 Reference Substances .................................................................... F-11
F3
Candidate Reference Substances .............................................................. F-27
Appendix G Statement of Work (SOW).......................................................................G-1
G1
A Validation Study For In Vitro Basal Cytotoxicity Testing .......................G-3
G2
Procedures for Acquisition, Preparation, Solubility Testing, andDistribution of Test Chemicals for a Validation Study for In Vitro
Basal Cytotoxicity Testing .......................................................................G-57

xiii

In Vitro Cytotoxicity Test Methods BRD Table of Contents

November 2006

Appendix H Rat and Mouse Oral LD50 Database........................................................H-1
H1
Rat and Mouse Oral LD50 Database ........................................................... H-3
H2
Evaluation of the Candidate Reference Oral LD50 Data ............................ H-39
Appendix I
I1
I2
I3
I4

In Vitro NRU Data .................................................................................... I-1
3T3 NRU Reference Substance Data........................................................... I-3
NHK NRU Reference Substance Data .......................................................I-59
3T3 NRU Positive Control (SLS) Data.....................................................I-111
NHK NRU Positive Control (SLS) Data ..................................................I-127

Appendix J
J1
J2
J3
J4
J5
J6
J7

LD50 and Toxicity Category Predictions ..................................................J-1
3T3 NRU Predictions: RC Millimole Regression ........................................ J-5
NHK NRU Predictions: RC Millimole Regression .................................... J-11
3T3 NRU Predictions: RC Rat-Only Millimole Regression....................... J-17
NHK NRU Predictions: RC Rat-Only Millimole Regression..................... J-21
3T3 NRU Predictions: RC Rat-Only Weight Regression ........................... J-25
NHK NRU Predictions: RC Rat-Only Weight Regression......................... J-29
Comparison of Millimole Regression with Weight Regression
Regarding Prediction of Toxicity (LD50) for Low or High Molecular
Weight Chemicals..................................................................................... J-34

Appendix K
K1
K2
K3
K4

IC50 and LD50 Data for Regressions ........................................................K-1
IC50 and LD50 Values Used for Laboratory-Specific Regressions ............... K-3
IC50 and LD50 Values Used for Combined-Laboratory Regressions.......... K-17
RC IC50 and LD50 Values for RC Substances with Rat Oral LD50 Data..... K-23
Individual Laboratory LD50 Predictions: RC Rat-Only Millimole
Regression ............................................................................................... K-33

Appendix L Outlier Information ................................................................................. L-1
L1
Outlier Characterization for the 3T3 and NHK NRU Test Methods
with the RC Millimole Regression ............................................................. L-3
L2
Discordant Substances for GHS Acute Oral Toxicity Category
Predictions Using the 3T3 and NHK NRU Test Methods and RC RatOnly Regressions ....................................................................................... L-9
L3
Analysis of Outliers by Halle (1998, 2003) for the RC Millimole
Regression ............................................................................................... L-17
Appendix M Acute Oral Toxicity Test Guidelines ...................................................... M-1
M1
OECD UDP Test Guideline ...................................................................... M-3
M2
EPA UDP Test Guideline........................................................................ M-31
M3
OECD ATC Method Test Guideline ....................................................... M-71
M4
OECD FDP Test Guideline ..................................................................... M-87
M5
OECD Guidance on Acute Oral Toxicity Testing...................................M-103
Appendix N UDP/ATC Simulation Modeling Results................................................. N-1
N1
UDP Simulation Results Using Starting Doses One Default Dose
Lower than the LD50 Predicted by the 3T3 and NHK NRU IC50 and the
RC Rat-Only Millimole Regression – 5000 mg/kg Upper Limit Dose ........ N-3

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In Vitro Cytotoxicity Test Methods BRD Table of Contents

N2
N3
N4

November 2006

UDP Simulation Results Using Starting Doses One Default Dose
Lower than the LD50 Predicted by the 3T3 and NHK NRU IC50 and the
RC Rat-Only Weight Regression – 5000 mg/kg Upper Limit Dose ..........N-13
ATC Simulation Results Starting at the Next Fixed Dose Below theLD50 Predicted by the 3T3 and NHK NRU IC50 and the RC Rat-Only
Millimole Regression - 2000 mg/kg Upper Limit Dose ............................N-23
ATC Simulation Results Starting at the Next Fixed Dose Below theLD50 Predicted by the 3T3 and NHK IC50 and the RC Rat-Only
Weight Regression – 2000 mg/kg Upper Limit Dose................................N-33

Appendix O Federal Register Notices ...........................................................................O-1
O1
70FR14473 - Request for Nominations for an Independent Peer Review
Panel To Evaluate In Vitro Testing Methods for Estimating Acute Oral
Systemic Toxicity and Request for In Vivo and In Vitro Data .....................O-3
O2
69FR61504 - Availability of Updated Standardized In Vitro
Cytotoxicity Test Method Protocols for Estimating Acute Oral
Systemic Toxicity; Request for Existing In Vivo and In Vitro Acute
Toxicity Data ...........................................................................................O-5
O3
69FR11448 - Notice of the Availability of Agency Responses to
ICCVAM Test Recommendations for the Revised Up-and-Down
Procedure for Determining Acute Oral Toxicity and In Vitro Methods
for Assessing Acute Systemic Toxicity ......................................................O-7
O4
66FR49686 - Report of the International Workshop on In Vitro
Methods for Assessing Acute Systemic Toxicity; Guidance Document
on Using In Vitro Data to Estimate In Vivo Starting Doses for Acute
Toxicity: Notice of Availability and Request for Public Comment .............O-9
O5
65FR57203 - Notice of an International Workshop on In Vitro
Methods for Assessing Acute Systemic Toxicity, co-sponsored by
NIEHS, NTP and the U.S. Environmental Protection Agency (EPA):
Workshop Agenda and Registration Information......................................O-11
O6
65FR37400 - Notice of an International Workshop on In Vitro
Methods for Assessing Acute Systemic Toxicity, co-sponsored by
NIEHS, NTP and the U.S. Environmental Protection Agency (EPA):
Request for Data and Suggested Expert Scientists ....................................O-15
Appendix P In Vitro Cytotoxicity Test Methods and the High Production
Volume (HPV) Challenge Program......................................................... P-1
P1
Supplemental Acute Toxicity Protocol ....................................................... P-3
P2
Office of Pollution Prevention and Toxics (OPPT) Letters to
Manufacturers/Importers ............................................................................ P-9
Appendix Q Additional UDP Simulation Modeling Results........................................Q-1
Q1
UDP Simulation Results for the RC Rat-Only Millimole Regression
Starting at the LD50 Predicted by the 3T3 and NHK NRU IC50 –5000 mg/kg Upper Limit Dose .................................................................. Q-3
Q2
UDP Simulation Results for the RC Rat-Only Weight Regression
Starting at the LD50 Predicted by the 3T3 and NHK NRU IC50 LD50 –5000 mg/kg Upper Limit Dose .................................................................Q-13
xv

In Vitro Cytotoxicity Test Methods BRD List of Figures

November 2006

LIST OF FIGURES
Figure 1-1
Figure 1-2
Figure 2-1
Figure 2-2
Figure 2-3
Figure 2-4
Figure 2-5
Figure 2-6
Figure 2-7
Figure 3-1
Figure 4-1
Figure 5-1
Figure 5-2
Figure 5-3
Figure 5-4
Figure 6-1
Figure 6-2
Figure 6-3
Figure 6-4
Figure 6-5
Figure 6-6
Figure 6-7
Figure 6-8
Figure 7-1
Figure 7-2
Figure 7-3
Figure 7-4
Figure 7-5

RC Millimole Regression for In Vitro Cytotoxicity (IC50x) and Rat andMouse Acute Oral LD50 Values for 347 Chemicals .....................................1-9
NICEATM/ECVAM Validation Study Phases .......................................... 1-13
Major Steps in the Performance of the NRU Test Methods .........................2-7
3T3 NRU OD for SLS as a Function of NR Concentration andDuration.................................................................................................... 2-26
SLS IC50 Values for Each NR Concentration and Incubation Duration(3T3 NRU) ............................................................................................... 2-27
Representative Concentration-Response for 2-Propanol in a 3T3 NRU
Range Finder Test..................................................................................... 2-30
Representative Concentration-Response for Aminopterin in a NHK
NRU Range Finder Test............................................................................ 2-31
Representative Concentration-Response for Aminopterin in a NHK
NRU Definitive Test ................................................................................. 2-32
Flow Chart for Determination of Reference Substance Solubility in
Medium, DMSO, or ETOH....................................................................... 2-38
The Fifty-Eight (58) Selected RC Reference Substances on the RC
Millimole Regression................................................................................ 3-27
Correlation of LD50 Values With the Reference LD50 Values for the
58 RC Chemicals ...................................................................................... 4-14
Reference Substance IC50 Results for the 3T3 NRU Test Method byLaboratory ................................................................................................ 5-24
Reference Substance IC50 Results for the NHK NRU Test Method byLaboratory ................................................................................................ 5-25
RC IC50 Values vs 3T3 NRU IC50 Values for 56 Substances in
Common ................................................................................................... 5-37
RC IC50 Values vs NHK NRU IC50 Values for 57 Substances in
Common .................................................................................................. 5-38
Combined-Laboratory 3T3 and NHK NRU Regressions .............................6-7
Regression for 47 RC Substances Using RC Data .......................................6-8
Regression for 47 RC Substances with the 3T3 and NHK Regressions........6-9
RC Rat-Only Millimole Regression (a) and RC Rat-Only Weight
Regression (b)........................................................................................... 6-19
Correlation of Dose-Mortality Slope to Hill Function Slope...................... 6-30
In Vitro – In Vivo Regressions for Phases Ib and II for ECBC ................... 6-37
In Vitro – In Vivo Regressions for Phases Ib and II for FAL...................... 6-38
In Vitro – In Vivo Regressions for Phases Ib and II for IIVS...................... 6-39
Mean Laboratory and Laboratory-Specific 3T3 and NHK NRU
Regressions.................................................................................................7-7
Frequency of Maximum:Minimum 3T3 NRU IC50 Ratios ......................... 7-39
Frequency of Maximum:Minimum NHK NRU IC50 Ratios....................... 7-40
Comparison of Maximum:Minimum NRU IC50 Ratios to Maximum:
Minimum LD50 Ratios .............................................................................. 7-41
SLS IC50 for Each Laboratory and Study Phase......................................... 7-45
xvi

In Vitro Cytotoxicity Test Methods BRD List of Tables

November 2006

LIST OF TABLES
Table ES-1
Table 1-1
Table 1-2
Table 2-1
Table 2-2
Table 2-3
Table 2-4
Table 2-5
Table 3-1
Table 3-2
Table 3-3
Table 3-4
Table 3-5
Table 3-6
Table 3-7
Table 3-8
Table 4-1
Table 4-2
Table 4-3
Table 4-4
Table 5-1
Table 5-2
Table 5-3
Table 5-4
Table 5-5
Table 5-6
Table 5-7
Table 5-8
Table 5-9
Table 5-10
Table 5-11
Table 6-1
Table 6-2
Table 6-3

Datasets Used for Validation Study Analyses.......................................... xxxvi
Summary of Current U.S. Legislation for Using Acute Toxicity Data
for Product Labeling .................................................................................1-14
Regulatory Classification Systems for Acute Oral Toxicity .......................1-15
Measured VC OD540 Values and Targets...................................................2-17
Refeeding/No Refeeding Data for the NHK NRU Test Method.................2-24
Error Rates in Phase Ib by Laboratory and Test.........................................2-28
Cell Seeding Densities ..............................................................................2-37
Comparison of Concentrations Tested in the Various Solubility
Protocols...................................................................................................2-39
GHS Classification Scheme for Acute Oral Toxicity ...................................3-3
Reference Substances Used in the 3T3 and NHK NRU Test Methods
Validation Study - Sorted by Toxicity .........................................................3-7
Distribution of Candidate Substances and Reference Substances by
Source and Toxicity Category ...................................................................3-26
Selected Substances: Distribution of RC Chemicals and RC Outliersby Toxicity Category.................................................................................3-26
Distribution of Chemical Class for the 72 Reference Substances by
Toxicity Category .....................................................................................3-29
Distribution of Product/Use Class for the 72 Reference Substances by
Toxicity Category .....................................................................................3-31
Reference Substances Metabolized to Active Metabolites .........................3-32
Reference Substances Tested in Phases Ib and II .......................................3-33
Internet-Accessible Databases Searched for LD50 Information.....................4-3
Rodent Acute Oral Reference LD50 Values Listed by GHS Category...........4-9
GHS Category Matches for the Rodent Acute Oral LD50 Initial andReference Values ......................................................................................4-12
Maximum:Minimum LD50 Ratios by GHS Toxicity Category ...................4-13
Validation Study Timetable.........................................................................5-4
Reference Substances Affected by the Stopping Rule................................5-10
Positive Control (PC) IC50 Results by Study Phase....................................5-11
3T3 NRU Test Method IC50 and Hill Slope Data by Laboratory................5-14
NHK NRU Test Method IC50 and Hill Slope Data by Laboratory..............5-19
Key to Validation Study Reference Substances .........................................5-26
Datasets Used for Validation Study Analyses............................................5-32
Comparison of 3T3 and NHK NRU IC50 Geometric Means.......................5-33
Frequency of 3T3:NHK IC50 Ratios for Reference Substances ..................5-36
Solubility Test Results (mg/mL)................................................................5-39
Reference Substances with Precipitate (PPT) and Volatility Issues............5-43
Datasets Used for Accuracy Analyses .........................................................6-4
Linear Regression Analyses of the 3T3 and NHK NRU and Rat Acute
Oral LD50 Test Results ................................................................................6-6
Outlier Substances for the RC and the 3T3 and NHK NRU Methods
When the RC Millimole Regression is Used..............................................6-10
xvii

In Vitro Cytotoxicity Test Methods BRD List of Tables

Table 6-4
Table 6-5
Table 6-6
Table 6-7
Table 6-8
Table 6-9
Table 6-10
Table 6-11
Table 6-12
Table 7-1
Table 7-2
Table 7-3
Table 7-4
Table 7-5
Table 7-6
Table 7-7
Table 7-8
Table 7-9
Table 7-10
Table 7-11
Table 7-12
Table 7-13
Table 7-14
Table 8-1
Table 8-2

November 2006

Substances With Mechanisms of Toxicity Not Expected to Be Activein the 3T3 or NHK Cells in Culture........................................................... 6-15
Linear Regression Analyses to Improve the Prediction of Rodent
Acute Oral LD50 Values from In Vitro NRU IC50 Using the RC
Database ................................................................................................... 6-17
Prediction of GHS Acute Oral Toxicity Category by the RC IC50
Values and the RC Millimole Regression .................................................. 6-21
Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK
NRU Test Methods and the RC Rat-Only Millimole Regression ............... 6-23
Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK
NRU Test Methods and the RC Rat-Only Weight Regression ................... 6-26
Comparison of Regressions and In Vitro NRU Test Methods for Their
Performance in Predicting GHS Acute Oral Toxicity Categories............... 6-28
Reference Substances with Dose-Mortality and NRU Hill Slopes ............. 6-29
Over- and Under- Prediction of Toxicity for Low and High Molecular
Weight Substances Using RC Rat-Only Weight and Millimole
Regressions............................................................................................... 6-33
Linear Regressions for 11 Substances Tested in Phases Ib and II............... 6-36
Reference Substances Excluded from Reproducibility Analyses
Because of Insufficient Cytotoxicity ...........................................................7-4
Number of Reference Substances Tested vs Number of Reference
Substances Yielding IC50 Values from Each Laboratory, by GHS
Acute Oral Toxicity Category .....................................................................7-4
Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU
Test Method................................................................................................7-8
Reference Substances with Significant ANOVA Differences Among
Laboratories for the 3T3 NRU Test Method.............................................. 7-20
Reproducibility of the IC50 Values from the NHK NRU Test Method ....... 7-22
Reference Substances with Significant ANOVA Differences Among
Laboratories for the NHK NRU Test Method............................................ 7-34
Summary of CV Results for the 3T3 and NHK NRU Test Methods .......... 7-34
Intralaboratory CV by Chemical Characteristics for the 3T3 and NHK
NRU Test Methods ................................................................................... 7-36
Interlaboratory 3T3 and NHK NRU Test Method CV Values Sorted by
Chemical Characteristics........................................................................... 7-38
CV Values for 3T3 and NHK NRU Test Method IC50 Values andNormalized IC50 Values ............................................................................ 7-43
ANOVA Results for the SLS IC50 Values in the 3T3 NRU Test
Method ..................................................................................................... 7-47
Linear Regression Analysis of SLS IC50 Values Over Time ...................... 7-48
ANOVA Results for the SLS IC50 Values in the NHK NRU Test
Method ..................................................................................................... 7-49
Solvent Determinations by Laboratory...................................................... 7-51
SMT-Recommended Documentation for FAL.............................................8-4
Phase III Error Rates in the Transfer of Data to the EXCEL® Template..... 8-10

xviii

In Vitro Cytotoxicity Test Methods BRD List of Tables

Table 8-3
Table 8-4
Table 8-5
Table 8-6
Table 9-1
Table 9-2
Table 9-3
Table 9-4
Table 10-1
Table 10-2
Table 10-3
Table 10-4
Table 10-5
Table 10-6
Table 10-7
Table 10-8
Table 10-9
Table 10-10
Table 11-1
Table 11-2
Table 11-3
Table 11-4

November 2006

Definitive Test and Positive Control (PC) Test Failure Rates in Phase
III..............................................................................................................8-10
Combined Definitive and Positive Control (PC) Test Success Rates forthe 3T3 and NHK Methods in Phase III.....................................................8-11
CV Values for Definitive Tests .................................................................8-11
GHS Acute Oral Toxicity Category Predictions by Laboratory..................8-13
Rat Acute Oral LD50 Ranges for Test Substances Used in Previous In
Vitro NRU Cytotoxicity Studies and the NICEATM/ECVAM Study ..........9-4
Chemical Classes Represented by the Substances Used in Published
Studies for Correlation of In Vitro NRU Cytotoxicity with Rodent
Acute Lethality ...........................................................................................9-4
EU Classes of Acute Oral Toxicity..............................................................9-8
Correct Identification of In Vivo Phototoxicants by the NHK NRU
Phototoxicity Assay ..................................................................................9-18
Change in Animal Use with Dose-Mortality Slope for the UDP ................10-8
Mean Animal Use in the UDP Using Starting Doses Based on the 3T3
and NHK NRU Test Methods with the Different Regressions .................10-10
Animal Use for the UDP by GHS Acute Oral Toxicity Category Using
Starting Doses Based on the 3T3 and NHK NRU Test Methods with
the RC Rat-Only Millimole Regression ...................................................10-12
Animal Use for the UDP by GHS Acute Oral Toxicity Category Using
Starting Doses Based on the 3T3 and NHK NRU Test Methods with
the RC Rat-Only Weight Regression .......................................................10-14
Animal Deaths in the UDP Using Starting Doses Based on the 3T3 andNHK NRU Test Methods ........................................................................10-15
Change in Animal Use with Dose-Mortality Slope in the ATC Method ...10-19
Animal Use for the ATC Method Using Starting Doses Based on the
3T3 and NHK NRU Test Methods with the Different Regressions ..........10-21
Animal Savings for the ATC Method by GHS Acute Oral Toxicity
Category Using Starting Doses Based on the 3T3 and NHK NRU Test
Methods with the RC Rat-Only Millimole Regression.............................10-23
Animal Savings for the ATC Method by GHS Acute Oral Toxicity
Category Using Starting Doses Based on the 3T3 and NHK NRU Test
Methods with the RC Rat-Only Weight Regression.................................10-25
Animal Deaths for the ATC Method Using Starting Doses Based on
the 3T3 and NHK NRU Test Methods.....................................................10-27
Costs for Major Laboratory Equipment .....................................................11-9
Costs for Cell Culture Materials and Commercial Laboratory In Vitro
Cytotoxicity Testing................................................................................11-10
Commercial Prices for Conducting In Vivo Acute Rat Toxicity Testing...11-11
Comparison of Time Needed for In Vitro and In Vivo Testing .................11-14

xix

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

November 2006

LIST OF ACRONYMS AND ABBREVIATIONS
A-CUTE-TOX
ADME
ANOVA
ASTDR
ASTM
ATC
ATCC
ATWG
BBB
BPE
BRD
o
C
CAS
CASRN
CCOHS
CDER
CESARS
CFU
CHRIS
CI
CICADSCIS
CNSCOLIPA
CPSC
CSF
CTFA
CV
DART®/ETIC
DEA
DHHS
DIMDI
DNA
DMEM
DMSO
D-PBS
DOD
DOT
EC
EC50

A-Cute-Tox Project (EU Research & Development Integrated Project)
Absorption, distribution, metabolism, and elimination
Analysis of varianceAgency for Toxic Substances and Disease Registry
American Society for Testing and Materials
Acute Toxic Class method
American Type Culture Collection
Acute Toxicity Working Group
Blood:brain barrier
Bovine pituitary extract
Background Review Document
Degrees Celsius
Chemical Abstracts Service
Chemical Abstracts Service Registry Number
Canadian Centre for Occupational Health and Safety (CCOHS)
U.S. FDA Center for Drug Evaluation and Research
Chemical Evaluation Search and Retrieval System
Colony forming units
Chemical Hazard Response
Confidence interval
Concise International Chemical Assessment Documents
ILO Occupational Safety and Health Information Centre
Central nervous system
The European Cosmetic Toiletry and Perfumery Association
U.S. Consumer Product Safety Commission
Colony stimulating factor
Cosmetic, Toiletries and Fragrance Association
Coefficient of variation
Developmental and Reproductive Toxicology/Environmental
Teratology Information Center
U.S. Drug Enforcement Administration
U.S. Department of Health and Human Services
Deutsches Institut fur Medizinische Dokumentation undInformation (The German Institute for Medical Documentation andInformation)
Deoxyribose nucleic acid
Dulbecco’s Modification of Eagle’s Medium
Dimethyl sulfoxide
Dulbecco’s phosphate buffered saline
U.S. Department of Defense
U.S. Department of Transportation
European Commission
Concentration of a substance that produces 50% of the maximum
possible response for that substancexx

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

ECBC
ECETOC
EC/HO
ECVAM
EDIT
EHC
EHS
EPA
ERG
ETOH
EU
EXTONET
FAL
FAO
FB1
FDA
FDP
FIFRA
FR
FRAME
GABA
GCCP
GHS
GLP
gm
HBSS
HPV
hr
HSDB
HSG
HTD
IARC
IC20
IC50
IC80
ICCVAM
ICSC
ID
ID50
IIVS
ILO
i.m.
INVITOXX

November 2006

U.S. Army Edgewood Chemical Biological Center
European Centre for Ecotoxicology and Toxicology of Chemicals
European Commission/British Home Office
European Centre for the Validation of Alternative Methods
Evaluation-guided development of new in vitro tests
Environmental Health Criteria
EPA’s Extremely Hazardous Substance list
U.S. Environmental Protection Agency
Emergency Response Guidebook
Ethanol (Ethyl alcohol)
European Union
The Extension Toxicology Network
FRAME Alternatives Laboratory
UN Food and Agriculture Organization
Fumonisin B1
U.S. Food and Drug Administration
Fixed Dose Procedure
U.S. Federal Insecticide, Fungicide, and Rodenticide Act
Federal Register
Fund for the Replacement of Animals in Medical Experiments
Gamma amino butyric acid
Good cell culture practices
Globally Harmonized System (of Classification and Labeling ofChemicals)
Good Laboratory Practices
Grams
Hanks’ balanced salt solution
High Production Volume
Hour(s)
Hazardous Substances Data Bank
Health and Safety Guides
Highest tolerated doseInternational Agency for Research on Cancer
Concentration producing 20% inhibition of the endpoint measured
Concentration producing 50% inhibition of the endpoint measured
Concentration producing 80% inhibition of the endpoint measured
Interagency Coordinating Committee for the Validation of Alternative
Methods
International Chemical Safety Cards
Insufficient data
Index of cytotoxicity; dose producing a 50% reduction in protein value
Institute for In Vitro Sciences
International Labour Organisation
Intramuscular
In Vitro Techniques in Toxicology (ERGATT FRAME ECVAM Data
bank)

xxi

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

IOM
i.p.
IPCS
IRAG
IRPTC
ISO
IUCLID
i.v.
JECFA
JMPR
KBM®
kg
Kow
L
LC
LD50
LDH
MAS
MEIC
MeSH®
µL
µm
µM
mg
MIT
mL
mM
MMAS
mmol
MPE
MSDS
MTT
N
NA
NADH
NC
NCS
NCTR
n.d.
NHK
NICEATM
NIEHS
NIH
NIOSH
NLM

November 2006

Institute of Medicine
Intraperitoneal
International Programme on Chemical Safety
Interagency Regulatory Alternatives GroupInternational Register of Potentially Toxic Chemicals
International Standards Organization
International Uniform Chemical Information Database
Intravenous
Joint Expert Committee on Food Additives
Joint Meeting on Pesticide Residues
Keratinocyte basal medium
Kilogram
Octanol-water partition coefficient
Liter
Lethal blood concentration
Dose that produces lethality in 50% of test animals
Lactate dehydrogenase
Maximum average Draize score
Multicentre Evaluation of In Vitro Cytotoxicity
Medical Subject Heading
Microliters
Micrometers
Micromoles
Milligram
Metabolic inhibition test
Milliliter
Millimolar
Modified maximum average score
Millimoles
Mean photo effect
Material Safety Data Sheets
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
Number (of substances)
Not applicable
Nicotine adenine dinucleotide (reduced)
Not calculated
Newborn calf serum
U.S. FDA National Center for Toxicological ResearchNot detectable
Normal human epidermal keratinocytes
National Toxicology Program Center for the Evaluation of Alternative
Toxicological Methods
U.S. National Institute of Environmental Health Sciences
U.S. National Institutes of Health
U.S. National Institute for Occupational Safety and HealthNational Library of Medicine

xxii

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

NR
NRU
NTP
OAT
OD
OD540
OECD
OHM/TADS
OPP
OPPTS
ORD
OSHA
OTA
PBS
PC
PDS
pg
PG
PIF
PIMS
pK
PLS
PPIS
PPT
QA
QC
R2
rs
RC
REACH
RTECS®
RTK NET
SD
SIDS
SIS
SLS
SMT
SOP
3T3
TESS
TG
TRI
TSCA
UDP
UN

November 2006

Neutral red
Neutral red uptake
U.S. National Toxicology Program
Organic anionic transporters
Optical density
Optical density (absorbance) at a wavelength of 540 nm
Organisation for Economic Co-operation and Development
EPA Oil and Hazardous Materials/Technical Assistance Data
SystemU.S. EPA Office of Pesticide Programs
EPA Office of Prevention, Pesticides and Toxic Substances
U.S. EPA Office of Research and Development
U.S. Occupational Safety and Health Administration
Ochratoxin A
Phosphate buffered saline
Positive control
Pesticide Data Sheets
Picogram
Packing group
Photoinhibition factor
Poisons Information Monographs
Acid/base dissociation constantPartial Least Squares (analysis)
EPA Pesticide Product Information System
Precipitate
Quality assurance
Quality control
Coefficient of determination
Spearman correlation coefficient
Registry of Cytotoxicity
Registration, evaluation, authorisation and restriction of chemicals
Registry of Toxic Effects of Chemical Substances
The Right-to-Know Network
Standard deviation
OECD Screening Information Data Sets
Scientific Information Service
Sodium lauryl sulfate
Study management teamStandard operating procedure
BALB/c mouse fibroblasts, clone A31 (ATCC # CCL-163)
Toxic Exposure Surveillance System
Test guideline
U.S. EPA Toxics Release Inventory
Toxic Substances Control Act
Up-and-Down Procedure
United Nations

xxiii

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

UNEP
USP
UV
VC
WHO
XTT
ZEBET

November 2006

United Nations Environment Programme
U.S. Pharmacopoeia
Ultraviolet (light)
Vehicle control
World Health Organization
2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carboxanilide
Zentralstelle zur Erfassung und Bewertung von Ersatz- undErgänzungsmethoden zum Tierversuch (German Center forDocumentation and Evaluation of Alternative Methods to Animal
Experiments)xxiv

In Vitro Cytotoxicity Test Methods BRD Acknowledgements

November 2006

ACKNOWLEDGMENTS
The following individuals are acknowledged for their contributions to the in vitro acutetoxicity test method review process.

Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM) Acute Toxicity Working Group (ATWG)

Consumer Product Safety Commission(CPSC)

Food and Drug Administration (FDA)
Leonard Schechtman, Ph.D. (ICCVAM Chair)
Kenneth Hastings, Ph.D.
Abigail Jacobs, Ph.D.
David Morse, Ph.D.
Thomas Umbreit, Ph.D.

Kailash Gupta, D.V.M., Ph.D.
Cassandra Prioleau, Ph.D.
Marilyn Wind, Ph.D. (ATWG Chair,
ICCVAM Vice Chair)
Department of Energy (DOE)

National Institute for Occupational
Safety & Health (NIOSH)

Po-Yung Lu, Ph.D.
Steven Reynolds, Ph.D.
Environmental Protection Agency(EPA)

National Institute of Environmental
Health Sciences (NIEHS)

Karen Hamernik, Ph.D.
Masih Hashim, Ph.D.
Marianne Lewis
Elizabeth Margosches, Ph.D.
Deborah McCallJohn Redden, Ph.D.
Amy Rispin, Ph.D.

Rajendra Chhabra, Ph.D., D.A.B.T.
William Stokes, D.V.M., D.A.C.L.A.M.
(ICCVAM Executive Director; NICEATM
Director)
Raymond Tice, Ph.D. (NICEATM Deputy
Director)
European Centre for the Validation of
Alternative Methods (ECVAM)
Liaisons
Silvia Casati, Ph.D.
Pilar Prieto, Ph.D.

xxv

In Vitro Cytotoxicity Test Methods BRD Acknowledgements

November 2006

National Toxicology Program (NTP) Interagency Center for the
Evaluationof Alternative Toxicological Methods (NICEATM)

Dave Allen, Ph.D.
ILS, Inc.

Linda Litchfield
ILS, Inc.

Bradley Blackard, M.S.P.H.
ILS, Inc.

Deborah McCarley
NIEHS

Sue Brenzel
ILS, Inc.

Michael Paris
ILS, Inc.

Thomas Burns, M.S.
ILS, Inc.

William Stokes, D.V.M., D.A.C.L.A.M.
(Director)
NIEHS

Patricia Ceger, M.S.
ILS, Inc.

Judy Strickland, Ph.D., D.A.B.T.
ILS, Inc.

Jeffery Charles, Ph.D., M.B.A., D.A.B.T.
ILS, Inc.

Raymond Tice, Ph.D. (Deputy Director)
NIEHS

Neepa Choksi, Ph.D.
ILS, Inc.

Jim Truax, M.A.
ILS, Inc.

Frank Deal, M.S.
ILS, Inc.

xxvi

In Vitro Cytotoxicity Test Methods BRD Acknowledgements

November 2006

Participants in the In Vitro Cytotoxicity Validation StudyNICEATM gratefully acknowledges the generous contributions of the individuals who
directly participated in the NICEATM/ECVAM Validation Study. Their time and efforts are
greatly appreciated.
BioReliance Corp.
(Chemical Distribution)
Martin Wenk, Ph.D. – Principal
Investigator

Constella Group (Statistical Analyses)
Patrick Crockett, Ph.D.
Eric Harvey, Ph.D.
Wendell Jones, Ph.D.
Robert Lee, M.S.
Jessica L. Matthews, M.S.
Michael Riggs, Ph.D.
Janine Wilcox
Nicole Williams

Institute for In Vitro Sciences (IIVS)(Lead Laboratory – Protocols)
Hans Raabe, M.S. – Study Director
Greg Mun – Laboratory Manager
Angela Sizemore – Research Technician
Gregory O. Moyer – Research Technician
John Harbell, Ph.D. – Scientific Director

Statistical Consultant
Joseph Haseman, Ph.D.

U.S. Army Edgewood Chemical
Biological Center (ECBC)(Testing Laboratory)
Cheng Cao, Ph.D. – Study Director
Janna Madren-Whalley – ResearchTechnician
Chundakkadu Krishna, Ph.D. – ResearchTechnician
James J. Valdes, Ph.D. – Scientific
AdvisorNIEHS
Grace Kissling, Ph.D. – Contract Project
Officer
Molly Vallant - Contract Project Officer
Study Management Team (SMT)NICEATM
William Stokes, D.V.M., D.A.C.L.A.M.
(NIEHS) – Director, NICEATM
Raymond Tice, Ph.D. (NIEHS) – Deputy
Director, NICEATM – Advisor
Judy Strickland, Ph.D., D.A.B.T. (ILS,
Inc.) – Project Coordinator
Michael Paris (ILS, Inc.) – Assistant
Project Coordinator
Jeffrey Charles, Ph.D., D.A.B.T. (ILS,
Inc.) – Advisor

FRAME Alternatives Laboratory (FAL)
University of Nottingham, UK(Lead Laboratory – Software)
Richard Clothier, Ph.D. – Study Director
Nicola Bourne – Research Technician
Monika Owen – Research Technician
Rachel Budworth – Research Technician

ECVAM
Thomas Hartung, Ph.D., M.D. – Head of
Unit from 2002
Silvia Casati, Ph.D. – Task Leader
Michael Balls, D. Phil.– Head of Unit until
June 2002

xxvii

In Vitro Cytotoxicity Test Methods BRD Acknowledgements[This Page Intentionally Left Blank]xxviiiNovember 2006

In Vitro Cytotoxicity Test Methods BRD Preface

November 2006

PREFACEThe Institute of Medicine estimates that more than 4 million poisonings occur annually in the
United States (Institute of Medicine [IOM] 2004). In 2001, 30,800 deaths placed poisoning
as the second leading cause of injury-related death behind automobile accidents (42,433
deaths) (IOM 2004). In order to ensure that all potentially hazardous substances have proper
warning labels, regulatory agencies require determination of acute toxicity hazard potentialof substances and products. This determination for oral acute toxicity hazard is currently
made using a test that requires laboratory rats. Historically, lethality estimated by the LD50
(i.e., the dose of a test substance that produces death in 50% of the animals tested) has been a
primary toxicological endpoint in acute toxicity tests.
The conventional LD50 acute oral toxicity in vivo test method has been modified in various
ways to reduce and refine1 animal use in toxicity testing (OECD 2001a, c, d, e; EPA 2002a).
Most recently, the LD50 was replaced, for hazard classification testing purposes, with the
UDP, based on an Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM) technical evaluation and formal ICCVAM recommendations (ICCVAM
2000, 2001c). This method now reduces animal use by over 70% compared to the previous
method.
In 1999, at the request of the U.S. Environmental Protection Agency (EPA) Office ofPesticides, Prevention, and Toxic Substances, ICCVAM reviewed the validation status of invitro methods for estimating acute oral toxicity. This request was based on studies publishedin recent years that showed a correlation between in vitro and in vivo acute toxicity. In vitro
cytotoxicity methods have been evaluated as another means to reduce and refine the use ofanimals and these methods may be helpful in predicting in vivo acute toxicity. Since moving
the starting dose closer to the LD50 reduces the number of animals necessary for the acuteoral systemic toxicity test, the use of in vitro cytotoxicity assays to predict a starting doseclose to the LD50 may reduce animal use.
In October of 2000, the International Workshop on In Vitro Methods for Assessing Acute
Systemic Toxicity sponsored by the National Toxicology Program (NTP), the National
Institute of Environmental Health Sciences (NIEHS) and the EPA was convened in
Arlington, VA. The Organizing Committee invited 33 expert scientists from academia,
industry, and government agencies to participate in the Workshop. Invited scientific experts
and ICCVAM agency scientists were assigned to one of four Breakout Groups and preparedrecommendations on the following:• In Vitro Screening Methods for Assessing Acute Toxicity• In Vitro Methods for Toxicokinetic Determinations• In Vitro Methods for Predicting Organ Specific Toxicity• Chemical Data Sets for Validation of In Vitro Acute Toxicity Test Methods

1

A reduction alternative is a new or modified test method that reduces the number of animals required. A
refinement alternative is a new or modified test method that refines procedures to lessen or eliminate pain or
distress in animals or enhances animal well-being (ICCVAM 2003).
xxix

In Vitro Cytotoxicity Test Methods BRD Preface

November 2006

Workshop participants concluded that none of the proposed in vitro methods had beenformally evaluated for reliability and relevance, and that their usefulness and limitations forgenerating information to meet regulatory requirements for acute toxicity testing had notbeen adequately assessed. However, an in vitro approach proposed by the German Center forDocumentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET) was
recommended for rapid adoption so that data could be generated to establish its usefulness
with a large number of chemicals (ICCVAM 2001a). In addition, a separate GuidanceDocument on Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity(ICCVAM 2001b) was prepared to provide sample cytotoxicity protocols and instructions forusing in vitro data to predict starting doses for acute in vivo systemic toxicity tests.
ICCVAM, which is charged with coordinating the technical evaluations of new, revised, andalternative test methods with regulatory applicability (ICCVAM Authorization Act of 2000,
Public Law 106-545; available: http://iccvam.niehs.nih.gov/about/PL106545.pdf), agreed
that in vitro basal cytotoxicity test methods should have a high priority for evaluation. The
NTP Center for the Evaluation of Alternative Toxicological Methods (NICEATM)
collaborated with the European Centre for the Validation of Alternative Methods (ECVAM),
a component of the European Commission’s Joint Research Centre, to further characterize
the usefulness of in vitro cytotoxicity assays as predictors of starting doses for acute oral
lethality assays. NICEATM and ECVAM designed a multi-laboratory validation study toevaluate the performance of two standardized in vitro basal cytotoxicity test methods using
72 reference substances with the ZEBET approach of using the Registry of Cytotoxicity (RC)regression model. Based on the procedures described in the Guidance Document (ICCVAM
2001b), the validation study used two mammalian cell types (i.e., BALB/c 3T3 mouse
fibroblasts [3T3] and primary normal human epidermal keratinocytes [NHK]) for in vitro
basal cytotoxicity test methods with a neutral red uptake (NRU) cell viability endpoint topredict starting doses for acute oral systemic toxicity test methods. The inclusion of humancells in the validation study also implements another workshop recommendation, that ofevaluating whether cytotoxicity in human or rodent cells can be used to predict human acutetoxicity.
The objectives identified for the validation study were to:• Further standardize and optimize the in vitro NRU basal cytotoxicity protocols
using 3T3 and NHK cells to maximize test method reliability (intralaboratory
repeatability, intra- and inter-laboratory reproducibility)• Assess the accuracy of the two standardized in vitro 3T3 and NHK NRU basal
cytotoxicity test methods for estimating rodent oral LD50 values across thefive United Nations (UN) Globally Harmonized System of Classification andLabelling of Chemicals (GHS; UN 2005) categories of acute oral toxicity, as
well as unclassified toxicities• Estimate the reduction and refinement in animal use achievable from using thein vitro 3T3 and NHK NRU basal cytotoxicity test methods to identify starting
doses for in vivo acute oral toxicity tests, assuming that no other information
were availablexxx

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Develop high quality in vivo acute oral lethality and in vitro NRU cytotoxicity
databases that can be used to support the investigation of other in vitro test
methods necessary to improve the prediction of in vivo acute oral lethality

Scientists assembled for the ICCVAM-sponsored scientific peer review panel meeting(“Panel”) on May 23, 2006 independently assessed the usefulness and limitations of the invitro basal cytotoxicity test methods to predict starting doses for acute oral systemic toxicity
test methods. The Background Review Document (BRD) on the two in vitro NRU test
methods prepared by NICEATM and provided to the peer review panel and the publiccontains:
1. Comprehensive summaries of the data generated in the validation study
2. An analysis of the accuracy and reliability of the test method protocols
3. Related information characterizing the potential animal savings produced byusing the in vitro basal cytotoxicity test methods as adjuncts to specific acutesystemic toxicity test methods
The Panel also evaluated draft test method performance standards, protocols, and draft
ICCVAM recommendations for test method uses and future studies. The public was invited
to provide comments on the BRD and other documents and to attend the Panel meeting. Prior
to the Panel meeting, public comments provided about the documents were provided to the
Panel for their consideration. The BRD can be obtained from the ICCVAM/NICEATM Web
site (http://iccvam.niehs.nih.gov) or by contacting NICEATM.
Following the conclusion of the Panel meeting, the ICCVAM and its Acute Toxicity
Working Group (ATWG) considered the Panel report, the performance standards for the useof in vitro basal cytotoxicity test methods to predict starting doses for acute systemic toxicity
test methods, and any public comments in preparation of its final test method
recommendations for these in vitro basal cytotoxicity test methods. These recommendations
will be made available to the public and provided to the U.S. Federal agencies forconsideration, in accordance with the ICCVAM Authorization Act of 2000 (Public Law 106545).
On behalf of the ICCVAM, we gratefully acknowledge the many contributions of all who
participated in the in vitro cytotoxicity validation study and those who assisted in the
preparation of the documents evaluated at the peer review meeting. We extend a special
thanks to the participating laboratory Study Directors and scientists who worked diligently toprovided critical data and information. We also thank the ECVAM scientists who
participated in the management of the validation study and who provided valuable
information, comments, and opinions throughout the study. The efforts of the ATWG
members were instrumental in assuring a complete and informative BRD. The efforts of the
NICEATM staff in coordinating the validation study, providing timely distribution ofxxxi

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information, and preparing the various documents are acknowledged and appreciated. We
especially acknowledge Dr. Judy Strickland and Mr. Michael Paris for their coordination of
the validation study and preparation of the BRD and other documents.
William S. Stokes, D.V.M. D.A.C.L.A.M.
RADM, U.S. Public Health Service
Director, NICEATM
Executive Director, ICCVAM
Leonard Schechtman, Ph.D.
Deputy Director for Washington Operations
National Center for Toxicological Research
U.S. Food and Drug Administration
Chairman, ICCVAM

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EXECUTIVE SUMMARY
This Background Review Document (BRD) reports the results of a validation study,organized and managed by the National Toxicology Program (NTP) Interagency Center for
the Evaluation of Alternative Toxicological Methods (NICEATM) and the European Centre
for the Validation of Alternative Methods (ECVAM), conducted to characterize two in vitro
basal cytotoxicity tests for determining starting doses for rodent acute oral toxicity assays. In
conducting this validation study, the protocols for two in vitro neutral red uptake (NRU)
assays using BALB/c mouse fibroblast 3T3 cells (3T3) and normal human epidermal
keratinocytes (NHK) were standardized and optimized, and the LD50 values for the reference
substances were refined. The accuracy and reliability of the two in vitro NRU test methods
were determined using 72 reference substances of various toxicities. Computer simulations
were used to estimate the potential reduction in animal usage that could be accomplished by
the use of either of these in vitro test systems. One outcome of this effort has been thegeneration of high quality in vivo lethality and in vitro cytotoxicity reference databases thatwill be useful in the development of other in vitro toxicity tests.
The validation study showed that the 3T3 and NHK NRU test methods are not sufficiently
accurate as stand-alone methods to correctly predict rodent acute oral toxicity. However,based on computer simulations for the reference substances tested in this study, the use ofeither of these two in vitro basal cytotoxicity test methods for the selection of starting doses
for rodent acute oral toxicity testing has the potential to reduce the number of animals used
per test and, in some cases, the number of substance-induced animal deaths.
Introduction and Rationale
Although in vitro basal cytotoxicity test methods are not currently regarded as suitablereplacements for rodent acute oral toxicity tests (Spielmann et al. 1999; ICCVAM 2001a),such methods have been examined as a possible approach to reduce and refine2 the use ofanimals for such testing. An international Multicentre Evaluation of In Vitro Cytotoxicity(MEIC) was initiated in 1983 to evaluate the relationship between in vitro cytotoxicity andacute human toxicity. Tests of 50 substances in 61 in vitro assays by multiple laboratories led
to the identification of a battery of three human cell line assays whose cytotoxicity responses
were highly correlated to human lethal blood concentrations (Bondesson et al. 1989;
Clemedson et al 1996, 1996a; Ekwall et al. 1998a, 1998b, 2000). The Registry ofCytotoxicity (RC), initially published in 1998, is a database of 347 substances that currently
consists of acute oral toxicity data from rats and mice and in vitro cytotoxicity data fromstudies using various mammalian cell types with a number of different toxic endpoints (Halle
1998, 2003). A regression formula, the RC millimole regression, constructed from these data
was proposed by ZEBET, the German National Centre for the Documentation and Evaluationof Alternative Methods to Animal Experiments, as a method to reduce animal use byidentifying the most appropriate starting doses for acute oral toxicity tests (Halle 1998, 2003;
Spielmann et al. 1999).
2

A reduction alternative is a new or modified test method that reduces the number of animals required. A
refinement alternative is a new or modified test method that refines procedures to lessen or eliminate pain or
distress in animals, or enhances animal well-being (ICCVAM 2003).
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These, and other, initiatives to use in vitro cytotoxicity test methods to reduce animal use inacute toxicity testing were evaluated at the International Workshop on In Vitro Methods forAssessing Acute Systemic Toxicity, in October 2000 (“Workshop 2000”; ICCVAM 2001a).
This workshop was organized by the U.S. Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM) and NICEATM. Pursuant to this workshop,
ICCVAM recommended (ICCVAM 2001a) further evaluation of the use of in vitro
cytotoxicity data as one of the approaches that could be used to estimate the starting doses forrodent acute oral toxicity studies. The recommendations are based on preliminary
information suggesting that this approach could reduce the number of animals used in suchstudies (i.e., reduction), minimize the number of animals that receive lethal doses (i.e.,
refinement), and avoid underestimating hazard. To assist in the adoption and implementationof the ZEBET approach, the Guidance Document on Using In Vitro Data to Estimate In Vivo
Starting Doses for Acute Toxicity (hereafter referred to as Guidance Document; ICCVAM
2001b) was prepared by ICCVAM with the assistance of the workshop participants.
In its recommendations for further evaluations, ICCVAM concurred with the Workshop 2000
recommendation that near-term validation studies should focus on two standard basal
cytotoxicity assays: one using a human cell NHK system and one using a rodent cell (3T3)system. Historical data for in vitro cytotoxicity testing using mouse 3T3 cells are available(e.g., Balls et al. 1995; Brantom et al. 1997; Gettings et al. 1991, 1994a, 1994b; Spielmann etal. 1991, 1993, 1996), as are historical data for in vitro basal cytotoxicity testing using NHK
cells (e.g., Gettings et al. 1996; Harbell et al. 1997; Sina et al. 1995; Willshaw et al. 1994).
NICEATM, in partnership ECVAM, designed an international, multi-laboratory validation
study to evaluate the reduction or refinement in animal use that could result from using
cytotoxicity data from the 3T3 and NHK NRU test methods to estimate starting doses for tworodent acute oral toxicity test methods, the Up-and-Down Procedure (UDP; OECD 2001a;
EPA 2002a) and the Acute Toxic Class (ATC) method (OECD 2001d). The NRU protocols,
as presented in the Guidance Document, were the initial basis of the NICEATM/ECVAM
validation study protocols. These protocols were originally derived from the BALB/c 3T3
Cytotoxicity Test, INVITTOX Protocol No. 46 (available at the FRAME-sponsored
INVITTOX database [http://embryo.ib.amwaw.edu.pl/invittox/]), the 3T3 cell studies byBorenfreund and Puerner (1984, 1985) and the rat epidermal keratinocyte study of Heimann
and Rice (1983). A detailed description of the 3T3 and NHK NRU test method protocols
used in the NICEATM/ECVAM validation study is provided in Section 2.
Protocol Components
Many protocol components used in the validation study are similar for the 3T3 and NHK
cells. The following procedures are common to both cell types:• Testing was performed in four phases (Ia, Ib, II, and III)• Preparation of reference substances and positive control• Cell culture environment conditions• Determination of test substance solubility• Configuration of 96-well plates for testing samples• 48-hour exposure to test substance• Range finder and definitive testingxxxiv

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Microscopic evaluation of cell cultures for toxicity
Measurement of NRU
Data analysis

The main differences in the test methods for the two cell types are:
• The conditions of propagation of the cells in culture• The cell growth medium components• The volumes of reference substance added to the 96-well plateThree laboratories participated in testing the 72 reference substances in both cell types:• ECBC: The U.S. Army Edgewood Chemical Biological Center (Edgewood,MD)• FAL: Fund for the Replacement of Animals in Medical Experiments
Alternatives Laboratory (Nottingham, UK)• IIVS: The Institute for In Vitro Sciences (Gaithersburg, MD)
BioReliance Corporation (Rockville, MD) procured and distributed the coded reference
substances and performed solubility tests prior to their distribution to the testing laboratories,
but did not perform any of the in vitro tests.
Validation Reference Substances
The 72 reference substances were selected to represent: (1) the complete range of in vivo
acute oral toxicity (encompassing all five GHS acute oral toxicity categories as well as lower
toxicities [GHS; UN 2005]); (2) the types of substances regulated by various regulatory
authorities; and (3) substances with human toxicity data and/or human exposure potential. To
ensure that the complete range of toxicity was covered, 12 substances were selected for eachof the five acute oral toxicity categories, with an additional 12 substances with lower
toxicities (i.e., LD50 >5000 mg/kg). A discussion of the characteristics and sources of thereference substances can be found in Section 3. The selected reference substances had thefollowing characteristics:• 58 (81%) of the 72 substances were also included in the RC, and 38% (22/58)
of these were outliers with respect to the RC millimole regression.
• 27 (35%) of the substances were pharmaceuticals, 17 (22%) were pesticides, 8(10%) were solvents, and 5 (6%) were food additives. The remaining
substances were used for a variety of manufacturing and consumer products.
The number of assigned uses (77) is greater than the number of selected
substances because some of the substances have more than one use.
• 57 (79%) were organic compounds and 15 (21%) were inorganic; wellrepresented classes of organic compounds included heterocyclics, carboxylic
acids, and alcohols.
• 22 (31%) substances were known, or expected to have, toxicologically active
metabolites.
• Many of the selected substances had multiple target organs/effects; including
neurological, liver, kidney, and cardiovascular effects.

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Table ES-1 reports the number of substances that were tested and the number of substancesused for the various analyses performed.
Table ES-1

Datasets Used for Validation Study Analyses1
Use

TestingComparison of laboratory IC50-LD50
regressions to one another
Comparison of combined-laboratory IC50LD50 regressions to a regression
calculated with RC data
Prediction of GHS accuracy using IC50
values in IC50-LD50 regressions;prediction of starting doses for acute oral
toxicity test (UDP and ATC) simulations
Reproducibility of acceptable rat oral
LD50 values
Reproducibility of IC50 values

3T3
NRU1
72

NHK
NRU1
72

47

51

47

47

67

68

NA

NA

64

68

Characteristics of Dataset
Substances tested
RC substances with IC50 values from alllaboratories and reference rat oral LD50
valuesRC substances with IC50 values for bothtest methods from all laboratories and rat
oral reference LD50 values
Substances with IC50 values from at least
one laboratory
62 substances with more than one
acceptable rat oral LD50 value
Substances with IC50 values from alllaboratories

Abbreviations: RC=Registry of Cytotoxicity; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes;
NRU=Neutral red uptake; NA=Not applicable.
1
Number of substances.

Rodent Acute Oral Toxicity Reference Data
Because the 3T3 and NHK NRU test methods are intended to be used as adjuncts to rodent
acute oral toxicity test methods, the LD50 values from rodent acute oral toxicity tests are themost appropriate reference data for evaluating the in vitro IC50 values (i.e., the test chemical
concentration that reduces cell viability by 50%). Rodent acute oral LD50 reference data forthe 72 reference substances were obtained from the literature. It was not possible to limit thedata to studies conducted under Good Laboratory Practice (GLP) guidelines (OECD 1998;
EPA 2003a, 2003b; FDA 2003) because only 2% of the published data retrieved were fromsuch studies. Although mouse toxicity data were initially considered for inclusion in the
database, the accuracy analyses were restricted to rat data. A total of 459 acute rodent oral
LD50 values were identified for the reference substances. Reference LD50 values for each
substance were identified by excluding studies with the following characteristics:• Feral rats
• Rats <4 weeks of age• Anesthetized rats
• Test substance administered in food or capsule• LD50 reported as a range or an inequality
For substances with multiple LD50 values (i.e., from different sources), the rodent reference
LD50 values for use in the validation study were determined by calculating a geometric meanof the available values for each reference substance. The reference LD50 values for 19 (26%)
of the 72 substances varied sufficiently from the initial LD50 values that came from the RC

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database and other summary sources, that the substances were reclassified into different GHS
categories.
The reliability of the calculated rat acute oral LD50 reference values was assessed bycomparison to other evaluations of the performance of rodent acute oral toxicity tests. For the
62 reference substances that had more than one LD50 value, the maximum:minimum ratios
ranged from 1.1 to 25.9, with most below an order of magnitude.
Test Method Accuracy
Although the 3T3 and NHK NRU test methods are not intended to be used as replacements
for rodent acute oral toxicity tests, they were evaluated for their ability to correctly predictthe reference LD50 values (i.e., accuracy3). The rationale for evaluating the accuracy of LD50
predictions is that the current acute oral toxicity test methods (i.e., UDP, ATC, and Fixed
Dose Procedure [FDP; OECD 2001c]) call for starting doses to be placed as close as possible
and just below the true LD50. When the starting dose is close to the true LD50 for a test
substance, fewer animals are needed. When the starting dose is below the true LD50, there is
reduced pain and suffering because doses tend to be lower, and the test outcome bias is moreconservative (i.e., higher toxicity). Regression models developed using IC50 and LD50 values
were used to derive estimated LD50 values from 3T3 or NHK NRU IC50 values.
A number of different analyses were performed in an attempt to improve the estimation of
the rat acute oral LD50. IC50-LD50 regressions (in millimole units) were calculated for each invitro cytotoxicity test method and participating laboratory using the 3T3 and NHK IC50
values. Because the regressions for each NRU test method among laboratories were notsignificantly different from one another (for each NRU test method, p >0.5), the regression
for each NRU test method was based on data pooled across the laboratories. This combinedlaboratory regression was then compared to the RC data using a regression based on RC IC50
and LD50 data for the 47 substances common to the validation study and the RC, with rat
acute oral LD50 reference values, and with both 3T3 and NHK IC50 values produced by all
three participating laboratories. The statistical comparison of slope and intercept(simultaneously) using an F test showed that neither the 3T3 regression nor the NHK
regression was significantly different from the RC regression for the 47 substances (p =0.61
and 0.76 respectively). These outcomes support use of the RC millimole regression.
Reference substances that fit the RC millimole regression poorly (i.e., outliers) were
evaluated to determine whether there were relationships between their outlier status and their
physical or chemical characteristics. Because the IC50-LD50 regressions for the 3T3 and NHK
NRU test methods yielded results that were not different from the RC regression for 47
substances, the RC millimole regression was preferred for analysis of outliers because it was
based on a much larger data set and because it had established acceptance limits (Halle 1998,
2003). Certain chemical structural classes, boiling points, molecular weights, and log Kow
values were related with outliers, but solubility in the 3T3 or NHK medium and the cells’lack of xenobiotic metabolic capability did not correlate with outlier status. Because these in3

Accuracy is the agreement between a test method result and an accepted reference value (ICCVAM 2003).

xxxviiIn Vitro Cytotoxicity Test Methods BRD Executive Summary

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vitro NRU test methods are based upon basal cytotoxicity, the mechanism of toxicity was
also considered as a characteristic to explain the presence of outliers. Twenty-two reference
substances were neurotoxic or cardiotoxic and were not expected to be active in the 3T3 andNHK cell cultures. Of these 22 substances, 13 (59%) were outliers (i.e., they fit the RC
millimole regression poorly) using the 3T3 NRU and 12 (55%) were outliers using the NHK
NRU. These substances represented 13/28 (46%) and 12/31 (39%) of the outliers for the 3T3
and the NHK NRU test methods, respectively. More information on the outlier analysis ispresented in Section 6.2.
The potential variation produced by combining the LD50 values of two rodent species in the
RC millimole regression was eliminated by developing a regression based solely on RC
substances with rat LD50 data (i.e., the RC rat-only millimole regression). The RC rat-only
data were also converted to a weight basis for an additional regression, the RC rat-only
weight regression, for applicability to mixtures or to substances for which molecular weightis unknown.
The accuracy of the in vitro NRU test methods when used with each of the IC50-LD50
regressions was characterized by determining the proportion of reference substances forwhich their GHS categories (based on rat acute oral LD50 data) were correctly predicted. The
accuracy of the RC rat-only millimole regression was 31% (21/67 reference substances) and
29% (20/68 reference substances) with the 3T3 and the NHK NRU test methods,
respectively. The accuracy of the RC rat-only weight regression was similar, 31% with the
3T3 NRU test method (21/67 reference substances) and 31% with the NHK NRU test method(21/68 reference substances). The poor accuracy is due, in part, to the skewness of thereference substance set with respect to the fit of the reference substances to the regressions
and to the differences between cell cultures and whole animal exposures. Each regression
showed a general trend to underpredict the toxicity of the most toxic chemicals, and to
overpredict the toxicity of the least toxic chemicals. A detailed discussion of the accuracy
analyses is presented in Section 6.4.
Test Method Reliability
Reproducibility is the consistency of individual test results obtained in a single laboratory(intralaboratory reproducibility) or among different laboratories (interlaboratory
reproducibility) using the same protocol and test samples. Reproducibility was evaluatedusing results from the 64 reference substances tested in 3T3 cells and the 68 substancestested in NHK cells that yielded replicate IC50 values in all three laboratories. Intra- andinter-laboratory reproducibility of the 3T3 and NHK NRU IC50 data was assessed using
analysis of variance (ANOVA), coefficient of variation (CV) analysis, comparison of thelaboratory-specific IC50-LD50 regressions, and comparison of maximum:minimum mean
laboratory IC50 values. Reproducibility was generally better with the NHK NRU test method.
Although ANOVA results for the positive control (sodium lauryl sulfate [SLS]) IC50 values
from the 3T3 NRU test method indicated that there were significant differences amonglaboratories (p =0.006) but not between study phases within laboratories (p >0.01), the data
show (see Figure 7-5) that laboratory means and standard deviations from each testing phaseoverlap , and that the IC50 was stable between testing phases. The interlaboratory CV values
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for the various study phases ranged from 2 to 16%. ANOVA results for the SLS IC50 fromthe NHK NRU test method showed significant differences among laboratories (p <0.001) andamong study phases within laboratories (p ≤0.001). The use of a different cell culture method
at FAL was responsible for SLS IC50 differences among the laboratories in Phases Ia and Ib.
After harmonization of culture methods across laboratories, the laboratory means andstandard deviations were similar for Phases II and III (see Figure 7-5). Interlaboratory CV
values for the NHK NRU for Phases Ia and Ib, were 39% and 21%, respectively.
Interlaboratory CV values for Phases II and III were 31% and 8%, respectively. The linear
regression analyses of the SLS IC50 over time (within each laboratory) for both NRU test
methods indicated that IC50 values generated over the 2.5-year duration of the study werestable.
For the reference substances, the similarity among the laboratories’ LD50 predictions (viaregression) from IC50 values (see Figure 7-1) was considered significant with respect to thereproducibility analyses because these in vitro NRU test methods are proposed for use indetermining starting doses for acute oral toxicity tests using the predicted LD50. ANOVA
showed significant laboratory differences for 23 substances with the 3T3 NRU test method(see Table 7-4) and six substances with the NHK NRU test method (see Table 7-6). Mean
intralaboratory CV values were 26% for both NRU test methods, but the NHK NRU test
method had a lower mean interlaboratory CV (28% vs. 47%). An analysis to determine therelationship, if any, between reference substance attributes and interlaboratory CV indicatedthat chemical class, physical form, solubility, and volatility had little effect. The CV seemed
to be related instead to the GHS hazard category, the IC50, and boiling point (see Section
7.2.3). However, the usefulness of these relationships is not known. Mean interlaboratory CV
values were larger for substances in the most toxic GHS hazard categories than for
substances in the other toxicity categories, especially with the 3T3 NRU test method. The
mean interlaboratory CV for substances in the LD50 ≤5 mg/kg (72%) and 5 < LD50 ≤50
mg/kg (78%) categories were larger than the mean overall interlaboratory CV (47%) with the
3T3 NRU test method. When the NHK NRU test method was used, the mean interlaboratory
CV was 37% for substances with LD50 ≤5 mg/kg, and 41% for substances with 5 < LD50 ≤50
mg/kg, and the mean overall interlaboratory CV was 28%. A Spearman correlation analysis
indicated that the IC50 was inversely correlated to interlaboratory CV for both the 3T3 (p=0.015) and NHK (p =0.014) NRU test methods, and that boiling point was positively
correlated to interlaboratory CV (p =0.007) for the 3T3 but not the NHK (p =0.809) NRU
test method.
The maximum:minimum mean laboratory IC50 ratios for the 3T3 NRU test method ranged
from 1.1 to 21.6, with 37 of 64 (58%) reference substances having ratios less than 2.5. The
maximum:minimum mean laboratory IC50 ratios for the NHK NRU test method ranged from
1.0 to 107.6, with 58 of 68 (85%) reference substances having ratios less than 2.5.
Data Quality
The laboratories reported no significant deviations from the protocols, and deviations that did
occur were acknowledged and addressed by the Study Directors. Tests that had deviations
affecting the data were rejected by the Study Directors and repeated. The computation of test
method and data collection errors showed that the non-GLP laboratory consistently had thexxxix

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highest error rate and the lowest intralaboratory reproducibility for IC50 results; however, thelaboratory’s GHS acute oral toxicity category predictions were comparable to that for theother laboratories.
An electronic copy of all data for the validation study can be obtained from NICEATM uponrequest by mail, fax, or e-mail to Dr. William S. Stokes, NICEATM, NIEHS, P. O. Box12233, MD EC-17, Research Triangle Park, NC, 27709, (phone) 919-541-2384, (fax) 919541-0947, (e-mail) niceatm@niehs.nih.gov.
Other Scientific Reports and Reviews
3T3 and NHK NRU methods have been evaluated for purposes other than the prediction ofstarting doses for acute toxicity studies (e.g., ocular irritancy; human lethal bloodconcentrations, in vivo phototoxicity). In vitro NRU cytotoxicity test methods using various
cell types have been evaluated for their correlation with rodent lethality endpoints (e.g.,
rat/mouse intravenous[i.v.], intraperitoneal [i.p.], and oral toxicity). Peloux et al. (1992) andFautrel et al. (1993) showed good correlations (r =0.88) of in vitro cytotoxicity with rodent
i.p./i.v. and i.v. toxicity data, respectively. A 3T3 NRU test method has been validated by
ECVAM for the identification of in vivo phototoxic potential.
No in vitro test methods have been validated for the prediction of acute oral toxicity.
Estimations of animal savings using in vitro cytotoxicity data to estimate starting doses for
the UDP did not use actual in vitro cytotoxicity data. Instead, animal savings were estimatedby assuming that the in vivo starting dose equals the true LD50, which is an approach thatassumes that cytotoxicity data can perfectly predict in vivo lethality. These theoreticalpredictions of animal savings in the UDP ranged from 25-40% (ICCVAM 2001a), as
compared with the average animal savings of 5.3-7.8% predicted using computer simulation
modeling of the UDP for the reference substances tested in the NICEATM/ECVAM study.
Halle et al. (1997) used the in vitro cytotoxicity data in the RC to determine that an animalsavings of 32% can be attained for the ATC method by using the LD50 predicted by the RC
millimole regression as the starting dose. For the reference substances tested in the
NICEATM/ECVAM validation study, most of which were a poor fit to the RC millimole
regression, the average animal savings for the ATC, as determined by computer simulation
modeling, was 4.8-10.2%.
Animal Welfare Considerations: Reduction, Refinement, and Replacement
Computer models were used to simulate testing of the reference substances using to the UDP
and ATC test methods. In principle, animal savings with the FDP could be estimated eventhough death is not the primary endpoint, but the validation study did not include thisanalysis. The number of animals that would be used, and the number of animals that would
survive or die during the UDP or ATC procedure, were determined for the default starting
doses and compared with those when starting dose was based on LD50 values determinedfrom IC50 values for each reference substance using the RC rat-only regressions.
Computer simulation of UDP testing showed that, for the reference substances used in thisvalidation study, using the 3T3 or NHK NRU test methods and the RC rat-only millimole
regression to identify the starting dose resulted in the use of fewer animals per test by anxlIn Vitro Cytotoxicity Test Methods BRD Executive Summary

November 2006

average of 5.3% (0.50 animals) to 6.6% (0.53 animals), depending upon the assumed
mortality-response slope and in vitro NRU test method used. The RC rat-only weight
regression predicted mean animal savings of 6.0% (0.56 animals) to 7.8% (0.62 animals).
When substances were grouped by GHS acute oral toxicity category, there were no animalsavings for substances in the 50 <LD50 ≤300 mg/kg category because the default starting
dose is in this range. The greatest animal savings were observed for substances with 2000 <LD50 ≤5000 mg/kg and LD50 >5000 mg/kg because the limit test, which would be used forsuch substances, uses fewer animals that the main test. Animal savings for these toxicity
categories using the RC rat-only millimole regression ranged from 11.3% (1.21 animals) to
20.3% (1.58 animals) per test. Use of the RC rat-only weight regression produced animalsavings of 12.8% (1.38 animals) to 21.0% (1.63 animals) per test. Although the use of the
3T3 and NHK NRU test methods to estimate starting doses for the simulated UDP decreased
the numbers of animals used per test, it did not change the numbers of animals that died.
Computer simulation of ATC testing showed that, for the reference substances used in thisvalidation study, using the 3T3 or NHK NRU test methods and the RC rat-only millimole
regression to identify the starting dose resulted in a savings of 4.8% (0.51 animals) to 7.3%
(0.80 animals) per test, depending upon the assumed mortality-response slope and the in vitro
NRU test method used. The use of the RC rat-only weight regression produced animalsavings of 8.6% (0.91 animals) to 10.2% (1.09 animals). When substances were grouped by
GHS acute oral toxicity category, there were no animal savings for substances in the 300 <LD50 ≤2000 mg/kg category because this category contains the default starting dose for the
ATC method. Animal savings were highest for substances with 5 < LD50 ≤50 mg/kg and
LD50 >5000 mg/kg. The mean animal savings for both in vitro NRU test methods for
substances with 5 < LD50 ≤50 mg/kg ranged from 9.8% (1.15 animals) to 11.4% (1.33
animals) per test for the RC rat-only millimole regression. The greatest reduction in animal
use would be for substances with LD50 >5000 mg/kg because the limit test used fewer
animals than the main test. Animal savings for these substances ranged from 17.1%, (2.03
animals) to 22.2% (2.66 animals) per test for the RC rat-only millimole regression. When the
RC rat-only weight regression was used, the mean animal savings with both in vitro NRU
test methods for substances with 5 < LD50 ≤50 mg/kg ranged from 10.8% (1.25 animals) to
13.0% (1.51 animals) per test. Mean animal savings for substances with LD50 >5000 mg/kg
ranged from 24.8% (2.94 animals) to 27.7% (3.33 animals) per test. The use of IC50 values toestimate starting doses for the ATC tests refined animal use by producing fewer animal
deaths by approximately 0.5 to 0.6 animals per test.
Simulations for the UDP and ATC method showed that the use of cytotoxicity results toestimate starting doses did not significantly alter the GHS categorizations compared with thecategories determined using default starting doses. This concordance was 97 to 99% for the
3T3 and NHK NRU test methods.
Practical Considerations
Practical issues with respect to the implementation of these in vitro NRU test methods
include the need for, and availability of, appropriate cell culture equipment, training andexpertise, cost, and time expenditure. The ECVAM Good Cell Culture Practice Task Force
Report 1 (Hartung et al. 2002) encourages the establishment of laboratory practices andxliIn Vitro Cytotoxicity Test Methods BRD Executive Summary

November 2006

principles that will reduce uncertainty in the development and application of in vitro test
methods.
All equipment and supplies are readily available, and the in vitro NRU test methods areeasily transferable to laboratories experienced with mammalian cell culture techniques. Muchof the training and expertise needed to perform the 3T3 and NHK NRU test methods arecommon to people with mammalian cell culture experience. Additional technical training
would not be intensive because these methods are similar in general performance to other invitro mammalian cell culture assays. GLP training should be provided to laboratory
personnel (including study directors and principal investigators) to ensure proper adherence
to test protocols and data documentation and verification procedures.
Prices for commercial in vitro NRU cytotoxicity testing to determine the IC50 for one
substance ranged from $1120 to $1850. It is not clear if the price of an in vivo test would be
reduced if it were preceded by an in vitro cytotoxicity test to set the starting dose. Thus, useof these test methods may not reduce the overall cost of rodent acute oral toxicity testing andmay increase the cost, but their use has the potential to reduce the number of animals and thetime needed for a study. The greatest savings in time and animals will occur if the IC50 data
determine that the rodent acute oral toxicity limit test should be performed, rather than themain test. Based on the cost and technical procedures associated with cell culture
maintenance, the 3T3 NRU test method is less expensive and less complicated to conduct
than the NHK NRU test method.

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1.0

INTRODUCTION AND RATIONALE FOR THE USE OF IN VITRO
NEUTRAL RED UPTAKE CYTOTOXICITY TEST METHODS TO
PREDICT STARTING DOSES FOR IN VIVO ACUTE ORALTOXICITY TESTING............................................................................................. 1-3

1.1

Historical Background and Rationale for the Use of In Vitro Cytotoxicity
Assays to Predict Starting Doses for Rodent Acute Oral Toxicity Tests ............ 1-4
1.1.1 The Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) Program ........ 1-4
1.1.2 An International Evaluation of Selected In Vitro Toxicity Test Systems
for Predicting Acute Systemic Toxicity......................................................... 1-6
1.1.3 The Registry of Cytotoxicity (RC) ................................................................ 1-7
1.1.4 The ZEBET Initiative to Reduce Animal Use ............................................... 1-9
1.1.5 The International Workshop on In Vitro Methods for Assessing Acute
Systemic Toxicity ....................................................................................... 1-10
1.1.6 The NICEATM/ECVAM In Vitro NRU Cytotoxicity Validation Study .... 1-11

1.2

Regulatory Rationale and Applicability for the Use of In Vitro
Cytotoxicity Test Methods to Predict Starting Doses for Acute Oral
Toxicity Testing in Rodents................................................................................... 1-14
1.2.1 Current Regulatory Testing Requirements for Acute Oral Toxicity ........... 1-14
1.2.2 Intended Regulatory Uses for In Vitro Cytotoxicity Test Methods............. 1-16
1.2.3 Similarities and Differences in the Endpoints of In Vitro Cytotoxicity
Test Methods and Rodent Acute Oral Toxicity Test Methods .................... 1-17
1.2.4 Use of In Vitro Cytotoxicity Test Methods in the Overall Strategy ofHazard Assessment ...................................................................................... 1-18

1.3

Scientific Basis for the In Vitro NRU Test Methods ........................................... 1-18
1.3.1 Purpose and Mechanistic Basis of In Vitro NRU Test Methods ................. 1-19
1.3.2 Similarities and Differences in the Modes/Mechanisms of Action for
In Vitro NRU Test Methods Compared with the Species of Interest .......... 1-20
1.3.3 Range of Substances Amenable to the In Vitro NRU Test Methods........... 1-20

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1.0

November 2006

INTRODUCTION AND RATIONALE FOR THE USE OF IN VITRO
NEUTRAL RED UPTAKE CYTOTOXICITY TEST METHODS TO
PREDICT STARTING DOSES FOR IN VIVO ACUTE ORAL TOXICITYTESTING

Poisoning is a more serious public health problem than generally recognized. The Institute ofMedicine (IOM) estimates that more than 4 million poisoning episodes occur annually in the
United States (IOM 2004). In 2001, poisoning (30,800 deaths) placed second behind
automobile accidents (42,433 deaths) as the leading cause of injury-related death (IOM
2004). To reduce the risk for accidental poisonings, various regulatory agencies in the UnitedStates (e.g., the Environmental Protection Agency [EPA], the Consumer Products Safety
Commission [CPSC]), require the testing of marketed products for acute oral toxicity inrodents. Increasing societal concerns about animal use have lead to the development andevaluation of alternative in vitro test methods that might refine, reduce, or replace acute oral
toxicity test methods1.
The purpose of this background review document (BRD) is to:• Describe a validation study organized and managed by the National
Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM) and the European Centre for
the Validation of Alternative Methods (ECVAM) to evaluate the ability of
two in vitro basal cytotoxicity test methods to predict starting doses for rodent
acute oral toxicity tests• Provide the results of an evaluation of the accuracy and reliability of the two
in vitro basal cytotoxicity test methods, as well as of the animal savings thatwould occur if these test methods were used to predict the starting dose.
The structure of the BRD follows the requested structure of the ICCVAM2 Guidelines for the
Nomination and Submission of New, Revised, and Alternative Test Methods (ICCVAM
2003).
This section provides:• A historical perspective of scientific efforts to develop and evaluate the abilityof in vitro cytotoxicity test methods to refine, reduce, or replace acute oral
toxicity test methods• A general review of reported correlations between in vitro cytotoxicity andacute oral lethality in rodents• The regulatory requirements for rodent acute oral toxicity testing• The scientific basis of using in vitro basal cytotoxicity test methods to predict
the starting doses for rodent acute oral toxicity assays
1

A reduction alternative is a new or modified test method that reduces the number of animals required. A
refinement alternative is a new or modified test method that refines procedures to lessen or eliminate pain or
distress in animals or enhances animal well-being (ICCVAM 2003).
2

The Interagency Coordinating Committee for the Validation of Alternative Methods
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In Vitro Cytotoxicity Test Methods BRD Section 1•
1.1

November 2006

The intended regulatory uses and applicability of in vitro basal cytotoxicity
test methods

Historical Background and Rationale for the Use of In Vitro Cytotoxicity
Assays to Predict Starting Doses for Rodent Acute Oral Toxicity Tests

This section provides the historical background and rationale for the NICEATM/ECVAM
validation study by summarizing several major studies promoted by the European Union(EU) to investigate the properties and capabilities of cell-based methods to predict acutetoxicity. The Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) Program was initiatedin 1983 to compare in vitro methods to acute oral lethality in humans (Section 1.1.1). In
1992-1993, the Fund for the Replacement of Animals in Medical Experiments (FRAME)
conducted an international evaluation of selected in vitro toxicity test systems for predicting
acute systemic toxicity (Section 1.1.2). Dr. Willi Halle published a monograph regarding thedevelopment of the Registry of Cytotoxicity (RC) database to evaluate whether basal
cytotoxicity data could accurately predict acute oral lethality in rats and mice (Section 1.1.3).
ECVAM organized a workshop in 1994 to evaluate the use of in vitro data for theclassification and labeling of chemicals and reviewed the assessment of acute oral toxicity
using in vitro data. Workshop participants suggested that the use in vitro data to determine
starting doses for acute oral toxicity tests would reduce the use of animals. The German
Center for Documentation and Evaluation of Alternative Methods to Animal Experiments(ZEBET) then recommended that in vitro basal cytotoxicity data be used with the RC
millimole regression, which is referred to as the ZEBET approach (Section 1.1.4), to
determine starting doses for acute oral toxicity tests. Section 1.1.5 provides background on
an international workshop that reviewed and evaluated the EU studies above and Section
1.1.6 describes the NICEATM/ECVAM in vitro cytotoxicity validation study that expands
upon the EU studies.
1.1.1
The Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) Program
The Scandinavian Society for Cell Toxicology established the MEIC program in 1983 toinvestigate the ability of in vitro cytotoxicity test methods to predict acute oral lethality inhumans (Bondesson et al. 1989). MEIC was based on the following assumptions:• In vitro cell culture systems could be used to model in vivo acute oral toxicity.
• The basal cytotoxicity detected by these in vitro test methods is responsible
for a large proportion of in vivo toxic effects3.
The MEIC program was an open study that invited laboratories worldwide to participate in
testing 50 reference substances using laboratory-specific in vitro cytotoxicity assays.
Although the MEIC management team requested that all participating laboratories test
chemicals with high purity, no effort was made to assure that the substances tested werepurchased from the same supplier or were of the same purity (Clemedson et al. 1996a).
Minimal methodological directives were provided so as to maximize protocol diversity
among the 96 participating laboratories.
3

Basal, or general, cytotoxicity was described as toxicity resulting from interference with basic cellular
structures and functions, such as cell membranes, metabolism, ion regulation, and cell division that are common
to all human and animal cells.

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In Vitro Cytotoxicity Test Methods BRD Section 1

November 2006

The reference substances were selected to represent different chemical classes for whichreference acute oral lethality data existed in humans (i.e., lethal doses, kinetics, and lethal
blood/serum concentrations [LC]) and rodents (oral median lethal dose [LD50] values)(Bondesson et al. 1989). The MEIC management team collected human data from clinicaland forensic toxicology handbooks and case reports of human poisonings (Ekwall et al.
1998a). The resulting data were presented and analyzed in a series of 50 MEIC Monographs.
Rat and mouse oral LD50 data were collected from the Registry of Toxic Effects for
Chemical Substances (RTECS®)4.
The 50 reference substances were tested in as many as 61 different in vitro assays (Ekwall etal. 1998b). The metric of interest was the IC50 (i.e., the concentration that inhibited the
response measured by 50%) for the endpoint measured. Of the 20 test methods that usedhuman-derived cells, 18 used cell lines and two used primary cell cultures. Of the 21 test
methods that used mammalian (but other than human) cells, 12 used cell lines and nine usedprimary cell cultures. Eighteen test methods were ecotoxicological in nature and two usedcell-free systems. Cell viability and/or cell growth were the endpoints of choice in the
majority of the cell-based systems. The chemical exposure duration ranged from 5 minutes to
6 weeks, but most frequently was 24 hours (Clemedson et al. 1996).
The ability of the in vitro IC50 data to predict human acute oral lethality was assessed using
human LC values compiled from three different data sets (Ekwall et al. 2000):• Clinically measured acute lethal serum concentrations• Acute LC values measured post-mortem• Peak LC values derived from approximate LC50 curves over time after
exposure
A partial least squares (PLS) analysis indicated that the IC50 data generated from as many as
61 test methods predicted the three sets of LC data well (R2=0.77, 0.76, and 0.83, Q2=0.74,
0.72, and 0.81, respectively, where R2 is the determination coefficient and Q2 is the predictedvariance according to cross-validation in the PLS model used). A two component PLS modelusing rat and mouse oral LD50 values less accurately predicted human LC values (R2=0.65,
Q2=0.64). These results suggested that in vitro basal cytotoxicity assays might be more
effective in estimating human acute oral lethality than rodent acute oral toxicity test methods.
Because the MEIC study showed that the in vitro test methods with the best predictivity
generally used human cell lines (Ekwall et al. 1998b), the MEIC management team identified
a battery of in vitro assays using three human cell lines that had maximal performance forpredicting peak acute LC values in humans (R2=0.79 and Q2=0.76) (Ekwall et al. 2000).
However, it was concluded that improvements in the prediction of human acute oral lethality
were necessary before in vitro cytotoxicity assays could replace animal tests. To adjust forlethality produced by mechanisms other than basal cytotoxicity, the Evaluation-guided
Development of New In Vitro Tests (EDIT) program was proposed to address targeted
4

RTECS® was originally published by the U.S. National Institute for Occupational Safety and Health (NIOSH)
and is currently licensed to MDL Information Systems, Inc.
1-5

In Vitro Cytotoxicity Test Methods BRD Section 1

November 2006

development of in vitro test methods for other endpoints, including biokinetics (e.g., gut
absorption, distribution, clearance), biotransformation, and target organ toxicity (Clemedson
et al. 2002).
1.1.2

An International Evaluation of Selected In Vitro Toxicity Test Systems for
Predicting Acute Systemic Toxicity
FRAME organized an international collaborative study conducted in 1992 - 1993 to evaluate
the prediction of rodent acute oral lethality by in vitro test methods (Fentem et al. 1993)5.
The objective of the study was to identify in vitro systems and strategies that could be used
for the classification and labelling of new chemicals, thereby reducing, and possibly
replacing, the use of animals for acute oral toxicity testing.
The 42 substances tested in the study comprised a diverse group of organic and inorganic
chemical classes, including surfactants, pharmaceuticals, and pesticides (Fentem et al. 1993).
In vitro toxicity assays using different mammalian cell lines, exposure periods, and toxicity
endpoints were evaluated, including:• Two cell proliferation assays (total protein in mouse BALB/c 3T3 fibroblast
cells and MTT6 reduction in Chinese hamster fibroblastoid V79 cells after a
72-hour exposure period)• Two cytolethality assays (MTT reduction in V79 cells and lactate
dehydrogenase [LDH] release from primary rat hepatocytes after a 24-hour
exposure period)• A cell function assay (myotube contractility inhibition in rat skeletal muscle
cells)The resulting in vitro IC50 data were linearly regressed against the lowest available rat or
mouse oral LD50 values for each test substance. There were no significant differences among
the IC50-LD50 regressions for the different in vitro test methods.
A subset of 26 to 40 of the 42 test substances, based on the availability of European Union(EU) hazard classification data, was used to evaluate two approaches for using in vitro IC50
data to classify chemicals into the four hazard categories used by the EU for acute oral
toxicity labelling (Fentem et al. 1993). One approach used the IC50 values obtained from thefive different in vitro test methods for each test substance to predict the LD50 value andhazard category from the IC50-LD50 regression. The accuracy of hazard classification for thefive in vitro tests was from 43 to 65%. The other approach used toxicokinetic parameters for
31 to 38 substances to convert the IC50 values to effective dose (i.e., ED50) values. Hazard
classification accuracy was 43 to 55%.

5

The collaborative study was conducted by the Institute of Toxicology, Kiel, Germany; the University of
Nottingham, United Kingdom; and the Gesellschaft für Strahlen- und Umweltforschung, Neuherberg, Germany(Society for Radiological and Environmental Research, which later changed its name to Center for
Environmental and Health Research [Forschungszentrum für Umwelt und Gesundheit])
6
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide is metabolized by the mitochondrial
succinate dehydrogenase of proliferating cells to yield a purple formazan reaction product.
1-6

In Vitro Cytotoxicity Test Methods BRD Section 1

November 2006

In addition, to ensure that a variety of toxic mechanisms were evaluated during in vitro
testing, the lowest predicted LD50 or ED50 from the results of a battery of three tests: a cellproliferation assay (total protein for 3T3 cells); a cytotoxicity/cytolethality assay using
primary rat hepatocytes (LDH release); and the rat skeletal muscle cell contractility assay,
was used also. The lowest predicted LD50 or ED50 of the three tests was then used to predict
toxicity classification. The accuracy of classification using this approach was 48% for the
ED50 and 45% for the predicted LD50 values.
Based on the results obtained, a battery of in vitro tests was recommended for classifying
chemicals for their acute lethal potency in rodents (Fentem et al. 1993). The first order test in
the battery measures basal cytotoxicity. This study observed no major differences in theperformances of the in vitro test methods that measure inhibition of cell growth regardless ofthe cell line (V79, 3T3-L1, or BALB/c 3T3), exposure duration (24-72 hours), or endpoint
measurement technique (MTT reduction, neutral red uptake [NRU], or protein
concentration). The second order test in the battery assesses hepatocyte-specific toxicity andthe role of biotransformation in cytotoxic activity. Co-cultures of rodent hepatocytes withproliferating cells such as 3T3 cells were recommended because the use of hepatocytes alone
would not indicate that a chemical requires bioactivation to produce its toxic effects. The
third order test in the battery detects chemicals that interfere with electrically excitable
membranes at non-cytotoxic concentrations (e.g., a contractility assay using primary culturesof rat muscle cells) (Fentem et al. 1993).
1.1.3
The Registry of Cytotoxicity (RC)The RC is a database of acute oral LD50 values for rats and mice obtained from RTECS®, andpublished IC50 values from in vitro cytotoxicity assays that used a variety of cell lines andcytotoxicity endpoints for substances with known molecular weights (Halle 1998, 2003). The
main purpose for compiling the RC was to evaluate, using data from substances with a wide
range of rodent acute oral toxicities, whether basal cytotoxicity (averaged over various cell
types, cell lines, and/or toxicity endpoints) accurately predicted acute oral lethality in rats andmice. The RC currently contains data for 347 different substances (Halle 1998, 2003) andefforts are underway to increase the number to 500 (ICCVAM 2001a). The RC does notcontain data on chemical mixtures.
The RC contains cytotoxicity data for substances that met the following criteria (Halle 1998,
2003):• At least two different IC50 values needed to be available, from studies using
either different cell types, different cell lines, or different cytotoxicity
endpoints• Data had to be generated using mammalian cells only (although data fromstudies using hepatocytes or related cells were excluded)• The chemical exposure duration had to be at least 16 hours, with no upper
limit
The following cytotoxicity endpoints were accepted:• Cell proliferation: cell number; cell protein; DNA content; DNA synthesis;
3
H-thymidine intake; colony formation

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In Vitro Cytotoxicity Test Methods BRD Section 1•••

November 2006

Cell viability/metabolic indicators: metabolic inhibition test (MIT-24);
mitochondrial reduction of tetrazolium salts into an insoluble (MTT) or
soluble (2,3-bis(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5carboxanilide [XTT]) dye
Cell viability/membrane indicators: NRU; trypan blue exclusion; cellattachment; cell detachment
Differentiation indicators, such as functional and/or morphological changes
among and within cells

IC50 values (1,912) for 347 substances were obtained from 157 original publications (Halle
1998, 2003). The two to 32 IC50 values for each substance were averaged as geometric means
to produce one IC50x value for each substance. The rodent LD50 values used in the RC wereobtained from RTECS®. For the first 117 substances, designated as the training data set (RCI), LD50 values were not revised when subsequent issues of RTECS® reported lower values7.
For the most recent 230 substances, designated as the verification set (RC-II), the LD50
values were taken from the 1983/84 RTECS® publication. Whenever obtainable, oral LD50
data from rats were used (282 values). If rat data were unavailable, LD50 data from mice wereused (65 values). Combining rat and mouse data in the regression was deemed to be justified
when separate regressions for the mouse and rat LD50 values against the IC50x values did not
result in significant differences between the slopes and intercepts of the two regressions(Halle 1998, 2003).
To develop a model for the prediction of acute oral LD50 values from IC50x values, Halle(1998, 2003) calculated a linear regression from pairs of the log-transformed IC50x values (inmM) and log transformed rodent oral LD50 values (in mmol/kg) (see Figure 1-1). Molar
concentrations were used to allow for a comparison among chemicals based on the number ofmolecules rather than formula weights. The regression, referred to here as the RC millimole
regression, has the following formula:log LD50 (mmol/kg) = 0.435 x log IC50x (mM) + 0.625
To identify an acceptability range for practical use and research purposes, the acceptableprediction interval for the LD50 was empirically defined as approximately one-half an orderof magnitude on either side of the best-fit linear regression (i.e., ± log 5, or ±0.699) (Halle
1998, 2003). This interval was based on eight linear regressions calculated for in vitro
mammalian cell cytotoxicity data using various endpoints and oral LD50 values from rat,mouse, or rat and mouse from five publications. The prediction interval approximates thepredicted LD50 range for the eight regressions across about eight orders of magnitude of IC50
values. When this approach was used, 73% (252/347) of the RC substances fall within theprediction interval.

7

RTECS® published the lowest LD50 reported for a substance and updates the information periodically.
1-8

In Vitro Cytotoxicity Test Methods BRD Section 1

Figure 1-1

November 2006

RC Millimole Regression for In Vitro Cytotoxicity (IC50x)
and Rat and Mouse Acute Oral LD50 Values for 347 Chemicals

103

102

101

100

10-1

10-2

10-3

10-4
10-5

10-4

10-3

10-2

10-1

100

101

102

103

IC50x (mM)

Abbreviations: RC=Registry of Cytotoxicity; IC50x=Geometric mean (of multiple endpoints and cell types) test
substance concentration that reduces cell viability by 50%; LD50=Dose producing death in 50% of the animals
tested.
The heavy line shows the fit of the data to a linear regression model, log (LD50) = 0.435 x log (IC50x) + 0.625;
r=0.67. The thinner lines show the empirical prediction interval (± log 5, or ±0.699) that is based on the
anticipated precision for the prediction of LD50 values from cytotoxicity data (Halle 1998, 2003).

1.1.4
The ZEBET Initiative to Reduce Animal Use
ECVAM organized a workshop in 1994 to evaluate the use of in vitro data for theclassification and labeling of chemicals (Seibert et al. 1996). Workshop participants reviewed
information on the assessment of acute oral toxicity using in vitro data and concluded that,for in vitro data to be used most effectively, the following information would be necessary:• The active concentration in vitro (i.e., the actual concentration available to thecultured cells)• The in vitro concentrations that produce basal cytotoxicity, hepatocyte
toxicity, and selective cytotoxicity (i.e., effects on cell-specific functions suchas transport processes or cell-to-cell communication)• The effect of biokinetic processes on acute oral toxicity in rodents• In vitro tests that provide the physicochemical parameters needed to estimate
equivalent body doses from in vitro data

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In Vitro Cytotoxicity Test Methods BRD Section 1

November 2006

The concept that in vitro data could be used to determine the starting doses for rodent acuteoral toxicity tests, so as to reduce the number of animals used, was first discussed at thisworkshop (Seibert et al. 1996). At that time, draft Organisation for Economic Co-operation
and Development (OECD) sequential rodent acute oral toxicity test guidelines (TGs) wereavailable; these included the:• Acute Toxic Class method (ATC; OECD draft Test Guideline [TG] 423[ICCVAM 2001a])• Up-and-Down Procedure (UDP; OECD draft TG 425 [ICCVAM 2001a])• Fixed Dose Procedure (FDP; OECD draft TG 420 [ICCVAM 2001a])
Final OECD TGs now exist for these rodent acute oral toxicity tests. The number of animals
needed depends upon the choice of the starting dose because the number of consecutive
dosing steps, and thus the number of animals used, is reduced as the starting dose moreclosely approximates the true toxicity class for the ATC or the FDP, or the true LD50 for the
UDP.
The ZEBET approach involves using an IC50 value from an in vitro basal cytotoxicity testwith the RC millimole regression to predict an LD50 value for use as a starting dose for the
ATC or UDP (Spielmann et al. 1999). Using simulation results performed to evaluate thedraft UDP test method, ZEBET predicted that the use of in vitro cytotoxicity assays topredict a starting dose equivalent to the LD50 had the potential to reduce animal use in the
UDP by 25-40%, depending upon the slope of the concentration response curve and thestopping rule applied (Spielmann et al. 1999; ICCVAM 2001a).
1.1.5

The International Workshop on In Vitro Methods for Assessing Acute Systemic
Toxicity
In 2000, the U.S. National Institute of Environmental Health Sciences (NIEHS), the NTP,
and the EPA jointly sponsored an International Workshop on In Vitro Methods for Assessing
Acute Systemic Toxicity (hereafter known as Workshop 2000). This workshop evaluated:• The ZEBET approach using the RC millimole regression to estimate LD50
values and set starting doses for in vivo testing• A testing strategy proposed by the European Center for the Validation of
Alternative Methods (ECVAM) (Siebert et al. 1996)• Other initiatives for reducing animal use in rodent acute oral toxicity testingby using in vitro cytotoxicity test methods (ICCVAM 2001a)
The Workshop 2000 participants concluded that no in vitro cytotoxicity test methods (orbattery of in vitro cytotoxicity test methods) existed that could replace the current in vivo
acute oral toxicity test methods (ICCVAM 2001a). Furthermore, they concluded that none of
the in vitro models reviewed had been adequately evaluated for reliability and relevance, andtheir usefulness and limitations for generating information for acute toxicity testing had notbeen assessed. However, there was agreement that: (1) in the near-term, in vitro basal
cytotoxicity test methods would be useful for estimating the starting dose for rodent acuteoral toxicity studies, and (2) further development, optimization, and validation of in vitro test
methods that considered target organ specificity and in vivo factors like adsorption,
distribution, metabolism, and excretion (ADME) that modulate the lethality of a xenobiotic
were needed (ICCVAM 2001a). Furthermore, the approach proposed by ZEBET (i.e., the use
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In Vitro Cytotoxicity Test Methods BRD Section 1

November 2006of in vitro basal cytotoxicity test methods to predict the starting dose for the sequential rodent
acute oral toxicity test methods) (Halle 1998, 2003; Spielmann et al. 1999) was
recommended for rapid adoption so that data could be generated to establish its usefulness
with a larger number of substances (ICCVAM 2001a). To assist in the adoption andimplementation of the ZEBET approach, several workshop participants prepared the
Guidance Document on Using In Vitro Data to Estimate In Vivo Starting Doses for Acute
Toxicity (hereafter referred to as Guidance Document; ICCVAM 2001b).
The Guidance Document recommended testing 10 to 20 RC substances (of high purity) fromthe RC in a candidate in vitro basal cytotoxicity assay to be used for predicting starting doses
for acute oral lethality tests (ICCVAM 2001b). The substances were to cover a wide range of
toxicities and fit the RC prediction model (i.e., the linear regression line) as closely as
possible. The in vitro test methods recommended and provided as examples were NRU
assays using 3T3 and normal human epidermal keratinocytes (NHK) cells. The IC50 resultsfrom testing the selected substances would be used to calculate a regression against the LD50
values used by the RC. If the resulting regression were parallel to the RC millimole
regression and within the ± log 5 (i.e., ±0.699) prediction interval for the RC, the GuidanceDocument recommended using the in vitro cytotoxicity assay to predict starting doses for
LD50 assays. If the regression from the in vitro assay did not meet these criteria, then the
Guidance Document advised either (a) adjusting the slope or (b) using the NRU protocols
offered in the Guidance Document (considered the most efficient approach).
Based on the conclusions and recommendation of the Workshop 2000 participants, ICCVAM
subsequently recommended that near-term validation studies should focus on two in vitro
basal cytotoxicity assays: one using human cells and one using rodent cells. Human cells areof interest because a principal aim of rodent acute oral toxicity testing is to predict potential
lethality in humans, while rodent cells may be a better predictor of lethality in rats and mice
(ICCVAM 2001a).
1.1.6
The NICEATM/ECVAM In Vitro NRU Cytotoxicity Validation Study
In response to the ICCVAM recommendation, NICEATM and ECVAM designed an
independent8 multi-laboratory validation study to evaluate in vitro basal cytotoxicity,
measured as NRU, as a predictor of acute oral lethality in rodents and potentially in humans.
Based on historical in vitro cytotoxicity data for mouse BALB/c 3T3 fibroblast cells (e.g.,
Balls et al. 1995; Brantom et al. 1997; Gettings et al. 1991, 1994a, 1994b; Spielmann et al.
1991, 1993, 1996) and NHK cells (e.g., Gettings et al. 1996; Harbell et al. 1997; Sina et al.
1995; Willshaw et al. 1994), it was decided that these two cells types should be the focus of
this validation effort.
The primary aim of this validation study was to determine if the NRU IC50 concentration of atest substance in either 3T3 or NHK cells could be used to estimate the rodent LD50, as ameans for predicting the starting doses for rodent acute oral toxicity studies. A secondary aim
was to determine the extent to which the NRU IC50 in either 3T3 or NHK cells could be used
8

“Independent” is used here to indicate that neither NICEATM nor ECVAM, nor its members, had a monetaryinterest in the test methods.

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In Vitro Cytotoxicity Test Methods BRD Section 1

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to estimate the blood serum concentrations associated with acute oral lethality in humans.
This evaluation will be the focus of a future ECVAM report.
The specific objectives for this validation study were to:• Further standardize and optimize the in vitro NRU basal cytotoxicity protocols
using 3T3 and NHK cells to maximize test method reliability (intralaboratory
repeatability, intra- and inter-laboratory reproducibility)• Assess the accuracy of the two standardized in vitro 3T3 and NHK NRU basal
cytotoxicity test methods for estimating rodent oral LD50 values across thefive United Nations (UN) Globally Harmonized System of Classification andLabelling of Chemicals (GHS; UN 2005) categories of acute oral toxicity, as
well as unclassified toxicities• Estimate the reduction and refinement in animal use achievable from using thein vitro 3T3 and NHK NRU basal cytotoxicity test methods to identify starting
doses for in vivo acute oral toxicity tests, assuming that no other information
were available• Develop high quality in vivo acute oral lethality and in vitro NRU cytotoxicity
databases that can be used to support the investigation of other in vitro test
methods necessary to improve the prediction of in vivo acute oral lethality
1.1.6.1 Study Design
The planning phase of the validation study included the selection of reference substances fortesting, which is described in Section 3, and the identification of rodent oral LD50 values forthe reference substances, which is described in Section 4. The validation study proceeded inseveral phases (see Figure 1-2) so that the Study Management Team (SMT) could evaluate
the reproducibility of results after each phase and refine the protocols, if necessary, before
proceeding to the next phase. The resulting NRU data collected were used to evaluate linear
regression formulas for the prediction of LD50 values from IC50 values (see Section 6).
Computer simulation modeling of acute oral toxicity test outcomes was then performed todetermine potential animal savings using the NRU-predicted starting doses compared withthe default starting dose for the UDP and the ATC (see Section 10). Study management andstudy participant information is provided in Appendix A.

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Figure 1-2

November 2006

NICEATM/ECVAM Validation Study Phases
Phase Ia: Laboratory Evaluation•••

Development of a positive control database for each laboratory
Perform at least 10 replicate NRU tests of the positive control substance (sodium laurylsulfate [SLS]) with each cell type.
Calculate mean IC50 value ±2 standard deviations for each cell type for each laboratory.
Establish acceptance criteria for positive control performance in future assays.
⇓
Phase Ib: Laboratory Evaluation••

Limited substance testing to demonstrate the reliability of the protocol
Each laboratory tests the same three coded substances three times with each cell type.
There was one substance each from low, medium, and high GHS toxicity categories.
Refine protocols and repeat, if necessary, until acceptable intra- and inter-laboratory
reproducibility is achieved.
⇓
Phase II: Laboratory Qualification

•
•
•
•

Evaluation of protocol refinements
Each laboratory tests nine coded substances covering the range of GHS toxicity
categories, with three replicate tests per substance in each test method.
Assure that corrective actions taken in Phase I have achieved the desired results.
Further refine protocols and re-test, if necessary, to achieve acceptable reliability.
Finalize protocols for Phase III.
⇓
Phase III: Laboratory Testing Phase•

Test of optimized protocols
Each laboratory tests 60 coded substances in three replicate tests using the finalized
protocol for each test method.

__________________________________________________________________________
Abbreviations: NRU=Neutral red uptake; GHS=Globally Harmonized System of Classification and Labelling ofChemicals (UN 2005)

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1.2

November 2006

Regulatory Rationale and Applicability for the Use of In Vitro Cytotoxicity
Test Methods to Predict Starting Doses for Acute Oral Toxicity Testing in
Rodents

1.2.1
Current Regulatory Testing Requirements for Acute Oral Toxicity
The major regulatory need for acute oral toxicity testing is for the hazard classification andlabeling of products, which is intended to alert handlers and consumers to potential toxicity
hazards. The LD50 values from acute oral toxicity tests using rodents are used to placesubstances in various toxicity categories that, in turn, invoke the associated hazard phrases tobe used on product labels. Table 1-1 shows the current U.S. legislation requiring the use of
acute oral toxicity testing for product labeling, and the substances regulated. Table 1-2
shows the statutory test protocol requirements and classification systems used by each U.S.
regulatory agency. Also included in this table is the UN Harmonized Integrated
Classification System for Human Health and Environmental Hazards of Chemical Substancesand Mixtures, which provides guidance to regulatory agencies on the use of the GHS (UN2005) as an internationally comprehensible system for hazard communication (OECD
2001b).
Table 1-1

Summary of Current U.S. Legislation for Using Acute Toxicity Data for
Product Labeling

Legislation(Year of Initial Enactment)
Federal Insecticide, Fungicide andRodenticide Act (FIFRA; 1947)
Federal Hazardous Substances Act (1964)
Occupational Safety and Health Act (1970)
Federal Hazardous Material Transportation
Act (1975)

U.S. Regulatory
AgencySubstances Regulated

EPA

Pesticides

CPSC
OSHA

Household products
Workplace materials

DOT

Transported substances

Abbreviations: EPA=U.S. Environmental Protection Agency; CPSC=U.S. Consumer Product Safety Commission;
FIFRA=Federal Insecticide, Fungicide, and Rodenticide Act; OSHA=U.S. Occupational Safety and Health Administration;
DOT=U.S. Department of Transportation.
Note: The U.S. Food and Drug Administration (FDA) does not require data for from acute lethality testing, and discourages
the use of animals for such testing (FDA 1993).

In addition to classification and labeling, acute oral toxicity test results may be used for:• Establishing dosing levels for repeated dose toxicity studies or other toxicity
studies• Identifying potential target organs• Providing information related to the mode of toxic action• Aiding in the diagnosis and treatment of toxic reactions• Providing information for comparison of toxicity and dose response among
substances in a specific chemical or product class• Aiding in the standardization of biological products• Aiding in judging the consequences of single, high accidental exposures in theworkplace, home, or from accidental release• Serving as a standard for evaluating alternatives to animal tests

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Table 1-2

November 2006

Regulatory Classification Systems for Acute Oral Toxicity

Regulatory Agency(Authorizing Act)
EPA (FIFRA)CPSC (Federal Hazardous
Substances Act)

Animals
Use current
EPA orOECD
protocolWhite rats,
200-300 g

OSHA (Occupational
Safety and Health Act)
DOT (Federal Hazardous
Material Transportation
Act)

Albino rats,
200-300 g
Male andfemale young
adult albinoratsOECD Guidance for Useof GHS (2001b)

Protocols not
specifiedEndpoint
Death

1

Death1 within 14 daysfor ≥ half of a group of≥10 animals
Death1, duration not
specified.
Death1 within 14 daysof half the animals
tested. Number ofanimals tested must be
sufficient for
statistically valid
results.
Not specified

Classification
I - LD50 ≤50 mg/kg
II - 50 < LD50 ≤500 mg/kg
III - 500 < LD50 ≤5000 mg/kg
IV - LD50 >5000 mg/kg
Highly toxic - LD50 ≤50 mg/kg
Toxic - 50 mg/kg < LD50 <5 g/kg
Highly toxic - LD50 ≤50 mg/kg
Toxic - 50 < LD50 <500 mg/kg
Packing Group 1 - LD50 ≤5 mg/kg
Packing Group II - 5 < LD50 ≤50 mg/kg
Packing Group III - LD50 <500 mg/kg (liquid)
LD50 <200 mg/kg (solid)

I - LD50 ≤5 mg/kg
II - 5 < LD50 ≤50 mg/kg
III - 50 < LD50 ≤300 mg/kg
IV - 300 < LD50 ≤2000 mg/kg
V - 2000 < LD50 ≤5000 mg/kg
Unclassified - LD50 >5000 mg/kg

Abbreviations: EPA=U.S. Environmental Protection Agency; OECD=Organisation for Economic Co-operation and Development; LD50=Dose
producing death in 50% of the animals tested; CPSC=U.S. Consumer Product Safety Commission; FIFRA=Federal Insecticide, Fungicide, andRodenticide Act; OSHA=U.S. Occupational Safety and Health Administration; DOT=U.S. Department of Transportation; GHS=Globally
Harmonized System of Classification and Labelling of Chemicals (UN 2005).
1
Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety
Evaluation calls for humane killing of moribund animals (OECD 2000). Moribund animals that are humanely euthanized are accepted as deaths.

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1.2.1.1 Test Methods for Assessing Acute Oral Toxicity
The current internationally recognized test methods for acute oral toxicity testing are the FDP(OECD 2001c), the ATC (OECD 2001d), and UDP (OECD 2001a; EPA 2002a) (seeAppendix M for test method guidelines). Information on toxic doses and signs of acutetoxicity and target organs can be obtained using any of these three methods. All threemethods are sequential tests in which the outcome of testing one or more animals at the firstdose is used to determine the second dose that should be tested. The FDP differs from the
UDP and ATC in that it involves using more animals per dose, and the primary endpoint ofinterest is evident toxicity9 rather than lethality. Both the FDP and the ATC methods provide
a range estimate of the LD50 for classification purposes. The UDP generally provides a point
estimate of the LD50 with a confidence interval (EPA 2002a).
Each of the test method guidelines includes a limit test in which up to five or six animals aretested at the limit, or upper bound, dose depending on the dose chosen (OECD 2001a, c, d, e;
EPA 2002a). The limit test can be performed using 2000 or 5000 mg/kg, depending on theregulatory need.
1.2.2
Intended Regulatory Uses for the In Vitro Cytotoxicity Test Methods
In vitro cytotoxicity test methods currently cannot serve as replacements for acute oral
toxicity tests in animals. However, such test methods can be used as adjuncts for rodent acuteoral toxicity tests. The current test guidelines for acute oral toxicity tests recommend using
information from structurally-related substances and the results of any other toxicity tests(EPA 2002b), including in vitro cytotoxicity test method (OECD 2001a, c, d; EPA 2002a) toselect the starting in vivo dose. The 3T3 and NHK NRU test methods may be used as part ofthis weight-of-evidence approach to select starting doses in order to reduce and refine the useof animals for acute oral toxicity testing.
Section 10 presents computer simulation analyses that characterize the extent of animal
reduction and refinement that may occur by using the in vitro NRU test methods to estimate
the starting doses for the UDP and the ATC method, by estimating the numbers of animals
used and the numbers of animal that die. These simulations determined (1) the numbers ofanimals used when using the default starting dose and, (2) the number of animals used whenusing a starting dose determined from the in vitro NRU test methods. These calculations
determined the reduction in animal use that can be achieved when using the in vitro NRU test
methods. To characterize the extent of refinement produced using the NRU-determined
starting dose, the number of animals that would have died with the NRU-determined starting
dose was compared with the number of animals that would have died when using the default
starting dose. Because there is a lack of information for specific substances about the dose atwhich evident toxicity occurs in relationship to the LD50, the FDP will not be consideredfurther in this document. However, the use of in vitro cytotoxicity data to determine starting
doses may also reduce the use of animals in the FDP.

9

Evident toxicity is a general term describing clear signs of toxicity following administration of the test
substance, such that the next highest fixed dose would result in the development of severe toxic signs, andprobably mortality (ICCVAM 2000).
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1.2.3

Similarities and Differences in the Endpoints of the In Vitro Cytotoxicity Test
Methods and Rodent Acute Oral Toxicity Test Methods
The endpoint measured in the in vitro NRU test methods is cell death. Neutral red dye istaken up and accumulated only by live cells; the primary measure of interest is the IC50 (i.e.,
the test substance concentration that causes a 50% inhibition of NRU). In contrast, theendpoint measured in acute oral toxicity assays is usually animal morbidity or death. Cell
death and animal death may have similar mechanistic bases because all cells, regardless ofwhether they are in animals or in vitro cell cultures, have similar cellular mechanisms; forexample, energy production and maintenance of cell membrane integrity.
Death of an animal death and death of a cultured cell due to toxicity both involve interferencewith vital cell processes or physical injury. Cell death in a culture system involves the deathof a single cell type, but through mechanisms that also operate in the animal. In contrast,
cellular injury in an animal, if sufficiently widespread or in a critical process, can lead to
injury or loss of function of other cell types in a tissue not directly affected by the treatment,
resulting in organ failure. Major organ system failures (e.g., liver and kidney failure),gastrointestinal corrosion, and bone marrow depression, can be fatal. Examples ofmechanisms leading to such organ failures are disruption of membrane structure or function,
inhibition of mitochondrial function, disturbance of protein turnover, and disruption ofenergy production (Gennari et al. 2004). Alternatively, the tissue injury could affect nonexposed vital organs or tissues through interference with homeostatic signaling mechanisms(Gennari et al. 2004). For example, respiratory depression leading to death may be due todepression of the central nervous system (CNS) rather than a direct assault on the respiratory
system itself.
Animal and cell culture systems are also different with respect to how a substance or toxicant
is delivered to the cell and how it is distributed within the cell, metabolized, and excreted.
After oral administration, animals must absorb the toxicant from the gastrointestinal tract,which involves the passage through membranes, many of which are selective with respect towhat molecules they will allow to pass. The toxicant may or may not be bound to serum
proteins, thereby reducing its availability to the target organ. The toxicant may be
metabolized before, during, and/or after its distribution to the target organs, or the toxicant or
its metabolites may be excreted before reaching the target organ or reacting with its
components. As a consequence, the most critical target organs may not be exposed to theactive metabolite, or be exposed for only a limited time or to a relatively small fraction of theadministered dose.
In contrast, in a cell culture system, the test substance is applied directly to the target cells
and the only membranes that must be passed are those of the target cell and its subcellularorganelles. No absorption and distribution by other cellular systems is required. Cell culture
systems may or may not include serum proteins, which could reduce the availability of
toxicant to the target site. For example, the 3T3 cell culture medium includes serum while theNHK cell culture medium does not. 3T3 and NHK cells have little to no capacity tometabolize xenobiotic compounds, and added cell-free metabolic activation systems, such as
rat liver homogenates, may not accurately mimic all phases of in vivo metabolism. Excretionfrom the cell culture milieu is not a consideration because anything excreted from the cell

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remains in the culture medium and is available to the other cells in the culture. As a result,the cells in culture (as opposed to cells in an animal) may be exposed to a test substance forthe entire duration of the test protocol.
Animals and cell culture systems may also differ with respect to the target on which a
toxicant acts. If a toxicant acts in a specialized organ system in vivo, it may not produce a
toxic effect by the same mechanism in cultured cells that are derived from a tissue different
from the target organ. For example, a substance that affects a neuroreceptor-mediatedpathway in animals would not be expected to produce a similar toxicity in 3T3 or NHK cells,
which are derived from fibroblasts and skin cells, respectively, and do not contain similar
neuroreceptors; if toxicity is seen in these cell cultures, it may be from a different mechanism
or in a different concentration relationship than in vivo. Even if a neurotoxin were applied toneuronal cells in culture, the cultured cells may not respond in the same way as neuronalcells in an animal because cells in culture, especially cell lines, may not retain the samefunctionalities as cells in vivo.
1.2.4

Use of In Vitro Cytotoxicity Test Methods in the Overall Strategy of Hazard
Assessment
In the overall strategy of hazard or safety assessment, the intended regulatory use of the invitro NRU test methods is to reduce and refine the use of animals in current acute toxicity
assays. The in vitro systems would serve as adjuncts to the in vivo test methods but are notintended as replacements for the rodent acute oral toxicity test methods. For the OECD
alternative acute oral toxicity assays (the ATC and UDP), the number of animals used
depends on the starting dose. The number of dosing steps (and animals) is reduced if thestarting dose is close to the true toxicity class (ATC) or the true LD50 (UDP) (Spielmann etal. 1999; ICCVAM 2001b).
As noted earlier, Spielmann et al. (1999) and the Guidance Document (ICCVAM 2001b)
suggest that the RC millimole regression analysis be used with in vitro cytotoxicity data to
predict starting doses for the ATC and UDP. The RC millimole regression cannot be applied
to unknown substances or to mixtures (e.g., product formulations) because such materials
cannot be assigned molecular weights. Therefore, the NICEATM/ECVAM validation study
also evaluated the classification accuracy and the reduction in animal use associated with aregression based on weight units (with IC50 in µg/mL and LD50 in mg/kg) (see Section 10).
This regression would potentially be appropriate for predicting the starting dose for mixturesand undefined substances.
1.3

Scientific Basis for the In Vitro NRU Test Methods

Cytotoxicity has been defined as the adverse effects resulting from interference withstructures and/or processes essential for cell survival, proliferation, and/or function (Ekwall1983). Ekwall (1983) described the concept of "basal cell functions" (mitochondrial activity,
plasma membrane integrity, etc.) that virtually all cells possess and suggested that, for mostsubstances, toxicity is a consequence of non-specific alterations in those cellular functions,which may then lead to adverse effects on organ-specific functions and/or death of theorganism. These effects may involve the integrity of membranes and the cytoskeleton,
cellular metabolism, the synthesis and degradation or release of cellular constituents or
products, ion regulation, and cell division.
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Ekwall (1983) and others (e.g., Grisham and Smith 1984) concluded that, because the actionsof substances that produce injury and death are ultimately exerted at the cellular level, in
vitro cytotoxicity assays might be useful for the prediction of acute lethality potency, as well.
Considerable research has been undertaken to develop and evaluate in vitro tests for use as
screens and as potential replacements for rodent LD50 tests, and numerous groups havereported good agreement between in vitro cytotoxicity and animal lethality (see reviews by
Phillips et al. 1990; Garle et al. 1994; Guzzie 1994). However, none of the proposed in vitro
models have been evaluated in any formal studies for reliability and relevance, and theirusefulness and limitations for generating information to meet regulatory requirements for
acute toxicity testing data have not been assessed.
1.3.1
Purpose and Mechanistic Basis of the In Vitro NRU Test Methods
A number of basal cytotoxicity endpoints can be used to measure cell death or interferencewith cell proliferation. The NRU test methods were chosen for the NICEATM/ECVAM
validation study because they were recommended in the Guidance Document for the purposeof obtaining cytotoxicity information to determine starting doses for rodent acute oral
toxicity assays (ICCVAM 2001b). Both the 3T3 and NHK NRU test methods werereproducible in previous validation studies (ICCVAM 2001b). In addition, both cell types areeasily obtainable from commercial sources and the Guidance Document provided
preliminary evidence that these assays could reproduce the RC millimole regression.
Additionally, the assays can be automated and they require no radioactivity or highly
dangerous reagents (see Section 2 for protocol discussion and Appendix B for protocols).
Neutral red is a weakly cationic water-soluble supravital dye that stains living cells(Borenfreund and Puerner 1985). It readily diffuses through the plasma membrane andconcentrates in lysosomes where it electrostatically binds to the anionic lysosomal matrix.
Toxicants can alter the cell surface or the lysosomal membrane to cause lysosomal fragility
and other adverse changes that gradually become irreversible. Thus, cell death and/or
inhibition of cell growth decreases the amount of neutral red retained by the culture.
Borenfreund and Puerner (1985) were the first to publish a protocol for the NRU assay using
3T3 cells as a method to objectively quantify toxicity previously assessed by subjective,
visual observation. The NRU assay, which was standardized for a 96-well plate format,
correlated two measurements of toxicity from the exposure of 3T3 cells to six surfactants: (1)a visual morphological evaluation of the cells under an inverted phase microscope, and (2) aquantitative measurement of NRU. The visual evaluation was designed to identify the highest
concentration of toxicant that causes only minimal morphological changes (i.e., the highest
tolerated dose [HTD]). Because Borenfreund and Puerner (1985) found that the HTD in the
NRU test was comparable to the concentration that produced 10% inhibition (i.e., the IC10)compared with the controls, the IC10 value was deemed to be a good index for comparing therelative toxicities of experimental agents. The assay was described as a rapid, reliable,
inexpensive, and reproducible in vitro test method for screening potentially toxic agents(Borenfreund and Puerner 1985). Furthermore, the authors suggested that the test method
was a good candidate for inclusion in a battery of assays for toxicity screening with the
purpose of reducing the use of animals for toxicity tests.

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1.3.2

Similarities and Differences in the Modes/Mechanisms of Action for the In Vitro
NRU Test Methods Compared with the Species of Interest
Although the ultimate species of interest for acute oral toxicity concerns is humans, labelingand hazard identification requirements are based on rodents. There are differences between
humans and rodents in terms of absorption, distribution, metabolism, excretion, and the
intrinsic sensitivity of target organs to xenobiotic compounds. The differences are largely
substance-specific and quantitative, although there are a number of substances where thehuman may produce metabolites not seen in the rodent and vice versa. In vitro cytotoxicity
studies have also noted differences in sensitivity between human cells and other mammaliancells (Clemedson et al. 1996b). It is important to note that, for certain chemicals, there canalso be large differences in sensitivity among different human cell types and cell lines(Clemedson et al. 1996b, 1998a, b).
Because of the differences in sensitivity between humans and rodents, it might be likely that
cultured human cells would predict human lethality better than cultured rodent cells and that
cultured rodent cells would predict rodent lethality better than human cells. Ekwall et al.
(1998b) showed that in vitro cytotoxicity test methods using human cell lines generally
predicted human toxicity more accurately than did test methods using nonhuman mammaliancells.
In addition to being derived from different species, there are several other differences
between 3T3 and NHK cells, all of which may contribute to differences in sensitivity.
• 3T3 cells are an immortal line, while the NHK cells are primary cells.
• The cells originate from different tissues; 3T3 cells are derived fromembryonic fibroblasts, while the NHK cells are isolated from neonatal
foreskin tissue.
• NHK cells grow more slowly in culture than the 3T3 cells (i.e., after seeding
into 96-well plates, NHK cells require 48-72 hours for growth to theappropriate confluence while 3T3 cells require approximately 24 hours; see
Appendix B).
• NHK cells have greater ability to metabolize xenobiotic compounds, in thatthey exhibit minimal cytochrome P450 activity (Babich et al. 1991), whereas
3T3 cells have practically no ability to metabolize xenobiotic compounds(INVITTOX 1991).
1.3.3
Range of Substances Amenable to the In Vitro NRU Test Methods
The in vitro NRU test methods can be applied to a wide range of substances as long as they
can be dissolved in the cell culture medium or in a nontoxic solvent (at the concentration
used), and do not react with the culture medium. Although these test methods may to be
applicable to mixtures, none were evaluated in this validation study. The toxicity of
substances that act by mechanisms not expected to be active in 3T3 or NHK cells (e.g., thosethat are specifically neurotoxic or cardiotoxic) will likely be underpredicted by these test
methods. Therefore, until more appropriate cell lines are developed, the results from basal
cytotoxicity testing with such substances may not be relevant for predicting in vivo effects.

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Insoluble substances or those unstable in aqueous environments are not compatible with thetest systems. Volatile substances may yield acceptable results if CO2 permeable plastic film
is used to seal the test plates. Testing for corrosive substances is unnecessary since there is no
regulatory requirement for acute oral toxicity testing for known corrosives. The 3T3 NRU
test method may underestimate the toxicity of substances that are highly bound to serum
proteins because the culture medium contains 5% serum during substance exposure. The
toxicity of substances that specifically affect lysosomes may be overestimated because they
may affect NRU binding, and therefore, retention, in the cell. Red substances (and othercolored substances) that absorb light in the optical density range of NR may interfere withthe test if they remain inside the cell in sufficient amounts after washing and are soluble in
the NR solvent.

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In Vitro Cytotoxicity Test Methods BRD Section 2

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2.0

TEST METHOD PROTOCOL COMPONENTS OF THE 3T3 AND
NHK IN VITRO NRU TEST METHODS............................................................2-3

2.1

Basis for Selection of In Vitro NRU Cytotoxicity Test Methods .........................2-4
2.1.1 Guidance Document Rationale for Selection of In Vitro NRU
Cytotoxicity Test Methods ..........................................................................2-4
2.1.2 Guidance Document Rationale for Selection of Cell Types .........................2-4

2.2

Overview of the 3T3 and NHK NRU Test Methods ............................................2-5
2.2.1 The 3T3 NRU Test Method.........................................................................2-6
2.2.2 The NHK NRU Test Method ......................................................................2-8
2.2.3 Measurement of NRU in the 3T3 and NHK Test Methods...........................2-9

2.3

Descriptions and Rationales of the 3T3 and NHK NRU Test Methods..............2-9
2.3.1 Materials, Equipment, and Supplies ............................................................2-9
2.3.2 Reference Substance Concentrations/Dose Selection.................................2-12
2.3.3 NRU Endpoints Measured.........................................................................2-13
2.3.4 Duration of Reference Substance Exposure ...............................................2-14
2.3.5 Known Limits of Use ................................................................................2-14
2.3.6 Basis of the Response Assessed.................................................................2-16
2.3.7 Appropriate Vehicle, Positive, and Negative Controls ...............................2-16
2.3.8 Acceptable Ranges of Control Responses..................................................2-17
2.3.9 Nature of Experimental Data Collected .....................................................2-18
2.3.10 Data Storage Media...................................................................................2-19
2.3.11 Measures of Variability.............................................................................2-19
2.3.12 Methods for Analyzing NRU Data ............................................................2-20
2.3.13 Decision Criteria for Classification of Reference Substances.....................2-20
2.3.14 Information and Data Included in the Test Report .....................................2-20

2.4

Proprietary Components of the In Vitro NRU Test Methods ...........................2-22

2.5

Basis for the Number of Replicate and Repeat Experiments for the
3T3 and NHK NRU Test Methods .....................................................................2-22

2.6

Basis for Modifications to the 3T3 and NHK NRU Test Method
Protocols..............................................................................................................2-22
2.6.1 Phase Ia: Laboratory Evaluation Phase......................................................2-22
2.6.2 Phase Ib: Laboratory Evaluation Phase......................................................2-25
2.6.3 Phase II: Laboratory Qualification Phase...................................................2-27
2.6.4 Phase III: Laboratory Testing Phase ..........................................................2-35

2.7

Differences Between the 3T3 and NHK NRU Test Method Protocols
for the Validation Study and the Guidance Document Standard Protocols .....2-36

2.8

Overview of the Solubility Protocol ...................................................................2-37

2.9

Basis of the Solubility Protocol...........................................................................2-38
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In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006

2.9.1 Initial Solubility Protocol Development .................................................... 2-38
2.9.2 Basis for Modification of the Phase II Protocol ......................................... 2-39
2.10

Components of the Solubility Protocol .............................................................. 2-40
2.10.1 Medium, Supplies, and Equipment Required............................................. 2-40
2.10.2 Data Collection ......................................................................................... 2-41
2.10.3 Variability in Solubility Measurement....................................................... 2-41
2.10.4 Solubility Issues During the Testing of the Reference Substances ............. 2-41
2.10.5 Analysis of Solubility Data ....................................................................... 2-41

2.11

Summary............................................................................................................. 2-42

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In Vitro Cytotoxicity Test Methods BRD Section 2

2.0

November 2006

TEST METHOD PROTOCOL COMPONENTS OF THE 3T3 AND NHK IN
VITRO NRU TEST METHODSThe Guidance Document (ICCVAM 2001b) recommended that the following be incorporated
into any in vitro cytotoxicity protocol used to predict rodent acute oral lethality:• A cell line (or primary cells) that divides rapidly (e.g., with a doubling time of<24 hours)• An initial seeding density that allows for exponential cell growth throughout
the exposure period• An exposure period that spans at least one cell cycle• Appropriate positive control (PC) and vehicle control (VC) substances forwhich toxicity and lack of toxicity, respectively, has been well characterized
by the performing laboratory• Solvents that are used only at concentrations that do not cause significant
toxicity to the cell system over the entire period of the assay• A well-established, quantifiable cytotoxicity endpoint that has goodinterlaboratory reproducibility• Tests that are compatible with at least 96-well plates and equipment (e.g.,spectrophotometric microplate reader) that allow a quick and precise
measurement of the endpoint of interest• Use of a progression factor in the concentration-response experiment thatyields graded effects between 0% and 100% cytotoxicity
Section 2.1 provides the basis for the selection of the in vitro 3T3 and NHK NRU test
methods. Section 2.2 provides descriptions of the NRU protocols applicable to this validation
study. Section 2.3 provides details for performing the 3T3 and NHK NRU test methods andexplains the rationales for the various test method components, and Section 2.4 describes any
3T3 and NHK NRU test method proprietary aspects. Section 2.5 discusses the basis for the
replicate and repeat tests conducted during validation of these two test methods. Section 2.6
details the modifications and revisions made during the first two phases of the validation
study which contributed to the development of the final protocol used in Phase III. Section
2.7 describes the differences between the protocols used in this study and the protocols
outlined in the Guidance Document. Sections 2.8, 2.9, and 2.10 provide details on thesolubility protocol evaluated during the validation study and used to identify the appropriate
solvent for dissolving the reference substances.
The 3T3 and NHK NRU test method protocols were provided to the three laboratories thatparticipated in the validation study (see Section 5.6.3 for additional laboratory information).
These were:• The U.S. Army Edgewood Chemical Biological Center (ECBC)• The FRAME Alternatives Laboratory (FAL)• The Institute for In Vitro Sciences (IIVS)
A fourth laboratory (BioReliance Corporation, Rockville, MD) was used to procure anddistribute the coded reference substances, and to perform solubility tests on the validation
study reference substances prior to their distribution to the participating laboratories.

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2.1

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Basis for Selection of the In Vitro NRU Cytotoxicity Test Method

As stated in Section 1, in agreement with the recommendations of the Workshop 2000
participants (ICCVAM 2001a), ICCVAM made the following recommendations andforwarded them to U.S. Federal agencies along with the Workshop 2000 Report (ICCVAM
2001a) and Guidance Document (ICCVAM 2001b).
“ICCVAM concurs with the Workshop recommendation that near-term
validation studies should focus on two standard cytotoxicity assays: one
using a human cell system and one using a rodent cell system. Since themurine BALB/c 3T3 cytotoxicity assay has been evaluated for only alimited number of chemical classes, there is merit in determining its
usefulness with a broader array of chemical classes. Cell lines establishedfrom the rat rather than the mouse might also be considered, as most acuteoral toxicity testing is conducted in this species. Human cell lines should
also be considered since one of the aims of toxicity testing is to makepredictions of potential toxicity in humans. Future validation studies
should therefore compare rodent and human in vitro data with one another,with rodent in vivo data, and with human in vivo data. Correlations
between in vitro and in vivo data might help in selecting cytotoxicity
assays for further evaluation”. (ICCVAM 2001a)
Based on this recommendation and the Guidance Document recommendation, NICEATM
and ECVAM selected the 3T3 and NHK NRU basal cytotoxicity test methods for validation.
2.1.1

Guidance Document Rationale for Selection of In Vitro NRU Cytotoxicity Test
Methods
The Guidance Document (ICCVAM 2001b) provided the basic approach for the use of invitro NRU basal cytotoxicity test methods to predict starting doses for rodent acute oral
toxicity assays using the RC millimole regression. The 3T3 and NHK NRU test method
protocols used in the validation study were derived from those proposed in the GuidanceDocument.
2.1.2
Guidance Document Rationale for Selection of Cell Types
The Workshop 2000 participants (ICCVAM 2001a) concluded that there were no significant
differences between the basal cytotoxicity results obtained using permanent mammalian celllines, primary human cells (e.g., NHK cells), or the IC50x approach of Halle and Spielmann(Halle 1998, 2003; Spielmann et al. 1999; Halle and Spielmann 1992). Further, the GuidanceDocument recommended that in vitro basal cytotoxicity test methods not use hepatocytes (orrelated metabolically competent cells) or other types of highly differentiated cells because
they may not give the best prediction of acute lethality for the large variety of chemicals
likely to be tested (ICCVAM 2001b). However, it was recognized that, ultimately, simple
predictive systems (in vitro or in silico) would be needed for early identification of those
substances likely to be metabolized to more toxic or less toxic species than the parent
chemical as well as those that were likely to exhibit cell-specific toxicity (e.g., Fentem et al.
1993; Seibert et al. 1996; Curren et al. 1998; Ekwall et al. 1999).

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Established rodent cell lines were recommended for validation because (ICCVAM 2001b):• It was assumed that such cells would give the best prediction of rat and mouseacute oral lethality (i.e., like correlates with like).
• The use of a readily available, easy to culture, immortalized cell line for invitro cytotoxicity testing would accelerate the development of a database thatcan be used to analyze the usefulness of this approach.
Human cells also offer potential advantages. As determined in the MEIC project, the in vitro
test methods with the best predictivity for peak acute LC values in humans generally usedhuman cell lines (Ekwall et al. 1998b). Thus, a long-term advantage of using human cells isthat in vitro human cell cytotoxicity data can be added to human toxicity databases tofacilitate the development of test methods that may better predict acute oral human lethality.
3T3, an immortalized mouse fibroblast cell line, and NHK, primary human cells, wereselected as representative rodent and human cells, respectively, for the NICEATM/ECVAM
validation study. Historical data for the 3T3 NRU test were available from a variety ofstudies, including controlled and blinded validation studies, indicating the reliability of thistest method (Gettings et al. 1991, 1992, 1994a, 1994b; Spielmann et al. 1991, 1993, 1996;
Balls et al. 1995; Brantom et al. 1997). NHK cells have also been used in validation studies
for basal cytotoxicity test methods with good results (Willshaw et al. 1994; Sina et al. 1995;
Gettings et al. 1996; Harbell et al. 1997).
2.2

Overview of the 3T3 and NHK NRU Test Methods

The Guidance Document (ICCVAM 2001b) includes a proposed 3T3 NRU test method
protocol based on the 3T3 Cytotoxicity Test (INVITTOX Protocol No. 46; available from the
FRAME-sponsored INVITTOX database [http://embryo.ib.amwaw.edu.pl/invittox/]), which
in turn was based on the Borenfreund and Puerner (1985) protocol, as elaborated on in
Spielmann et al. (1991, 1996). This protocol was updated based on experience obtained
during the validation of the 3T3 NRU Phototoxicity Test (INVITTOX Protocol No. 78; alsoavailable at the FRAME INVITTOX database). The RC millimole regression for predictionof acute oral rat and mouse toxicity (Halle 1998, 2003; Spielmann et al. 1999) was included
as the prediction model (ICCVAM 2001b; see Section 1.1.2).
The NHK NRU protocol provided in the Guidance Document was based on the protocol usedby IIVS, which was based on a NRU protocol of Borenfreund and Puerner (1984) and a rat
epidermal keratinocytes protocol (Heimann and Rice 1983). Formulations for the media andsolutions, and general NHK cell culture techniques, correspond to Clonetics products fromthe CAMBREX Corporation.
The protocol components for the 3T3 and NHK NRU test methods used in this validation
study are similar (see Figure 2-1). The nature of the NRU response is described in Section
1.3.1. Figure 2-1 provides an overview to the major steps for performance of the in vitro
NRU test methods. The following procedures are common to both cell types:•
Preparation of substances and the PC•
Cell culture environmental conditions•
Determination of test substance solubility•
96-well plate configuration for testing samples
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In Vitro Cytotoxicity Test Methods BRD Section 2

•
•
•
•

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Range finder and definitive tests
Microscopic evaluation of cell cultures for toxicity based on morphologicalalterations
Procedures for measurement of NRU
Data analysis procedures

The main protocol differences between the two cell lines are:•
The conditions of propagation of the cells in culture (e.g., time needed toreach appropriate confluence)•
The growth media components•
The volumes of substances applied to the 96-well plates•
The number of cell divisions undergone by each cell line during exposure to atest substance
2.2.1

The 3T3 NRU Test Method

2.2.1.1 Initiating and Subculturing 3T3 Cells
Each laboratory initially prepared a large pool of 3T3 cells (described further in Section
2.3.1.1), cryogenically preserved multiple ampules of these cells in liquid nitrogen, andperiodically removed an ampule when needed. Although the NRU protocols used for eachstudy phase provided cell culture density guidelines for subculturing the cells, eachlaboratory refined the final seeding density to achieve optimal growth.
Cryopreserved 3T3 cells were thawed, resuspended in a culture medium containing
Dulbecco’s Modification of Eagle’s Medium (DMEM) supplemented with non-heatinactivated 10% newborn calf serum (NCS), transferred into tissue culture flasks (25 or 7580 cm2), and incubated at 37 °C ±1 °C, 90% ±5% humidity, and 5.0% ±1% CO2/air. Whencells reached 50 to 80% confluence (as estimated from a visual inspection of cell density),
they were removed from the flask by trypsinization. A single-cell suspension was added to
new flasks for propagation and the cells were passaged/subcultured at least two times1 before
seeding into 96-well plates for testing. This study did not evaluate the potential effects thatcell passage number may have on the performance of the 3T3 NRU test method.

3T3 cells were maintained in culture for approximately two months (approximately 18 passages) and used for
the NRU test. The Guidance Document (ICCVAM 2001b) did not provide a rationale for using 18 passages as
the limit, but it was probably recommended to maintain homogeneity of the 3T3 cell population (i.e., decrease
the potential of the population to drift genetically). The more passages the cells undergo, the more likely their
response to chemical stress may change.
1

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Figure 2-1

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Major Steps in the Performance of the NRU Test Methods(1) Cells (3T3 or NHK) are seeded into 96-well plates to form a sub-confluent

monolayer; plates are incubated at 37 ºC (24 hours for 3T3 cells; 48-72 hours
for NHK cells)⇓(2) Culture medium is removed (3T3 cells only)⇓(3) Reference substances in the appropriate solvents are added to the cells; cells
are exposed for 48 hours at 37 ºC over a range of eight (8) concentrations⇓(4) Cells are evaluated microscopically for toxicity based on morphologicalappearance⇓(5) Treatment medium is removed; cells are washed once with Dulbecco’s
Phosphate Buffered Saline (D-PBS); Neutral Red (NR) dye medium is added(3T3 cells: 25 µg/mL NR dye; NHK cells: 33 µg/mL NR dye); plates are
incubated for 3 hours at 37 ºC⇓(6) NR medium is discarded; cells are washed once with D-PBS; NR desorbing
fixative is added to the wells⇓(7) Plates are shaken for 20 minutes at room temperature⇓(8) NR absorption is measured at optical density (OD) 540 ±10 nm⇓(9) NRU is calculated as a percent of vehicle control values to define IC20, IC50,
and IC80 concentrations (µg/mL)2_________________________________________________________________________
Abbreviations: NRU=Neutral red uptake; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal
keratinocytes; IC20, IC50, IC80=Substance concentration that reduces cell viability by 20, 50, and 80%,
respectively.

IC50 values are used for estimating the LD50 value of a reference substance. The IC20 and IC80 values were
determined for possible use in estimating human lethal concentrations in blood.
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2.2.1.2 Preparation of Cells for 96-well Plate Assays
3
After subculturing the cells, 100 µL of the cell suspension (2.0 – 3.0x10 cells/well) wereplaced in the appropriate wells and 100 µL of cell-free culture medium were dispensed into
the 36 peripheral wells (blanks). The peripheral wells were in rows 1 and 8 and columns 1
and 12 (See Figure 1 in Appendix B1 or B2). Peripheral wells were used only for blanks
because they may be subjected to more evaporation than interior wells. The GuidanceDocument authors (and the SMT and Study Directors) concluded that such conditions would
ultimately affect cell growth in these wells. One plate was prepared for each reference
substance. The cells were incubated for 24 ±2 hours at 37 ºC and checked visually to be surethat approximately a 50% confluent monolayer was present at the time of substanceapplication.
2.2.1.3 Reference Substance Application
After the appropriate incubation period to achieve a half-confluent monolayer, the medium
was removed and 50 µL of culture medium with 10% NCS were added to each well. Then,
50 µL treatment medium containing the appropriate substance concentrations were added fora final concentration of 5% NCS. The cells were then incubated at 37 ºC for 48 ±0.5 hours.
At the end of the incubation period, the cells were microscopically evaluated for changes inmorphology and their appearance was documented (as per Visual Observation Codes in the
protocol) prior to measurement of NRU.
2.2.2

The NHK NRU Test Method

2.2.2.1 Initiating and Subculturing NHK Cells
Cryopreserved NHK cells (ampules of cryopreserved cells were obtained from CAMBREX
Corporation and stored in liquid nitrogen until needed) were thawed, resuspended in serumfree keratinocyte complete growth medium (see Section 2.3.1.4 for components of themedium), transferred into tissue culture flasks (25 cm2 without fibronectin-collagen coating),
and incubated at 37 °C ±1 °C, 90% ±5% humidity, and 5.0% ±1% CO2/air. When the cells
reached 50 to 80% confluence (as estimated from a visual inspection of cell density), they
were removed from the flask by trypsinization and prepared for subculturing into the 96-wellplates. Care was taken to prevent the keratinocyte cultures from becoming 100% confluent as
this may lead to cell differentiation, which would alter the intrinsic sensitivity of these cells
to cytotoxic substances. To minimize potential sources of experimental variability, thelaboratories used the same lot of Clonetics® cells throughout the validation study, the samebrand of growth medium and supplements (and concentrations of supplements), and cells
were not used beyond their second passage. The protocols for each study phase provided cell
culture density guidelines, but each laboratory refined the final seeding densities to achieve
appropriate growth.
2.2.2.2 Preparation of Cells for 96-well Plate Assays
3
After subculturing the cells, 125 µL of the cell suspension (2.0 – 2.5x10 cells/well) wereplaced in the appropriate wells and 125 µL of cell-free culture medium were dispensed into
the peripheral wells (blanks). One plate per reference substance was prepared. The cells wereincubated at 37 ºC for 48-72 hours and checked to be sure that cultures were at 20 to 50%
confluence at the start of exposure to the reference substance.

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2.2.2.3 Reference Substance Application
To add the reference substances, 125 µL of culture medium containing the appropriate
reference substance concentrations were added to the existing 125 µL of culture medium inthe test wells. The cells were then incubated at 37 ºC for 48 ±0.5 hours. At the end of the
exposure period, the cells were microscopically evaluated for changes in morphology andtheir appearance was documented (as per Visual Observation Codes in the protocol [seeAppendices B1 and B2]) prior to measurement of their NRU.
2.2.3
Measurement of NRU in the 3T3 and NHK Test Methods
The treatment medium was removed from the 96-well plates, the cells were rinsed withphosphate buffered saline (PBS), and 250 µL NR dye medium was added to the wells (25 µg
NR/mL for 3T3 cells; 33 µg NR/mL for NHK cells). The plates were then incubated (37 °C±1 °C, 90% ±5% humidity, and 5.0% ±1% CO2/air) for three hours. After incubation, the NR
medium was removed, the cells were rinsed with PBS, and 100 µL of the desorb solution
were applied. The plates were shaken on a microtiter plate shaker for 20 to 45 minutes toextract NR from the cells and to form a homogeneous solution. The optical density (OD) of
the resulting colored solution was measured (within 60 minutes of adding the desorb
solution) at 540 nm ±10 nm (OD540) in a spectrophotometric microtiter plate reader, using theblank wells as reference. Data from the plate reader were transferred to a Microsoft®
EXCEL® (Microsoft Corporation, Redmond, WA, USA) spreadsheet template (hereafter
know as EXCEL® template) designed by the SMT and the testing laboratories for statisticalanalyses.
2.3

Descriptions and Rationales of the 3T3 and NHK NRU Test Methods

The protocols used in Phases I, II, and III of the validation study (Appendices B and C) are
modifications of the protocols reported in the Guidance Document (ICCVAM 2001b). The
participating laboratories provided comments and recommendations during the developmentof these protocols. The following information is specific to the protocols used in thisvalidation study.
2.3.1

Materials, Equipment, and Supplies

2.3.1.1 3T3 Cells
The CCL-163, 3T3 BALB/c mouse fibroblast, cell line, clone 31 from the American Type
Culture Collection (ATCC), Manassas, VA, USA, was used. The 3T3 cells, an immortalized
mouse fibroblast cell line, were procured from the ATCC by IIVS at passage 64. IIVS
cultured the cells to expand their number and cryogenically preserved them as a pool atpassage number 69. ECBC and FAL received frozen ampules of cells at passage number 69
from IIVS, propagated the cells, and cryopreserved multiple ampules of cells at a slightly
higher passage number to establish their working cell banks for use throughout the study.
Each laboratory determined the doubling time for the 3T3 cell line prior to NRU testing inPhase Ia as required by the protocol in Appendix C1. The following doubling times werereported: 18.6 hours by ECBC; 17 hours by FAL; and 17 hours by IIVS. No other doublingtime measurements were made. The extent of cell confluence was monitored during the study
to identify when the cultures were in exponential growth.

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2.3.1.2 NHK Cells
A single lot of pooled donor, primary neonatal foreskin keratinocyte (NHK) cells (Clonetics®
# CC-2507; lot # 1F0490N) from CAMBREX Bio Science Walkersville, Inc., Walkersville,
MD, USA, was used throughout the validation study. Keratinocytes from other sources
would be acceptable if they meet the growth requirements identified in the protocols. Eachlaboratory determined the doubling time for the NHK cells prior to testing in Phase Ia (asrequired by the protocol in Appendix C2). The following doubling times were reported: 21hours by ECBC; 10 hours by FAL; and 15.8 hours by IIVS. No other doubling timemeasurements were made. The extent of cell confluence was monitored during the study toidentify when the cultures were in exponential growth.
2.3.1.3 Tissue Culture Materials and Supplies
The 3T3 and NHK NRU test methods require general tissue culture materials and supplies(see Appendices B1 and B2 [protocols] for formulations, and concentrations of solutions andmedia). Both test methods used the same materials for solubility testing (Section 2.8.1).
Freshney (2000) provides information on all aspects of cell culture, including materials,
supplies, and equipment needed. The following materials were needed for both test methods:• Trypsin (0.05%)• PBS• Hanks’ Balanced Salt Solution (HBSS) without Ca2+ and Mg2+
• NR dye• Glacial acetic acid
• Dimethyl sulfoxide (DMSO) [analytical grade]• Ethanol (ETOH) [100% non-denatured for test substance preparation]• Distilled water
2.3.1.4 Cell Culture Materials
Laboratory items needed include the following:• Sterile, disposable tissue culture plasticware (e.g., 25 cm2,75-80 cm2 flasks;
multiwell/microtiter [96-well] plates; petri dishes) [Note: The laboratories inthis study used tissue culture plasticware from various suppliers.]• Cryogenic ampules• Pipettes, pipettors, pipette tips
• Multichannel solution reservoirs• Centrifuge tubes• Microporous sterilization filters• General plastic containers• Glass tubes (for preparation of substance dilutions)2.3.1.5 Equipment
Performance of the NRU tests requires a laboratory equipped with a designated cell culturearea. Essential equipment for cell culture work and the NRU test methods include:• Incubator (37 ºC ±1 ºC, 90% ±5% humidity, 5.0% ±1% CO2/air)• Laminar flow clean bench/cabinet (standard: "biological hazard")• Water bath (37 ºC ±1 ºC)• Inverted phase contrast microscope (with 10X to 40X objectives)• Centrifuge (capable of 220 x g)
• Laboratory balance (capable of measuring to 10 mg)
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•
•
•
•
•
•
•
•
•
•
•
•
•

November 2006

Spectrophotometer for reading 96-well plates (i.e., microtiter plate reader)equipped with 540 nm ±10 nm filter
Shaker for microtiter plates
Cell counter or hemocytometer
Pipetting aid (e.g., vacuum pipettor unit)
Pipettes, pipettors (multi-channel and single channel, multichannel repeater
pipette)Waterbath sonicator
Refrigerator
Freezer (to at least -70 ºC)
Cryostorage container (and liquid nitrogen supply)
Magnetic stirrer
Antistatic bar ionizer
Personal computer
Osmometer
pH meter

2.3.1.6 Culture Medium
For 3T3 Cells
DMEM containing high glucose (4.5 gm/L) and supplemented with NCS, L-glutamine,
penicillin, and streptomycin was used for the 3T3 cells. Heat-inactivated serum was not usedin this study. Heat-inactivation of serum is often used to destroy heat-labile components suchas complement factors, and microbial contaminants such as mycoplasma (Hyclone® 1996;
Mediatech, Inc. 2006). However, some heat-labile complement factors can also beinactivated by the standard cell culture practice of warming serum-containing medium to 37°C prior to use, and mycoplasma can be eliminated by filtering the medium (e.g., using 0.1µm pore-size rated filters). Heating serum to 56 °C (heat-inactivation temperature) can
destroy other heat-labile components such as growth factors, vitamins, amino acids, andhormones. Loss of these components can diminish the capacity of the serum to promote
attachment of cells to culture vessel surfaces and to support cell growth. An additional
confounding factor is that the procedure for heat-inactivation is highly precise, and deviationfrom the basic protocol can create additional issues such as protein denaturation and serum
turbidity.
For NHK Cells
Although the contents of the NHK basal culture medium are proprietary, the formulation isbased on a commercially available, non-proprietary basal medium (MCDB 153 medium
formulation [Tsao et al. 1982]; e.g., MCDB 153 medium - SIGMA-ALDRICH productnumber #M 7403 http://www.sigmaaldrich.com/sigma/datasheet/m7403dat.pdf). The
laboratories recommended this medium for use with the CAMBREX Clonetics® NHK cells
because they all had access to this supplier. Other media are acceptable for NHK NRU
testing if the performance standards prescribed in the media prequalification protocol are met(see Appendix B4 and Section 2.6.3.5).
The serum-free culture medium used for NHK cells was Clonetics® keratinocyte basal
medium (KBM®) supplemented with KBM® SingleQuots® (epidermal growth factor, insulin,hydrocortisone, antimicrobial agents, bovine pituitary extract [BPE]) and Calcium SingleQuots®
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(calcium) [all from CAMBREX Corporation] to make keratinocyte complete growth medium.
Although the keratinocyte complete growth medium is a defined serum-free medium, it
contains BPE collected from bovine pituitary glands. BPE contains growth factors andhormones, and is added to serum-free medium as a mitogenic supplement. Variability in thecomposition of the BPE could be a factor in cell growth kinetics. However, it is suggested
that the undefined BPE components could be replaced with defined growth supplements,
such as insulin, epidermal growth factor, and fibroblast growth factor, without adversely
affecting the cellular proliferation rates and general physiology of human keratinocytes (LifeTechnologies, Inc. 1997).
2.3.2
Reference Substance Concentrations/Dose Selection
Each laboratory weighed and dissolved the reference substances on the same day as the startof the exposure period. The highest concentration of dissolved reference substance was
identified using the solubility protocol and designated as the 2X stock solution. All reference
substance dilutions for an assay were serially derived from this stock solution (see GuidanceDocument for serial dilution methods).
2.3.2.1 Range Finder Test
A range finder 3T3 or NHK NRU test was performed to determine the concentrations of areference substance to be used for the definitive (concentration-response) test (see Section
2.3.2.2). The range finder test used eight concentrations of the reference substance preparedby diluting the stock solution using log intervals to cover a large concentration range (e.g.,
1:10, 1:100, 1:1000, etc.; up to eight orders of magnitude). The highest concentrations
applied to the cells were 10 mg/mL for substances dissolved in culture medium and 1 mg/mL
in medium for substances dissolved in DMSO, unless precluded by solubility. ETOH was not
used as a solvent for any of the substances in the validation study (see Sections 2.8, 2.9, and2.10).
If the range finder test did not produce cytotoxicity, then a second range finder test was
conducted at higher concentrations (e.g., the highest concentration would be >10 mg/mL if in
medium, >1 mg/mL if in DMSO) unless precluded by solubility. If the substance being tested
was insoluble or poorly soluble, then more stringent solubility procedures were employed toincrease the stock concentration (to the maximum concentration specified in Appendices B1
and B2). If the range finder test produced a biphasic dose-response curve3 for NR uptake, theconcentrations selected for the definitive tests covered the response range that included thelowest concentration that reduced viability by 50% (see Section 2.6.3.2).
2.3.2.2 Definitive Test
The concentration-response determination is referred to as the definitive test because it isused to determine the IC50 value of the substance being tested. The concentration closest to
the calculated IC50 value in the range finder test served as the midpoint of the eight
concentrations tested in the definitive test. In the absence of other information (e.g.,knowledge of the slope of the toxic response), the recommended dilution factor was 1.47
(6√10), which divides a log interval into six equidistant steps (e.g., 10, 14.7, 21.5, 31.6, 46.4,
68.1, 100). The Guidance Document considered a progression factor of 1.21 (12√10) to be the
A biphasic dose-response curve is a dose-response in which cytotoxicity increases (as dose increases),plateaus, and then increases again.
3

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smallest factor practically achievable, and this was the lowest required concentration interval.
The PC was tested similarly to the reference substances in the definitive test and the same
recommended dilution factors were used (dilution factor at the discretion of the Study
Director).
A definitive test was considered successful if it met all of the test acceptance criteria outlinedin the NRU protocols. Definitive tests were repeated as per the protocols if the test failed tomeet all of the test acceptance criteria. Section 2.5 addresses the basis for replicate testing.
If minimal or no cytotoxicity was observed in the range finder test, the maximum
concentration for the definitive test was determined as follows:• For Substances Prepared in NHK or 3T3 Medium: A review of the RC
chemicals used in this validation study showed that, among water-soluble
chemicals, glycerol had the highest reported IC50 value (57 mg/mL). To
capture this value, and that of other relatively non-toxic chemicals, the highest
concentration of a substance applied to the cells in the definitive test waseither 100 mg/mL (using 200 mg/mL 2X stock) or the maximum soluble dose
if the substance was not soluble at that concentration.
• For Substances Prepared in DMSO: Based on the maximum concentration of
DMSO that could be added to culture medium without causing cytotoxicity(i.e., 0.5%), the highest concentration of a substance that could be applied tothe cells in the definitive test was 2.5 mg/mL. In the event that the reference
substance was not soluble at this concentration, the highest soluble
concentration was used.
2.3.3

NRU Endpoints Measured

2.3.3.1 NRU and Measurement
After cells were exposed to the reference substance or the controls (PC; VC) for 48 hours,
they were washed and incubated with the NR dye at 37 ºC for an additional three hours. The
dye was eluted from the cells using a desorb solution and the OD of the resulting solutions
were measured using a spectrophotometric microtiter plate reader. Because NR is absorbed
by healthy cells, the amount of dye eluted, as measured by the spectrophotometer, is
proportional to NRU and thus to the number of live cells present at culture termination. The
OD data from the spectrophotometer were recorded on the EXCEL® template. Relative cell
viability for each reference substance and the PC was determined using six replicate wells(six wells [minimum of four scorable] in the 96-well plate) per concentration. Cells treatedwith the VC were considered to have 100% cell viability (i.e., the mean OD of the VC wells= 100% viability). Cell viability in other test wells was computed in reference to the meanVC OD value (i.e., [well OD/mean VC OD] x 100 = % viability).
2.3.3.2 Determination of IC50, IC20, and IC80 Values
IC50 values were determined from the concentration-response curve using a Hill function,
which is a four parameter logistic mathematical model relating the concentration of a
substance to the response (typically following a sigmoidal shape). Modifications to the Hillfunction used in later phases of the study are described in Section 2.6.3.

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Data from the EXCEL® template were transferred to a template designed by the SMT for
GraphPad PRISM® 3.0, a commercially available statistical software (GraphPad Software,Inc., San Diego, CA, USA – hereafter known as PRISM® template). The PRISM® templateused the Hill function to calculate the IC50, IC20, and IC80 concentrations, reported as µg/mLof reference substance in solution. IC20 and IC80 data were collected for potential use indesigning a prediction model for estimating human lethal blood concentrations.
2.3.4
Duration of Reference Substance Exposure
The SMT and laboratory representatives reevaluated the reference substance exposure
duration recommended in the Guidance Document (ICCVAM 2001b) before initiating thestudy. The Guidance Document recommended an exposure of 24 hours for the 3T3 cells and48 hours for the NHK cells. However, Riddell et al. (1986) showed large differences incytotoxicity for 3T3 cells in response to some chemicals, depending on whether the exposure
duration was 24 or 72 hours. Although the toxicity induced by substances that damage, forexample, cell membranes is likely to be observed in a relatively shorter time, the toxic effectsof substances that interfere with cell functions/processes specifically relating to DNA
replication (e.g., protein and nucleic acid synthesis) and cell division (e.g., mitotic spindle
formation) are more pronounced after longer exposure periods. This occurs because cells areaffected only at certain phases of the cell cycle.
IIVS conducted studies to evaluate the effect of exposure durations of 24, 48, and 72 hours
and of 48 and 72 hours on the sensitivity of 3T3 cells and NHK, respectively, to six
chemicals selected from the list in Riddell (1986). Because the closest fit to the RC millimole
regression occurred when a 48-hour exposure duration was used, this exposure duration was
selected for use with both cell types in the validation study (Curren et al. 2003) (seeAppendix E).
2.3.5

Known Limits of Use

2.3.5.1 Solubility/Precipitation/Volatility
In vitro test methods cannot be used for substances that cannot be dissolved in media,
DMSO, or ETOH at a sufficiently high concentration to induce cytotoxicity in excess of
50%. Also, chemicals that are unstable or exothermic in water cannot be adequately tested
with these in vitro test methods (as well as in vivo methods).
Precipitation of a test substance in the dosing solution or in the culture medium after thesubstance to be tested has been added can affect the concentration-response and thus reduce
the accuracy of the calculated IC50. Some reference substances used in the validation study
had precipitates in their medium/DMSO 2X concentrations prior to dilution for application to
the test wells. Precipitates were also observed for some substances in a number of test wells
after addition of the media/DMSO 1X solutions (see Section 5.8 and Table 5-11) to thecultures and/or at the end of the exposure period.
Volatility was detected for a number of reference substances during the range finder tests byobservance of cross contamination (i.e., high cytotoxicity) in VC wells. Plate sealers wereused during the definitive tests to control volatility (see Section 2.6.3 – Testing Volatile
Reference Substances), and could be used during the range finder tests if the Study Director
suspected that the reference substance might be volatile. The use of plate sealers required
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additional laboratory training, and some volatile substances were difficult to test even withthe use of plate sealers. Furthermore, some test substances (e.g., organic solvents) may reactchemically with the plastic in the sealers.
2.3.5.2 Biokinetic Determinations
The Workshop 2000 report (ICCVAM 2001a) discussed the role of chemical biokinetics invivo vis-a-vis acute toxicity, as illustrated in the following quote:“Results obtained from in vitro studies in general are often not directly
applicable to the in vivo situation. One of the most obvious differences
between the situation in vitro and in vivo is the absence of processes
regarding absorption, distribution, metabolism and excretion (i.e.,
biokinetics) that govern the exposure of the target tissue in the intact
organism. The concentrations to which in vitro systems are exposed may notcorrespond to the actual situation at the target tissue after in vivo exposure. In
addition, the occurrence of metabolic activation and/or saturation of specificmetabolic pathways or absorption and elimination mechanisms may also
become relevant for the toxicity of a compound in vivo. This may lead tomisinterpretation of in vitro data if such information is not taken into
account. Therefore, predictive studies on biological activity of compounds
require the integration of data on the mechanisms of action with data on
biokinetic behavior.”
The 3T3 and NHK NRU test methods do not account for biokinetics.
2.3.5.3 Organ-Specific Toxicity
The Workshop 2000 report also addressed concerns about the in vitro prediction of organspecific toxicity, and identified the organ systems for which failure after acute exposure
could lead to lethality (i.e., liver, central nervous system, kidney, heart, lung, andhematopoietic system) (ICCVAM 2001a). Each organ system was reviewed individually.
Although the 3T3 and NHK NRU test methods do not assess organ-specific toxicity, they
may be useful in a test method battery such as that proposed by the Workshop 2000
participants (see Section 2.3.5.4).
2.3.5.4

The Role of Cytotoxicity Tests in an In Vitro Battery Approach for Possible
Replacement of In Vivo Acute Toxicity Testing
A five-step in vitro testing scheme was proposed for a test battery that may eventually bedemonstrated to be an adequate replacement for rodent acute oral toxicity test methods forregulatory purposes (ICCVAM 2001a).
Step 1: Perform a physico-chemical characterization and biokinetic modeling.
Step 2: Evaluate basal cytotoxicity using, for example, the 3T3 or NHK NRU test
methods.
Step 3: Evaluate the potential that metabolism will mediate the basal cytotoxicity
effect.
Step 4: Assess the test substance’s effect on energy metabolism.
Step 5: Assess the ability of the test substance to disrupt epithelial cell barrier
function.

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The Workshop 2000 participants suggested that implementation of the 5-step testing scheme
would require the following:• Identification of the most appropriate cell culture systems to use based on
accuracy, reproducibility, cost, and availability• Development of a standardized protocol for each test method used in each of
the five steps, and validation of each test method using that protocol• Development of prediction models for the relevant human toxic levels
required by regulatory agencies• Evaluation of the test battery using substances that are appropriate for all
endpoints, and then test sufficient substances to develop a prediction model• Validation of the entire testing scheme and the prediction model
2.3.6
Basis of the Response Assessed
Neutral red is a weakly cationic, water-soluble, supravital dye that stains living cells byreadily diffusing through the cell membranes and concentrating in lysosomes. The intensityof the dye desorbed from the cells in a culture is directly proportional to the number of living
cells. Cell death and/or growth inhibition decreases the amount of neutral red taken up by theculture (see Section 1.3.1).
2.3.7

Appropriate Positive, Vehicle, and Negative Controls

2.3.7.1 Positive Control
The Guidance Document recommended sodium lauryl sulfate (SLS; Chemical Abstracts
Service Registry Number [CASRN] 151-21-3) as an appropriate PC for in vitro cytotoxicity
test methods (ICCVAM 2001b), and historical data are available (e.g., Spielmann et al.
1991). A PC test plate was included with every 3T3 and NHK NRU test method assay andwas treated the same as any reference substance assay plate.
The historical mean PC IC50, standard deviation (SD), and acceptance limits, were
determined separately for each laboratory (see Table 5-3), based on their individual
historical databases (see Figure 1-2). The acceptable range for the PC IC50 was based on thestatistical approach recommended in the Guidance Document. In Phase Ib, the IC50 limits
accepted for the PC tests were within two SD of the historical mean PC IC50 value. In the
Phase II studies, the IC50 limits for PC tests were within 2.5 SD of the historical mean value(i.e., from Phases Ia and Ib). In Phase III, the IC50 limits used for the PC were within 2.5
standard deviations of the mean PC IC50 from Phases I and II. The exception to this was the
FAL NHK data, where only the Phase II data were used as the basis for establishing theacceptable PC range. The SLS data produced by FAL during Phase I was not used insubsequent historical database compilations because FAL used a modified cell culture
protocol in Phase II (see Section 2.6.2.6).
2.3.7.2 Vehicle Control
The VC consisted of complete DMEM (see Appendix B1) for 3T3 cells and complete
growth medium (Clonetics® KBM® with supplements [see Appendix B2]) for NHK cells
when the reference substances were dissolved in culture medium. For reference substancesdissolved in DMSO, the VC consisted of medium with the same amount of DMSO (0.5%[v/v]) as was applied to the 96-well test plate.

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2.3.7.3 Negative Control
Negative control cultures (i.e., those that were not exposed to the solvent) were not used inthis validation study. Neither DMSO, at the concentration used, nor the culture medium
affected the performance of the 3T3 and NHK NRU test methods.
2.3.8
Acceptable Ranges of Control Responses
The Guidance Document established an absolute value (i.e., uncorrected for blankabsorbance) range of the OD540 for the VC to indicate whether the cells seeded in the 96-wellplate had grown with a normal doubling time during the assay. A mean OD540 ≥0.3 wasrecommended as the acceptable range of VC responses and was made a test acceptancecriterion for both cell types at the start of the study. However, prior to Phase II, this was
rescinded as a test acceptance criterion. The protocols for Phases II and III provide a range of
OD values for use as guidance in future studies with these test methods (Table 2-1).
Table 2-1
Laboratory

Measured VC OD540 Values1 and Targets
Phase Ia

Phase Ib

Phase II

Phase III

3T3 NRU Test Method
Target Range2

0.3≤ OD ≤1.1

0.30≤ OD ≤0.80

0.103≤ OD ≤0.813

0.103≤ OD ≤0.813

ECBC

0.326 – 0.457

0.214 – 0.839

0.217 – 0.730

0.191 – 0.797

FAL

0.490 – 0.780

0.247 – 0.742

0.289 – 0.768

0.126 – 1.161

IIVS

0.336 – 0.538

0.319 – 0.598

0.307 – 0.578

0.256 – 0.544

NHK NRU Test Method
Target Range2

0.3≤ OD ≤1.1

0.60≤ OD ≤1.70

0.35 ≤ OD ≤ 1.50

0.205 ≤ OD ≤ 1.645

ECBC

0.863 – 2.312

0.788 – 1.282

0.139 – 1.175

0.114 – 1.344

FAL

0.484 – 1.698

0.146 – 1.706

0.110 – 1.292

0.183 – 1.347

IIVS

0.550 – 1.883

0.487 – 1.001

0.201 – 0.841

0.430 – 0.834

Abbreviations: VC=Vehicle control; OD540=Optical density at 540 nM; 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red
uptake; NHK=Normal human epidermal keratinocytes; ECBC=Edgewood Chemical Biological Center; FAL=Fund for
Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences.
1
Lowest to highest OD values for tests that meet test acceptance criteria.
2Ranges used for all laboratories. Ranges for Phases Ia and Ib were test acceptance criteria. Ranges for Phases II and III
were used as target ranges, rather than as test acceptance criteria.

In Phase III, 99.5% (914/919) of all 3T3 mean VC OD values and 97% (913/944) of all NHK
mean VC OD values were within the target ranges. Most OD values outside the ranges werefrom range finding tests and were usually the result of volatile reference substances affecting
the VC cells adjacent to the highest reference substance concentration wells.
The VC OD values had a tendency to be lower in Phases II and III as compared to Phases Ia
and Ib. Protocol revisions made throughout Phases Ia, Ib, and II (as listed below) most likely
contributed to the differences in the OD values. Possible explanations for changes in OD
values for the 3T3 cells include:• Some tests in Phases Ia and Ib exhibited NR crystals that caused higher OD
readings.
• Cell seeding densities were revised from 2.5 x 103 cells/well to a range of 2.0– 3.0 x 103 cells/well.

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Possible explanations for changes in OD values for the NHK cells include:• The minimum percent confluence of cells necessary before the reference
substance could be applied was reduced from 30% to 20% confluence.
• Cell growth was reduced in some tests in the later study phases as a result of
medium and supplement issues (e.g., certain lots of basal medium andmedium supplements for NHK cells did not provide optimum growth
conditions for the keratinocytes).
2.3.8.1 Vehicle Controls as a Quality Control Tool
To check for systematic cell seeding errors and reference substance volatility, VCs wereplaced both at the left side (row 2) and the right side (row 11) of the 96-well plate (seeFigure 1 in Appendix B1). Volatile reference substances generally affected the left side VC,
which was next to the highest reference substance concentration in the 96-well plate. The test
acceptance criterion for the VC was that the means for the left and the right set of VCs had tobe within 15% of the mean of all VCs. This criterion, which was adopted from the protocols
in the Guidance Document (ICCVAM 2001b), was used for reference substances and the PC
in all phases of the validation study.
2.3.9
Nature of Experimental Data Collected
Each laboratory maintained a study workbook to document all aspects of the study andincluded the raw data for all steps of each assay (e.g., cell growth, test substance treatment,
weighing and dilution of reference substances), as well as for all solubility studies.
2.3.9.1 NRU OD Measurements
At the conclusion of the NRU desorb step, the OD of the resulting colored solution in eachwell of the 96-well plates was measured at 540 ±10 nm in a spectrophotometric microtiterplate reader. Each laboratory followed its in-house Standard Operating Procedure (SOP) for
use of the microplate readers. These SOPs included instructions for operation and calibrationof the instruments. Critical specifications such as alignment, accuracy, reproducibility, andlinearity were included as standard parameters for review and routine calibration. Raw OD
data from the plate reader was electronically transferred to the EXCEL® template. The
template converted the raw data from each treatment well (six wells/reference substanceconcentration) to derived data by subtracting the mean blank OD value (two blankwells/reference substance concentration) from each reference substance well OD. There were
12 VC wells and 20 associated blank wells. The corrected VC OD values were used to
calculate the mean VC OD, which was then used to calculate relative viability (% of meanVC OD) in each test well for the reference substance or PC. The percent viability values
were then transferred to the PRISM® template to calculate the IC20, IC50, and IC80 values.
2.3.9.2 Information and Data Collected
Originals of the raw data (i.e., the Study Workbook and computer printouts of absorbancereadings from the plate reader) and copies of other raw data, such as instrument logs, werecollected and archived under the direction of the Study Director according to Good
Laboratory Practice (GLP)-compliant procedures.
The Study Director/technicians entered the following information into the EXCEL® template:•Testing identification for: test facility, chemical code, study number, 96-wellplate number, experiment number

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•

•
•
•
•
•

•

•

November 2006

Reference substance preparation: solvent used, solvent concentration in
dosing solutions, highest stock concentration, dilution factor, pH of 2X dosing
solutions, medium clarity/color, presence/absence of precipitate in 2X
solutions, PC concentration range
Cell line/type: cell supplier, lot number, cryopreserved passage number,passage number in assay
Cell culture conditions: medium, supplements, suppliers and lot numbers,
serum concentrations
Timeline: dates of cell seeding, dose application, OD540 determination
Raw data: OD values from each well from the microtiter plate reader
Test results: mean corrected OD540 value, Hill function R2 value, logs of IC20,
IC50, and IC80 (PRISM® template presents data as logs of the ICx; EXCEL®
converts values to µg/mL)
Test acceptance criteria: acceptable number of values on each side of the IC50
(i.e., number of points >0 and ≤50% viability, and >50 and <100% viability),
acceptable percent difference for the VCs, acceptable Hill function R2 value(coefficient of determination) and calculated IC50 concentration for the PC
Visual observations: protocol codes for cell culture conditions for all reference
substance concentrations (i.e., relative level of cell cytotoxicity, cellmorphology, presence of precipitate)

2.3.10
Data Storage Media
Raw and derived data from the NRU tests were saved in the EXCEL® template file format
provided by the SMT. All EXCEL® and PRISM® files were copied and transferred tocompact disks. NICEATM and the laboratories printed copies of all data sheets (stored at
NICEATM and at the testing facilities), and included copies in the laboratories’ final reports.
2.3.11
Measures of Variability
Each 96-well plate used in the NRU tests had three main measures of variability.
1) Each plate contained VCs on each end of the plate (columns 2 and 11) (seeFigure 1 in Appendix B1 for plate map). The difference between the mean
NRU OD for each VC column and mean of the pooled VC wells was used as atest acceptance criterion. The Study Director rejected the test if the difference
was greater than 15%, which indicated cross-contamination from a volatile
substance or possible cell seeding errors.
2) A mean relative viability was determined for each concentration of thesubstance tested along with the SD and coefficient of variation(%CV=SD/mean x 100).
3) Macros were included in the EXCEL® template to perform an outlier test
(Dixon and Massey 1981) on the data for the six replicate wells for eachconcentration. Outliers (i.e., individual well values that exceeded the 99%
confidence interval [CI] for the replicate wells) were highlighted and could be
excluded from the resulting analysis to improve curve fit. The Study Director
made the decision as to whether or not to remove outliers and provided ajustification for the decision.

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Other test-to-test measures of variability were considered in this study.
• Each set of assays for reference substances included a PC plate. If the SLS PC
test did not meet test acceptance criteria, then the tests for the associatedreference substances were rejected. The SMT recommended testing amanageable number of definitive test plates (e.g., 4 to 6) with each PC to limit
the number of definitive NRU tests rejected for PC failure. In this validation
study, 4.2% of all definitive tests performed were rejected because the PC
failed (i.e., the PC IC50 was outside the acceptable confidence limits).
• SDs and CVs were determined for mean IC50 values from replicate tests.
Replicate testing included three definitive tests for each reference substance,each performed on a different day.
2.3.12
Methods for Analyzing NRU Data
Relative cell viability for each reference substance concentration was calculated using theODs of the six replicate values (minimum of four acceptable replicate wells) per test
concentration. Relative cell viability was expressed as a percentage of the mean VC OD.
Absolute OD data from the microtiter plate reader was transferred to the EXCEL® templatefor performance of these calculations. Where possible, the concentration range (eight
concentrations) tested for each reference substance ranged from no effect to 100% toxicity.
The IC20, IC50, and IC80 values were determined from the concentration-response curve using
the PRISM® template and applying a Hill function to the % viability data. The IC20 and IC80
values were calculated for potential use in the development of a human prediction model(reported elsewhere).
2.3.13
Decision Criteria for Classification of Reference Substances
The 3T3 and NHK NRU test methods will not be used to classify reference substances inhazard categories but rather to aid in setting the starting dose for sequential rodent acute oral
toxicity test methods (i.e., the UDP and ATC) (see Section 10 for an analysis of theestimated animal savings). The RC millimole regression procedure was used to predict arodent LD50 value from an NRU IC50 value. Section 6.3 addresses the accuracy of the 3T3
and NHK NRU test methods for predicting GHS hazard categories when used with IC50-LD50
regressions, calculated using a subset of the RC data (i.e., substances with rat oral LD50 data).
2.3.14
Information and Data Included in the Test Report
Test and Control Substances
With the exception of the PC, the laboratories tested coded substances and had
minimal information about the test substances’ properties (see Section 3.3 for thereference substance information provided to the laboratories). The following
describes the test and test substance information that should be included in an NRU
test method report.
• Chemical name(s) and synonyms, if known• The CASRN, if known• Formula weight, if known• Purity and composition of the substance or preparation (in percentage[s] byweight)• Physicochemical properties (e.g., physical state, volatility, pH, stability,
chemical class, water solubility)
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Solubilization of the test/control substances (e.g., vortexing, sonication,
warming, grinding) prior to testing, if applicable
Information Concerning the Sponsor and the Test Facility• Name and address of the sponsor, test facilities, study director, andparticipating laboratory technicians• Justification of the test method and specific protocol used
Test Method Integrity• The procedure used to ensure the integrity (i.e., accuracy and reliability) of thetest method over time (e.g., use of the PC data)
Criteria for an Acceptable Test
• Acceptable VC differences between each column of wells and the mean ofboth columns• Acceptable concurrent PC ranges based on historical data (include thesummary historical data)• Number of toxic points on either side of the IC50 (i.e., number of points >0and ≤50% viability and >50 and <100% viability)
Test Conditions• Experiment start and completion dates• Details of test procedures used
• Test concentration(s) used and how they were derived• Cell type used and source of cells• Description of modifications made to the test procedure• Reference to historical data of the test model (e.g., solvent and PCs)
• Description of the evaluation criteria used
Results• Tabulation of data from individual test samples (e.g., IC50 values for thereference substance and the PC, absolute and derived OD readings, reported intabular form, including data from replicate repeat experiments as appropriate,
and the means and standard deviations for each experiment)
Description of Other Effects Observed• Cell morphology, precipitate, NR crystals, etc.
Discussion of the Results
Conclusion
Quality Assurance (QA) Statement for GLP-Compliant Studies• A statement describing all inspections and other QA activities during thestudy, and the dates results were reported to the Study Director. This
statement will also serve to confirm that the final report reflects the raw data.
•

During the validation study, the GLP-compliant laboratories, IIVS and ECBC, followed
additional reporting requirements provided in the relevant GLP guidelines (e.g., OECD 1998;
EPA 2003a, b; FDA 2003).
The SMT and laboratories developed standard forms for data collection (i.e., EXCEL® and
PRISM® templates). The solubility test form was derived from a standard form provided by
IIVS. The EXCEL® template was an adaptation of a template format presented in the
Guidance Document (ICCVAM 2001b).

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2.4

November 2006

Proprietary Components of the In Vitro NRU Test Methods

The only proprietary components used in these test methods are the NHK cells and the NHK
basal culture medium obtained from CAMBREX Clonetics®. All other components arereadily available through various scientific product suppliers.
Section 2.3.1.2 describes the NHK cells used in the study and provides the only commercial
source. All laboratories throughout the entire study used cells from the same lot. Procedures
used to verify the integrity of the NHK cells included comparison of positive control data
across laboratories and observations of cell growth throughout the study. If a laboratory
reported a problem with the cells, the SMT and Study Directors evaluated the testing
parameters to decide if the problem was cell-oriented or if other factors influenced the
problem. Section 2.6.3.5 provides information concerning the resolution of cell-related issues
and revisions made to the protocols to address such difficulties.
Section 2.10.1.1 and Appendices B2 and B4 provide information about the NHK growth
medium, supplements, and commercial source. Problems arose with the keratinocyte growth
medium during the study and resolutions and outcomes are addressed in Sections 2.6.3.5,
2.6.3.6, 5.3.4, and 11.1.2.2.
Although this study used proprietary components for the NHK NRU test method, cells andmedium from the commercial source used in the study are not required for implementation ofthis test method.
2.5

Basis for the Number of Replicate and Repeat Experiments for the 3T3 andNHK NRU Test Methods

The study protocols required each laboratory to test each coded reference substance in atleast one range finding test using a log dilution factor, and in at least three definitive tests on
three different days using a smaller dilution factor than used in the range finding test. Assays
were performed over a number of days to evaluate day-to-day variation. Laboratories tested
each coded reference substance until three definitive tests met the test acceptance criteria.
Additional testing was often dictated by:• Chemical issues (low toxicity, volatility, insolubility, and precipitation)• PC failure• Technical difficulties such as NR crystal formation
A stopping rule for insoluble reference substances was incorporated into the protocols for
Phase III to limit the number of retests (see Appendices B1 and B2):
“If the most rigorous solubility procedures have been performed and theassay cannot achieve adequate toxicity to meet the test acceptance criteria
after three definitive tests, then the Study Director may end all testing forthat particular chemical.”
2.6

Basis for Modifications to the 3T3 and NHK NRU Test Method Protocols

2.6.1
Phase Ia: Laboratory Evaluation Phase
All protocol revisions were implemented during Phase Ia unless otherwise stated.

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2.6.1.1 NR Dye Crystals
NR dye crystals formed in the 96-well test plates when used at 50 µg/mL (OD values
measured in the blanks increased from ~ 0.05 to 0.10) in both NRU test procedures.
Troubleshooting efforts included incubating the NR medium overnight; centrifuging andfiltering the NR medium prior to application to the 96-well plates; and reducing theconcentration of NR dye. The laboratories performed tests using a reduced NR concentrationof 33 µg/mL. Since there were no quantitative differences in results between tests with 50µg/mL and tests with 33 µg/mL NR, the SMT accepted tests with both concentrations.
Protocol Revision: The NR dye concentration was reduced to 33 µg/mL for both cell types insubsequent test Phases.
2.6.1.2 3T3 Cell Growth
The growth rate of 3T3 cells (as determined by monolayer confluence) was slower than
expected. As a result, the cells required more time in culture to obtain the proper density after
seeding.
Protocol Revision: The 3T3 cells must be passaged 2-3 times after thawing before being used
for the test. The protocol also emphasized attainment of the appropriate percentage of cell
confluence (not more than 50% for 3T3 cells) required at the time the cells were exposed to
the reference substance, rather than using the time in culture as the guide.
2.6.1.3 NHK Cell Growth
The NHK cells had an additional growth problem that manifested as a ring of dead/dying
cells around the center of the wells. Troubleshooting efforts included evaluating various
brands of 96-well plates (laboratories were not required to use the same brand of plates) andeliminating the change of medium prior to reference substance treatment. All laboratories
participated in evaluating the effect of changing (i.e., refeeding) or not changing (i.e., norefeeding) the medium by performing a small study with the PC (SLS). Tests were
performed: 1) after refeeding the cells with fresh medium, and 2) by adding SLS to themedium already on the cells. Control ODs were generally higher in the tests in which themedium was not replenished, but sensitivity to SLS was generally unchanged (see Table 22). FAL was experiencing difficulties in NHK cell growth at this stage of the study which
may account for the difference in the refeeding and no refeeding SLS IC50 values. The SMT
accepted tests with refeeding and those without refeeding (for Phase Ia) as long as they met
the test acceptance criteria.
IIVS presented detailed information on the ring of dead cells issue (Raabe 2004). The
laboratory showed that the ring of cell death coincided with the formation of a meniscus
resulting from the residual medium left in the well after removal of the spent medium. The
problem was resolved by eliminating the removal of medium before applying test chemicalrather than requiring a standard brand of 96-well plates.
Protocol Revision: Step 2 of the NHK NRU test method was eliminated (change of medium
prior to addition of reference substance). The volume of medium (with cells) was changed
from 250 µL/well to 125 µL/well.

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Table 2-2

November 2006

Refeeding/No Refeeding Data for the NHK NRU Test Method
RefeedECBC
No Refeed

IIVS
RefeedNo Refeed

RefeedFAL
No Refeed

Number of Test Plates

4

4

6

6

2

4

Absolute OD1 for VC

0.265±0.151

0.621±0.3220.885±0.0571.12±0.0331.41±0.1271.24±0.430OD1 for SLS IC50

0.102
±0.0790.282±0.1650.415±0.0290.533±0.0170.696±0.0650.606±0.217SLS IC50 (µg/mL)1

3.33
±0.47

3.23
±0.61

3.41
±0.58

3.49
±0.39

6.21
±0.88

8.14
±0.40

Abbreviations: NHK=Normal human epidermal keratinocyte; NRU=Neutral red uptake; ECBC=Edgewood Chemical
Biological Center; IIVS=Institute for In Vitro Sciences; FAL=Fund for the Replacement of Animals in Medical Experiments
Alternatives Laboratory; VC=Vehicle control; OD=Optical density; SLS=Sodium lauryl sulfate.
Note: OD values for SLS IC50 were extrapolated from the concentration-response curve data
1
Mean ± standard deviation (uncorrected for blank absorbance

FAL, in contrast to the other two laboratories, used 80 cm2 culture flasks for culturing thethawed cells from the ampules of cryogenically-preserved pool of cells and encountereddifficulties in obtaining a satisfactory number of adhering NHKs.
Protocol Revision (FAL only): Culture flasks were coated with fibronectin-collagen to
promote cell adherence.
2.6.1.4 Vehicle Control OD Limits
In Phase I, the acceptable range of VC OD values designated in the protocols (0.3 ≤ OD
≤1.1) were frequently unattainable in both test methods. Despite this, the Study Directors
reported that the cells were adequately responsive. The SMT withdrew the VC OD limits as atest acceptance criterion.
Protocol Revision for Phase Ib: OD ranges were provided as guidelines for each cell typebased on OD data from all laboratories, a review of the concentration-response data, and theability of each test to pass the other test acceptance criteria. Each laboratory developed its
own VC OD acceptability range based on its historical data.
2.6.1.5 Precipitate Formation
During solubility testing, it was observed that some substances, when tested at the same
concentrations, precipitated in the 3T3 medium but not in the NHK medium. When a liquid
reference substance (i.e., 2-propanol) produced this effect, the precipitate was attributed tothe protein in the serum in the 3T3 medium rather than insolubility.
Protocol Revision: The reference substances were dissolved in 3T3 medium without NCS to
make the 2X solutions. The dissolved 2X reference substance was added to medium
containing 10% NCS to reach the final 5% NCS and 1X reference substance concentrations.
2.6.1.6 Dilution Factor
After a range finder test was performed, the definitive tests were to be performed using a6√10=1.47 dilution scheme centered on the IC50 that was calculated from the range finder. In
Phase Ia, the Study Directors, for various reasons related to the specific substance being
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tested, sometimes deviated from this requirement and used other dilution factors. The SMT
agreed that the dilution factor requirements should be modified to allow more flexibility insetting up tests. The SMT accepted data generated using dilution factors other than therecommended 1.47 for definitive tests if all other test acceptance criteria were met. The useof smaller dilution factors generally increased the number of concentrations in the 10% to
90% viability range, which improved the precision of the IC50 calculation.
Protocol Revision: The 6√10=1.47 dilution scheme was a suggested starting range, rather
than a specific test acceptance criterion in subsequent test Phases.
2.6.1.7 Test Acceptance Criteria
The test acceptance criteria at the beginning of Phase Ia were:• The IC50 for SLS had to be within the 95% CI of the historical PC mean
established by the Test Facility (rescinded after commencement of Phase Ia)• The OD540 of the VCs (with blank subtracted) had to be ≥0.3 and ≤1.1
(rescinded after commencement of Phase Ia)• Mean OD values of the left and right VCs (columns 2 and 11 in the 96-welltest plate) must not differ by more than 15% from the mean of all VC OD
values• At least two cytotoxicity values, one on either side of the IC50 but between
10% and 90% viability, needed to be present (added after commencement ofPhase Ia)
• The Hill function curve fits (R2 >0.9 or 0.8 < R2 <0.9) were evaluated on acase by case basis for acceptability by the SMT (added after commencementof Phase Ia).
2.6.2
Phase Ib: Laboratory Evaluation Phase
All protocol revisions developed during Phase Ia were implemented during Phase Ib unlessotherwise stated.
2.6.2.1 NR Crystal Formation
FAL and ECBC routinely observed NR crystals forming in the 96-well test plates in the 3T3
NRU tests when 33 µg/mL NR was used. All laboratories tested 25 and 33 µg/mL NR
concentrations and 2- and 3-hour NR incubation periods to determine which NR
concentration and incubation period would provide optimal NRU measurements without
crystal formation. In addition to determining whether NRU had reached a plateau at theseconcentrations and incubation times, the laboratories also determined whether the response to
SLS differed under these conditions. Crystals were observed only at 33 µg/mL NR whenpresent for three hours. Figure 2-2 shows that the average OD results were similar for all NR
concentrations and incubation periods tested. Figure 2-3 shows that the SLS IC50 values
were equivalent at the different NR concentrations and incubation periods. To minimizechanges to the 3T3 protocol, the NRU concentration was lowered from 33 to 25 µg/mL,
while the NR incubation period was maintained at three hours. The NR concentration and the
incubation period for the NHK NRU test method remained at 33 µg/mL and three hours,
respectively.
Protocol Revision for Phase II: The NR concentration for the 3T3 NRU test method was
reduced to 25 µg/mL for the three-hour incubation period. Revised methods for preparation
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In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006of the NR dye solution included filtration of the solution, maintenance of the solution at 37ºC prior to application to the cells, and application of the NR solution to the cells within 15
minutes after removing it from 37 ºC. Also, cells were observed during the NR incubation
period to monitor possible crystal formation.
2.6.2.2 Heating of Reference Substance Solutions
The laboratories had difficulty solubilizing arsenic trioxide, one of the reference substancesused in Phase Ib. Heating and mechanical applications for increasing the laboratory’s ability
to solubilize substances into culture medium were reviewed and revised.
Protocol Revision for Phase II: The duration range for heating a stock solution at 37 ºC (ifheating is needed) was increased from 5 to 10 minutes to 5 to 60 minutes.
Figure 2-2

3T3 NRU OD for SLS as a Function of NR Concentration and Duration

Abbreviations: OD=Optical density; NR=Neutral red; SLS=Sodium lauryl sulfate; 3T3=BALB/c 3T3
fibroblasts; NRU=Neutral red uptake; h=Hours.
Note: Error bars are one standard deviation.

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In Vitro Cytotoxicity Test Methods BRD Section 2

Figure 2-3

November 2006

SLS IC50 Values for Each NR Concentration and Incubation Duration(3T3 NRU)

Abbreviations: SLS=Sodium lauryl sulfate; IC50=Test substance concentration that reduces cell viability by
50%; NR=Neutral red; 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red uptake; ECBC=Edgewood Chemical
Biological Center; IIVS=Institute for In Vitro Sciences; FAL=Fund for the Replacement of Animals in Medical
Experiments Alternatives Laboratory.
Note: SLS range is mean IC50 value ± one standard deviation.

Protocol Revision for Phase II: The duration range for heating a stock solution at 37 ºC (ifheating is needed) was increased from 5 to 10 minutes to 5 to 60 minutes.
2.6.2.3 Growth of Untreated Cells
VC OD values were frequently lower than specified in the Phase I acceptance criteria. Phases
Ia and Ib incorporated the acceptance limits shown in Table 2-1 for the VC, but the limits
were rescinded as test acceptance criteria for Phase II because the laboratories frequently
failed to meet them even though cell growth and responsiveness to SLS was adequate.
Protocol Revision for Phase II: The specified VC OD range was eliminated as a test
acceptance criterion. The OD data (all laboratories combined) from the VCs for both cell
types was used to calculate OD ranges that would serve as guidelines for other tests (seeSection 2.2.9).
2.6.2.4 Correction of Reference Substance OD Values
Each reference substance concentration was applied to six treatment wells and to two cellfree wells (i.e., blank wells) used to generate the background OD540 values to adjust forpotential interference with the NR dye. The mean blank well OD (absolute OD) for eachreference concentration was subtracted from the reference substance concentration ODs toprovide the corrected OD for each replicate well.

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In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006

2.6.2.5 Laboratory Error Rates
The SMT determined the Phase 1b error rates (number of tests with errors/total number oftests conducted) for each laboratory (Table 2-3) and compiled a list of the types of errors
encountered. The vast majority of errors were transcriptional and typographical errors in thedata sheets provided to the SMT.
Table 2-3

Error Rates1 in Phase Ib by Laboratory and Test
NRU Test Method

Laboratory
3T3

NHK

ECBC

1/9 (10%)

4/17 (23%)

FAL

42/45 (93%)

12/29 (41%)

IIVS

1/20 (5%)

1/20 (5%)

Abbreviations: NRU=Neutral red uptake; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes;
ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals In Medical Experiments
Alternatives Laboratory; IIVS=Institute for In Vitro Sciences
Note: Most errors were transcriptional and typographical and not technical.
1
Number of tests with errors/total number of tests (some data files had more than one error)

2.6.2.6 Resultant Protocol Changes for Phase II
Following the completion of Phases Ia and Ib, IIVS sponsored a weeklong laboratory training
exercise for the cytotoxicity testing laboratories to help standardize the level of training
among the technical staff and to identify any further 3T3 and NHK NRU protocol revisions
that might be needed. Protocol revisions made because of this exercise included:• Multi-channel repeater pipettes can be used for dispensing cells into the 96well plates and dispensing plate rinse solutions, NR medium, and desorb
solution but are not accurate enough to dispense the PC or the reference
substances to the treatment wells.
• Use of 8-channel reservoirs for applying dosing solutions to the wells so thatmulti-channel single delivery pipettes could be used
• Use of a standardized length of time that the HBSS rinse remains on the cellmonolayers in flasks during the cell subculture step
• Protection of plates from light during the shaking step for NR extraction; alllaboratories will cover plates with a light-impermeable barrier (e.g., aluminumfoil) during this step
• Allow plates to stand for at least five minutes after the shaking step iscomplete and eliminate any bubbles in media observed in the wells before
measuring the OD• Change the allowable seeding density range for 3T3 NRU test method from
2.5x103 cells/well to 2 – 3x103 cells/well• Change the NHK culture flask size used at FAL for start-up of cryopreserved
cells from 80 cm2 to 25 cm2 (the size the other laboratories had been using),and discontinue using a fibronectin-collagen coating.

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In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006

2.6.2.7 Test Acceptance Criteria
The test acceptance criteria were revised as follows:• The IC50 for SLS (PC) should be within 2 SDs (approximately 95%) of thehistorical mean established by each laboratory in Phase Ia.
• The mean OD values of the left and right VCs (columns 2 and 11 in the 96well test plate) should not differ by more than 15% from the mean of all VC
OD values on that plate.
• At least one calculated cytotoxicity value should be between 10% and 50%
viability, and one value between 50% and 90% viability.
• The Hill function curve fit (R2 >0.9 or 0.8 < R2 <0.9) should be evaluated on acase-by-case basis for acceptability by the SMT.
• VC OD criteria were based on Phase Ia data (mean ± two SDs): 0.3 to 0.8 forthe 3T3 test method, and 0.6 to 1.7 for the NHK NRU test method(requirement for use of VC OD criteria as test acceptance criteria wasrescinded after commencement of Phase Ib)
2.6.3
Phase II: Laboratory Qualification Phase
All protocol and acceptance criteria revisions were implemented during Phase II unlessotherwise stated.
2.6.3.1 Testing of Volatile Reference Substances
When 2-propanol was tested in 3T3 and NHK cells, vapors from the highest concentration
wells contaminated the adjacent VC wells and also appeared to affect some lower
concentration wells (i.e., the wells exhibited unexpectedly reduced levels of NRU). An
example range finder concentration-response curve is shown in Figure 2-4. Such tests failed
the VC criterion. When lower concentrations were used to avoid contaminating the VC wells
adjacent to the highest concentration, the toxicity was inadequate to produce an IC50. To
address this problem, IIVS repeated their tests using film plate sealers, which isolated
individual wells from one another; this was sufficient to prevent the cross-wellcontamination, and acceptable results were obtained. Based on these data, the SMT
recommended to the other two laboratories that film plate sealers be used when testing 2propanol.
FAL had previous experience layering mineral oil on the culture media in a well to prevent
volatile substances from escaping, and provided 2-propanol test data where mineral oil had
been added to each well. The data showed that the average oil vs. film IC50 values were notsignificantly different. However, there was less variability in the NRU data when using thefilm sealer so the SMT recommended this methodology.
A >15% difference between the mean VC OD of all VC cells and the mean OD of each VC
columns on opposite ends of the test plate was used as a general indicator of substancevolatility in the test if the VC adjacent to the highest test concentration had a significantly
reduced OD value.
Protocol Revision: The SMT included the use of film sealers in the Phase III protocols when
testing suspected volatile compounds.

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In Vitro Cytotoxicity Test Methods BRD Section 2

Figure 2-4

November 2006

Representative Concentration-Response for 2-Propanol in a 3T3 NRU
Range Finder Test

A
B
C
D
E
F
G
H

1
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

2
Blank
VC1
VC1
VC1
VC1
VC1
VC1
Blank

A
B
C
D
E
F
G
H

1
0.000
0.002
-0.0010.003
0.003-0.004-0.0040.004

2-0.002
0.080
0.067
0.058
0.077
0.068
0.071
0.000

3
Blank
C1
C1
C1
C1
C1
C1
Blank

4
Blank
C2
C2
C2
C2
C2
C2
Blank

96-WELL PLATE MAP
5
Blank
C3
C3
C3
C3
C3
C3
Blank

6
Blank
C4
C4
C4
C4
C4
C4
Blank

7
Blank
C5
C5
C5
C5
C5
C5
Blank

8
Blank
C6
C6
C6
C6
C6
C6
Blank

9
Blank
C7
C7
C7
C7
C7
C7
Blank

10
Blank
C8
C8
C8
C8
C8
C8
Blank

CORRECTED ABSORBANCE (Sample OD540 - Mean Blank OD540)
3-0.001-0.001
0.004
0.003
0.001
-0.002
0.003
0.001

4
-0.001
0.070
0.059
0.056
0.067
0.050
0.053
0.001

5
0.000
0.124
0.109
0.110
0.106
0.110
0.122
0.000

6
-0.003
0.206
0.171
0.163
0.092
0.164
0.147
0.003

7
0.001
0.296
0.284
0.243
0.218
0.216
0.204
-0.001

8
0.002
0.389
0.334
0.271
0.252
0.289
0.226
-0.002

9
0.002
0.291
0.237
0.246
0.328
0.336
0.263
-0.00210-0.001
0.301
0.308
0.251
0.250
0.267
0.295
0.001

11
Blank
VC2
VC2
VC2
VC2
VC2
VC2
Blank

12
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

11
-0.002
0.343
0.337
0.283
0.290
0.281
0.330
0.001

12
-0.0030.002-0.0040.002
0.003-0.001-0.003-0.002Abbreviations: 3T3=BALB/c 3T3 fibroblasts; VC=Vehicle control; C1 to C8=Test substance concentrations (C1-highest concentration, C8lowest concentration); OD540=Optical density at 540 nm; A to H=Row identification.
Note: %Difference of the two VC columns from the average VC was 63%. The mean corrected optical density (OD) for VC1, adjacent to
the highest 2-propanol concentration, was 0.070, while that for VC2, adjacent to the lowest 2-propanol concentration, was 0.310. Setting themean VC OD to 100% viability shifted the toxicity curve such that lower concentrations of 2-propanol seemed to be less toxic to the cells
than the VCs (i.e., >100%).
Error bars are ±1 standard deviation.

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November 2006

2.6.3.2 Atypical Concentration-Responses
Atypical concentration-responses are defined for this study as response curves that differ
from a basic sigmoidal shaped curve. Curves that show a biphasic response as well as thosethat exhibited a plateau-like response at toxicity levels than 100% were considered atypical.
Two of the laboratories observed biphasic concentration-responses in the range finder tests
for aminopterin and colchicine. When the range finder tests produced a biphasic response(see Figure 2-5 for an example), the SMT advised the laboratories to focus the definitive
tests on the lowest concentrations that produced at least a 50% loss in viability. Although
doing so eliminated the biphasic response in the definitive tests, the highest tested
concentrations did not reduce cell viability to 0% (see Figure 2-6). This effect withcolchicine was very reproducible across laboratories in the NHK NRU test, but only FAL
achieved this biphasic type of response with colchicine in the 3T3 NRU test. Aminopterin
produced similar concentration-responses in the NHK NRU test at ECBC and FAL, but not
at IIVS. In the 3T3 NRU test, only FAL obtained a biphasic response with aminopterin.
Figure 2-5

Representative Concentration-Response for Aminopterin in a NHK NRU
Range Finder Test

Abbreviations: NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake.
Representative dose-response for aminopterin in a NHK range finder test. Laboratories were instructed tofocus the definitive tests on the lowest concentration that produced a 50% reduction in viability in the range
finder test.

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In Vitro Cytotoxicity Test Methods BRD Section 2

Figure 2-6

November 2006

Representative Concentration-Response for Aminopterin in a NHK
NRU Definitive Test

Abbreviations: NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake.
Note that the maximum reduction in cell viability plateaued at about 75%

Biphasic concentration-responses are not uncommon. Calabrese (2005) states that numerous
mechanistic explanations (including hormesis4) could account for biphasic response curves.
Such concentration-responses could be because the substance acts through more than one
mechanism of action (e.g., one mechanism that is active at low test substance concentrations
and other mechanism[s]) that are effective at higher concentrations). Conolly and Lutz
(2004) also provide examples of pharmacological and toxicological data sets of biologically
based mechanisms that could explain biphasic responses. These examples include:• Membrane receptor subtypes with opposite downstream effects• Receptor-mediated gene expression• Induction of DNA repair and “co-repair” of background DNA damage• Modulation of the cell cycle
Although non-linear responses could also be due to technical error (e.g., improper dosing,unacceptable media, contamination), the responses seen in this study were reproducible, andthere was no evidence to suggest that technical errors were involved. The SMT assumed thatthese responses were based on the chemicals’ mechanisms of action. For example, colchicine
binds to microtubular protein and interferes with function of mitotic spindles, which arrests
cell division (NLM 2003). Aminopterin blocks the use of folic acid by the cells, inhibiting
metabolism, RNA production, and protein synthesis, which is lethal during the S phase of thecell cycle by (NLM 2002). The variability of IC50 results for these substances among thelaboratories may be due to different levels of cell confluence in the cultures at the time oftreatment.

Hormesis is a dose-response characterized by a compound’s ability to produce an opposite effect at low doses
compared with its effect at high doses (e.g., stimulatory at low doses and inhibitory at high doses).
4

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In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006

2.6.3.3 Hill Function
The Hill function used in the various phases of this study was defined as follows:

Y = Bottom +Top " Bottom
1+ 10 (logEC50"logX)HillSlope

where Y=response (i.e., % viability), X is the substance concentration producing the
response, Bottom is the minimum response (0% viability, maximum toxicity), Top is the
maximum response!(maximum viability), EC50 is the substance concentration at the response
midway between Top and Bottom, and HillSlope describes the slope of the curve. When
Top=100% viability and Bottom=0% viability, the EC50 is the equal to the IC50.
Responses that do not achieve 100% cytotoxicity with increasing substance concentration do
not fit the Hill function well. The R2 values from such tests often failed the acceptancecriterion. To obtain a better model fit, the Bottom parameter was estimated without
constraints (the previous practice was to use Bottom=0). However, when Bottom≠0, the EC50
reported by the Hill function was not the same as the IC50 because the Hill function relies on
EC50, which is defined as the point midway between the Top and Bottom responses. Thus,
the Hill function calculation using the Prism® software was rearranged to calculate the IC50
as follows:# Top " Bottom &log%"1($ Y " Bottom'
logIC 50 = logEC 50 "HillSlope

where IC50 is the concentration producing 50% toxicity, EC50 is the concentration producing
a response midway between the Top and Bottom responses; Top being the maximum! survival), Bottom is the minimum response (0% viability, maximum
response (maximum
toxicity), Y=50 (i.e., 50% response), and HillSlope describes the slope of the response. The
X from the standard Hill function equation is replaced, in the rearranged Hill function
equation, by the IC50.
IIVS performed the recalculations for their NHK NRU colchicine tests and the SMT
performed the necessary recalculations for the other laboratories. Tests that were recalculatedby the SMT are noted in the data summaries.
Protocol Revision: The protocol was revised to state that if a range finding test produces abiphasic response, then the concentrations selected for the subsequent tests should cover themost toxic dose-response range.
2.6.3.4 Insoluble Reference Substances
Lithium carbonate was insoluble in 3T3 medium. Only ECBC managed to expose 3T3 cells
to sufficient lithium carbonate to produce three tests that met the acceptance criteria.
Precipitate was reported for two of those tests at the three highest concentrations in the wells.
Because the third highest concentration, 510.2 µg/mL, was approximately the IC50 (average
was 564 µg/mL), the true IC50 for lithium carbonate may actually be lower than was
2-33

In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006

calculated, and therefore the LD50 value would be underestimated. However, the data werereproducible and were not discarded.
Protocol Revision for Phase III: The protocol was revised to allow an increase in thestirring/rocking duration in an incubator from one to three hours if cytotoxicity in the range
finder test was limited by solubility. Also, a Stopping Rule for Insoluble Chemicals wasadded (see Section 2.5 and Appendices B1 and B2) so that the laboratories would notcontinue repeated testing of insoluble substances in order to obtain three acceptable definitive
tests.
2.6.3.5 Inadequate Cell Growth in NHK Medium
IIVS and FAL had several NHK NRU test failures that were attributed to poor cell growth.
The SMT compiled KBM and SingleQuot lot numbers that the laboratories were using,along with the laboratory assessments of NHK cell growth. The information was used to
identify the lots that produced adequate growth. The SMT also obtained quality assurance
and quality control test results from CAMBREX Clonetics on the lots of KBM, but the
information provided was inadequate for determining how the medium would perform in the
NHK NRU test method.
Resolution: A protocol for prequalifying the medium was developed (see Appendix B4). For
Phase III, the SMT asked IIVS to prequalify new lots of KBM and SingleQuots for use by
all laboratories.
2.6.3.6 Performance Standards for Media to Support NHK Growth
A prequalification-of-medium protocol (Appendix B4) was developed and used by IIVS totest several different lots of medium and supplements to find combinations that maintained
the typical growth characteristics of the NHK cells used in this study. The laboratories thenreserved samples of the acceptable lots at CAMBREX so that testing would not beinterrupted due to unavailability of adequate materials.
Test Acceptance Criteria for Prequalifying Media Using SLS• The fit of the SLS dose-response to the Hill model should be R2 ≥0.85 (i.e.,from PRISM® software).
• The difference between the mean of all VCs and (a) the left mean VC, and (b)the right mean VC should be ≤15%.
• At least one concentration should exhibit >0% and ≤50% viability and at least
one should exhibit >50% and <100% viability.
• After meeting all other acceptability criteria, the SLS IC50 must be within thehistorical range (±2.5 SD) established by the laboratory.
Other Criteria for Prequalifying Media (for consideration by a Study Director)• General observations: rate of cell proliferation; percent confluence; number of
mitotic figures per field; colony formation; distribution of cells in the flask;
absence or presence of contamination• Cell morphology observations should include overall appearance (e.g., good,
fair, poor), and presence of abnormal cells

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In Vitro Cytotoxicity Test Methods BRD Section 2•••
2.6.3.7

November 2006

Mean corrected OD540 of the VCs (e.g., are the values high/low whencompared to historical data)
Cell morphology and confluence of the VC wells at the end of the 48-hourtreatment
Cell doubling time, as compared to the doubling time with the previous
batches of medium

Test Acceptance Criteria for Phase II• The IC50 for SLS (PC) should be within 2.5 SDs of the historical mean
established by the laboratory (Phases Ia and Ib)• Mean OD values of the left and right VCs (columns 2 and 11 in the 96-welltest plate) do not differ by more than 15% from the mean of all VC well OD
values. At least one calculated cytotoxicity value ≥10% and ≤50% viability
and at least one value >50% and ≤90% viability• R2 ≥0.90. The test fails if R2 <0.80. If the 0.80 ≤ R2 <0.90, the SMT evaluates
the model fit (Note: The Study Director makes this determination for nonvalidation studies.)

2.6.4
Phase III: Laboratory Testing Phase
The changes below were made in the Phase III protocols based on the data and results in
Phase II.
2.6.4.1 Required Cytotoxicity Values
Obtaining at least one calculated cytotoxicity value >0% and ≤50% viability and at least one
that is >50% and <100% viability may be difficult or unattainable for substances with steep
dose responses.
Protocol Revision: The test acceptance criterion was qualified so that tests with only one
concentration between 0 and 100% viability were acceptable if the smallest practical dilution
factor (i.e., 1.21) was used and all other test acceptance criteria were met.
Tests for three reference substances were accepted that met this new criterion in the 3T3
NRU test method: diquat dibromide (1/9 tests); epinephrine bitartrate (2/9 tests); 1,1,1trichloroethane (2/8 tests). No NHK tests required the use of these criteria (i.e., one point
between 0% and 100% viability at the lowest dilution factor).
2.6.4.2 Revisions to Data Analysis Procedures
The following revisions to data analysis procedures were made in Phase III NRU protocols:• If the Bottom parameter of the Hill function was fit to a value <0%, then theparameter was set to zero (0) for the IC calculations.
• If toxicity plateaued above 20% viability (i.e., toxicity was <80%), the IC80
was not determined. The IC20 and IC50 values were calculated from the rangeof available toxic responses.
• The requirement for substance dose-responses to fit the Hill equation with R2≥0.90 was rescinded. The Hill equation was used to characterize the shape ofthe response rather than to establish an acceptance criterion. The PC
acceptance criterion was modified to R2 ≥0.85.

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In Vitro Cytotoxicity Test Methods BRD Section 2

2.7

November 2006

Differences Between the 3T3 and NHK NRU Protocols for the Validation
Study and the Guidance Document Standard Protocols

As the validation study progressed through Phases I and II, the protocols provided in the
Guidance Document (ICCVAM 2001b) were optimized to address problems that wereencountered during the validation study phases. Changes to the Guidance Document
protocols are described below.
• 3T3 cell seeding density for 96-well plates was decreased from 1x104
cells/well to 2.0 – 3.0x103 cells/well.
• The calcium concentration in NHK medium was changed from 0.15 mM to
0.10 mM. The test laboratories had expressed concern that cell differentiation
would occur at the higher concentration and requested a lower concentration.
CAMBREX Clonetics®, the supplier of the NHK cells and NHK medium usedin this study, normally grows NHK cells in 0.15 mM calcium and has seen no
differentiation. The supplier agreed that the cells would grow well at 0.10 mM
but should not be cultured at concentrations <0.10 mM in order to avoid
morphological and growth rate changes (CAMBREX technical division,
personal communication).
• NHK cells were subcultured once prior to being distributed to the test wells,
rather than for three passages. The laboratories expressed concern about thepossibility of cell differentiation with subsequent passages in culture.
• The highest recommended final concentrations of DMSO and ETOH in theculture media were reduced from 1% to 0.5%. IIVS performed experimentswith both cell types to determine the concentration necessary to avoid solvent
toxicity. 3T3 cells were tested with 0.5, 1, and 2% ETOH and DMSO at 0.1,
0.2, 0.3, 0.4, 0.5, 1, and 2% concentrations. The 0.5% concentrations of bothsolvents were chosen as optimal because that concentration of ETOH
produced no toxicity. Although 0.5% DMSO produced slight toxicity (i.e.,cells were 91% viable as compared to the control cells; See Appendix E1),
this concentration was chosen by the SMT and laboratories as an acceptabletrade-off between slight toxicity and the ability to test substances at higher
concentrations, and was used throughout the study for all reference substancesthat needed solvents other than culture medium (see Curren et al. 2003).
DMSO was the preferred solvent if the test substance was not soluble inculture medium, and ETOH was not used in this study.
• The pH of the reference substance solutions was not adjusted with NaOH or
HCl regardless of whether solutions became acidic or basic (optimum
mammalian cell culture pH is approximately 7.4 [Freshney, 2000]) upon
addition of the test substance because some of the basal cytotoxicity produced
by test substances may be due to pH effects. See Appendix F1 for pH valuesof the reference substances in culture medium.
• The CO2 concentration in the incubator was reduced from 7.5% to 5.0%
because the laboratories were already set up to use 5% CO2, which is a typicaloptimum CO2 concentration for mammalian cell culture.
• Washing and fixing the cells with a formaldehyde solution prior to NR elution
from the cells was eliminated. Formaldehyde disposal was problematic in
FAL’s regulatory environment. The SMT and the laboratories agreed that the2-36

In Vitro Cytotoxicity Test Methods BRD Section 2••

Table 2-4

November 2006

use of formaldehyde was unnecessary because the NR desorb solution (1%glacial acetic acid, 50% ETOH, and 49% H2O) adequately fixed the cells to
the test plate (INVITTOX 1991).
Reference substance exposure time for the 3T3 cells was extended from 24hours to 48 hours (see Section 2.2.4 and Appendix E1).
Cell culture seeding densities for subculture were provided as guidance, rather
than as strict cell number ranges. The laboratories determined adequate celldensities (see Table 2-4) based on their own experience with the growth ofthe cells in the wells, and the time needed to reach the appropriate level ofconfluence needed for addition of the test substance, the VC, and PC.
Cell Seeding Densities1

ProtocolGuidance Document2

3T3 cells/cm2
subculture toflasks1.25x104

3T3 cells/well
96-well Plate
2.5x103

NHK cells/cm2
subculture toflasks
3.5x103

NHK cells/well
96-well Plate2 – 2.5x103

Phase Ia

0.42 – 1.68x10

42.5x103

2.5 – 9x103

2 – 2.5x103

Phase Ib

0.42 – 1.68x104

2.5x103

2.5 – 9x103

2 – 2.5x103

Phase II

0.42 – 1.68x104

2 – 3x103

2.5 – 9x103

2 – 2.5x103

Phase III0.42 – 1.68x104

2 – 3x103

2.5 – 9x103

2 – 2.5x103

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes
1
Cell numbers determined by Coulter Counter or hemocytometer
2
ICCVAM (2001b)

2.8

Overview of the Solubility Protocol

The SMT, with assistance from the laboratories, developed a solubility protocol to provide
guidance for determining the most appropriate solvent for each test substance. The solubility
protocol was based on an EPA guideline (EPA 1998) that involved testing for solubility in aparticular solvent, beginning at a relatively high concentration and proceeding tosuccessively lower concentrations by adding more solvent as necessary for dissolution.
Testing stopped when, upon visual observation, the procedure produced a clear solution withno cloudiness or precipitate. The order of selection priority was culture medium, DMSO, andETOH. Each laboratory tested the solubility of each reference substance using this protocol
and provided the data to the SMT prior to initiating cytotoxicity testing. The SMT analyzed
the solubility data provided by BioReliance and each testing laboratory, and designated thesolvent to be used by all laboratories for each reference substance. This eliminated one
potential variable in the NRU test results among laboratories.
The solubility protocol used by the in vitro laboratories required the sequential testing ofreference substances in the various solvents at concentrations that would be equivalent to theconcentration that would be applied to the cell cultures. The solubility flow chart in Figure
2-7 shows, for example, that 2 mg/mL medium and 200 mg/mL DMSO or ETOH wereequivalent concentrations because they yielded 1 mg/mL in cell culture. Medium was diluted
by one-half when applied to cultures. The 0.5% [v/v] final concentrations were achieved bydiluting DMSO and ETOH by 200-fold. At each concentration, the following mixing
procedures were employed, as necessary, to completely dissolve the reference substance in
2-37

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November 2006

the sequence: vortex (1 to 2 minutes); sonication (up to 5 minutes); warming to 37 °C (5 to
60 minutes [NRU protocols allow warming to be extended to three hours if cytotoxicity inthe range finder test was limited by solubility]). If the reference substance was still notdissolved, the next lower concentration, or a different solvent, was tested.
Figure 2-7

Flow Chart for Determination of Reference Substance Solubility in
Medium1, DMSO, or ETOH

Tier

1

Concentration in
3T3 and NHK
Media

Start Here20 mg/mL2

3

2 mg/mL

0.20 mg/mL

20 mg/mL

Incomplete
solubility

Concentration
on Cells

200 mg/mL

10 mg/mL

2 mg/mL

0.2 mg/mL

Incomplete
solubility

200 mg/mL

Concentration in
Ethanol5

Incomplete
solubility

Incomplete
solubility

Concentration in
DMSO

4Incomplete
solubility

Incomplete
solubility

1 mg/mL

20 mg/mL

0.1 mg/mL

Incomplete
solubility

Incomplete
solubility

2 mg/mL

0.01
mg/mL

Incomplete
solubility

Incomplete
solubility

0.2 mg/mL
End

0.001
mg/mL

Abbreviations: DMSO=Dimethyl sulfoxide; ETOH=Ethanol; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal
human epidermal keratinocytes.
Note: DMSO is U.S.P. analytical grade. ETOH is U.S.P. analytical grade (100% non-denatured).
1
3T3 Medium - DMEM (Dulbecco’s Modification of Eagle’s Medium) with supplements; NHK medium KBM (Keratinocyte Basal Medium) with supplements (from CAMBREX Clonetics).

2.9

Basis of the Solubility Protocol

The solubility protocol used by BioReliance, which tested solubility of the reference
substances prior to testing by the in vitro laboratories, is provided in Appendix G. The
protocol is based largely on information from the literature and Internet searches forsolubility procedures, the experience of the SMT and IIVS, and solubility and IC50
information from the RC chemicals database (Halle 1998, 2003). The only formal solubility
protocol discovered was the EPA Product Properties Test Guideline, OPPTS 830.7840 Water
Solubility Column Elution Method; Shake Flask Method (EPA 1998).
2.9.1
Initial Solubility Protocol Development
BioReliance evaluated the solubility of each reference substance in cell culture media at
2000, 400, and 200 mg/mL, and if not soluble at those concentrations, in DMSO and then
ETOH, at the same concentrations (initial protocol). It was apparent that these concentrations
were not low enough when the laboratory was unable to achieve solubility for arsenic
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trioxide. The solubility protocol was revised twice to lower the range of concentrations tested(see Table 2-5). An extra tier of concentrations ≤1 mg/mL was added for poorly soluble andinsoluble substances. The protocol used by the laboratories was further revised to reduce thenumber of steps required (by testing in log units) and to test in tiers using concentrations thatreflected the concentrations anticipated in the cell cultures (see Figure 2-7).
Table 2-5

Comparison of Concentrations Tested in the Various Solubility Protocols

Solubility
Protocol Version

Concentrations Tested (mg/mL)
Step 1

Step 2

Step 3

Step 4

Step 5

Steps 6-10

BioReliance (1 )(4/26/02) andPhase Ia

2,000

400

200

NA

NA

NA

BioReliance (2nd)(9/17/02)

200

40

20

10

2

NA

BioReliance (3rd)(10/11/02)

200

40

20

10

2

1, 0.5, 0.25,
0.125, 0.05

Phases Ib, II, III for
cytotoxicity
laboratories

20
Medium

2 Medium
200 DMSO
200 ETOH

0.2 Medium
20 DMSO
20 ETOH

2 DMSO
2 ETOH

0.2 DMSO
0.2 ETOH

stNA

Abbreviations: DMSO=Dimethyl sulfoxide; ETOH=Ethanol; Medium=Cell culture medium; NA=Not applicable
Note: DMSO is U.S.P. analytical grade. ETOH is U.S.P. analytical grade (100% non-denatured).

In Phases Ib and II, the SMT used the data from BioReliance to select the solvents to be used
for testing the various chemicals. When it became apparent that the laboratories sometimes
obtained different solubility results than those reported by BioReliance, the SMT used thecytotoxicity results from the laboratories to determine the solvents to be used for Phase III
reference substances.
The final protocol provided a tiered approach for determining the 2X stock concentration foreach reference substance (see Figure 2-7). This protocol had the advantage of reducing thenumber of steps for testing (compared to that used by BioReliance) (see Appendix B3).
2.9.2
Basis for Modification of the Phase II Protocol
All three testing laboratories found arsenic trioxide (tested in Phase Ib) less soluble (seeTable 5-10) than was reported by BioReliance (BioReliance values: 0.25 mg/mL in 3T3
medium and 0.05 mg/mL in NHK medium). This chemical was not soluble using theprocedures in the initial solubility protocol. IIVS warmed the stock solution (at least 200µg/mL for 2X) for longer than the protocol specified (i.e., 30 to 50 minutes) but still had
persistent, small, undissolved particles. ECBC obtained a clear solution (highest 2X
concentration was 30 to 50 µg/mL), but found precipitated particles after the solution stood atroom temperature. Sonication time was increased to 15 to 30 minutes, and heating time toapproximately 30 minutes to get a finer suspension. This procedure achieved a morehomogeneous mixture, resulting in more uniform serial dilutions and a more even applicationof the reference substance to the cells. FAL stirred the suspension (approx. 20 to 90 µg/mL)in the CO2 incubator for 1.5 to 2 hours to get clear medium.

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Protocol Revision for Phase II: The duration of the heating step was altered from 5 to 20
minutes to 5 to 60 minutes.
2.10

Components of the Solubility Protocol

2.10.1

Medium, Supplies, and Equipment Required

2.10.1.1 Medium and Chemical Supplies• 3T3 culture medium: DMEM without L-glutamine and containing Hanks’salts and high glucose [4.5gm/l]; L-glutamine, 200 mM; NCS• NHK culture medium: Keratinocyte Basal Medium without Ca++ (KBM®,
Clonetics® CC-3104); KBM® SingleQuots® medium supplements (Clonetics®
CC-4131): epidermal growth factor, insulin, hydrocortisone, bovine pituitary
extract; Calcium SingleQuots® (Clonetics® CC-4202); penicillin/streptomycin
solution (antimicrobial agents)• United States Pharmacopoeia (U.S.P.) analytical grade DMSO
• U.S.P. analytical grade (100%, non-denatured) ETOH
2.10.1.2 Equipment• Waterbath (37 °C)• Sonication apparatus• Vortex mixer• Micropipettors• Balance (capable of weighing 10 mg)• pH meter
2.10.1.3 Procedures
The Phase III solubility protocol required the dissolving of approximately 10 mg of reference
substance in approximately 0.5 mL medium (both 3T3 and NHK media were used) for a final
concentration of 20 mg/mL (see Appendix B3). In order, the mixture was vortexed for 1 to 2
minutes, sonicated for up to 5 minutes, and warmed to 37 °C for 5 to 60 minutes, asnecessary, to dissolve the substance. The endpoint for dissolution was a clear solution withno noticeable precipitate. If the reference substance was not soluble in medium at 20 mg/mL,then more medium was added to a concentration of 2 mg/mL (i.e., a total volume of approx.
5 mL) (Step 2). The mixing procedures were repeated as necessary to dissolve the reference
substance. If the reference substance did not dissolve, approximately 10 mg reference
substance was added to approximately 0.5 mL DMSO in an attempt to dissolve it at aconcentration of 200 mg/mL (Step 3). If the reference substance was not dissolved, the same
concentration was attempted in 100% ETOH (Step 4). Step 5 began in the same way, with
0.2 mg/mL medium and then progressed to 20 mg/mL DMSO, and then 20 mg/mL ETOH.
Determination of reference substance solubility was limited to visual observation of theresulting solution. If a solution appeared clear, then solubility testing ceased. If particles werevisible or if the solution appeared cloudy, then more stringent mixing and/or heatingprocedures were employed. If necessary, the solubility procedure proceeded to the next
solvent/concentration tier. The duration of the solubility test was dependent on theprocedures used to achieve solubility. Some reference substances were immediately
solubilized (e.g., liquids) and others required up to 60 minutes of heating and agitation orsonication.
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2.10.2
Data Collection
All laboratories (including the reference substance distribution laboratory, BioReliance) useda worksheet designed to capture the solubility information for each reference substance. The
endpoint for each step was a visual observation of the solution, a documented comment
describing the observation, the concentration, and a conclusion of soluble or insoluble. Eachworksheet contained:• Reference substance code number and physical description• Solvent used (3T3 medium, NHK medium, DMSO, ETOH)• Amount of reference substance (mg) used in the initial stage• Volume of solvent added and final volume (mL)• Test substance concentration (µg/mL) in the solvent• pH and color of the solution• Mechanical procedures used (vortexing, sonication, heating), duration, andtemperature• Comments (soluble/insoluble at the particular concentration; visualobservations; reactivity with solvent)The solubility test information and data from the laboratories were transferred via email to
the SMT and stored on the NICEATM server and as hard-copy printouts. Each laboratory
also maintained electronic and hard-copy files of its data.
2.10.3
Variability in Solubility Measurements
Solubility determinations were not replicated because within-laboratory results were notexpected to vary. Comparison of the results to determine inter-laboratory concordance for the
72 reference substances (see Section 5.8 for results) provided a measure of variability among
the laboratories and information about the reproducibility of the solubility determinations(see Section 7.4).
2.10.4
Solubility Issues During the Testing of the Reference Substances
Substance solutions were monitored throughout all aspects of the test procedures, andobservations were documented. The lowest concentration of the substance in a 2X solutionthat contained observable precipitates, particles, globules, or oily droplets, was documented
in the EXCEL® template. After substance exposure, all wells of the 96-well test plates wereobserved microscopically and scored using a visual observation code. The code addressed
growth characteristics and the presence or absence of precipitates (see Appendix B [testmethod protocols] for the observation codes used). For solubility issues, the Study Directors
made determinations of test acceptance based on the recommended concentration levels andthe presence of precipitates, their scientific expertise, and test acceptance criteria.
2.10.5
Analysis of Solubility Data
During Phase III, the SMT used the solubility data from all laboratories to determine thesolvents to be used for each chemical (see Section 5.8 for solubility results and SMT
selections). If the solubility of an individual reference substance was different in 3T3
medium and NHK medium, the same solvent would be used for both test methods, rather
than having different solvents for each method. For example, if solubility in one culture
medium was ≥2 mg/mL and solubility in the other was <2 mg/mL, and the substance wassoluble in DMSO at 200 mg/mL, the SMT would select DMSO as the solvent for both test
methods (each test method using its respective culture medium).
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Solubilizing sufficient reference substance to produce cytotoxicity was challenging for
relatively insoluble, low toxicity, substances such as lithium carbonate (in the 3T3 NRU test
method) but generally was not a problem for toxic substances that did not require as high aconcentration to kill cells. Some insoluble and highly toxic reference substances were
problematic, however, because the amount of powdered reference substance added to solvent
was very small, and laboratory personnel found it difficult to determine the presence ofsolute particles in solution. Arsenic trioxide is an example of such a solute (see Section
2.9.2).
2.11

Summary

The Guidance Document NRU protocols were used as the basis of the validation study
protocols. The SMT and participating laboratories made initial modifications to the protocols
prior to implementation of the study. Other protocol modifications were made after
commencement of testing and were the result of recommendations from the laboratories andthe SMT, based on their experience with the initial protocols. The resulting optimized
protocols were used in the main testing phase (Phase III) of the study.
The protocol components used in the validation study were similar for the 3T3 and NHK
cells. The following procedures were common to the NRU protocols for both cell types:• Testing was performed in four phases (Phases Ia, Ib, II, and III)• Preparation of reference substances and positive control• Cell culture environment conditions• Determination of test substance solubility• Configuration of 96-well plates for testing samples• 48-hour exposure to test substance• Range finder and definitive testing• Microscopic evaluation of cell cultures for toxicity• Measurement of NRU• Data analysis
The main differences in the test methods for the two cell types were:• The conditions of propagation of the cells in culture• The cell growth medium components• The volumes of reference substance added to the 96-well plate
A solubility protocol was developed which allowed the laboratories to identify the mostappropriate solvent and appropriate limit concentrations for each test substance.
Three laboratories participated in testing the 72 reference substances in both cell types andone additional laboratory procured and distributed the coded reference substances andperformed solubility tests prior to their distribution to the testing laboratories.

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3.0

REFERENCE SUBSTANCES USED FOR VALIDATION OF THE 3T3
AND NHK NRU TEST METHODS ....................................................................3-3

3.1

Rationale for the 72 Reference Substances Selected for Testing ........................3-3
3.1.1 Reference Substance Selection Criteria .......................................................3-3
3.1.2 Candidate Reference Substances .................................................................3-4
3.1.3 Selection of Reference Substances for Testing ............................................3-5

3.2

Characteristics of the Selected Reference Substances.........................................3-6
3.2.1 Source Databases Represented by the Selected Reference Substances .........3-6
3.2.2 Chemical Classes Represented by the Selected Reference Substances .......3-28
3.2.3 Product/Use Classes Represented by the Selected Reference
Substances ................................................................................................3-28
3.2.4 Toxicological Characteristics of the Selected Reference Substances ..........3-28
3.2.5 Selection of Reference Substances for Testing in Phases Ib and II.............3-32
3.2.6 Unsuitable and Challenging Reference Substances ....................................3-34

3.3

Reference Substance Procurement, Coding, and Distribution .........................3-34
3.3.1 Exceptions ................................................................................................3-35

3.4Reference Substances Recommended by the Guidance Document ...................3-35

3.5

Summary.............................................................................................................3-36

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3.0

REFERENCE SUBSTANCES USED FOR VALIDATION OF THE 3T3
AND NHK NRU TEST METHODS

3.1

Rationale for the 72 Reference Substances Selected for TestingThis section describes the procedures used to select the 72 reference substances selected fortesting in Phase Ia of the validation study.
3.1.1
Reference Substance Selection Criteria
The SMT (see Appendix A) selected reference substances for testing using a process basedon general recommendations made by Workshop 2000 participants (ICCVAM 2001a). The
following criteria were used:• The toxicities of the reference substances should be evenly distributed acrossthe expected range of rodent LD50 values, using the GHS classification for
acute oral toxicity as a guide (UN 2005).
• The reference substances should cover a wide range of structural and use
classes, and be relevant to the needs of the various user communities.
• Substances with human toxicity data and/or human exposure potential (i.e.,
substances of interest to society) should be included. Substances with humanacute toxicity data were particularly important to ECVAM for determining therelationship of the NRU IC50 values to human blood/serum LC.
Table 3-1 shows the GHS scheme for classifying substances into six toxicity categories (fivewith measured LD50 ranges and an unclassified category with LD50 values greater than 5000
mg/kg) based on acute rodent oral LD50 values (UN 2005). The SMT used this scheme forthe classification of candidate substances to assure that the reference substances selected forthe validation study represented the full range of acute oral toxicity.
Table 3-1

GHS Classification Scheme for Acute Oral Toxicity

Category

LD50 (mg/kg)

1

LD50 ≤52

5 < LD50 ≤50

350 < LD50 ≤300

4300 < LD50 ≤2000

5

2000 < LD50 ≤5000

Unclassified

LD50 >5000

Abbreviations: UN=United Nations; GHS=Globally Harmonized System ofClassification and Labelling of Chemicals (UN 2005).
LD50=Dose that produces lethality in 50% of the test animals.

For the purposes of the initial toxicity classification, the rodent oral LD50 values for theindividual substances were obtained from readily available toxicological databases. Theserodent oral LD50 values were re-evaluated in Section 4 for the purpose of identifying themost appropriate reference LD50 values to use for the accuracy analyses (i.e., determine to3-3

In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

what extent there is agreement between a test method result and an accepted reference value[see Section 6.3]). Rat LD50 data were preferred because:• The current acute oral toxicity test guidelines recommend using rats (OECD2001a, c, d; EPA 2002a)• The majority of LD50 data used in the RC millimole regression were fromstudies using rats (282 rat data points and 65 mouse data points) (Halle 1998,
2003)• The great majority of acute oral systemic toxicity testing is performed withrats
Mouse oral LD50 values were used (10 substances) for the initial toxicity classification when
rat data were unavailable, however, mouse data were not used in the regression analyses
presented in Section 6. The toxicological databases, in order of preference, were:• The RC, which contains LD50 values that came largely from the 1983/84
RTECS® (Halle 1998, 2003). The RC is a database of acute oral LD50 values
for rats and mice obtained from RTECS® and IC50 values from in vitro
cytotoxicity assays using multiple cell lines and cytotoxicity endpoints forchemicals with known molecular weights.
• The current RTECS® (MDL Information Systems 2001, 2002)• The current Hazardous Substances Data Bank (HSDB; U.S. National Libraryof Medicine [NLM] 2001, 2002).
To insure that a wide range of structural and use classes were selected, reference substancesof interest to the various U.S. regulatory agencies, as determined from substance lists
received from the various agencies, were included. Substances with human toxicity data
and/or human exposure potential were chosen by mining publicly available databases (e.g.,
the NTP test database, the MEIC database) for potential candidates.
3.1.2
Candidate Reference Substances
The process of identifying the 72 reference substances started with the compilation of adatabase of 116 candidates. The intent of the SMT was to compile a database with at least 12
substances in each GHS toxicity category that also met the other selection criteria, and thento prioritize the substances within each category to select the 72 to be tested. As
recommended by Workshop 2000 (ICCVAM 2001a), the following publicly available
databases and other sources were used to identify candidate substances:• The MEIC program, which collected human toxicity data and in vitro toxicity
data from 61 test methods for 50 substances (Ekwall et al. 1998)• The EDIT program, which targeted development of in vitro test methods forendpoints other than basal cytotoxicity; includes 20 chemicals that are asubset of the MEIC chemicals• The RC (Halle 1998, 2003), which contains in vitro cytotoxicity and in vivo
rodent LD50 data for 347 substances• The Toxic Exposure Surveillance System (TESS) (Litovitz et al. 2000), which
compiles reports of toxic human exposures from poison control centersthroughout the United States• Pesticides recommended for consideration by the EPA Office of Pesticide
Programs (OPP)

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In Vitro Cytotoxicity Test Methods BRD Section 3•••

November 2006

The Guidance Document (ICCVAM 2001b), which reported in vitro NRU
results for 11 RC substances using protocols similar to those to be used in thevalidation study
The U.S. NTP test database, which contains information on the toxicity of
substances relevant to human exposure (NTP 2002)
The EPA High Production Volume (HPV) Challenge Program list ofchemicals. The HPV is a voluntary testing program to provide the public witha complete set of baseline health and environmental effects data for eachchemical that is manufactured within or imported into the United States atamounts >1 million pounds/year (EPA 2000a)

The candidate substances from the list of 116 that were not selected as reference substances
to use in the validation study are listed in Appendix F3, grouped by GHS category, alongwith the rat or mouse oral LD50 value, the database(s) or other source(s) used to identify thesubstance as a potential candidate, and the type of product and/or use for the substance.
3.1.3
Selection of Reference Substances for Testing
Using the candidate substance database, 72 reference substances (12 GHS-unclassified
substances and 12 substances from each of the five GHS acute oral toxicity hazard
categories) were selected. This number of substances per GHS category was consideredadequate by the ICCVAM Acute Toxicity Working Group (ATWG), ICCVAM, ECVAM,
and the SMT to accurately evaluate the performance of these two in vitro NRU test methods
for identifying the starting dose for rodent acute oral toxicity tests across the range of toxic
levels that would be encountered during testing. The criteria used for prioritizing thecandidate substances were:• The availability of rodent acute oral toxicity data
• The availability of human acute oral toxicity data and/or relevance for human
exposure• The level of volatility (because the cells are exposed for 48 hours while
incubated at 37 °C in 96-well plates, volatilization from wells containing avolatile reference substance would affect the accuracy of the IC50 calculationand potentially contaminate other wells)• Not a controlled substance according to the U.S. Drug Enforcement Agency(DEA). Excluding substances that are listed in DEA Schedules I and II fromconsideration obviates the requirement for U.S. laboratories to obtain a DEA
license and adhere to the DEA substance storage and control procedures• Practical considerations such as cost and disposal
If more than 12 candidate substances in a GHS category met the above criteria, then selection
was based on two further considerations. One consideration was the distribution of substancetoxicities within each toxicity category so as to select substances that represented the entire
range of toxicity within each category. Another consideration, which applied only to
candidate substances selected from the RC database, was the fit of the toxicity to the RC
millimole regression. Substances with the best fit to the RC millimole regression werepreferentially selected to prevent the entire set of reference substances from having
proportionally more “outlier” substances (i.e., greater than one-half log from the RC
millimole regression) than the entire RC database.
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In Vitro Cytotoxicity Test Methods BRD Section 3

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The final list of selected reference substances is sorted by GHS acute oral toxicity category inTable 3-2.
3.2

Characteristics of the Selected Reference Substances

The physical/chemical and toxicological information in Appendix F may be useful forcharacterizing the performance of the in vitro NRU test methods for various chemical types(e.g., chemical class, toxic effect class). Appendix F1 lists the reference substances inalphabetical order with information on the CASRN, purity, supplier, pH (of the highest
concentration tested in NRU), and concentrations tested. Appendix F2 provides thereference substances in alphabetical order, and information on physical/chemical
characteristics such as molecular weight, chemical class, water solubility, acid/basedissociation constant (pK), boiling point, and octanol-water partition coefficient (log Kow), a
measure of lipid solubility. Although test substance concentration and toxicity may beheavily influenced by molecular charge and surface activity (ICCVAM 2006), these
attributes were not characterized because this type of information is not readily available.
Appendix F2 also includes the major toxic effects attributed to each chemical, ability to pass
the blood:brain barrier (BBB), metabolic activation/inactivation (whether or not it ismetabolized, or the identification of the metabolites), and mechanism of lethality (where
known) for each of the reference substances. The remainder of this section summarizes
selected characteristics of the reference substances.
3.2.1
Source Databases Represented by the Selected Reference Substances
The primary sources of substances were well represented in the final list of reference
substances. Table 3-3 shows the distribution of reference substances by GHS category fromeach of the source lists. Forty-two (58%) of the 72 substances were MEIC chemicals (17 of
the 42 MEIC chemicals [40%] were also EDIT chemicals), 46 (64%) were involved inhuman poisonings as reported by TESS, 51 (71%) have been evaluated by the NTP, and 18(25%) are listed in the EPA’s HPV Challenge Program. Some substances were present inmore than one database.
The other major source of reference substances was the RC, which contributed 58 (81%) of
the 72 chemicals, as shown in Table 3-4. Because the RC millimole regression was used to
identify outlier substances (see Section 6.2), the fit of the RC substances to this regression
was relevant (Halle 1998, 2003). Halle (1998, 2003) defined outliers as those chemicals withlog IC50-log LD50 points that were >0.699 (i.e., log 5) from the RC millimole regression.
Table 3-4 shows the number of RC outliers selected for testing and the corresponding
number of outliers in the RC. Although the percentage of outliers in several GHS categories
is similar to the percentage in the RC, the total percentage of RC outliers in the set ofreference substances (i.e., 38% [22/58]) is greater than the percentage in the RC (i.e., 27%[95/347]). This occurred because the fit to the RC millimole regression was not the major
deciding factor during selection of the 72 reference substances.

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Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Molecular
log Kow5
Weight (g/mol)

Chemical Class6

Molecular Structure

Cl——Hg

LD50 ≤5 mg/kg

Mercury II chloride

1

MEIC, EDIT,
RC (outlier),
TESS, NTP

Preservative;
Manufacturing;
Insecticide

271.500.22

Inorganic compound;
Mercury compound;
Chlorine compound

Triethylenemelamine1

RC (outlier),
NTP

Manufacturing;
Insect chemosterilant

204.23–0.54

Organic compound;
Heterocyclic
compound

Sodium selenate

2**

TESS, NTP

Feed additive

188.90NA

Inorganic compound;
Sodium compound;
Selenium compound

Busulfan2

RC (outlier),
NTP

Pharmaceutical(antineoplastic)

246.31–0.52

Organic compound;
Alcohol;
Acyclic hydrocarbon;
Sulfur compound

3-7

In Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Cycloheximide2

RC (outlier),
NTP

Antibiotic
Fungicide2

RC (outlier),
EPA, NTP

Pesticide(insecticide)

Parathion2

RC (outlier),
EPA, NTP

Pesticide(insecticide)

Strychnine

2*MEIC, TESS,EPA

Pesticide(rodenticide)

Disulfoton

Molecular
log Kow5
Weight (g/mol)

0.55

Organic compound;
Heterocyclic
compound4.02

Organic compound;
Organophosphorouscompound;Sulfur compound

291.28

3.83

Organic compound;
Organophosphorouscompound;Sulfur compound

334.401.93

Organic compound;
Heterocyclic
compound281.40274.423-8

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)**Molecular
log Kow5
Weight (g/mol)

Source3

Product/Use4

Chemical Class6

RC

Pharmaceutical(antineoplastic);
Pesticide(rodenticide)476.45NA

Organic compound;
Heterocyclic
compoundAminopterin

3

Phenylthiourea

3

RC (outlier),
NTP

Pesticide(rodenticide)

152.200.71

Organic compound;
Sulfur compound;
Urea

Epinephrine bitartrate

4**

RC (outlier),
NTP (HCl salt)

Pharmaceutical(adrenergic)

333.30–1.52

Organic compound;
Alcohol;
Amine

Physostigmine*

EHS

Pharmaceutical(anticholinesterase)NA

Organic compound;
Carboxylic acid;
Heterocyclic
compound5

275.40

3-9

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Molecular
log Kow5
Weight (g/mol)

Product/Use4

Chemical Class6

5 < LD50 ≤50 mg/kg

Colchicine

6**MEIC, RC,
TESS, NTP

Pharmaceutical(gout suppressant)399.45

1.03

Organic compound;
Polycyclic compound

Potassium cyanide

10

MEIC, EDIT,
RC (outlier),
TESS

Electroplating

65.12

NA

Inorganic compound;
Potassium compound;
Nitrogen compound

Dichlorvos

17*

TESS, EPA,
NTP, HPV

Pesticide(insecticide)220.981.43, 1.45

Organic compound;
OrganophosphorouscompoundDigoxin

18**

MEIC, EDIT,
RC (outlier),
TESS

Pharmaceutical(antiarrhythmic)

780.901.26

Organic compound;
Polycyclic compound;
Carbohydrate

3-10

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Fenpropathrin

18*

EPA

Pesticide(insecticide)

Endosulfan

18*

TESS, EPA,
NTP

Pesticide(insecticide)

Arsenic III trioxide

20

MEIC, EDIT,
RC, TESS,EPA, NTP

Thallium I sulfate

29**

MEIC, EDIT,
RC (outlier),
TESS

Molecular
log Kow5
Weight (g/mol)

Chemical Class6

6.0 @
20° C

Organic compound;
Nitrile; Ester; Ether

406.91

3.83

Organic compound;
Heterocyclic
Compound;Sulfur compound

Pesticide(insecticide)

197.80NA

Inorganic compound;
Arsenical

Pesticide(rodenticide/insecticide)504.80NA

Inorganic compound;
Metal;
Sulfur compound

349.43

3-11

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Sodium arsenite

41*

TESS, NTP

Pesticide(herbicide, insecticide,fungicide)129.90NA

Inorganic compound;
Arsenical;
Sodium compound

Triphenyltin hydroxide

44

RC, EPA, NTP,
HPV

Pesticide(fungicide/insecticide)367.02NA

Organic compound;
Organometallic
compound

Sodium dichromate
dihydrate50

RC, EPA, GD,
NTP

Oxidizing agent

298.00NA

Inorganic compound;
Sodium compound;
Chromium compound

50

MEIC, EDIT,
RC (outlier),
TESS, EPA,
NTP

Pharmaceutical(stimulant)

162.020

1.17

Organic compound;
Heterocyclic
compoundNicotine

Molecular
log Kow5
Weight (g/mol)

3-12Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Molecular
log Kow5
Weight (g/mol)

Product/Use4

Chemical Class6

50 < LD50 ≤300 mg/kg

Paraquat

58

MEIC, EDIT,
RC (outlier),
TESS, EPA

Pesticide(herbicide)257.20–4.22 @
pH 7.4

Organic compound;
Heterocyclic
compoundHexachlorophene

61

MEIC, RC,
TESS, NTP

Disinfectant

406.91

6.91

Organic compound;
Cyclic hydrocarbon;
PhenolLindane76

MEIC, EDIT,
RC (outlier),
EPA, NTP

Pesticide(insecticide)290.803.72

Organic compound;
Halogenatedhydrocarbon

Cadmium II chloride

88

RC, TESS, GD,
NTPConsumer;Industrial products

183.31

NA

Inorganic compound;Cadmiumcompound3-13

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Verapamil HCl

108

MEIC, EDIT,
RC (outlier),
TESS, NTP

Pharmaceutical(antiarrhythmic)

491.08

3.79

Organic compound;
Amine

Haloperidol

128*

MEIC, TESS

Pharmaceutical(antipsychotic)

375.903.36

Organic compound;
KetoneSodium oxalate

155

MEIC, EDIT,
RC, TESS, NTP

Paints;Cleaners

134.00NA

Organic compound;
Carboxylic acid;
Sodium compound

Phenobarbital

163

MEIC, RC(outlier), TESS,NTP

Pharmaceutical(anticonvulsant)232.23

1.47

Organic compound;
Heterocyclic
compoundMolecular
log Kow5
Weight (g/mol)

3-14

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Sodium I fluoride

180

MEIC, RC,
TESS, EPA,
NTPElectroplating;Water fluoridation

41.99

NA

Inorganic compound;
Sodium compound;
Fluorine compound

Caffeine

192

MEIC, RC(outlier), TESS,NTP, HPV

Pharmaceutical(stimulant);
Food additive

194.20–0.07

Organic compound;
Heterocyclic
compoundDiquat dibromide

231

MEIC, RC,TESS

Pesticide(herbicide)362.10–3.05

Organic compound;
Heterocyclic
compoundCupric sulfate * 5 H2O

300

MEIC, RC,
TESS, EPA,
NTP

Pesticide(insecticide/fungicide)249.70NA

Inorganic compound;
Sulfur compound;
Metal

Molecular
log Kow5
Weight (g/mol)

3-15

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Molecular
log Kow5
Weight (g/mol)

Chemical Class6

300 < LD50 ≤2000 mg/kg

Amitriptyline HCl

319

MEIC, EDIT,
RC, TESS

Pharmaceutical(antidepressant)313.905.04

Organic compound;
Polycyclic
compoundPhenol414

MEIC, RC,
TESS, EPA,
NTP, HPVDisinfectant

94.11

1.46

Organic compound;
Phenol

Propranolol HCl

470**

MEIC, RC,
TESS, GD

Pharmaceutical(antiarrhythmic)

295.803.09

Organic compound;
Alcohol; Amine;
Polycyclic compound

Chloral hydrate

479

MEIC, RC,
TESS, NTP

Pharmaceutical(sedative)

165.400.99

Organic compound;
Alcohol

3-16

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Glutethimide

600

MEIC, RC,TESS

Pharmaceutical(sedative)

217.301.9

Organic compound;
Heterocyclic
compoundAtropine sulfate

623

MEIC, EDIT,
RC, TESS

Pharmaceutical(antimuscarinic)

694.801.83

Organic compound;
Heterocyclic
compoundValproic acid1695 **RC, MEIC,
TESS, NTP

Pharmaceutical(anticonvulsant)144.202.75

Organic compound;
Carboxylic acid;
LipidsMolecular
log Kow5
Weight (g/mol)

3-17

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Meprobamate

794*

MEIC, TESS

Pharmaceutical(antidepressant)218.30NA

Organic compound;
Carboxylic acid

Acetylsalicylic acid

1000

MEIC, EDIT,
RC, TESS, NTP

Pharmaceutical(analgesic)

180.201.19

Organic compound;
Carboxylic acid;
PhenolLithium I carbonate

7

1187

MEIC, RC,
TESS, NTP (Clsalt)

Pharmaceutical(mood stabilizer)

73.89

NA

Inorganic compound;
Lithium compound;
Alkylies;
Carbon compound

Procainamide

1950*

MEIC, TESS

Pharmaceutical(antiarrythmic)

271.79NA

Organic compound;
Carboxylic acid;
Amide

Molecular
log Kow5
Weight (g/mol)

3-18

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Carbamazepine

1957*

MEIC, TESS

Pharmaceutical(antiepileptic)

Molecular
log Kow5
Weight (g/mol)

236.30Chemical Class6

2.45

Organic compound;
Heterocyclic
compoundMolecular Structure2000 < LD50 ≤5000 mg/kg

Acetaminophen

2404

MEIC, EDIT,
RC, TESS, NTP

Pharmaceutical(analgesic)

151.200.8

Organic compound;
Amide

Potassium I chloride

2602

MEIC, RC,
TESS, NTP

Pharmaceutical(electrolyte);
Manufacturing

74.55

NA

Inorganic compound;
Potassium compound;
Chlorine compound

Boric aid

2660*

TESS, EPA,
NTP

Pesticide(insecticide)

61.83

NA

Inorganic compound;
Boron compound;
Acids

3-19

K+

Cl-

In Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Carbon tetrachloride

2799

MEIC, RC,
TESS, NTP,
HPV

Solvent

153.82

2.83

Organic compound;
Halogenatedhydrocarbon

Dimethylformamide

2800

RC, GD, NTP,
HPV

Solvent

73.10

–1.01

Organic compound;
Amide;
Carboxylic acid

Sodium chloride

2998

MEIC, EDIT,
RC, TESS,EPA, NTP

Pharmaceutical(electrolyte);
Food additive

58.44

NA

Inorganic compound;
Sodium compound;
Chlorine compound

Citric Acid

3000*

EPA, NTP,
HPV

Food additive

192.10–1.72

Organic compound;
Carboxylic acid

Molecular
log Kow5
Weight (g/mol)

3-20

Chemical Class6

Molecular Structure

Na+

Cl-

In Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity
RodentOral LD502(mg/kg)Source3

Product/Use4

Chloramphenicol

3393

MEIC, RC,
NTP

Pharmaceutical(antibiotic)

323.14

1.14

Organic compound;
Alcohol;
Cyclic hydrocarbon;
Nitro compound

Lactic acid

3730

RC, NTP, HPV

Food additive

90.08

–0.72

Organic compound;
Carboxylic acid

Acetonitrile

3798

RC, NTP, HPV

Solvent

41.05

–0.34

Organic compound;
NitrileXylene(mixed isomers)

4300

MEIC, RC,
TESS, NTP,
HPV

Solvent

106.17

3.12 – 3.2

Organic compound;
Cyclic hydrocarbon

GHS
Category1/Substance

Molecular
log Kow5
Weight (g/mol)

3-21

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Trichloroacetic acid

4999

RC, NTPFixative

Molecular
log Kow5
Weight (g/mol)

163.40Chemical Class6

1.33

Organic compound;
Carboxylic acid

LD50 >5000 mg/kg

2-Propanol

5843

MEIC, RC,
TESS, EPA,
NTP, HPVDisinfectant

60.10

0.05

Organic compound;
Alcohol

Gibberellic acid

6305

RC, EPA, NTP

Plant growth regulator

346.380.24

Organic compound;
Polycyclic compound

Propylparaben

6326**RC (outlier),
NTP

Food additive

180.203.04

Organic compound;
Carboxylic acid;
Phenol

3-22

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

5-Aminosalicylic acid7749**RC (outlier),
NTP

Pharmaceutical(antibiotic)

153.101.32

Organic compound;
Carboxylic acid;
PhenolEthylene glycol

8567

MEIC, EDIT,
RC, TESS,
NTP, HPV

Antifreeze

62.07

–1.36

Organic compound;
Alcohol

Diethyl phthalate

8602

RC (outlier),
NTP, HPV

Plasticizer

222.202.47

Organic compound;
Carboxylic acid

NA

Inorganic compound;
Sodiumcompound;
Oxygen compound;
Chlorine compound

Sodium hypochlorite

8

8910

TESS, NTP

Molecular
log Kow5
Weight (g/mol)

Disinfectant

74.44

3-23

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

1,1,1-Trichloroethane

10298

MEIC, RC,
NTP, HPV

Solvent

133.41

2.49

Organic compound;
Halogenatedhydrocarbon

Dibutyl phthalate

11998

RC (outlier),
NTP, HPV

Plasticizer

278.30

4.9

Organic compound;
Carboxylic acid

Glycerol12691

RC, GD, NTP,
HPV

Solvent

92.09

-1.76

Organic compound;
Alcohol

Methanol

13012

MEIC, EDIT,
RC, TESS,
NTP, HPV

Solvent

32.04

–0.77

Organic compound;
Alcohol

Molecular
log Kow5
Weight (g/mol)

3-24

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

Ethanol

November 2006

RodentOral LD502(mg/kg)Source3

Product/Use4

14008

MEIC, RC(outlier), TESS,EPA, NTP,
HPV

Solvent

Molecular
log Kow5
Weight (g/mol)

46.07

–0.31

Chemical Class6

Molecular Structure

Organic compound;
AlcoholAbbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); LD50=Dose that produces lethality in 50% of the test animals; K ow=Octanol:water partition
coefficient; EDIT=Evaluation-guided Development of New In vitro Test Batteries (substances in EDIT program are a subset of the MEIC substance set); EPA=Pesticides registered with theEnvironmental Protection Agency; EHS=EPA’s Extremely Hazardous Substance list; HPV=High Production Volume chemicals (i.e., those that are imported into or produced in the United States inamounts >1,000,000 lbs/year); GD=Guidance Document (ICCVAM 2001b); MEIC=Multicentre Evaluation of In Vitro Cytotoxicity; NA=Non applicable; NTP=National Toxicology Program;
RC=Registry of Cytotoxicity with the chemicals classified as regression outliers shown in parentheses; TESS=Toxic Exposure Surveillance System (Litovitz et al. 2000); HSDB=Hazardous Substances
Data Bank; RTECS®=Registry of Toxic Effects of Chemical Substances.
*
From RTECS® (MDL Information Systems 2002).
**
Mouse.
1
GHS category designation for the substance (e.g., LD50 <5 mg/kg)
2
LD50 data are from the Registry of Cytotoxicity (Halle 1998, 2003) and are for rats, unless otherwise noted. The LD50 values are rounded to the nearest whole number.
3
Sources used to identify candidate chemicals.
4
Product/use categories from HSDB (NLM 2002) or RTECS®(MDL Information Systems 2002). Pharmaceutical uses from Gilman et al. (1985) or Thomson PDR® (2004).
5
From HSDB (NLM 2001, 2002) or Material Safety Data Sheets.
6
Based on Medical Subject Heading [MeSH ®] descriptors (NLM 2005).
7
Mouse data for lithium sulfate (Halle 1998, 2003).
8
From HSDB (NLM 2002).

3-25

In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

Distribution of Candidate Substances and Reference Substances by Source1 and Toxicity Category

Table 3-3

GHS Category(mg/kg)
LD50 ≤5

Reference Substances/ MEIC Reference/
Candidate Substances MEIC Candidates
12/13
2/2

EDIT Reference/
EDIT Candidates
1/1

TESS Reference/
TESS Candidates
3/3

NTP Reference/
NTP Candidates
5/9

HPV Reference/
HPV Candidates
0/0

5 < LD50 ≤50

12/15

6/6

5/5

9/10

8/11

2/5

50 < LD50 ≤300

12/26

11/17

4/5

11/19

9/18

1/3

300 < LD50 ≤2000

12/38

12/29

3/5

12/27

5/23

1/5

2000 < LD50 ≤5000

12/12

6/6

2/2

6/6

12/12

6/6

LD50 >5000

12/12

5/5

2/2

5/5

12/12

8/8

Total

72/116

42/65

17/20

46/70

51/85

18/27

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); LD50=Dose that produces lethality in 50% of the test animals;
MEIC=Multicentre Evaluation of In Vitro Cytotoxicity; EDIT=Evaluation-Guided Development of In vitro Tests; TESS=Toxic Exposure Surveillance System; NTP=U.S.
National Toxicology Program; HPV=U.S. Environmental Protection Agency (EPA) High Production Volume program.
1
Substances may have been selected from more than one source (see Table 3-2 and Appendix F3).

Selected Substances: Distribution of RC Chemicals and RC Outliers1 by Toxicity Category

Table 3-4

GHS Category(mg/kg)RC Outliers/
Total Chemicals

LD50 ≤55 < LD50 ≤50
50 < LD50 ≤300
300 < LD50 ≤2000
2000 < LD50 ≤5000
LD50 >5000

10/11 (91%)
15/26 (58%)
24/70 (34%)
14/139 (10%)
12/57 (21%)
20/44 (45%)

Candidate
Substances
13
15
26
38
12
12

Total

95/347 (27%)

116

Candidate and Selected Substances
RC Reference /RC Reference Outliers/
RC Candidates
RC Reference Chemicals
9/10
8/9 (89%)
8/10
4/8 (50%)
11/18
5/11 (45%)
9/29
0/9 (0%)10/10
0/10 (0%)
11/11
5/11 (45%)
58/88

22/58 (38%)

Abbreviations: RC=Registry of Cytotoxicity; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); LD50=Dose
that produces lethality in 50% of the test animals.
1
Chemicals falling outside the log 5 (i.e., > ±0.699) prediction interval for the RC millimole regression (Halle 1998, 2003).

3-26

In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

Among the 58 RC substances selected for use in the validation study, 22 (38%) were outliers
for the RC millimole regression. Toxicity1 was underpredicted for 17 (77%) of these outlier
substances and overpredicted (i.e., predicted LD50 was lower than measured in vivo LD50) for
the remaining five (23%). For the 95 outlier substances in the RC, the number of substances
for which toxicity was over- or under-predicted was approximately the same. Toxicity was
underpredicted for 49 (52%) outliers and overpredicted for 46 (48%) outliers (Halle 1998,
2003). Figure 3-1 shows the 58 RC chemicals selected for testing, in addition to the 289 RC
chemicals that were not selected, and the RC millimole regression. In the figure, the outliers
are those points outside the RC prediction interval. For the 58 RC substances selected fortesting, the majority (17/22) of the outliers are below the RC millimole regression line.
Figure 3-1

The Fifty-Eight (58) Selected RC Reference Substances on the RC
Millimole Regression

103

102

101

100

10-1

10-2

10-3

10-4
10-6

10-5

10-4

10-3

10-2

10-1

100

101

102

103

IC50x(mM)
Abbreviations: RC=Registry of Cytotoxicity; LD50=Dose that produces lethality in 50% of the test animals;
IC50=Test substance concentration that reduces cell viability by 50%.
The 58 RC chemicals tested in the NICEATM/ECVAM validation study are shown by *. The RC regression,log (LD50) = 0.435 x log (IC50x) + 0.625, is shown by the bold line. The lighter lines show the ± log 5 (i.e.,±0.699) prediction interval (Halle 1998, 2003). The open boxes represent the 289 chemicals not included in thevalidation study.

1

Toxicity is inversely proportional to LD50. High LD50 values reflect low toxicity and low LD50 values reflect
high toxicity
3-27

In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

3.2.2
Chemical Classes Represented by the Selected Reference Substances
Medical subject heading (MeSH®) descriptors from the NLM were used to determine
chemical class designations for the selected substances. Of the 72 reference substances, 57(79%) were organic and 15 (21%) were inorganic. The number of substances in the organic(79) and inorganic (31) subclasses is greater than the number of substances in each classbecause some of the substances are classified in more than one subclass. The most commonly
represented classes of organic compounds were heterocyclics (14/57, 25%), carboxylic acids(14/57, 25%), and alcohols (10/57, 18%). Table 3-5 shows the distribution of the substancesamong the GHS toxicity categories. The 14 heterocyclics were evenly distributed among thefirst four GHS toxicity categories for LD50 ≤2000 mg/kg with the majority of theheterocyclics (11/14) in the categories for LD50 <300 mg/kg. The majority of the carboxylic
acids (12/14) and alcohols (8/10) had an LD50 >300 mg/kg, while the majority of the
inorganics (10/15) had an LD50 <300 mg/kg.
3.2.3
Product/Use Classes Represented by the Selected Reference Substances
Product and use information was obtained from HSDB (NLM 2002) or RTECS® (MDL
Information Systems 2002). The number of assigned uses (77) is greater than the number of
selected substances because some of the substances have more than one use. Table 3-6
shows the distribution of products and uses of the selected substances according to their GHS
categories. Pharmaceutical (27/77; 35%) and pesticide (17/77; 22%) uses were observed
most frequently. The toxicity category of 300 < LD50 ≤2000 mg/kg had the highest numberof pharmaceuticals. Every toxicity category except LD50 >5000 mg/kg had at least four
substances with pharmaceutical uses. The majority of pesticides (16/17; 94%) had an LD50
<300 mg/kg. The next most frequent uses were as solvents (8/77; 10%) and food additives(5/77; 6%); LD50 >2000 mg/kg contained most of the substances with solvent (8/8; 100%)and food additive (4/5; 80%) uses.
3.2.4

Toxicological Characteristics of the Selected Reference Substances

3.2.4.1 Corrosivity
The intent of the SMT was to prioritize only those substances with low corrosivity because
guidelines for acute systemic toxicity testing indicate that corrosive or severely irritating
substances need not be tested (OECD 2001a, c, d). The UN and U.S. Department ofTransportation Packing Group (DOT PG) classification system was used to classify the
corrosivity hazard associated with the candidate substances. However, after substanceselection was completed and testing had begun, the SMT learned that the PG classification
system was also based on hazards other than corrosivity (e.g., dermal and inhalation toxicity,
flammability, etc.). Therefore, the selected substances were not actually prioritized bycorrosivity. Subsequent information on the corrosivity of the selected substances was
obtained from HSDB (NLM 2004) and the Material Safety Data Sheets (MSDS) provided
with the purchased substances. Seven substances that were not identified by the DOT PG
classification system had corrosive notations. The MSDS notations for lactic acid, sodium
hypochlorite, sodium oxalate, and trichloroacetic acid indicated that these substances should
carry a corrosive label. Chloral hydrate, mercury II chloride, and potassium cyanide werenoted by HSDB to be corrosive to eyes or skin.

3-28

In Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-5

November 2006

Distribution of Chemical Class for the 72 Reference Substances by Toxicity Category

Chemical Class1

GHS Acute Oral Toxicity Category (mg/kg)
50 < LD50 ≤300 300 < LD50 ≤2000 2000 < LD50 ≤5000

LD50 >5000

Total

LD50 ≤55 < LD50 ≤50

Carboxylic acid

1

0

1

4

4

4

14

Heterocyclic compound

5

2

4

3

0

0

14

Alcohol

2

0

0

2

1

5

10

Phenol

0

0

1

2

0

2

5

Polycyclic compound

0

2

0

2

0

1

5

Sulfur compound

4

1

0

0

0

0

5

Amine

1

0

1

1

0

0

3

Cyclic hydrocarbon

0

0

1

0

1

1

3

Halogenated hydrocarbon
Organophosphorouscompound
Amide

0

0

1

0

1

1

3

2

1

0

0

0

0

3

0

0

0

1

2

0

3

Nitrile

0

1

0

0

1

0

2

Acyclic hydrocarbon

1

0

0

0

0

0

1

Carbohydrate

0

1

0

0

0

0

1

Ester

0

1

0

0

0

0

1

Ether

0

1

0

0

0

0

1

Ketone

0

0

1

0

0

0

1

Lipid

0

0

0

1

0

0

1

Nitro compound

0

0

0

0

1

0

1

Organometallic compound

0

1

0

0

0

0

1

Sodium compound

0

0

1

0

0

0

1

Urea

1

0

0

0

0

0

1

Total Organics

17

11

11

16

11

14

79

Organic

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In Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-5

November 2006

Distribution of Chemical Class for the 72 Reference Substances by Toxicity Category

Chemical Class1

GHS Acute Oral Toxicity Category (mg/kg)
50 < LD50 ≤300 300 < LD50 ≤2000 2000 < LD50 ≤5000

LD50 >5000

Total

LD50 ≤55 < LD50 ≤50

Sodium compound

1

2

1

0

1

1

6

Chlorine compound

1

0

1

0

2

1

5

Arsenical

0

2

0

0

0

0

2

Metal

0

1

1

0

0

0

2

Potassium compound

0

1

0

0

1

0

2

Sulfur compound

0

1

1

0

0

0

2

Acid

0

0

0

0

1

0

1

Alkalies

0

0

1

0

0

0

1

Boron compound

0

0

0

0

1

0

1

Cadmium compound

0

0

1

0

0

0

1

Carbon compound

0

0

0

1

0

0

1

Chromium compound

0

1

0

0

0

0

1

Fluorine compound

0

0

1

0

0

0

1

Lithium compound

0

0

0

1

0

0

1

Mercury compound

1

0

0

0

0

0

1

Nitrogen compound

0

1

0

0

0

0

1

Oxygen compound

0

0

0

0

0

1

1

Selenium compound

1

0

0

0

0

0

1

Total Inorganic

4

9

7

2

6

3

31

Inorganic

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005).
1
Based on the Medical Subject Heading [MeSH®] descriptor (NLM 2005). Some substances are counted more than once because they appear in more than one subclassunder the organic or inorganic classes.

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In Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-6

November 2006

Distribution of Product/Use1 Class for the 72 Reference Substances by Toxicity Category

Product/Use Class1

GHS Acute Oral Toxicity Category (mg/kg)
50< LD50 ≤300 300< LD50 ≤2000 2000< LD50 ≤5000
0
0
0

5< LD50 ≤50
0Antifreeze

0

0

0

0

0

1

1

Consumer/industrial products

0

0

1

0

0

0

1

Disinfectant

0

0

1

1

0

2

4

Electroplating

0

2

0

0

0

0

2

Fluoridation

0

0

1

0

0

0

1

Feed additive

1

0

0

0

0

0

1

Fixative

0

0

0

0

1

0

1

Food additive

0

0

1

0

3

1

5

Manufacturing

1

0

0

0

1

0

2

Oxidizing agent

0

1

0

0

0

0

1

Paints, cleaners

0

0

1

0

0

0

1

Pesticide

5

7

4

0

1

0

17

Pharmaceutical

4

3

4

11

4

1

27

Plant growth regulator

0

0

0

0

0

1

1

Plasticizer

0

0

0

0

0

2

2

Preservative

1

0

0

0

0

0

1

Solvent

0

0

0

0

4

4

8

Antibiotic/fungicide

LD50 >5000
0

Total

LD50 ≤51

1

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005).
1
Product/use information from Hazardous Substances Data Bank (NLM 2002) or Registry of Toxic Effects of Chemical Substances ([RTECS®], MDL Information Systems 2002).
Some substances are counted more than once because they appear in more than one use category.

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In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

3.2.4.2 Toxicity Targets
As shown in Appendix F2, the most common toxicological effects in humans or rodents
were neurological (40 substances); 26 cause central nervous system (CNS) depression, sevenproduce CNS stimulation, four produce CNS affects such as encephalopathy, and three affectthe peripheral nervous system. Other common target systems include the liver (17substances), kidney (15 substances), and cardiovascular system (10 substances). No target
organ information was available for gibberellic acid. Among the 72 reference substances, 27
had more than one toxicity target.
3.2.4.3 Metabolism
Table 3-7 shows the 22 reference substances that are known or expected to produce
active/toxic metabolites in vivo. In contrast, dichlorvos, fenpropathrin, meprobamate,phenylthiourea, and sodium dichromate are rapidly metabolized to less toxic compounds.
Because the NHK and 3T3 cells have little (Babich 1991) or no (INVITTOX 1991)
metabolic capability, respectively, metabolites of these compounds would not be expected to
be present in vitro. Appendix F2 provides for more information on the metabolism(activation/inactivation) of the selected reference substances.
Table 3-7

Reference Substances Metabolized to Active Metabolites
Active Metabolites
Expected

Known to Have Active Metabolites
Acetaminophen

Carbamazepine

Digoxin

Methanol

Carbon tetrachloride

Acetonitrile

Chloral hydrate

Disulfoton

Parathion

Triethylenemelamine

Acetylsalicylic acid

Cycloheximide

Ethanol

Procainamide HCl

Valproic acid

Amitriptyline HCl

Dibutyl phthalate

Ethylene glycol

Verapamil HCl

Busulfan

Diethyl phthalate

Glutethimide

3.2.5
Selection of Reference Substances for Testing in Phases Ib and II
Based on the Guidance Document (ICCVAM 2001b) recommendation that 10 to 20
substances be tested to qualify candidate in vitro cytotoxicity tests for determining starting
doses for rodent acute oral toxicity assays, 12 reference substances were chosen from amongthe 72 reference substances for testing in Phases Ib and II (see Table 3-8). The criteria forchoosing these reference substances, in order of importance, were:• Two reference substances must be included from each of the five GHS
toxicity categories and the unclassified category.
• The log LD50 (mmol/kg) must be within the prediction interval (±0.699) of the
RC millimole regression. The Guidance Document (ICCVAM 2001b)
recommends that reference substances for evaluating an in vitro basal
cytotoxicity test to use with the RC millimole regression fit the regression as
closely as possible.
• MEIC chemicals must be included. Cytotoxicity data from these phases (andPhase III of this study), and the available human toxicity information for the
MEIC chemicals, could be used to build a prediction model for estimating

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In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

human LC values. The Phase Ib reference substances arsenic trioxide andethylene glycol are also EDIT chemicals (subset of MEIC chemicals).
If more than two substances in a GHS category met the above criteria, reference substanceswere selected so that the LD50 was as close to the RC millimole regression as possible and/or
to represent the full range of toxicity in each GHS category.
Table 3-8

Reference Substances Tested in Phases Ib and II

Reference Substances

Aminopterin
Sodium selenate
Colchicine
Arsenic III trioxide

RC
MEIC
Reference Reference
No.
No.
LD50 ≤5 mg/kg
54-62-63NA13410-01-0
NANA
5 < LD50 ≤50 mg/kg

CASRN

64-86-81327-53-3

6
153

60
26

Rodent Oral
LD501
(mg/kg)Observed –Predicted
log LD502

3
1.63-0.652NA64
20-0.593-0.591

88
180

0.011
-0.1094704
11874,5-0.023-0.2564

2602
3393

0.085
0.441

5843
8567

0.396
0.321

50 < LD50 ≤300 mg/kg
Cadmium II chloride
Sodium I fluoride

10108-64-2
7681-49-4

81
106

NA
14

300 < LD50 ≤2000 mg/kg
DL-Propranolol HCl
Lithium I carbonate

350-60-90
544-13-2

54
3274

23
20

2000 < LD50 ≤5000 mg/kg
Potassium I chloride
Chloramphenicol
2-Propanol
Ethylene glycol

7447-40-7
56-75-7

346
50
91
45
LD50 >5000 mg/kg
67-63-0
128
10
107-21-1
360
7Abbreviations: CASRN=Chemical Abstracts Service Registry Number; RC=Registry of Cytotoxicity; MEIC=Multicentre
Evaluation of In Vitro Cytotoxicity; NA=Not applicable (i.e., substances not included in the RC and/or MEIC studies);
RTECS®=Registry of Toxic Effects of Chemical Substances.
1
From the RC (Halle 1998, 2003) unless otherwise indicated. Data are for rats unless otherwise indicated.
2
Available only for substances included in the RC. This figure characterizes the log LD50 deviation from the RC regression.
Outliers are > ±0.699 from the regression line.
3
RTECS® (MDL Information Systems 2002).
4
Mouse data.
5
For lithium sulfate.

Only nine of the 72 reference substances met all three criteria. In the most toxic category(i.e., LD50 ≤5 mg/kg), only one RC chemical, aminopterin, was within 0.699 of the RC
millimole regression. Sodium selenate was selected as the second reference substance in thiscategory even though its fit to the RC millimole regression was not known. Neither
aminopterin nor sodium selenate were MEIC chemicals. For the 50 < LD50 ≤300 mg/kg
category, cadmium chloride was selected over the MEIC chemicals cupric sulfate 5H2O,
diquat dibromide, sodium oxalate, and hexachlorophene because it fit the RC millimole
regression better than the four MEIC chemicals (the observed LD50 minus log predicted LD50
values were -0.534 to -0.337).

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In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

3.2.6
Unsuitable and Challenging Reference Substances
Several reference substances could not be adequately tested for cytotoxicity in 3T3 cells
and/or NHKs in from one to all three of the laboratories. The following reference substances
did not produce sufficient toxicity at soluble concentrations for calculation of an IC50 at thehighest concentrations tested under the testing conditions used in the study (see also Tables
5-2, 5-4, and 5-5):• Carbon tetrachloride (no 3T3 or NHK NRU IC50 data from ECBC, FAL, or
IIVS)• Xylene (no 3T3 or NHK NRU IC50 data from ECBC or FAL)
• Methanol (no 3T3 NRU IC50 data from ECBC, FAL, or IIVS; no NHK NRU
IC50 data from ECBC)• Lithium carbonate (no 3T3 NRU IC50 data from FAL or IIVS)• 1,1,1-Trichloroethane (no 3T3 NRU IC50 data from FAL or IIVS; no NHK
NRU IC50 data from ECBC)• Valproic acid (no 3T3 NRU IC50 data from ECBC or FAL; no NHK NRU
IC50 data from ECBC, FAL, or IIVS)
Other reference substances were difficult to test because of volatility or lack of toxicity, butthree acceptable tests could be obtained after a number of trials.
• Acetonitrile and 2-propanol were highly volatile and nontoxic, so that evenwith the use of film plate sealers, from one to seven tests failed the VC anddata points test acceptance criteria at each laboratory.
• Disulfoton failed at least one test in both test methods at ECBC and FAL
because of inadequate toxicity (i.e., an IC50 could not be detected) andinsolubility. All laboratories reported precipitate in the test plates for 3T3 andNHK NRU tests. IIVS had no failed tests in either test method.
• Dibutyl phthalate failed one 3T3 NRU test at ECBC and one NHK NRU testat FAL because of inadequate toxicity and solubility.
• Lindane failed one 3T3 NRU test at FAL because of inadequate toxicity andsolubility and one because of its volatility.
• Parathion failed one test because of inadequate toxicity and solubility in bothtest methods and one NHK NRU test because of volatility at FAL.
• Diethyl phthalate failed one NHK NRU test because of volatility at FAL.
• Digoxin (all laboratories), gibberellic acid (ECBC and FAL), and strychnine(ECBC and FAL) failed at least one 3T3 NRU test because of inadequate
toxicity and solubility.
3.3

Reference Substance Procurement, Coding, and Distribution

BioReliance collected information from the suppliers of the reference substances on theiranalytical purity, composition, and stability (see Appendix F1), tested the reference
substances for solubility, packaged them into 4 g aliquots for shipment to the testinglaboratories, and archived two additional samples. All reference substances were given arandom number code that was unique for each testing facility to conceal the identities fromthe testing laboratories. Approximately 100 g of the PC substance, SLS, was distributed,
uncoded, to each laboratory and one additional sample was archived.

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In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

Reference substances were packaged so as to minimize damage during transit, and shipped
under appropriate storage conditions and according to the appropriate regulatorytransportation procedures. Testing facilities were notified upon shipment in order to prepare
for receipt. With the exception of the PC substance which was shipped directly to the Study
Directors, the reference substances were shipped to the test facility Safety Officers.
Shipments were accompanied by a sealed information packet containing the appropriate
health and safety procedures (i.e., MSDS or equivalent documentation with information
regarding the proper protection for handling, procedures for dealing with accidental ingestion
or contact with skin or eyes, and for containing and recovering spills), and a code disclosure
key. Also provided was a data sheet giving a minimum of essential information needed by
the testing laboratory for each reference substance, including color, odor, physical state,weight or volume of sample, specific density for liquid reference substances, and storage
instructions. The shipment directed the Safety Officer to:• Notify BioReliance and the SMT upon receipt of reference substances• Retain the health and safety package and provide the coded reference
substances and chemical data sheets with minimum essential information to
the laboratory Study Director without revealing the identities of the test
substances• Notify the SMT if test facility personnel open the health and safety packet atany time, for any reason, during the study• Return the unopened health and safety package to BioReliance after testing iscompleted
3.3.1
Exceptions
The Safety Officer for ECBC required the information on reference substance codes before
the substances were shipped in order to satisfy the facility’s environmental procedures andrequirements. The reference substance codes were stored in a classified safe located in the
Safety Office which was in a building separate from the cytotoxicity testing laboratory, andwere to be opened only by the Safety Officer. The ECBC Safety Officer opened the sealed
health and safety packets for lithium carbonate and ethanol upon receipt of those substances
because the code information for these substances was not included in the list originallyprovided. ECBC cytotoxicity testing personnel did not have direct access to the reference
substance codes.
3.4

Reference Substances Recommended by the Guidance Document

The Guidance Document specifically recommended testing the following 11 substances tovalidate candidate in vitro basal cytotoxicity assays: sodium dichromate dihydrate, cadmium
chloride, p-phenylenediamine, DL-propranolol HCl, trichlorfon, ibuprofen, nalidixic acid,salicylic acid, antipyrene, dimethylformamide, and glycerol (ICCVAM 2001b). Of these 11
substances (see Appendix F3 and Section 3.1.2), five (sodium dichromate dihydrate,cadmium chloride, DL-propranolol HCl, dimethylformamide, and glycerol) were chosen for
testing after the candidate substances were prioritized as described in Section 3.1.3. The
seven that were not selected did not satisfy the selection criteria (e.g., not MEIC chemicals,
not identified as high exposure risk in TESS)

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In Vitro Cytotoxicity Test Methods BRD Section 3

3.5

November 2006

Summary

Seventy-two reference substances were selected for testing in the NICEATM/ECVAM
validation study. These substances were selected to represent: (1) the complete range of invivo acute oral LD50 values; (2) the types of substances regulated by the various regulatory
authorities; and (3) those with human toxicity data and/or human exposure potential. To
insure that the complete range of toxicity was covered, the GHS (UN 2005) was used to
select 12 substances for each acute oral toxicity category and 12 unclassified substances. The
set of selected reference substances had the following characteristics:• Thirty-five percent (27/77 uses) were pharmaceuticals, 22% (17/77 uses) werepesticides, 10% (8/77 uses) were solvents, and 6% (5/77 uses) were foodadditives. The remaining substances were used for a variety of manufacturing
and consumer products.
• In terms of relevance of the substances to human exposure, 58% (42/72) were
included in the MEIC study (substances chosen because of availability ofhuman lethality data), 24% (17/72) were included also in the EDIT program(EDIT substances are a subset of the MEIC substances), 64% (46/72) had
human exposure data reported by TESS, 71% (51/72) had been evaluated by
NTP, and 25% (18/72) were on the EPA HPV list.
• Eighty-one percent (58/72) of the substances were in the RC and 38% (22/58)
of these were outliers with respect to the RC millimole regression. The RC
millimole regression underpredicted the toxicity of 77% (17/22) of the outliers
and overpredicted the toxicity of 23% (5/22). For the 95 outlier substances in
the RC, however, the number of substances for which toxicity was over- or
under-predicted was approximately the same (i.e., toxicity was underpredicted
for 49 [52%] outliers and overpredicted for 46 [48%] outliers [Halle 1998,
2003]).
• Seventy-nine percent (57/72) were organic compounds and 21% (15/72) were
inorganic. The most commonly represented classes of organic compounds
were heterocyclics (25%, 14/57), carboxylic acids (25%, 14/57), and alcohols(18%, 10/57).
• Nineteen substances (26%, 19/72,) were known to have active metabolites andthree others were expected to have active metabolites based on their chemical
structures.
• Many of the substances produced toxicity in more than one organ system. The
most common target systems were neurological (40 substances), liver (17substances), kidney (15 substances), and cardiovascular (10 substances). No
target organ information was available for one substance (gibberellic acid).

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In Vitro Cytotoxicity Test Methods BRD Section 4

November 2006

4.0

RODENT ACUTE ORAL LD50 REFERENCE VALUES USED TO
ASSESS THE ACCURACY OF THE 3T3 AND NHK NRU TESTMETHODS ............................................................................................................... 4-3

4.1

Methods Used to Obtain Rodent Acute Oral LD50 Reference Values................. 4-3
4.1.1 Identification of Candidate Rodent Acute Oral LD50 Reference Data .......... 4-3
4.1.2 Criteria Used to Select Candidate Rodent Acute Oral Data for
Determination of LD50 Reference Values...................................................... 4-5

4.2

Final Rodent Acute Oral LD50 Reference Values ................................................. 4-7

4.3

Relevant Toxicity Information for Humans .......................................................... 4-8

4.4

Accuracy and Reliability of the Rodent Acute Oral LD50 Reference
Values ...................................................................................................................... 4-13
4.4.1 Variability Among the Acceptable LD50 Values ......................................... 4-13
4.4.2 Comparison of Rodent Acute Oral LD50 Reference Values with theCorresponding RC LD50 Values .................................................................. 4-14
4.4.3 Comparison of the Variability Among Acceptable LD50 Values toThose Obtained in Other Studies ................................................................. 4-15

4.5

Summary................................................................................................................. 4-16

4-1

In Vitro Cytotoxicity Test Methods BRD Section 4

November 2006

[This Page Intentionally Left Blank]

4-2

In Vitro Cytotoxicity Test Methods BRD Section 4

4.0

November 2006

RODENT ACUTE ORAL LD50 REFERENCE VALUES USED TO ASSESS
THE ACCURACY OF THE 3T3 AND NHK NRU TEST METHODS

The procedures and analyses presented in this section were designed to identify the mostaccurate rodent acute oral LD50 values for the 72 reference substances used in the validation
study. These values were needed to ensure that the reference substances were correctly
placed within the different GHS toxicity categories and to provide a data set against which tocompare the predicted LD50 values estimated using the IC50 data obtained from the 3T3 andNHK NRU test methods (see Section 6). The predicted LD50 values are used to determine thestarting dose for rodent acute oral toxicity tests and the more accurate the prediction, thefewer the number of rodents that would be used in an acute oral toxicity test (see Sections
1.0 and 1.2.2).
4.1

Methods Used to Obtain Rodent Acute Oral LD50 Reference Values

4.1.1
Identification of Candidate Rodent Acute Oral LD50 Reference Data
No animal testing was performed to obtain the rodent oral acute LD50 reference data for thisvalidation study. To identify reference data for the 72 substances, rat acute oral LD50 studies
were located using literature searches, secondary references, and electronic databasesearches. Literature searches were conducted in PubMed (U.S. NLM) and the Institute of
Scientific Information (ISI) Web of Science® (Thomson Scientific, Philadelphia, PA) using
each chemical name and “lethal dose 50” as search terms. Secondary references included
NTP technical reports, Toxicological Profiles from the Agency for Toxic Substances andDisease Registry (ATSDR), Cosmetic Ingredient Reviews by the Cosmetics Industry
Council, pesticide handbooks, the Merck Index, and various other summary sources. Table
4-1 lists the electronic databases searched to locate references for rat oral LD50 values. Rat
LD50 data were preferred because:• The current acute oral toxicity test guidelines recommend using rats (OECD2001a, c, d; EPA 2002a)• The majority of LD50 data used in the RC millimole regression were fromstudies using rats (282 rat data points and 65 mouse data points) (Halle 1998,
2003)• The majority of acute oral systemic toxicity testing is performed with rats
Table 4-1

Internet-Accessible Databases Searched for LD50 Information

Database/Source1
Agency for Toxic Substances and Disease Registry(ATSDR)
Center for Drug Evaluation and Research (CDER)
CHEMFINDER
Chemical Carcinogenesis Research Information
System (CCRIS); National Cancer Institute (NCI)
WebsiteSponsor(s)U.S. Department of Health and Human Services (DHHS)
U.S. Food and Drug Administration (FDA)
CambridgeSoft Corporation
NCI; National Institutes of Health (NIH); DHHS
Michigan Department of Natural Resources; Ontario
Ministry of the Environment; Canadian Centre for
Occupational Health and Safety (CCOHS)
CHEMpendium
U.S. Coast Guard

Chemical Evaluation Search and Retrieval System(CESARS)
Chemical Hazard Response (CHRIS)
4-3

In Vitro Cytotoxicity Test Methods BRD Section 4

Table 4-1

November 2006

Internet-Accessible Databases Searched for LD50 Information
Database/Source1

Sponsor(s)U.S. Environmental Protection Agency (EPA) Office ofPesticide Programs (OPP); California EPA Department of
Pesticide Regulation(CCOHS) CHEMpendiumChemical Ingredients Database
CHEMINDEX; CHEMINFOChemRTK High Production Volume (HPV)
Challenge Program; OPPT Chemical Fact Sheets;
Chemical Information Collection and Data
Development

EPA Office of Pollution Prevention and Toxics (OPPT)World Health Organization (WHO) International
Programme on Chemical Safety (IPCS); CCOHS;
International Labour Organisation (ILO) Occupational
Safety and Health Information Centre (CIS)
WHO IPCS; CCOHS; ILO; United Nations Environment
Programme (UNEP)
U.S. Consumer Product Safety Commission (CPSC)
Zentralstelle zur Erfassung und Bewertungvon Ersatz- und
Erganzungsmethoden zum Tierversuch (ZEBET) [German
Centre for the Documentation and Validation of Alternative
Methods]
EPA; The National Library of Medicine (NLM); The
National Institute of Environmental Health Sciences(NIEHS); National Center for Toxicological Research(NCTR)
Transport Canada; U.S. Department of Transportation(DOT); Secretariat of Communications and Transportationof Mexico

CIS Chemical Information
Concise International Chemical Assessment
Documents (CICADS)
Consumer Product Safety Commission Website
Deutsches Institut fur Medizinische Dokumentation
und Information (DIMDI) [The German Institute for
Medical Documentation and Information];
Registry of Cytotoxicity (RC)
Developmental and Reproductive
Toxicology/Environmental Teratology Information
Center (DART®/ETIC)
Emergency Response Guidebook (ERG 2000)
Environmental Health Criteria (EHC) monographs;
Health and Safety Guides (HSG); International
Agency for Research on Cancer (IARC)
European Centre for the Validation of Alternative
Methods (ECVAM) Scientific Information Service(ECVAM SIS)
HAZARDTEXT®; MEDITEXT®; INFOTEXT®;
SARATEXT®; REPROTEXT®; REPROTOX®
Integrated Risk Information System (IRIS)International Chemical Safety Cards (ICSC)
IPCS/EC Evaluation of Antidotes Series
International Uniform Chemical Information
Database (IUCLID)
Joint Expert Committee on Food Additives(JECFA); Joint Meeting on Pesticide Residues(JMPR); Pesticide Data Sheets (PDS)
Material Safety Data Sheets (MSDS)
Multicentre Evaluation of In Vitro Cytotoxicity(MEIC)The National MSDS Repository
National Toxicology Program (NTP) Chemical
Health and Safety Database
National Transportation Library
New Jersey Hazardous Substance Fact Sheets
Oil and Hazardous Materials/Technical Assistance

WHO IPCS; CCOHS
European Commission Joint Research Centre
TOMES Plus®, MICROMEDEX, Greenwood Village, CO
EPA Office of Research and Development (ORD)
WHO IPCS; CCOHS; Commission of the European Union(EU)
European Chemicals Bureau
WHO IPCS; CCOHS; Food and Agriculture Organization(FAO) of the United Nations
Interactive Learning Paradigms, Incorporated
Scandinavian Society for Cell Toxicology
MSDSSEARCH, Inc.
NIEHS
DOT
New Jersey Department of Health and Senior Services
EPA Office of Waste and Water Management

4-4

In Vitro Cytotoxicity Test Methods BRD Section 4

Table 4-1

November 2006

Internet-Accessible Databases Searched for LD50 Information

Database/Source1
Data System (OHM/TADS)
Organisation for Economic Co-operation andDevelopment (OECD) Screening Information Data
Sets (SIDS)
Pesticide Action Network Pesticide Database
Pesticide Product Information System (PPIS)
Poisons Information Monographs (PIMs)
Registry of Toxic Effects of Chemical Substances(RTECS®);NIOSH Pocket Guide to Chemical
Hazards
SCORECARD

Sponsor(s)IPCS; CCOHS; International Register of Potentially Toxic
Chemicals (IRPTC); UNEP
Pesticide Action Network North America
EPA Office of Pesticide Programs (OPP)
IPCS; CCOHS
National Institute for Occupational Safety and Health(NIOSH)
Environmental Defense
University of California, Davis; Oregon State University;
Michigan State University; Cornell University; Universityof Idaho

The EXtension TOXicology NETwork(EXTOXNET)

Office of Management and Budget Watch; Center for
Public Data access

The Right-to-Know Network (RTK NET)
Toxic Chemical Release Inventory (TRI);
GENE-TOX
Toxic Substances Control Act Test Submissions(TSCATS)TOXLINE®; Hazardous Substances Data Bank(HSDB); ChemIDplus

The National Library of Medicine (NLM)
EPA OPPTNLM (TOXNET)

Abbreviations: LD50=Dose lethal to 50% of the animals tested
1
Includes public and proprietary databases

A total of 195 references containing LD50 data retrieved through these searches werereviewed and evaluated. Information regarding the materials, animals, and methods used toderive the 491 LD50 values reported by these references were compiled and are provided inAppendix H1. Appendix H2 provides a narrative characterization and evaluation of theLD50 values.
4.1.2

Criteria Used to Select Candidate Rodent Acute Oral Data for Determination ofLD50 Reference Values
This effort was to designed to derive a set of high quality reference oral LD50 values fromdata that were collected using standardized protocols, accompanied by documentation
showing that established testing procedures were followed in compliance with national andinternational GLP guidelines (OECD 1998; FDA 2003; EPA 2003a,b). After a review of thecollected data, the SMT determined that a requirement for GLP compliance would eliminate
99% (452 of the 459 values remaining after exclusion of 30 duplicate values and twoerroneous values) of the oral LD50 values.
The SMT then considered limiting the selection of LD50 values to those from studies thatused the specifications for animals recommended by the current acute oral toxicity test
guidelines. The current guidelines recommend using young adult rats, 8 to 12 weeks of age,of a common laboratory strain (e.g., Sprague-Dawley) and the most sensitive sex (OECD2001a, c, d; EPA 2002a). Female animals are recommended if there is no information fromwhich to determine the most sensitive sex. A limited number of LD50 values were available
4-5

In Vitro Cytotoxicity Test Methods BRD Section 4

November 2006

from animals that fit this description; only 3% (14/459) of the oral LD50 values were
determined using 8 to 12 week old female laboratory rats. An additional 15 LD50 values wereobtained from female rats in an appropriate weight range (age not provided in the reference)for that age range (~ 176-250 g according to Charles River [http://www.criver.com], Harlan[http://www.harlan.com/us/index.htm], and Taconic Farms[http://www.taconic.com/anmodels/spragued.htm] websites). Thus, only 6% (29/459) of theacute oral LD50 values in the database, covering 21 of the 72 reference substances (29%),
were from studies that used the strain, sex, and age of rats recommended by current test
guidelines (OECD 2001a; EPA 2002a).
4.1.2.1 Final Exclusion Criteria
Because so few studies met the initial criteria (i.e., GLP compliance and use of animals
recommended by current acute oral toxicity test guidelines), the database was reviewed andevaluated to derive alternative criteria for the development of reference LD50 values. For thisevaluation, the SMT looked for commonalities among the data records that, when selected,
provided a comparable data set for each chemical. Review of the available data indicated thatthe majority of acute oral toxicity tests were conducted by gavage to unanesthetized, young
adult laboratory rats of both genders. Thus, the selection process was revised to exclude
studies that reflected the following, less typical, materials, animals, and methods in order tocompile a homogenous set of reference LD50 values for each chemical. The studies excluded
were those with:• Feral rats
• Rats <4 weeks of age• Anesthetized rats
• Test chemical administered in food or capsule• LD50 reported as a range or inequality
Data from feral rats were excluded because the health status and age of these animals was
uncertain. All laboratory rat strains/stocks were deemed acceptable on the assumption thatthey were healthy and provided with adequate care and housing during testing. Data fromneonates and weanlings were excluded because their sensitivity to chemical toxicity may
differ from that of adults. Four weeks was considered the minimum acceptable age because
rats are typically weaned at approximately three weeks of age (Barrow 2000). Data fromfeeding experiments or experiments that involved administration of the chemical in capsules
were also excluded because gavage is the most common mode of administration for acuteoral studies and the rate of gastrointestinal absorption for these other methods is likely to bedifferent (Nebendahl 2000). Because LD50 point estimates are required for the prediction
model, LD50 values reported as ranges or inequalities were unacceptable.
4.1.2.2 Assumptions Regarding Materials, Animals, and Methods
The level of detail for describing the materials, animals, and methods for the LD50 studies
varied greatly. For example, some studies reported only that white rats were used, whileothers provided complete information on stock/strain, gender, and age of animals. Details onother protocol components such as the number of animals tested per dose group, method ofadministration, doses administered, clinical signs, and times of death varied as well. In order
to use as much of the available data as possible, the following assumptions were made if astudy report did not state otherwise:
4-6

In Vitro Cytotoxicity Test Methods BRD Section 4•••

November 2006

Rats were young adults of a common laboratory strain
Rats were not anesthetized
Oral route of administration was by gavage

4.1.2.3 Calculation of Reference LD50 Values
If a substance had multiple LD50 values after the application of the exclusion criteria, theoutliers at the 99% level (Dixon and Massey 1981) were excluded. A geometric mean and
95% confidence limits were calculated from the remaining values, and used as the reference
LD50. A geometric mean was used because it is the antilog of the mean of the logarithm of
the values and is less affected than the arithmetic mean by extreme values. The use of ageometric mean also corresponds with the approach used for the RC millimole regression toderive a single IC50 value from multiple IC50 values (Halle 1998, 2003), and with theapproach used to derive the IC50 value for each chemical for the in vitro - in vivo regressions
evaluated in the NICEATM/ECVAM validation study (see Section 6).
In addition to the statistical evaluation of outliers, an extreme value, which was not a
statistical outlier but was based on biological plausibility, was identified for trichloroacetic
acid. This chemical had five reported LD50 values ranging from 400-8900 mg/kg after
applying the exclusionary criteria. The lowest value (400 mg/kg) was rejected as biologicallyimplausible because up to 1000 mg/kg/day had been used in an oral chronic rodent
carcinogenicity study with no, or only minimal, toxicity (EPA 1996).
4.1.2.4 Use of Rat and Mouse Data
If no rat oral LD50 values could be found for a reference substance, mouse acute oral LD50
values were evaluated using the same approach as was used for rat values. Because an IC50LD50 regression model using only rat data was preferable, the three reference substances (i.e.,epinephrine bitartrate, colchicine, and propylparaben) for which mouse values only wereavailable were not used for the evaluations of accuracy (Section 6) or animal reduction(Section 10).
4.2

Final Rodent Acute Oral LD50 Reference Values

After the application of the exclusionary criteria, there were 385 acceptable rodent acute oral
LD50 values from which to calculate reference LD50 values. Table 4-2 shows the reference
LD50 value for each substance in descending order of toxicity, presented both as mg/kg andas mmol/kg. Data are presented as mmol/kg in order to be consistent with the RC approach.
The RC millimole regression used units of mmol/kg for the LD50 and mM for the IC50 (seeSection 1.1.3). Also shown for each substance are the 95% confidence limits around thegeometric mean, the ratio of the maximum to the minimum acceptable value, the number ofLD50 values used to calculate the reference value, the number of LD50 values available (notincluding duplicate values or erroneous values), and the LD50 value initially used for hazard
classification of the reference substance (see Table 3-2).
Table 4-2 lists the reference substances grouped by GHS acute oral toxicity category (UN2005) using the reference LD50 values that were derived as described above. The initialcategorization for this study, which used the LD50 values in the far right column of Table 4-2(i.e., values reported in Table 3-2, which come from the RC unless otherwise specified),
placed 12 substances in each toxicity category. Table 4-3 compares the number of substancesin each GHS toxicity category based on their reference LD50 values with the number in each
4-7

In Vitro Cytotoxicity Test Methods BRD Section 4

November 2006

category based on the initial LD50 values. The initial and reference LD50 values placed 53(74%) of the substances in the same GHS category. Nineteen substances (26%) werereclassified based on the reference LD50 values (this value is the sum of the numbers in the
discordant cells in Table 4-3). Compared with the initial LD50 value, the reference LD50
value was higher for 18 (25%) and lower for only one (1%) of the substances.
Of the 19 reference substances that were reclassified because of the reference LD50 values,
five substances originally assigned to the most toxic, LD50 ≤5 mg/kg, category (i.e.,aminopterin, mercury chloride, busulfan, parathion, and strychnine) were moved to the next,
less toxic, category (5< LD50 ≤50 mg/kg). In the 5< LD50 ≤50 mg/kg category, four
substances (dichlorvos, fenpropathrin, sodium dichromate dihydrate, and nicotine) moved to
the less toxic 50< LD50 ≤300 mg/kg category, and one (triphenyltin hydroxide) moved two
categories to 300< LD50 ≤2000 mg/kg. In the 50< LD50 ≤300 category, four substances(haloperidol, caffeine, copper sulfate pentahydrate, and sodium oxalate) moved to a lower
toxicity category (300< LD50 ≤2000 mg/kg). Only carbamazepine moved from the 300<
LD50 ≤2000 mg/kg category to the 2000< LD50 ≤5000 mg/kg category. In the 2000< LD50
≤5000 mg/kg category, citric acid, trichloroacetic acid and dimethylformamide moved to thenext lower toxicity category (LD50 >5000 mg/kg). In the LD50 >5000 mg/kg category, 5aminosalicylic acid moved to the higher toxicity, 2000< LD50 ≤5000 mg/kg category. This
was the only substance that moved to a more toxic category
4.3

Relevant Toxicity Information for Humans

The relevance of rodent acute oral LD50 data to human LC values was assessed by the MEIC
program (Ekwall et al. 1998b), which used mouse and rat oral LD50 data from RTECS®
(Ekwall et al. 1998a). Mean lethal doses in humans were collected primarily from handbooks
containing human clinical toxicity information (Ekwall et al. 1998a) supplemented, whennecessary, by an in-house compendium from the Swedish Poisons Information Centre.
Ekwall et al. (1998b) calculated least squares linear regressions for the prediction of themean human LC values by rat and/or mouse oral LD50 data for the 50 MEIC substances using
units of log mol/kg. They reported a correlation of R2 =0.607 for the rat oral LD50 predictionof mean human LC values and R2 =0.653 for the mouse oral LD50 prediction of mean human
LC values. It is important for comparisons of MEIC data with rodent LD50 values to note that
the MEIC human values are not lethal doses, and therefore not equivalent to LD50 values.
Many of the values (if not the majority) are blood concentrations that were associated withmorbidity or mortality, and usually do not reflect the actual dose consumed by the patient.
These are not necessarily the peak blood concentrations, but only the concentrations at thetime of ascertainment, which could have ranged from immediately after onset of medical
treatment to post-mortem. The MEIC organizers readily admitted that they could not relate
the blood concentrations to the administered dose.
The relevance of the NRU data collected in the NICEATM/ECVAM validation study to theprediction of human acute toxicity will be addressed elsewhere by ECVAM in a separate
evaluation.

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In Vitro Cytotoxicity Test Methods BRD Section 4

Table 4-2

November 2006

Rodent Acute Oral Reference LD50 Values Listed by GHS Category1

GHS Category1/
Reference Substance

Reference
Acute Oral
LD502,3(mg/kg)95%
ConfidenceInterval4(mg/kg)Cycloheximide
Phenylthiourea
Sodium selenate
Epinephrine bitartrate
Triethylenemelamine
Physostigmine
Disulfoton

2
3
3
4 (mouse)
4
5
5

NC
NC
NC
NC
1-25
NC
2-10

Parathion
Strychnine
Aminopterin
Potassium cyanide
Busulfan
Colchicine
Thallium I sulfate
Arsenic III trioxide
Endosulfan
DigoxinMercury II chloride
Sodium arsenite

6
6
7
7
12
15 (mouse)
25
25
28
28
40
44

3-12
NC
NC
5-10
NC
NC
NC
10-64
NC
NC
27-60
36-53

51

44-58

34.17-64.5

59
70
76
82
93
100
111

40-88
68-72
57-10068-98
65-13278-129NC

17-97.5
68-71
48.5-16456-21557-11588-125108-114

Sodium dichromate
dihydrate
Dichlorvos
Nicotine
Fenpropathrin
Hexachlorophene
Paraquat
LindaneVerapamil HCl

Reference
Reference
Acute Oral
Acute Oral
LD50 Range5
LD502(mg/kg)(mmol/kg)
LD50 ≤5 mg/kg (N =7)
1-2.5
0.0071130.0197
1.6-5.98
0.015940.01961-13
0.012050.0182
2.3-12.6
0.01825 < LD50 ≤50 mg/kg (N =12)
1.8-300.02092.35-16.2
0.0188
7
0.016
5-10
0.111
1.9-290.049
5.886-29
0.037525
0.049513-81.5
0.127
18-43
0.068
28
0.0362
12-92
0.148
36-53
0.336
50 < LD50 ≤300 mg/kg (N =12)

4-9

95%
ConfidenceInterval4(mmol/kg)

Maximum:
Minimum
Value6N

Initial Rodent
Acute Oral
LD503,7(mg/kg)

NC
NC
NC
NC
0.0037-0.12
NC
0.009-0.036

2.5
NC
3.7
NC
13.0
NC
5.5

3
1
2
1
4
1
6

2
3
28
4 (mouse)
158
20.010-0.041
NCNC
0.077-0.15
0.008-0.38
NCNC
0.050-0.32
NCNC
0.010-0.22
0.28-0.40

16.7
6.9
NC
2.0
15.3
4.9
NC
6.3
2.4
NC
7.7
1.5

10
3
1
7
4
3
1
5
2
1
10
5

2
28
3 (mouse)102
6 (mouse)
29 (mouse)
20
188
18 (mouse)
1
418

0.193

0.17-0.22

1.9

11

50

0.266
0.430
0.217
0.202
0.498
0.344
0.226

0.18-0.40
0.42-0.44
0.16-0.29
0.17-0.24
0.35-0.71
0.27-0.44
NC

5.7
1.0
3.4
3.8
2.0
1.4
1.1

9
4
9
19
5
4
2

178
50
188
61
58
76
108

In Vitro Cytotoxicity Test Methods BRD Section 4

Table 4-2

November 2006

Rodent Acute Oral Reference LD50 Values Listed by GHS Category1

Sodium I fluoride
Cadmium II chloride
Diquat dibromide
Phenobarbital

Reference
Acute Oral
LD502,3(mg/kg)
127
135
160
224

95%
ConfidenceInterval4(mg/kg)
92-17588-208NC
NC

Caffeine
Triphenyltin hydroxide
Haloperidol
Amitriptyline HCl
Propranolol HCl
Cupric sulfate ● 5 H2O
Phenol

310
329
330
348
466
474
548

256-374
208-520NCNCNC
269-836434-692

Lithium carbonate590

479-728

Glutethimide
Sodium oxalate
Chloral hydrate
Atropine sulfate
Valproic acid
Meprobamate
Acetylsalicylic acid
Procainamide HCl

600
633
638
819
995
1387
1506
1950

NC
NC
391-1040
641-1045NC
1291-1489
1224-1854
NC

Acetaminophen
Potassium I chloride
Carbamazepine
Boric aid
5-Aminosalicylic acid
Chloramphenicol
Acetonitrile
Lactic acid

2163
2799
2805
3426
3429
3491
3598
3639

GHS Category1/
Reference Substance

Reference
Reference
Acute Oral
Acute Oral
LD50 Range5
LD502(mg/kg)(mmol/kg)
64-2793.020
88-211
0.738
121-2310.466
162-3180.966
300 < LD50 ≤2000 mg/kg (N =16)
192-483
1.59
46.4-1200
0.896
128-8500.877
320-380
1.18
466
1.575
236.2-960
1.90
317-1500
5.82
525-710

7.98

600
2.76
558-7074.724
479-8633.86
600-1136
1.21
670-1480
6.91
1286-1522
6.35
616-2840
8.36
1950
8.286
2000 < LD50 ≤5000 mg/kg (N =11)
NC
1944-2404
14.3
NC
2600-3020
37.6
NC
1957-4025
11.9
2617-4486
2660-5140
55.4
NC
2800-4200
22.4
NC
2500-5000
10.8
2951-4375
1320-8120
87.6
NC
3543-3730
40.3

4-10

95%
ConfidenceInterval4(mmol/kg)
2.19-4.16
0.48-1.14
NC
NC

Initial Rodent
Acute Oral
LD503,7(mg/kg)
180
88
231
163

Maximum:
Minimum
Value6

N

4.4
2.4
1.9
2.0

12
5
3
3

1.32-1.93
0.57-1.42
NCNCNC
1.08-3.35
4.82-7.68

2.5
25.9
6.6
1.2
NC
4.1
4.7

10
15
2
2
1
6
14

6.5-9.9

1.4

4

NC
NC
2.36-6.29
0.95-1.54
NC
5.92-6.82
6.8-10.3
NC

NC
1.3
1.8
1.9
2.2
1.2
4.6
NC

1
211
4
7
2
6
1411
1

192
44
1288
319
470 (mouse)
300
414
1187 (mouse;
sulfate salt)600
155 (mouse)9479
623
1695 (mouse)
7948
1000
19508

NC
NC
NC
42.3-72.6
NCNC
71.9-107
NC

1.2
1.2
2.1
1.9
1.5
2.0
6.2
1.1

2
2
2
6
2
3
26
2

2404
2602
19578
26608
7749 (mouse)
3393
3798
3730

In Vitro Cytotoxicity Test Methods BRD Section 4

Table 4-2

November 2006

Rodent Acute Oral Reference LD50 Values Listed by GHS Category1

Carbon tetrachloride
Sodium chloride
XyleneReference
Acute Oral
LD502,3(mg/kg)
3783
4046
4667

95%
ConfidenceInterval4(mg/kg)3024-4732
2917-5623
1294-16827

2-Propanol
Trichloroacetic acid
Dimethylformamide
Citric Acid
Gibberellic acid
Propylparaben
Ethylene glycol
Methanol
Dibutyl phthalate
Diethyl phthalate
Sodium hypochlorite
Ethanol
1,1,1-Trichloroethane
Glycerol

5105
5229
5309
5929
6040
6332 (mouse)
7161
8710
8892
9311
10328
11324
12078
19770

4624-5636
2745-9961
3548-7925
NCNCNC
6266-8204
6223-12218
6180-12794
NCNC
8610-14894
10000-14588
10495-37154

GHS Category1/
Reference Substance

Reference
Reference
Acute Oral
Acute Oral
LD50 Range5
LD502(mg/kg)(mmol/kg)
2350-10054
24.6
3000-6140
69.3
1537-8620
43.9
LD50 >5000 mg/kg (N =14)
4500-5840
84.9
3320-8900
32.0
2800-7182
72.6
3000-11700
30.9
5780-6300
17.4
6332
35.1
4000-9900
115.4
5628-12880
272
7499-12436
31.9
8600-10100
41.9
8200-13000
62.8
7060-17775
245.7
9600-16000
90.5
12600-27650
215

95%
ConfidenceInterval4(mmol/kg)
20-31
50-96
12-15877-94
16.8-61.0
49-108NC
NC
NC
101-132
194-38122-46
NCNC
187-32375-109114-403

Maximum:
Minimum
Value6

N

4.3
2.0
5.6

15
5
4

Initial Rodent
Acute Oral
LD503,7(mg/kg)
2799
2998
4300

1.3
2.7
2.6
3.9
1.1
NC
2.5
2.3
1.7
1.2
1.6
2.5
1.7
2.2

6
4
6
2
2
1
16
6
4
2
2
8
6
4

5843
4999
2800
30008
6305
6326 (mouse)
8567
13012
11998
8602
891010
14008
10298
12691

Abbreviations: LD50=dose lethal to 50% of the animals tested; GHS=Globally Harmonized System of Classification and Labelling of Chemicals(UN 2005); N=Number of acceptable values used for geometric mean; NC=Not calculated.
1
Categorized using the reference oral LD50.
2
Based on a geometric mean of acceptable LD50 values from adult laboratory rats unless otherwise specified.
3
Values rounded to the nearest whole number.
4
For the geometric mean of the acceptable LD50 values, NC is used for substances with three acceptable values or less, which was considered
too few for calculation of a valid confidence interval.
5
Range of acceptable oral LD50 values.
6
Ratio of minimum acceptable LD50 to maximum acceptable LD50.
7Values rounded to the nearest whole number. Values are from the RC unless otherwise specified; rat data unless otherwise specified.
8
RTECS® (MDL Information Systems 2002).
9
RC reference for rat oral LD50 of 155 mg/kg is Shrivastava et al. (1992), which references Klinger and Kersten (1961). Klinger and Kersten(1961) indicate the value was determined by intraperitoneal administration to mice.
10
HSDB (NLM 2002).
11
An erroneous value obtained from the literature was not included.

4-11

In Vitro Cytotoxicity Test Methods BRD Section 4

Table 4-3

November 2006

GHS Category Matches for the Rodent Acute Oral LD50 Initial and Reference Values
Reference LD50 (mg/kg)

Initial LD50
(mg/kg1)

Total

Category
Match

Reference
LD50
Lower

Reference
LD50
Higher

LD50 ≤55 < LD50 ≤50

50 < LD50 ≤300

300 < LD50 ≤2000

2000 < LD50 ≤5000

LD50 >5000

LD50 ≤5

7

5

0

0

0

0

12

58%

0%

42% (5)

5 < LD50 ≤50

0

7

4

1

0

0

12

58%

0%

42% (5)50 < LD50 ≤300

0

0

8

4

0

0

12

67%

0%

33% (4)300 < LD50 ≤2000

0

0

0

11

1

0

12

92%

0%

8% (1)

2000 < LD50 ≤5000

0

0

0

0

9

3

12

75%

0%

25% (3)

LD50 >5000

0

0

0

0

1

11

12

92%

8%

0% (0)

Total

7

12

12

16

11

14

72

74%

1%

25% (18)

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); LD50=Dose lethal to 50% of animals tested.
Note: Shaded cells show the number of chemicals for which both LD50 categories agree.
1
Initial LD50 values were used for reference substance selection and were obtained from the RC (Halle 1998, 2003), RTECS® (MDL Information Systems
2002), and HSDB (NLM 2002) (see Table 3-2).

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In Vitro Cytotoxicity Test Methods BRD Section 4

4.4

November 2006

Accuracy and Reliability of the Rodent Acute Oral LD50 Reference Values

Accuracy (concordance) is the closeness of agreement between a test method result and an
accepted reference value (in this case to the rodent acute oral LD50 measurement) (ICCVAM
2003). Because there are insufficient data to permit a comparison between rodent and humanlethal doses, the accuracy of rodent acute oral LD50 values for predicting the oral LD50 inhumans cannot be determined. Acute toxicity testing in rodents leads to a relative ranking of
the toxicity of chemicals for regulatory purposes, with the default assumption that the rodent
values and ranking are predictive of the human values and ranking.
The among laboratory reproducibility of the reference LD50 values determined in this section
may be judged by evaluating the range of acceptable LD50 values for each reference
substance and by comparing the values (and their variability) with the variability of LD50
values derived from controlled acute oral toxicity studies.
4.4.1
Variability Among the Acceptable LD50 Values
The variability among the acceptable rodent acute oral LD50 values used to calculate thereference LD50 value for each reference substance was assessed by calculating the ratio of the
maximum to the minimum value (see Table 4-2). For the 62 reference substances with morethan one acceptable LD50 value, the maximum:minimum ratio ranged from 1.1 to 25.9, with amean of 4.3 and a median of 2.2. The maximum:minimum ratios were greater than 10 for
four substances: triethylenemelamine, parathion, busulfan, and triphenyltin hydroxide. The
low LD50 values for triethylenemelamine, busulfan, and parathion may have contributed tothe high maximum:minimum ratios. The four LD50 values for triethylenemelamine ranged
from 1 to 13 mg/kg, the four values for busulfan ranged from 1.9 to 29 mg/kg, and the 10
values for parathion ranged from 1.8 to 30 mg/kg.
Table 4-4 shows the maximum:minimum LD50 ratios by toxicity category. The more toxic
substances (i.e., LD50 ≤50 mg/kg) tended to have higher maximum:minimum ratios thansubstances with lower toxicity (i.e., LD50 >50 mg/kg). This is anticipated because small dayto-day, or laboratory-to-laboratory variations in weighing and dosing the lower
concentrations would have a higher impact on the chemicals being administered in low doses
than those being administered in the high dose range.
Table 4-4

Maximum:Minimum LD50 Ratios by GHS Toxicity Category

GHS Category(LD50 in mg/kg)
LD50 ≤55 < LD50 ≤50
50 < LD50 ≤300
300 < LD50 ≤2000
2000 < LD50 ≤5000
LD50 >5000

Mean
Maximum:Minimum
LD50 Ratio
6.2
7.1
2.4
4.6
2.6
2.3

MedianMaximum:Minimum
LD50 Ratio
4.6
6.3
1.9
2.2
2.0
2.3

Range ofMaximum:Minimum
LD50 Ratio
2.5 – 13.0
2.0 - 16.7
1.1 - 5.7
1.2 - 25.9
1.2- 22.3
1.1 - 3.9

N
4
9
12
13
11
13

Abbreviations: LD50=Dose lethal to 50% of animals tested; GHS-Globally Harmonized System of Classification andLabelling of Chemicals (UN 2005); N=Number of chemicals with more than one acceptable LD50 value after application of
the exclusion criteria described in Section 4.1.2.

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4.4.2

Comparison of Rodent Acute Oral LD50 Reference Values with the Corresponding
RC LD50 Values
The correspondence of the rodent acute oral LD50 reference values with the RC LD50 values
for the 58 reference substances in common with the RC are shown on a log scale in Figure 41. Not surprisingly, a Spearman correlation analysis for the two sets of log transformed
values yielded a significant correlation (p <0.0001) with a correlation coefficient, rs, of 0.97.
Figure 4-1 shows that the LD50 reference values tended to be higher than the RC LD50
values. One factor in this difference is that the majority of LD50 values used in the RC werefrom the 1983/84 RTECS®, which contains the lowest LD50 value found for a particularchemical without regard to the available methodological information, without considerationof whether it is an outlier with respect to the other available values, and without scientificreview before publication. Thus, because the reference LD50 values are based on thegeometric mean from multiple studies, it is not surprising that these values tended to behigher than the single values in the RC database.
Correlation of LD50 Values With the Reference LD50 Values for the 58
RC Chemicals

Figure 4-1
100000

10000

1000
Triphenyltinhydroxide100

Mercury
chloride
Busulfan

10

1

0.1
0.1

1

10

100

1000

10000

100000Registry of Cytotoxicity LD50 (mg/kg)
Abbreviations: LD50=Dose lethal to 50% of animals tested; RC=Registry of Cytotoxicity.
The diagonal line shows the 1:1 relationship.

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When comparing the reference LD50 values to the RC values, the substances with the largest
differences were busulfan, triphenyltin hydroxide, and mercury chloride (see Figure 4-1).
• The LD50 reference value for busulfan was six times that of the RC value (12mg/kg vs. 1.9 mg/kg). The RC value (from 1983/84 RTECS®) was from apaper by Schmahl and Osswald (1970) in which they cited a rat oral LD50 of
1.86 mg/kg. The literature also contained rat oral LD50 values of 28 and 29
mg/kg for male and female Sprague-Dawley rats, respectively (Matsuno et al.
1971).
• The LD50 reference value for triphenyltin hydroxide was 7.5 times the RC
LD50 (329 mg/kg vs. 44 mg/kg). The 15 LD50 values used to determine thereference value included the RC value, and had a wide range, 44-1200 mg/kg.
Because of the large variation in the data, which was evenly distributed
throughout the range neither the highest nor the lowest values were outliers.
• The LD50 reference value for mercury chloride was 40 mg/kg, while the RC
value was 1 mg/kg. The RC value was from a summary document thatreported the rat oral LD50 as a range of 1-5 mg/kg (Worthing and Walker
1991). Because it was reported as a range, it was excluded from thecalculation of the reference value (see Section 4.1.2.1). The remaining 11
values ranged from 12 to 160 mg/kg. The highest value (160 mg/kg) was
considered an outlier when compared to the other 10 values and therefore
excluded from the reference value calculation.
4.4.3

Comparison of the Variability Among Acceptable LD50 Values to Those Obtainedin Other Studies
The variation seen here for 62 reference substances is not atypical, considering the results ofother studies that examined the variation among rodent acute oral LD50 values derived for thesame substance. For example, Weil and Wright (1967) showed that LD50 values varied by asmuch as five-fold for the 10 substances tested in eight laboratories using exactly the same
protocol. Another international study involving 65 participating laboratories in eight
countries that did not control the LD50 protocols among laboratories, reported
maximum:minimum ratios from 3.6 to 11.3 (with LD50 values ranging from 44 to 5420
mg/kg) for five substances (Hunter et al. 1979). The chemicals tested, and the LD50 ranges
were:• PCP144-523 mg/kg• Sodium salicylate
800-4150 mg/kg• Aniline
350-1280 mg/kg• Acetanilide
805-5420 mg/kg• Cadmium chloride
70-513 mg/kg
The results of a follow-on study in which the same substances were tested by 100
laboratories in 13 countries showed that adherence to a specific protocol reduced the range ofmaximum:minimum LD50 ratios from 3.6 to 11.3 to 2.4 to 8.4 (Zbinden and Flury-Roversi
1981).

1

Compound undefined in the publication.
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Although the LD50 data collected from the literature for the NICEATM/ECVAM validation
study used various rat strains, sexes, observation durations, and calculation methods for
estimating the LD50, the variation in LD50 values for individual substances was similar to thedata of the earlier cited studies. The current study found four of the 62 substances withmultiple LD50 values had maximum:minimum LD50 values higher than that reported by
Hunter et al. (1979) (i.e., >11.3), and three of those were in the highest toxicity category.
Hunter et al. (1979) also observed that the largest variation was associated with the more
highly toxic substances.
4.5

Summary

To enable the comparison of in vitro NRU data with rodent acute oral toxicity data, LD50
reference values for the 72 reference substances were calculated using data obtained from theliterature, database searches, and secondary references. Rat acute oral LD50 values werepreferred, but mouse acute oral LD50 values were collected for three substances with noavailable or acceptable rat data. The 491 LD50 values that were retrieved comprised 485 rat
LD50 values and six mouse values. It was not possible to identify a high quality data set
produced under GLP guidelines because only 3% of the data records were in GLP
compliance. Instead, as described in Section 4.1.2.1, a homogenous set of LD50 values foreach substance was identified by applying specific exclusion criteria related to the materials,
animals, and methods used for each study.
After analysis of the acceptable values for outliers, the remaining 385 values were used toderive rodent acute oral LD50 reference values by calculation of a geometric mean of thevalues for each substance. As a result of this procedure, the LD50 reference values for 19 of
the 72 reference substances were sufficiently different from the values that were used in the
RC and other summary sources, so that they were reclassified into different GHS oral
toxicity categories.
Because there is no reference standard against which to evaluate the accuracy of the rodent
acute oral toxicity test, the reliability of the LD50 reference values was assessed bycomparison to other evaluations of the performance of this test method. The
maximum:minimum ratio of the acceptable values for the 62 reference substances that had
more than one LD50 value ranged from 1.1 to 25.9, and the ratios for four of the substanceswere greater than one order of magnitude.

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5.0

3T3 AND NHK NRU TEST METHOD DATA AND RESULTS........................ 5-3

5.1

Study Timeline and Participating Laboratories ................................................... 5-3
5.1.1 Statements of Work (SOW) and Protocols .................................................... 5-3
5.1.2 Study Timeline............................................................................................... 5-4
5.1.3 Participating Laboratories.............................................................................. 5-4

5.2

Coded Reference Substances and GLP Guidelines............................................... 5-5
5.2.1 Coded Reference Substances ......................................................................... 5-5
5.2.2 Lot-to-Lot Consistency of Reference Substances.......................................... 5-5
5.2.3 Adherence to GLP Guidelines ....................................................................... 5-5

5.3

3T3 and NHK NRU Test Method Protocols .......................................................... 5-5
5.3.1 Phase Ia: Laboratory Evaluation Phase.......................................................... 5-6
5.3.2 Phase Ib: Laboratory Evaluation Phase ......................................................... 5-6
5.3.3 Phase II: Laboratory Qualification Phase ...................................................... 5-7
5.3.4 Phase III: Main Validation Phase .................................................................. 5-9

5.4

Data Used to Evaluate Test Method Accuracy and Reliability ......................... 5-10
5.4.1 PC Data ........................................................................................................ 5-10
5.4.2 Reference Substance Data............................................................................ 5-12

5.5

Statistical Approaches to the Evaluation of 3T3 and NHK Data ...................... 5-26
5.5.1 Statistical Analyses for Phase Ia .................................................................. 5-27
5.5.2 Statistical Analyses for Phase Ib.................................................................. 5-28
5.5.3 Statistical Analyses for Phase II .................................................................. 5-28
5.5.4 Statistical Analyses for Phase III ................................................................. 5-29
5.5.5 Summary of the Data Used for Statistical Analyses.................................... 5-32

5.6

Summary of NRU Test Results............................................................................. 5-32

5.7

Availability of Data ................................................................................................ 5-38

5.8

Solubility Test Results ........................................................................................... 5-38
5.8.1 Solubility Data ............................................................................................. 5-43
5.8.2 Solubility and Volatility Effects in the Cytotoxicity Tests.......................... 5-43

5.9

Summary................................................................................................................. 5-44

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[This Page Intentionally Left Blank]

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In Vitro Cytotoxicity Methods BRD Section 5

5.0

November 2006

3T3 AND NHK NRU TEST METHOD DATA AND RESULTS

This section summarizes the IC50 results generated by testing 72 coded reference substances(see Section 3) in the 3T3 and NHK NRU test method protocols. These IC50 values wereused to evaluate the accuracy (also known as concordance - see Section 6) of the two in vitro
cytotoxicity test methods for predicting in vivo GHS acute oral toxicity categories and theirreliability (intra- and inter-laboratory reproducibility - see Section 7). The individual test
data for the passing and failing tests are provided in Appendix I for the reference substances
and the PC. The raw data for each test (in EXCEL® and PRISM® files) are available uponrequest from NICEATM on compact disk(s), as are the laboratory reports. Requests can bemade by mail, fax, or e-mail to Dr. William S. Stokes, NICEATM, NIEHS, P. O. Box 12233,
MD EC-17, Research Triangle Park, NC, 27709, (phone) 919-541-2384, (fax) 919-541-0947,(e-mail) niceatm@niehs.nih.gov.
Section 5.1 discusses the timeline for the validation study, the study participants, and theirroles in the study. Section 5.2 documents the use of coded reference substances and the GLP
compliance by the participating laboratories. Section 5.3 discusses the protocol revisions thatwere made during the study and the effect the revisions had on the results. Section 5.4
presents the IC50 data collected during each phase to assess the reliability and accuracy(relevance) of the NRU methods. Section 5.5 presents the statistical analyses performed.
Section 5.6 summarizes the results of IC50 comparisons of the 3T3 and NHK methods.
Section 5.7 offers information about the availability of all the data (e.g., raw OD data fromall tests, laboratory reports), and Section 5.8 presents the solubility test results for thereference substances from all laboratories.
5.1

Study Timeline and Participating Laboratories

5.1.1
Statements of Work (SOW) and Protocols
The SMT provided the laboratories with SOWs for each test method prior to initiation of
testing (see Appendix G), and proposed dates for completion of the various aspects of thestudy (e.g., transfer of data, provision of reports). The SOWs defined the following:• Project objectives• Management and key personnel• Required facilities, equipment, and supplies• Quality assurance requirements• Test phases and schedules• Products (e.g., reports) required• Report preparation
The SOW for BioReliance contained all of the above requirements, and also included
requirements for:• Reference substance acquisition, coding, preparation, and distribution• Solubility testing
The SMT, in consultation with the laboratories, prepared Test Method Protocols for eachphase of the study. Cytotoxicity testing in each phase of the validation study was initiated ineach laboratory when the SMT received a signed protocol specific for that phase from the
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Study Director. Solubility testing for the Phases I and II substances was performed prior tocytotoxicity testing for those substances; most of the solubility testing for the Phase III
substances was performed toward the end of Phase II and during the early part of Phase III.
5.1.2
Study Timeline
The actual timeline of the study is shown in Table 5-1. The SMT modified the original
timeline presented in the SOWs because of a number of factors, such as, protocol revisions,side studies, difficulties with acquisition of medium, etc.
Table 5-1

Validation Study Timetable

Event
Receipt of SOW from SMT
Procurement of Test
Substances
Solubility Testing
Completed
Distribution of Reference
Substances
Phase Ia
Phase Ib
Phase II
Phase III
Initiation of Phase Ia
Completion of Phase Ia
Initiation of Phase Ib
Completion of Phase Ib
Initiation of Phase II
Completion of Phase II
Initiation of Phase III
Completion of Phase III

BioRelianceJun 2002

ECBC
Jun 2002

FAL
Jun 2002

IIVS
Jun 2002

Jul 2002 - Jan 2003

NA

NA

NA

Jul 2002 - Jan 2003

Dec 2003

Dec 2003

Jan 2004

NA

NA

NA

Aug 2002
Nov 2002
Dec 2002
May 2003
Jun 2003
Nov 2003
Dec 2003
Dec 2004

Aug 2002
Nov 2002
Dec 2002
May 2003
Jun 2003
Nov 2003
Dec 2003
Dec 2004

Aug 2002
Oct 2002
Dec 2002
May 2003
Jun 2003
Nov 2003
Dec 2003
Jan 2005

Jul 2002
Sep 2002
Nov 2002
Feb - Mar 2003
NA
NA
NA
NA
NA
NA
NA
NA

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in Medical
Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; SOW=Statement of Work; SMT=StudyManagement Team; NA=Not applicable.
Note: BioReliance distributed the reference substances and performed solubility testing. ECBC, FAL, and IIVS tested thereference substances for solubility and in vitro cytotoxicity.

5.1.3

Participating Laboratories• BioReliance Corporation
14920 Broschart Road
Rockville, Maryland 20850-3349
Study Director: Dr. Martin Wenk
• U.S. Army Edgewood Chemical Biological Center (ECBC)
Molecular Engineering Team
Aberdeen Proving Ground, MD 21010
Study Director: Dr. Cheng Cao

5-4

In Vitro Cytotoxicity Methods BRD Section 5

•

•

5.2

November 2006

Institute for In Vitro Sciences (IIVS)
21 Firstfield Road Suite 220
Gaithersburg, MD 20878
Study Director: Mr. Hans Raabe
Fund for the Replacement of Animals in Medical Experiments Alternatives
Laboratory (FAL)
Queens Medical Centre, University of Nottingham
Nottingham NG7 2UH
United Kingdom
Study Director: Dr. Richard Clothier

Coded Reference Substances and GLP Guidelines

5.2.1
Coded Reference Substances
BioReliance acquired 73 substances (72 reference substances and one PC substance) from
reputable commercial sources (see Appendix F1). All but eight of the reference substanceswere >99% pure (see Section 8.1.2.1). BioReliance coded each substance with a unique,random identification number when repackaging them into smaller units for distribution to
the laboratories. These units were given an additional code unique to the respective
cytotoxicity laboratories, so that they could be provided in a blinded fashion (see Section 3.4
for distribution procedures). The coded substance units were packaged and shipped such that
their identities were concealed; however, all laboratories knew the identity of the positive
control. The SMT revealed the codes for each phase after all laboratories had submitted theirdata and reports for that phase. The laboratories periodically required additional aliquots ofreference substance, and BioReliance provided these aliquots from the original stock ofreference substance in the same manner that the original aliquots were provided.
5.2.2
Lot-to-Lot Consistency of Reference Substances
Each substance was purchased as a single lot, and each laboratory received aliquots from thissame lot throughout the validation study. The reference substance suppliers provided
certificates of analysis for each lot, along with the MSDS documents containing substance,
physical, and safety and handling information.
5.2.3
Adherence to GLP Guidelines
BioReliance, ECBC, and IIVS, followed GLP procedures for all testing, with the exceptionof tests designed to resolve technical challenges (e.g., formation of NR crystals; use of film
plate sealers for volatile substances; slow growth of cells). The laboratories submitted all data
to their respective quality assurance units (as per GLP requirements) and copies of the data
were submitted to NICEATM. FAL followed most of the GLP guidelines, but did not employ
independent quality assurance reviews of laboratory procedures or documentation. The Study
Director for FAL performed all data reviews and provided copies to NICEATM. Hard copy
printouts and electronic versions of all data are available at NICEATM.
5.3

3T3 and NHK NRU Test Method Protocols

The protocols for the 3T3 and NHK NRU test methods used during Phase III laboratory
testing were the result of modifications and revisions to the Guidance Document (ICCVAM
2001b) protocols, the optimization of the protocols used in the laboratory evaluation Phases
Ia and Ib, and the laboratory qualification phase (Phase II) (see Section 2.6). Figure 1-2

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provides an outline of the study phases, and identifies where repeated observations werecarried out to permit protocol evaluation and comparison. Sections 2.2 and 2.3 address thesimilarities and differences between the 3T3 and NHK protocols. The remaining subsectionsin Section 5.3 address the modifications to the protocols used in each phase, and how thosemodifications affected each data set.
5.3.1
Phase Ia: Laboratory Evaluation Phase
During Phase Ia, each laboratory established an historical database for the PC substance,
SLS. No reference substances were tested in this phase. Ten concentration-response tests
were performed using SLS and no more than two tests were performed/day. The resulting
data were used to calculate the acceptable response limits for the SLS IC50 for use during
Phase Ib testing.
Section 2.6.1 summarizes issues that occurred during Phase I and addresses protocol changes
made after the initiation of Phase Ia. The specific changes to the protocols for both cellsystems are summarized below, along with the impact these changes had on the test data.
Changes made in the protocols during Phase Ia were incorporated into the Phase Ib protocols.
5.3.1.1

Protocol Changes and the Effect on the Data
• NR Dye Crystals: Reduced the NR dye concentration for both cell types. No
subsequent tests failed because of NR crystal formation. The background OD
values decreased and this was not interpreted as a negative effect on the data.
• 3T3 Cell Growth: Modified cell culture conditions for 3T3 cells to improvecell growth characteristics. No apparent effect on the data was detected.
• NHK Cell Growth (96-well plates): Removed the cell culture refeeding step
performed prior to reference substance addition. Although the OD values forthe vehicle controls became higher, the SLS IC50 results were similar whether
or not the cells were re-fed.
• NHK Cell Growth (in culture flasks): FAL coated their culture flasks withfibronectin-collagen prior to seeding thawed cells. This may have affected the
SLS data from FAL because it had the highest SLS IC50 values of the threelaboratories (7.45 µg/mL vs. 4.03 µg/mL for ECBC and 3.68 µg/mL for
IIVS). The fibronectin-collagen coating procedure was eliminated, andsubsequent SLS data and IC50 results from FAL were comparable to the datafrom the other two laboratories.
• OD Limits: Eliminated the VC OD range as a test acceptance criterion. The
SMT decided to accept tests that had VC ODs outside the originally preset
range if all other test acceptance criteria were met. Test data were notadversely affected by relaxing this criterion.
• Dilution Factor: The SMT accepted data generated using dilution factors
other than the recommended 1.47 for definitive tests if all other test
acceptance criteria were met. The use of smaller dilution factors generally
increased the number of data points between 10 - 90% viability, and theprecision of the IC50 calculation was improved.

5.3.2
Phase Ib: Laboratory Evaluation Phase
Phase Ib was designed to determine whether the protocol revisions following Phase Ia wereeffective in improving intra- and inter-laboratory reproducibility, and to determine whether
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the laboratories could obtain reproducible results when testing coded reference substances ofvarious toxicities. Three coded reference substances representing the full range of toxicity
were tested: arsenic trioxide (high toxicity: 5< LD50 ≤50 mg/kg), propranolol HCl (medium
toxicity: 300< LD50 ≤2000 mg/kg), and ethylene glycol (low toxicity: LD50 >5000 mg/kg)(see Section 3.3.5 for the selection of substances to be tested in Phases Ib and II). Because
Phase Ib was part of the laboratory evaluation phase, the SMT decided that three substances
would be sufficient, and that it was not necessary to represent all GHS acute oral toxicity
categories. Each substance was tested in all laboratories at least once in a range finding
experiment, and then in three, acceptable definitive tests performed on three different days.
Section 2.6.2 summarizes the technical challenges that arose during this phase and addresses
protocol changes made after initiation of Phase Ib. The specific changes made in the 3T3 andNHK protocols, along with the effect the changes had on the test data, are summarized
below.
5.3.2.1

Protocol Changes and the Effect on the Data
• NR Dye Crystals: Reduced the concentration of NR in the 3T3 method. The
OD values and SLS IC50 results were similar in four exploratory experiments
regardless of the NR concentration or NRU incubation time. The eliminationof NR crystals reduced the background OD values without affecting thesensitivity of the procedure.
• VC OD Range: Used new VC OD ranges for guidance (e.g., as target values toassess cell growth), rather than as a test acceptance criterion, for the remainderof the study. This increased the number of tests that met the acceptancecriteria. Relative toxicities did not change. The test data were not adversely
affected by the removal of this criterion.

5.3.3
Phase II: Laboratory Qualification Phase
The results from Phase II were used to determine whether the protocol revisions from Phase
Ib were effective in improving intra- and inter-laboratory reproducibility, and whether thelaboratories could obtain reproducible results when testing a larger set of substances covering
a wider range of physical/substance characteristics and toxicities. Nine coded reference
substances were tested: aminopterin, cadmium chloride, chloramphenicol, colchicine, lithium
carbonate, potassium chloride, 2-propanol, sodium fluoride, and sodium selenate. These
substances (with the exception of sodium selenate) are included in the RC, and were selected
because they fit the RC millimole regression line (i.e., they were within the acceptanceintervals established by Halle [1998, 2003]). The RC is a database of acute oral LD50 values
for rats and mice obtained from RTECS® and IC50 values from in vitro cytotoxicity assays
using multiple cell lines and cytotoxicity endpoints for substances with known molecularweights (Halle 1998, 2003). Sodium selenate was selected because of its high toxicity,
despite the fact that it was not in the RC, because there were no other substances in thehighest GHS acute oral toxicity category, other than aminopterin, that were within the RC
millimole regression acceptance intervals. Each laboratory tested each substance at least oncein a range finding experiment, and then in three acceptable definitive tests performed on
different days.

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Section 2.6.2 summarizes the technical issues that arose during this phase and the protocol
changes made prior to Phase II. The specific changes made in the 3T3 and NHK NRU
protocols, along with the effect the changes had on the test data, are summarized below.
5.3.3.1

Protocol Changes and the Effect on the Data
• Blank Wells: Added reference substance to blank wells of the test plate todetermine if reference substance affected (i.e., increased OD values) compared
to medium-filled blank wells. There was no apparent effect on the test data as
there were no noticeable differences in OD values between blanks with culture
medium or culture medium and reference substance.
• VC OD Range: Eliminated the VC OD range as an acceptance criterion. There
was no apparent effect on test data from not restricting the OD values to a preset range.
• Harmonization of Laboratory Techniques: Made revisions to the Phase II
protocols as a result of the harmonization training by the testing laboratories(see Section 2.6.2.6). There was no apparent effect on the test data from IIVS
and ECBC, but there was an improvement in the FAL data quality (e.g., fewer
lost OD values due to cell seeding errors, more uniform OD values for six
replicate wells per reference substance).
• 3T3 Cell Seeding Density: Added a range of cell seeding densities to be usedby the laboratories. This optimized the cell confluence at the end of chemical
exposure and no apparent effects on the data were detected because of thismodification.
• NHK Cell Growth from Cryopreserved Stock Cells: Eliminated the use offibronectin-collagen coating of 80-cm2 flasks for the initial propagation of
NHK cells. By doing this, FAL achieved better cell growth, lower IC50 values
for the PC, and better agreement of the mean SLS IC50 values with those of
the other laboratories.
• Volatile Substances: Added the use of a CO2 permeable plate sealer to control
volatility (as identified by cross contamination of the control wells). The useof plate sealers for volatile substances was incorporated into the Phase III
protocols.
• R2 Acceptance Criterion: Relaxed the R2 criterion for the fit of the doseresponse data to the Hill function. Some tests that did not meet the original
criterion were accepted by the SMT after determining that even though thecurve fit was not optimum, it adequately conveyed the toxicity of thesubstance (i.e., an IC50 could be calculated with an adequate number of
toxicity points between 0 and 100% viability).
• Unusual Concentration-Response: Revised the Hill function calculation toaddress substances that produced a concentration-response in which toxicity
plateaued before reaching 0% viability. This modification allowed for a curve
fit to the Hill function for such substances, and thus a better estimation of their
IC50 values.
• PC IC50 Range: Expanded the SLS IC50 acceptable range, which resulted inadditional tests in Phase II being acceptable. Expanding the PC range reduced
the number of reference substance retests, and thereby qualified additional

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In Vitro Cytotoxicity Methods BRD Section 5

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definitive tests as acceptable because they would not fail simply because thePC was out of the pre-set range.
5.3.4
Phase III: Main Validation Phase
The purpose of Phase III was to generate high quality in vitro cytotoxicity data using the 3T3
and NHK NRU test methods with protocols that were optimized based on the experience andresults in Phases I and II. Sixty coded reference substances were tested; 46 of these were RC
substances that covered a broad range of toxicity. The reference substances in Phase III
spanned all five GHS toxicity categories and unclassified substances. Each substance wastested in each laboratory at least once in a range finding experiment, and then in three
acceptable definitive tests performed on different days.
Section 2.6.4 addresses protocol changes made before the initiation of Phase III. The specificchanges made in the 3T3 and NHK protocols, along with the effect the changes had on thetest data, are summarized below.
5.3.4.1

Protocol Changes and the Effect on the Data
• Prequalification of NHK Culture Medium: Included a protocol forprequalifying NHK culture medium and supplements. This prevented theparticipating laboratories from using medium and supplements that did notsupport adequate growth of the cells.
• Stopping Rule for Testing: Added this rule for reference substances that wereinsoluble (i.e., <200 µg/mL) and/or did not produce sufficient cytotoxicity forthe calculation of an IC50. This rule allowed testing to end for substances that
produced no IC50 data after three definitive tests. Substances for which an IC50
was not produced by one or more laboratories are presented in Table 5-2.
Carbon tetrachloride did not produce an IC50 in any of the laboratories ineither the 3T3 or the NHK NRU test methods, and methanol did not producean IC50 in the 3T3 NRU test method.
• Acceptable Range for Dose-Response Data Points: Modified the test
acceptance criterion for the number of data points required on the toxicity
curve. The criterion was changed from requiring a minimum of two points (atleast one >0% and ≤50% viability, and at least one >50% and <100%viability) to one point >0% and <100% viability, if the smallest practical
dilution factor (i.e., 1.21) was used, and all other test acceptance criteria were
met. This reduced the number of failed experiments for substances with very
steep concentration-response curves, without reducing the quality of the IC50
data. For the 3T3 NRU test method, diquat dibromide (1/9 definitive tests),
epinephrine bitartrate (2/9 definitive tests), and 1,1,1-trichloroethane (2/8
definitive tests) had such steep dose-responses that some acceptable tests met
these revised criteria. None of the NHK NRU tests needed the revised criteria.
• R2 Acceptance Criterion: Rescinded the R2 criterion for the fit of the Hillfunction. The SMT determined that the R2 criterion was best used tocharacterize the shape of the concentration-response curve rather than toestablish a criterion for test acceptability. This reduced the number of failed
experiments without affecting the calculation of the IC50 values as long as an

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Table 5-2

November 2006

adequate number of toxicity points between 0 and 100% viability wereobtained.
PC Acceptance Criteria: Modified the PC acceptance criterion for Hillfunction fit.
Hill Function Analysis: Altered the PRISM® template for the Hill function
analysis to perform calculations for ICx values in two ways: (1) constraining
Bottom parameter to zero, and (2) fitting the Bottom parameter. As a result of
the changes and efforts by the laboratories to use dilution schemes that
captured the entire concentration-response range, very few tests in Phase III
had R2 <0.9.
Biphasic Dose-Response in Range Finder Test: Provided guidance forproceeding with definitive testing when a biphasic dose-response wasobtained in the range-finder test. The definitive test was to focus on the lowest
concentrations that produced responses around 50% viability (See Section
2.6.3.2).
Reference Substances Affected by Stopping Rule1

Reference Substance
Carbon tetrachloride
Disulfoton
Gibberellic acid
Methanol
1,1,1-Trichloroethane
Valproic acid
XyleneTesting Stopped -- No IC50 Data
3T3 NRU Test Method
NHK NRU Test Method
ECBC
FAL
IIVS
ECBC
FAL
IIVS
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake;
ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in Medical Experiments
Alternatives Laboratory; IIVS=Institute for In Vitro Sciences.
1
Substances that did not provide sufficient cytotoxicity for the calculation of an IC50 in one or more laboratories (identifiedby X).

5.4

Data Used to Evaluate Test Method Accuracy and Reliability

This section first presents the acceptable PC data and IC50 results from each laboratory foreach phase of the validation study, and then presents the reference substance IC50 results andHill Slopes from each phase. The individual test data for both passing and failing tests areprovided in Appendix I for the PC and reference substances. Accuracy (concordance for theprediction of GHS acute oral toxicity category) and reliability assessments are provided in
Sections 6 and 7, respectively.
5.4.1
PC Data
A summary of the acceptable SLS data IC50 results used to calculate quality control
acceptance limits for each test method in each laboratory are provided in Table 5-3. The SLS
IC50 results were used to calculate acceptable limits for each laboratory to use in subsequent
study phases. One of the test acceptance criteria for each reference substance test was that theassociated SLS IC50 must be within the acceptance limits. The individual test data for bothpassing and failing PC tests are provided in Appendix I3 for the 3T3 and in Appendix I4 for
the NHK methods.
5-10In Vitro Cytotoxicity Methods BRD Section 5

Positive Control (PC)1 IC50 Results by Study Phase

Table 5-3
Study
Phase

November 2006

Mean
IC50
(µg/mL)

ECBC
Standard
Acceptance
Deviation
Limits(µg/mL)

N

Mean
IC50
(µg/mL)

FAL
Standard
Acceptance
Deviation
Limits(µg/mL)

N

Mean
IC50
(µg/mL)

IIVS
Standard
Acceptance
Deviation
Limits(µg/mL)N

3T3 NRU
Ia2
3

Ib

4

II

III

5

38.3

4.71

28.8 – 47.7

15

42.3

8.56

25.2 – 59.5

25

40.9

3.19

34.5 – 47.3

12

41.3

5.99

26.4 – 56.3

12

43.2

4.68

31.5 – 54.9

17

42.1

3.40

33.6 – 50.6

13

41.2

4.20

30.8 – 51.6

29

45.9

7.50

27.2 – 64.7

36

40.6

3.50

31.8 – 49.3

21

41.6

3.41

NA

65

41.1

6.23

NA

26

41.5

3.74

NA

22

4.03

1.32

1.40 – 6.67

15

7.45

3.07

1.34 – 13.6

18

3.68

0.555

2.57 – 4.79

30

NHK NRU
Ia2
3

Ib

4

II

III

5

6

3.65

0.98

1.22 – 6.10

11

5.35

2.32

0 – 11.1

15

3.57

0.59

2.10 – 5.04

17

3.59

1.41

0.07 – 7.11

22

3.20

1.05

0.57 – 5.82

15

3.78

0.73

1.94 – 5.61

26

3.03

0.75

NA

57

3.45

0.90

NA

35

3.12

0.53

NA

20

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; ECBC=Edgewood Chemical Biological Center;
FAL=Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; N=Number of acceptable tests; NA=Not
applicable1
PC was sodium lauryl sulfate (SLS).
2
Values generated from Phase Ia data were used as acceptance criteria for Phase Ib tests; Acceptance limits = Mean ±2 X standard deviation.
3
Values generated from Phases Ia and Ib data were used as acceptance criteria for Phase II tests; Acceptance limits = Mean ±2.5 X standard deviation.
4
Values generated from Phases Ia, Ib, and II data were used as acceptance criteria for Phase III tests; Acceptance limits = Mean ±2.5 X standard deviation.
5
Values generated from Phase III test data.
6
Calculation of lower limits yielded a negative value, so that lower limit was set at 0 and later revised to 0.1 µg/mL.

5-11

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

5.4.1.1 Phase Ib PC Data Acceptance Limits
The SLS IC50 acceptance limits for Phase Ib testing were calculated using the Phase Ia data.
The data sets from each laboratory were examined for outliers using the method of Dixon
and Massey (1981), but none were identified. The acceptance limits for the SLS IC50 values
for each laboratory and test method were the mean ±2 SD.
5.4.1.2 Phase II PC Data Acceptance Limits
The IC50 values from the Phase Ia and Ib SLS tests were used to calculate laboratory-specificand test method-specific quality control acceptance limits for Phase II. Phase Ib tests that had
SLS IC50 values outside of the acceptance limits were considered acceptable if they met allother test acceptance criteria. For any day during which there was more than one SLS test(for any one method and laboratory), the IC50 values were averaged to better reflect day-today variation and avoid overweighting the overall mean with multiple values from a single
day. Outliers at the 99% level were removed and the remaining values were used to calculate
the mean ±2.5 SD acceptance limits. The acceptance limits were expanded from 2 SD in
Phase Ib to 2.5 SD for Phase II to allow for the fact that the SDs decrease as more data arecollected.
5.4.1.3 Phase III PC Data Acceptance Limits
The IC50 values from the Phase I and II SLS tests were used to calculate laboratory-specificand method-specific quality control acceptance limits for Phase III data. The SLS IC50 values
outside the acceptance limits were considered acceptable if the tests met all other acceptancecriteria. For any day for which there was more than one SLS test (for any one method andlaboratory), the IC50 values were averaged to better reflect day-to-day variation and avoid
overweighting the overall mean with multiples values from a single day. ANOVA was used
to compare the Phase Ia, Ib, and II data within each laboratory to determine whether the SLS
IC50 for each method and laboratory was changing over the course of the study. For PC datathat were not significantly different from phase to phase at p <0.05, the IC50 values were used
to calculate the mean ±2.5 SD as the acceptance limits for Phase III. The only significant
differences in SLS values seen between study phases (p <0.0002) were the FAL results forNHK. This difference was attributed to the changes in cell culture practices between Phases
Ib and II (see Section 5.3.3). Thus, only the Phase II SLS IC50 values were used to calculate
the acceptance limits for Phase III NHK data at FAL.
5.4.2
Reference Substance Data
Reference substance data and results from the individual 3T3 and NHK tests (both acceptableand unacceptable) from each laboratory are presented in Appendices I1 and I2. Tables 5-4
and 5-5 summarize the IC50 and Hill Slope data from the acceptable 3T3 and NHK tests,
respectively, for each reference substance and laboratory. The Hill Slope data are provided
for supplemental information on the concentration-response characteristics for each reference
substance, but were not used for reliability or accuracy analyses. These tables are organizedalphabetically by substance name and provide substance class (based on the NLM Medical
Subject Heading [MeSH index]), arithmetic mean IC50 and SD for each laboratory, arithmetic
mean Hill Slope and SD for each laboratory, and the number of tests used to produce themean values. Figure 5-1 graphically presents the 3T3 IC50 data from Table 5-4, and Figure
5-2 presents the NHK IC50 data from Table 5-5. The reference substances in Figures 5-1 and
5-2 are ordered by ascending IC50 (lowest value [most toxic] to highest value [least toxic])
using the 3T3 IC50 values from IIVS (the lead laboratory for the study). This allows a simple
5-12

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

comparison of each reference substance value from each laboratory. Table 5-6 provides thenumerical key to the reference substances in Figures 5-1 and 5-2.
Because of their low toxicity and/or low solubility, some substances were not sufficiently
toxic for calculation of an IC50 value. For the 3T3 NRU test method, no IC50 values wereobtained for carbon tetrachloride or methanol in any laboratory (see Table 5-4). ECBC was
the only laboratory that obtained IC50 values for lithium carbonate, and IIVS was the only
laboratory that obtained IC50 values for xylene. Only one acceptable test (and IC50 value) was
obtained for disulfoton at FAL, for 1,1,1-trichloroethane at ECBC, and for valproic acid at
IIVS. FAL did not achieve sufficient toxicity for the calculation of an IC50 for gibberellicacid in any 3T3 NRU tests performed. For the NHK NRU test method (see Table 5-5), there
was insufficient toxicity in all tests in all laboratories for a calculation of an IC50 for carbon
tetrachloride. Only one laboratory achieved sufficient toxicity for the calculation of an IC50
for 1,1,1-trichloroethane (ECBC) and xylene (IIVS). One laboratory, ECBC, failed toachieve sufficient toxicity for the calculation of an IC50 for methanol. All of these substances,with the exception of methanol, produced precipitate in the cell culture medium. The solvent
used for methanol was DMSO, and because the amount of DMSO that could be used in thecell culture was limited to 0.5%, the amount of DMSO that could be used to dissolve
methanol was also limited. The differences among laboratories regarding their ability toattain a high enough concentration to achieve an IC50 for some substances may be due to thediffering perceptions of the laboratory personnel regarding whether or not the substance wassufficiently dissolved, or differences in the techniques used to dissolve the substances.

5-13

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-4

3T3 NRU Test Method IC50 and Hill Slope Data by Laboratory

Amide

Phase
in
which
TestedIII

Acetonitrile

NitrileAcetylsalicylic
acid

Carboxylic
Acid; Phenol

Aminopterin
5-Aminosalicylic
acid
Amitriptyline
HCl
Arsenic III
Trioxide
Atropine sulfate

Substance

ChemicalClass5

Acetaminophen

Boric acid

BusulfanCadmium II
chloride

November 2006

ECBC
2

FAL

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

40.8

9.12

3

III

6433

129

III

646

Heterocyclic

II

Carboxylic
Acid; Phenol

2

IIVS

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

-1.53

0.354

66.2

23.0

3

3

-2.29

0.648

9690

5634

61.5

3

-1.75

0.473

1234

0.005

0.001

3

-2.00

0.395

III

1467

203

3

-1.82

Polycyclic

III

6.03

1.38

3

Arsenical

Ib

2.41

0.782

Heterocyclic

III

54.1

III

Boron
compound;Acid
Alcohol;
Sulfurcompound;Acyclic
hydrocarbon
Cadmiumcompound;
Chlorinecompound

2

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

SD4

-1.23

0.503

43.4

11.4

3

-1.55

0.165

3

-1.55

0.196

9330

1217

3

-2.63

0.245

298

3

-1.99

0.393

401

62.0

3

-1.31

0.167

0.012

0.005

3

-3.36

1.59

0.005

0.001

3

-1.46

0.198

0.267

2070

334

3

-2.33

0.809

1557

179

3

-1.64

0.326

-2.47

0.668

7.86

2.20

3

-2.98

0.446

7.81

1.38

3

-4.48

0.916

4

-1.94

0.204

1.04

0.070

4

-3.02

2.09

4.09

2.23

3

-1.62

0.285

29.6

3

-1.32

0.480

133

41.1

3

-2.20

0.695

70.0

5.7

3

-1.27

0.165

1497

484

3

-1.14

0.039

3987

693

3

-1.86

0.654

1202

581

3

-1.71

0.677

III

40.4

19.3

3

-0.515

0.003

321

180

3

-1.14

0.802

43.7

1.77

3

-0.627

0.164

II

0.480

0.066

3

-1.85

0.529

0.400

0.129

3

-3.05

0.743

0.817

0.427

3

-2.45

0.449

Caffeine

Heterocyclic

III

133

13.3

3

-1.11

0.097

157

81.7

3

-0.866

0.250

191

14.4

3

-1.27

0.077

Carbamazepine

Heterocyclic

III

83.0

12.0

3

-1.94

0.539

152

56.9

3

-3.50

1.27

91.8

11.0

3

-2.34

0.307

Carbontetrachloride

Halogenatedhydrocarbon

III

NA

NA

-

NA

NA

NA

NA

-

NA

NA

NA

NA

-

NA

NA

Chloral hydrate

Alcohol

III

151

15.6

3

-1.73

0.172

241

25.1

3

-2.16

0.597

170

19.9

3

-1.68

0.084

II

55.3

12.4

4

-0.779

0.057

273

82.2

4

-1.16

0.249

156

27.9

3

-0.952

0.036

III

473

138

3

-1.89

0.423

1148

143

4

-3.68

0.407

865

160

3

-2.51

0.530

Chloramphenicol

Citric acid

Alcohol;Nitro
compound;Cyclichydrocarbon
Carboxylic
acid

5-14

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-4
Substance

November 2006

3T3 NRU Test Method IC50 and Hill Slope Data by Laboratory
ChemicalClass5

Phase
in
which
Tested
II

ECBC
2

FAL

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

0.021

0.002

4

2

IIVS

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

-1.69

0.049

0.093

0.042

3

2

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

SD4

-1.61

1.80

0.028

0.0003

3

-1.69

0.255

Colchicine

Polycyclic

Cupric sulfate
pentahydrate

Sulfurcompound;
Metal

III

82.7

3.18

3

-4.85

0.700

123

54.0

4

-17.7

15.5

5.72

1.75

3

-5.71

1.14

Cycloheximide

Heterocyclic

III

0.125

0.057

3

-1.19

0.167

0.647

0.451

3

-1.53

0.128

0.109

0.025

3

-0.937

0.158

III

23.5

3.98

3

-3.37

1.27

191

94.5

4

-0.965

0.140

20.7

1.37

3

-2.62

0.283

III

9.83

3.42

3

-1.32

0.297

32.8

2.07

3

-3.42

1.00

18.3

2.09

3

-2.13

0.439

III

85.5

29.0

3

-1.11

0.340

147

37.8

3

-2.03

0.422

106

25.3

3

-2.35

0.824

Dibutyl phthalate
Dichlorvos
Diethyl phthalate

Carboxylicacid
Organophosphorous
Carboxylicacid
Polycyclic;

III

351

137

3

-2.11

2.05

892

319

3

-3.26

2.21

317

67.9

2

-3.04

1.52

Dimethylformamide

Amide;
Carboxylic
acid

III

5343

515

3

-1.96

0.087

5483

517

3

-1.80

0.143

4900

183

3

-1.87

0.102

Diquat dibromide
monohydrate

Heterocyclic

III

3.87

0.887

3

-1.59

0.197

36.1

35.5

3

-11.5

10.1

5.39

1.36

3

-3.00

0.784

III

137

74.9

3

-2.06

1.88

11200

NA

1

-1.22

NA

60.4

52.5

3

-2.23

1.08

III

5.27

3.01

3

-0.669

0.243

15.2

11.9

4

-0.762

0.221

3.61

1.53

3

-0.871

0.636

III

51.5

6.16

3

-5.99

3.08

63.4

6.63

3

-45.1

32.0

63.4

1.91

3

-4.74

1.51

Digoxin

Disulfoton

Endosulfan
Epinephrine
bitartrate

Carbohydrate

Organophosphorous;
SulfurcompoundHeterocyclic
SulfurcompoundAlcohol;
Amine

Ethanol

Alcohol

III

5360

1754

3

-1.33

0.104

8420

1205

3

-1.88

0.128

6413

345

3

-1.99

0.372

Ethylene glycol

Alcohol

Ib

18325

1658

4

-3.79

4.08

31650

7453

4

-1.70

0.166

25900

3081

3

-1.67

0.079

Fenpropathrin

Nitrile;Ester; Ether

III

22.6

2.41

3

-2.54

0.350

42.4

26.8

4

-1.44

0.645

16.7

2.03

3

-2.53

0.495

Gibberellic acid

Polycyclic

III

8027

908

3

-1.95

0.678

NA

NA

-

NA

NA

7657

745

3

-1.66

0.087

Glutethimide

Heterocyclic

III

167

7.00

3

-1.3

0.045

284

20.7

3

-1.47

0.131

125

9.25

4

-1.20

0.163

Glycerol

Alcohol

III

20000

2987

3

-2.02

0.273

38878

28238

4

-2.27

1.29

27833

10882

3

-1.87

0.306

Haloperidol

Ketone

III

5.32

0.649

3

-2.34

0.445

7.99

0.655

3

-4.99

0.378

5.47

0.654

3

-1.86

0.048

5-15

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-4
Substance

Hexachlorophene
Lactic acid
Lindane
Lithium I
carbonate
Meprobamate
Mercury II
chloride

November 2006

3T3 NRU Test Method IC50 and Hill Slope Data by Laboratory
ChemicalClass5Cyclichydrocarbon
PhenolCarboxylic
acid
Halogenatedhydrocarbon
Alkalies;
Inorganiccarbon;LithiumcompoundCarboxylicacid
Mercury
compound;
Chlorinecompound

Phase
in
which
Tested

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

III

5.02

2.41

3

III

2943

315

III

125

II

ECBC
2

FAL
2

IIVS

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

-1.62

0.189

5.35

1.75

3

3

-4.13

1.54

3487

561

119

3

-0.737

0.231

266

564

67.6

3

-1.59

0.313

III

353

49.7

3

-1.16

III

3.45

0.177

3

-4.18

2

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

SD4

-1.17

0.322

3.06

0.289

3

-1.66

0.217

3

-6.62

3.23

2790

259

3

-3.64

1.09

94.8

4

-1.26

1.283

90.4

111

5

-1.46

0.262

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

0.438

877

128

4

-1.32

0.270

386

9.02

3

-1.12

0.133

0.988

5.99

1.87

3

-4.34

1.11

3.51

0.120

3

-4.16

1.31

MethanolAlcohol

III

NA

NA

-

NA

NA

NA

NA

-

NA

NA

NA

NA

-

NA

NA

Nicotine

Heterocyclic

III

272

65.3

3

-1.58

0.357

412

136

3

-12.0

6.99

450

54.7

3

-49.6

70.9

Paraquat

Heterocyclic

III

21.3

7.29

3

-1.32

0.341

24.9

16.5

3-

-4.10

3.13

23.7

15.2

3

-1.92

0.581

Parathion

Organophosphorous;
Sulfurcompound

III

22.7

12.1

3

-1.89

1.33

141

98.7

4

-1.62

0.520

22.0

4.94

3

-1.55

0.562

Phenobarbital

Heterocyclic

III

634

134

3

-1.43

0.177

726

255

3

-1.84

0.851

476

111

4

-1.67

0.418

Phenol

III

50.2

10.9

3

-1.46

0.318

104

24.8

3

-1.55

0.205

58.1

6.78

3

-1.41

0.259

III

30.1

19.8

3

-0.781

0.218

239

65.8

3

-0.890

0.206

89.0

21.9

3

-1.40

0.127

III

28.2

14.9

3

-1.51

0.595

37.8

1.93

3

-7.22

1.04

20.4

6.71

4

-1.70

0.157

II

3352

468

4

-3.32

1.17

3842

1198

5

-4.31

2.27

3710

417

3

-2.87

0.147

PhenolPhenylthiourea
Physostigmine
Potassium I
chloride

Sulfurcompound;
Urea
Carboxylic
acid;
Heterocyclic
Potassiumcompound;
Chlorinecompound

5-16

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-4
Substance

November 2006

3T3 NRU Test Method IC50 and Hill Slope Data by Laboratory
ChemicalClass5

Phase
in
which
Tested

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

III

15.3

3.76

3

III

400

15.3

ECBC
2

FAL
2

IIVS

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

-1.48

0.677

159

81.9

3

3

-12.4

1.91

431

4.73

2

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

SD4

-1.03

0.152

18.9

0.950

3

-3.43

0.488

3

-45.6

18.4

497

39.3

3

-19.9

13.1

Procainamide
HCl

Potassiumcompound;NitrogencompoundCarboxylicacid; Amide

2-Propanol

Alcohol

II

2610

240

2

-1.80

0.001

3970

139

3

-1.65

0.241

4110

161

3

-1.93

0.160

Propranolol HCl

Alcohol

Ib

13.6

4.37

4

-2.54

0.627

13.5

6.85

4

-3.31

2.53

17.6

3.78

3

-3.45

1.44

III

20.9

3.33

3

-1.23

0.259

51.8

14.8

3

-1.45

0.442

17.1

2.10

3

-1.24

0.245

III

0.496

0.028

3

-1.43

0.087

1.44

0.819

3

-3.79

1.22

0.683

0.117

3

-1.90

0.535

III

4790

233

3

-1.55

0.182

4625

611

4

-2.67

0.620

4877

457

3

-2.03

0.366

III

0.603

0.087

3

-1.64

0.136

0.657

0.244

3

-5.01

1.51

0.547

0.092

3

-1.93

0.194

II

61.3

5.55

3

-5.06

1.50

96.1

17.7

3

-4.40

0.971

82.0

5.81

3

-2.73

0.850

III

823

108

3

-2.57

1.12

805

367

3

-4.13

3.05

2005

872

4

-3.20

0.279

III

42.0

17.3

3

-1.83

0.380

31.0

8.66

3

-3.11

0.367

49.5

26.3

4

-2.32

0.592

II

12.7

1.62

3

-1.59

0.217

54.2

10.4

3

-3.76

0.968

36.5

5.23

3

-1.65

0.112

Potassium
cyanidePropylparaben
Sodium arsenite

Sodium chloride
Sodiumdichromate
dihydrate
Sodium I fluoride

Sodiumhypochlorite

Sodium oxalate

Sodium selenate

Carboxylicacid; Phenol
Sodiumcompound;Arsenical
Sodiumcompound;
Chlorinecompound
Sodiumcompound;
Chromiumcompound
Sodiumcompound;Fluorinecompound
SodiumcompoundOxygencompound;
Chlorinecompound
Sodiumcompound;Carboxylic
acid
Sodiumcompound;Seleniumcompound

5-17

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-4
Substance
Strychnine
Thallium I
sulfateTrichloroaceticacid
1,1,1-Trichloroethane
Triethylenemelamine
Triphenyltinhydroxide
Valproic acid
Verapamil HCl
XyleneNovember 2006

3T3 NRU Test Method IC50 and Hill Slope Data by Laboratory
ChemicalClass5

Phase
in
which
Tested
III

ECBC
2

FAL
IC50
µg/mL

SD
(IC50)N

Hill
Slope3

-2.51

0.728

124

20.3

3

3

-1.02

0.201

13.4

10.4

99.1

3

-1.66

0.118

1220

41100

NA

1

-2.38

NA

III

0.086

0.009

3

-0.567

III

0.026

0.004

3

III

547

67.1

Amine

III

32.2

Cyclichydrocarbon

III

NA

Heterocyclic
Organometallic
compoundCarboxylicacid; Lipids

SD(IC50)N

Hill
Slope3

389

80.9

3

III

2.81

0.671

III

762

III

IIVS

SD4

Sulfurcompound;
Metal
Carboxylic
acid
Halogenatedhydrocarbon

IC50
µg/mL

2

1

Heterocyclic

1

2

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

SD4

-5.85

0.922

83.5

5.35

3

-6.49

2.12

4

-0.714

0.302

6.27

1.75

3

-0.752

0.081

72.1

3

-2.22

0.089

801

114

3

-1.77

0.130

21250

2357

3

-31.5

32.1

9827

180

3

-21.8

8.47

0.018

1.45

0.265

3

-1.88

1.04

0.169

0.049

3

-0.615

0.138

-1.66

0.257

0.026

0.021

3

-4.78

3.37

0.015

0.008

3

-1.46

0.149

3

-2.24

0.742

1807

175

3

-4.07

0.766

574

NA

1

-1.24

NA

5.82

3

-4.43

1.362

34.6

1.72

3

-29.1

18.6

38.9

4.20

3

-5.00

0.935

NA

-

NA

NA

NA

NA

-

NA

NA

724

87.1

3

-1.91

0.473

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; SD=Standard deviation;
N=Number of data points; NA=Not available (i.e., IC50 values or Hill Slope values could not be generated [see notes in Appendix I for more information])
1
Arithmetic mean.
2Standard deviation of IC50.
3Arithmetic Mean of Hill Slope values.
4Standard deviation of Hill Slope values.
5
Chemical class assigned is based on the classification of the National Library of Medicine’s Medical Subject Heading (MeSH), http://www.nlm.nih.gov/mesh/meshhome.html.

5-18

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-5

November 2006

NHK NRU Test Method IC50 and Hill Slope Data by Laboratory
IC501
µg/mL

2

SD

Amide

Phase
in
which
TestedIII

558

Acetonitrile

NitrileIII

Acetylsalicylic
acid

Carboxylic
Acid; Phenol

Aminopterin
5-Aminosalicylic
acid
Amitriptyline
HCl
Arsenic III
Trioxide
Atropine sulfate

Substance

ChemicalClass5

Acetaminophen

Boric acid

BusulfanCadmium II
chloride

ECBC

FAL
SD

IC501
µg/mL

2

SD

-1.09

0.108

447

4

-2.61

0.424

19.9

3

-1.94

889

182

3

III

29.9

6.52

Polycyclic

III

10.8

Arsenical

Ib

Heterocyclic

SD

2

SD

N

Hill
Slope3

SD4

-1.09

0.646

571

79.0

3

-1.20

0.154

4

-5.95

3.34

9290

413

3

-2.79

0.306

98.3

3

-1.85

0.324

514

79.1

3

-1.97

0.083

545

42.2

3

-1.27

0.225

611

70.7

2

-1.72

0.547

0.806

78.2

42.3

3

-7.96

6.90

48.8

7.90

3

-3.66

0.629

-1.79

0.236

7.57

5.43

3

-1.43

0.479

10.9

1.04

3

-2.27

0.278

4

-2.67

0.470

2.55

1.92

6

-1.78

1.14

20.9

6.4

3

-2.02

0.338

10.5

3

-1.26

0.307

104

88.2

3

-2.90

3.48

83.2

21.0

3

-1.21

0.101

440

138

3

-1.19

0.233

517

378

3

-0.752

0.117

464

11

3

-1.33

0.194

III

253

68.2

3

-0.783

0.323

268

193

3

-1.50

0.357

313

37.2

3

-1.66

0.459

II

2.20

0.823

5

-4.01

1.25

1.88

1.22

3

-3.36

3.14

1.86

0.151

3

-4.65

1.38

N

80.7

3

10868

7824

III

631

Heterocyclic

II

Carboxylic
Acid; Phenol

Boron
compound;Acid
Alcohol;
Sulfurcompound;Acyclic
hydrocarbon
Cadmiumcompound;
Chlorinecompound

IIVS
IC501
µg/mL

Hill
Slope3N

Hill
Slope3

83.7

3

10153

1960

0.367

694

-2.03

0.375

3

-3.45

3.34

3

7.77

2.54

III

85.4

III

4

4

Caffeine

Heterocyclic

III

817

256

3

-1.44

0.504

591

186

3

-1.06

0.499

574

7.81

3

-1.28

0.117

Carbamazepine

Heterocyclic

III

66.1

8.4

3

-1.15

0.307

253

325

3

-2.57

2.53

63.9

5.27

3

-1.34

0.444

Carbontetrachloride

Halogenatedhydrocarbon

III

NA

NA

-

NA

NA

NA

NA

-

NA

NA

NA

NA

-

NA

NA

Alcohol

III

140

34.2

3

-1.55

0.378

159

50.1

3

-1.33

0.105

112

1.73

3

-1.42

0.123

II

318

142

3

-1.51

0.794

414

182

4

-1.16

0.091

367

79.7

3

-0.917

0.249

III

526

82.4

3

-1.62

0.158

312

51.6

4

-1.25

0.249

433

22.3

3

-1.62

0.080

Chloral hydrate

Chloramphenicol

Citric acid

Alcohol;Nitro
compound;Cyclichydrocarbon
Carboxylic
acid

5-19

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-5
Substance

November 2006

NHK NRU Test Method IC50 and Hill Slope Data by Laboratory
ChemicalClass5

Phase
in
which
Tested
II

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

0.005

0.002

3

-2.15

1.39

0.008

0.001

3

-3.16

1.96

0.008

0.002

3

-13. 8

11.0

ECBC
1

FAL
1

IIVS
1

Colchicine

Polycyclic

Cupric sulfate
pentahydrate

Sulfurcompound;
Metal

III

190

19.6

3

-6.16

3.16

195

12.5

3

-3.85

0.328

207

7.09

3

-5.69

0.871

Cycloheximide

Heterocyclic

III

0.053

0.012

3

-1.24

0.152

0.120

0.094

3

-0.850

0.388

0.071

0.013

3

-1.54

0.178

III

28.3

7.64

3

-1.40

0.295

47.4

34.3

3

-1.02

0.352

22.0

1.32

3

-1.33

0.197

III

8.56

2.28

3

-1.17

0.147

12.4

3.74

3

-2.29

2.33

12.2

0.416

3

-1.50

0.214

III

174

14.4

3

-2.21

0.358

71.5

67.3

3

-1.67

0.637

189

33.1

3

-1.97

0.242

III

0.00540.0007

3

-2.00

0.127

0.00010.00002

3

-1.38

0.684

0.004

0.0003

3

-4.59

1.73

Dibutyl phthalate
Dichlorvos
Diethyl phthalate
Digoxin

Carboxylicacid
Organophosphorous
Carboxylicacid
Polycyclic;
Carbohydrate

Dimethylformamide

Amide;
Carboxylic
acid

III

9353

155

3

-3.67

0.273

7817

100

3

-2.85

0.590

6397

202

3

-3.00

0.161

Diquat dibromide
monohydrate

Heterocyclic

III

3.59

0.825

3

-1.44

0.051

6.77

3.73

4

-1.38

0.488

3.84

0.313

3

-1.10

0.139

III

140

27.0

3

-1.65

1.15

808

213

3

-0.841

0.452

186

59.2

3

-0.836

0.209

III

3.44

0.573

3

-1.68

0.438

1.42

0.701

4

-1.19

0.369

2.19

0.437

3

-2.20

0.242

III

115

10.8

3

-7.37

2.10

81.7

28.4

3

-8.39

5.81

75.0

12.2

3

-4.90

2.81

Disulfoton

Endosulfan
Epinephrine
bitartrate

Organophosphorous;
SulfurcompoundHeterocyclic
SulfurcompoundAlcohol;
Amine

Ethanol

Alcohol

III

8290

390

3

-2.13

0.035

12013

2286

3

-1.82

0.635

10250

867

3

-2.29

0.185

Ethylene glycol

Alcohol

Ib

38000

4681

3

-3.22

0.650

49800

4371

3

-3.02

0.188

40000

5341

4

-2.56

0.444

Fenpropathrin

Nitrile;Ester; Ether

III

3.73

1.01

3

-1.42

0.486

2.23

0.616

3

-4.37

4.45

1.82

0.310

3

-1.78

0.617

Gibberellic acid

Polycyclic

III

2850

402

3

-2.45

0.372

2940

276

3

-5.90

2.69

2807

121

3

-3.30

1.104

Glutethimide

Heterocyclic

III

187

64.3

3

-1.47

0.616

170

24.1

3

-1.29

0.145

176

27.5

3

-1.54

0.237

Glycerol

Alcohol

III

34267

15399

3

-3.32

1.97

18023

8334

3

-1.62

0.521

29033

4596

3

-2.69

0.511

Haloperidol

Ketone

III

3.69

1.01

3

-0.964

0.206

3.72

1.81

3

-0.732

0.097

3.29

1.15

3

-0.840

0.100

5-20

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-5
Substance

Hexachlorophene
Lactic acid
Lindane

Lithium I
carbonate
Meprobamate
Mercury II
chloride

November 2006

NHK NRU Test Method IC50 and Hill Slope Data by Laboratory
ChemicalClass5Cyclichydrocarbon
PhenolCarboxylic
acid
Halogenatedhydrocarbon
Alkalies;
Inorganiccarbon;LithiumcompoundCarboxylicacid
Mercury
compound;
Chlorinecompound

Phase
in
which
Tested

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

III

0.027

0.004

3

-2.21

0.301

0.046

0.020

3

-2.91

0.662

0.021

0.002

3

-2.36

0.059

III

1290

52.9

3

-2.36

0.306

1320

60.8

3

-3.25

0.328

1313

138

3

-3.23

0.408

III

19.1

3.14

3

-3.02

0.969

23.2

7.09

3

-2.24

0.315

15.6

2.4

3

-2.61

0.265

II

411

119

3

-1.95

0.456

486

95.7

3

-1.78

1.31

535

31.6

3

-2.64

0.164

III

761

116

3

-1.90

0.695

163

189

3

-0.806

0.206

624

84.2

3

-2.04

0.170

III

6.87

1.04

3

-16.3

4.95

5.4

1.02

3

-17.8

13.1

5.35

0.09

3

-17.8

3.31

ECBC
1

FAL
1

IIVS
1

MethanolAlcohol

III

NA

NA

-

NA

NA

1133

213

3

-1.79

0.874

2100

226

3

-1.86

0.297

Nicotine

Heterocyclic

III

94.3

24.7

3

-0.654

0.092

134

78.4

3

-0.668

0.077

112

27.7

3

-0.7330.047

Paraquat

Heterocyclic

III

48.3

6.03

3

-1.04

0.158

96.6

37.2

3

-1.34

0.326

53.4

5.52

3

-1.47

0.034

Parathion

Organophosphorous;
Sulfurcompound

III

34.0

10.0

3

-1.60

0.640

31.2

11.9

3

-1.18

0.200

29.0

8.34

3

-1.85

0.956

Phenobarbital

Heterocyclic

III

693

180

3

-1.10

0.214

360

95.5

3

-0.976

0.229

381

69.9

3

-1.68

0.353

Phenol

III

59.1

21.4

3

-0.919

0.084

93.2

5.97

3

-1.15

0.209

80.8

5.12

3

-0.915

0.029

III

363

58

3

-1.55

0.726

401

83.6

3

-3.49

1.91

272

71.7

3

-1.00

0.053

III

164

5.51

3

-3.05

0.552

212

238

3

-3.81

2.44

139

8.74

3

-2.97

0.135

II

2560

432

3

-2.23

0.383

2287

631

3

-1.09

0.163

1990

161

3

-2.05

0.165

PhenolPhenylthiourea
Physostigmine
Potassium I
chloride

Sulfurcompound;
Urea
Carboxylic
acid;
Heterocyclic
Potassiumcompound;
Chlorinecompound

5-21

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-5
Substance

November 2006

NHK NRU Test Method IC50 and Hill Slope Data by Laboratory
ChemicalClass5

Phase
in
which
Tested

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

III

29.3

6.9

3

-1.21

0.241

89.0

100

3

-1.10

0.319

16.9

2.21

3

-1.37

0.154

III

1480

200

3

-3.56

0.813

1787

221

3

-4.22

1.57

2027

229

3

-4.42

0.459

ECBC
1

FAL
1

IIVS
1

Procainamide
HCl

Potassiumcompound;NitrogencompoundCarboxylicacid; Amide

2-Propanol

Alcohol

II

5263

583

3

-2.01

0.173

4273

1139

3

-2.31

0.211

7087

480

3

-3.01

0.406

Alcohol

Ib

38.3

4.54

3

-3.44

0.559

43.8

2.52

3

-2.72

1.461

28.6

3.28

4

-2.09

0.413

III

18.1

2.42

3

-1.18

0.122

18.6

2.84

3

-1.58

0.399

13.8

1.21

3

-1.20

0.065

III

0.79

0.248

3

-1.69

0.222

0.336

0.187

3

-1.54

0.317

0.470

0.066

3

-1.96

0.197

III

3583

263

3

-2.43

0.153

1118

1388

3

-1.96

0.371

3470

300

3

-2.47

0.208

III

0.784

0.113

3

-2.35

0.282

0.851

0.302

4

-3.52

1.49

0.576

0.100

3

-2.32

0.199

II

48.7

6.92

3

-2.50

0.263

39.7

9.61

3

-2.60

1.04

53.7

6.82

4

-2.71

0.150

III

1863

581

3

-5.19

1.14

1243

576

3

-2.78

1.27

1633

180

3

-3.86

0.211

III

355

54.9

3

-4.00

1.99

350

147

4

-6.10

6.40

360

94.6

3

-3.13

0.555

II

7.47

0.861

3

-1.78

0.529

16.1

9.55

3

-3.07

0.456

10.0

1.33

3

-1.75

0.226

Potassium
cyanidePropranolol HCl
Propylparaben
Sodium arsenite

Sodium chloride
Sodiumdichromate
dihydrate
Sodium I fluoride

Sodiumhypochlorite

Sodium oxalate

Sodium selenate

Carboxylicacid; Phenol
Sodiumcompound;Arsenical
Sodiumcompound;
Chlorinecompound
Sodiumcompound;
Chromiumcompound
Sodiumcompound;Fluorinecompound
SodiumcompoundOxygencompound;
Chlorinecompound
Sodiumcompound;Carboxylic
acid
Sodiumcompound;Seleniumcompound

5-22

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-5
Substance
Strychnine
Thallium I sulfate
Trichloroaceticacid
1,1,1-Trichloroethane
Triethylenemelamine
Triphenyltinhydroxide
Valproic acid
Verapamil HCl
XyleneNovember 2006

NHK NRU Test Method IC50 and Hill Slope Data by Laboratory
Phase
in
which
Tested
III

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

100

76.6

4

-1.30

0.729

52.5

28.0

3

-1.60

0.260

55.1

3.43

3

-1.47

0.466

III

0.198

0.100

3

-2.08

1.01

0.153

0.031

3

-2.64

0.639

0.127

0.020

3

-2.90

0.338

III

348

63.5

3

-1.36

0.241

541

150

3

-1.34

0.411

394

50.8

3

-1.48

0.103

III

8137

591

3

-14.0

6.08

NA

NA

-

NA

NA

NA

NA

-

NA

NA

III

1.69

0.950

3

-0.838

0.076

2.03

0.471

3

-1.37

0.471

2.13

0.480

3

-1.95

0.369

III

0.021

0.007

3

-2.46

0.698

0.007

0.007

3

-3.55

1.68

0.011

0.003

3

-3.34

0.396

III

468

116

3

-1.31

0.252

702

160

3

-1.83

0.455

430

71.5

3

-1.24

0.115

Amine

III

60.5

13.6

3

-1.72

0.238

79.4

33.9

3

-1.88

0.915

66.2

5.57

3

-2.53

0.221

Cyclichydrocarbon

III

NA

NA

-

NA

NA

NA

NA

-

NA

NA

486

185

3

-2.88

1.99

ChemicalClass5Heterocyclic
Sulfurcompound;
Metal
Carboxylic
acid
Halogenatedhydrocarbon
Heterocyclic
Organometallic
compoundCarboxylicacid; Lipids

ECBC
1

FAL
1

IIVS
1

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; SD=Standard deviation;
N=Number of data points; NA=Not available (i.e., IC50 values or Hill Slope values could not be generated [see notes in Appendix I for more information])
1
Arithmetic mean.
2Standard deviation of IC50.
3Arithmetic Mean of Hill Slope values.
4Standard deviation of Hill Slope values.
5
Chemical class assigned is based on the classification of the National Library of Medicine’s Medical Subject Heading (MeSH), http://www.nlm.nih.gov/mesh/meshhome.html.

5-23

In Vitro Cytotoxicity Methods BRD Section 5

Figure 5-1

November 2006

Reference Substance IC50 Results for the 3T3 NRU Test Method by Laboratory

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in Medical Experiments Alternatives
Laboratory; IIVS=Institute for In Vitro Sciences.
Points show the mean arithmetic IC50 (µg/mL) for each reference substance from each laboratory. Error bars show the standard deviation. Data were
sorted in ascending order of 3T3 IC50 values from IIVS (lead laboratory in the validation study). Table 5-6 provides the numerical key for reference
substance identification.

5-24

In Vitro Cytotoxicity Methods BRD Section 5

Figure 5-2

November 2006

Reference Substance IC50 Results for the NHK NRU Test Method by Laboratory

Abbreviations: ECBC=Edgewood Biological Center; FAL=Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory;
IIVS=Institute for In Vitro Sciences.
Points show the mean arithmetic IC50 (µg/mL) for each reference substance from each laboratory. Error bars show the standard deviation. Data were
sorted in ascending order of 3T3 IC50 values from IIVS (lead laboratory in the validation study). Table 5-6 provides the numerical key for reference
substance identification.

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In Vitro Cytotoxicity Methods BRD Section 5

November 2006

Key to Validation Study Reference Substances1

Table 5-6No

Reference SubstanceNo

Reference SubstanceNo

Reference SubstanceNo

Reference Substance1

Aminopterin

19

Propylparaben

37

Strychnine

55

Citric acid2

Triphenyltin hydroxide20

Propranolol HCl

38

Phenylthiourea

56

Boric acid3

Colchicine

21

Dichlorvos

39

Lindane57

5-Aminosalicylic acid4

Cycloheximide

22

Potassium cyanide

40

Carbamazepine

58

Sodium hypochlorite

5

Triethylenemelamine

23

Physostigmine

41

Diethyl phthalate

59

Lactic acid6

Sodium dichromate
dihydrate

24

Dibutyl phthalate

42

Glutethimide

60

Potassium I chloride

7

Sodium arsenite

25

Parathion

43

Chloramphenicol

612-Propanol

8

Cadmium II chloride

26

Paraquat

44

Chloral hydrate

62

Sodium chloride

9

Hexachlorophene

27

Sodium selenate

45

Caffeine

63

Dimethylformamide

10

Mercury II chloride

28

Verapamil HCl

46

Digoxin64

Ethanol

11

Endosulfan

29

Acetaminophen

47

Meprobamate

65

Gibberellic acid12

Arsenic III trioxide

30

Busulfan48

Acetylsalicylic acid66

Acetonitrile

13

Diquat dibromide
monohydrate

31

Sodium oxalate

49

Nicotine

67

1,1,1-Trichloroethane

14

Haloperidol

32

Phenol50

Phenobarbital

68

Ethylene glycol

15

Cupric sulfate
pentahydrate

33

Disulfoton

51

Procainamide HCl

69

Glycerol16

Thallium I sulfate

34

Epinephrine bitartrate

52

Valproic acid70

Lithium I carbonate

17

Amitriptyline HCl

35

Atropine sulfate

53

Xylene71

Carbon tetrachloride

18

Fenpropathrin

36

Sodium I fluoride

54

Trichloroacetic acid72

MethanolAbbreviations: No=Number.
1
As used in Figures 5-1 and 5-2.

5.5

Statistical Approaches to the Evaluation of 3T3 and NHK Data

The statistical approaches used for data evaluation are reviewed in the following sections foreach phase of the validation study. Section 2.2.3 discussed the endpoint measurements forthe 3T3 and NHK test methods. The OD values of each of six replicate wells ([minimum offour] in the 96-well plate) per test concentration (eight concentrations/reference substance orPC) were used to determine relative cell viability in relation to the mean VC OD on the sameplate. The cell viability values calculated for the replicate wells for each concentration wereused to determine the concentration-response curve (percent viability vs. log concentration)for each test. The IC50 value was determined from fitting the curve to a Hill function.

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In Vitro Cytotoxicity Methods BRD Section 5

November 2006

5.5.1
Statistical Analyses for Phase Ia Data
The laboratories reported the IC50 results for SLS in µg/mL. The SMT used the results fromthe acceptable tests to calculate means and SDs for each method at each laboratory.
5.5.1.1 Outlier Determination for Replicate Well Concentration Data
A test for outliers at the 99% level (Dixon and Massey 1981) was used to determine thepresence of outlier OD values among the six replicate wells for each reference substanceconcentration. The SMT applied the outlier test to the Phase Ia data when extreme values
were noted. Outliers were excluded from the data set, and the IC50 was recalculated. The raw
data files include all data provided by the laboratories, including the excluded outlier OD
values. Because the protocol required a minimum of four acceptable test wells per reference
substance concentration, no more than two wells of the six replicates could be excluded.
5.5.1.2 Curve Fit Criteria
After the completion of Phase Ia testing, a curve fit criterion was implemented for test
acceptance following a visual review of the fit of the OD data to the Hill function curve. The
SMT considered the fit of the concentration-response curve to the Hill function to be
acceptable when R2 >0.9. A fit of R2 <0.8 was considered unacceptable and the data fromthat test were rejected. Curves with a fit of 0.8 < R2 <0.9 were evaluated visually for
goodness of fit and accepted if the SMT concluded that there were sufficient data points
between 0 and 100% cytotoxicity, and a reasonable shape to the curve, to calculate areasonably accurate IC50 value. Each test with a curve fit in this range was analyzed on acase-by-case basis, and no standard pass/fail criterion was developed. [Note: The use of acurve fit criterion was reevaluated in Phases Ib and II, and was eliminated as a test
acceptance criterion for Phase III test results. An R2 value ≥0.85 was maintained as a test
acceptance criterion for the PC because its fit to the Hill function was well characterized.]
5.5.1.3 Reproducibility Analyses for PC IC50 Values
To evaluate reproducibility of the IC50 values for the PC for each test method, within andbetween the laboratories, the SMT considered the American Society of Testing and Materials(ASTM) Standard E691-99, Standard Practice for Conducting an Interlaboratory Study to
Determine the Precision of a Test Method (ASTM 1999). This method uses two statistics, hand k, to judge the consistency of means and variances between laboratories. However, aminimum of six laboratories is required for this type of analysis and the SMT decided that it
could not be appropriately applied to three laboratories. The variability of the PC IC50 results
obtained from each test and laboratory was assessed using CV analysis and one-way
ANOVA. Dividing the SD by the arithmetic mean IC50 value, and multiplying by 100
produced the CV. CV values were calculated for the acceptable tests within each laboratory
to determine intralaboratory reproducibility. To compare the variation among laboratories,
the CV was calculated using the arithmetic mean IC50 values from each of the threelaboratories. Although no criterion for an acceptable CV was determined for this study,
ECVAM recently used CV <30% as an acceptable range for both intra- and inter-laboratory
reproducibility (Zuang et al. 2002; Fentem et al. 2001). Although CV <30% was intended toreflect an acceptable maximum for normal biological variability, the range was not supported
by data.
For the ANOVA, IC50 values were first converted to mM units and then log-transformed toobtain normal distributions. One-way ANOVA was performed with SAS PROC GLM
5-27

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

software (SAS Institute 1999; see Appendix D1 for example SAS code). A significance levelof p <0.01 was used to test results between the laboratories in order to be conservative withrespect to identifying laboratory differences.
5.5.2

Statistical Analyses of Phase Ib Data

5.5.2.1 Outlier Determination for Replicate Well Concentration Data
For consistency of replicate well concentration data, the SMT applied the same outlier test
used for the Phase Ia data (Dixon and Massey 1981) when extreme OD values were noted. If
the extreme value was an outlier at the 99% level, it was excluded from the data set, and theIC50 was recalculated. All data are available in the data files provided by the laboratories,
including the excluded outlier OD values.
5.5.2.2 Reproducibility Analyses of the Reference Substance IC50 Values
One-way ANOVA and CV analyses were used to assess method reproducibility within andamong laboratories. For the ANOVA, the IC50 values were first converted to mM units andthen log-transformed to obtain normal distributions. One-way ANOVA was performed withSAS PROC GLM (SAS Institute 1999; see Appendix D1 for example SAS code). A
significance level of p <0.01 was used to test results between the laboratories in order to beconservative with respect to identifying laboratory differences. When the ANOVA detectedsignificant differences among the laboratories, contrast analyses were performed todetermine which laboratory was different from the others. These analyses compared theresults of each laboratory with those of the other two laboratories. A significant difference in
response among the laboratories was indicated by p <0.01.
CV values were calculated for each reference substance by dividing the SD by the arithmetic
mean IC50 value and multiplying by 100. CV values were calculated for the acceptable tests
in each laboratory to determine intralaboratory reproducibility. To compare the variation
among laboratories, the CV was calculated using the arithmetic mean IC50 values from eachof the three laboratories.
As an additional approach to the assessment of interlaboratory reproducibility for each test
substance, the maximum:minimum IC50 ratios (i.e., the maximum arithmetic mean laboratory
IC50 value compared to the minimum arithmetic mean laboratory IC50 value) were calculated.
This approach is similar to the calculation of maximum:minimum LD50 ratios for examining
reproducibility of reference LD50 values (see Section 4.4.1).
5.5.3

Statistical Analyses of Phase II Data

5.5.3.1 Outlier Determination for Replicate Well Concentration Data
The Dixon and Massey (1981) outlier test was incorporated into the EXCEL® templates toassess the consistency of replicate well data for each reference substance concentration.
Outliers at the 99% level were highlighted and the Study Director was offered the option ofremoving the value from subsequent calculations (e.g., mean OD of the six replicates; %viability; IC50).
5.5.3.2 Reproducibility Analyses of the Reference Substance IC50 Values
The intra- and inter-laboratory reproducibility of the IC50 values were assessed using theacceptable tests to calculate the mean IC50, SD, and CV for each substance, method, and5-28

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

laboratory, as described in Section 5.5.2.2. One-way ANOVAs and calculations ofmaximum:minimum IC50 ratios were performed as described in Section 5.5.2.2.
5.5.3.3 Comparison of 3T3 and NHK Test Results with the RC Millimole Regression
To compare the 3T3 and NHK test results for the reference substances to those of the RC
millimole regression, each IC50 value was transformed to mM units for the calculation ofgeometric mean IC50 values. The use of geometric means corresponded with the approach
used to obtain single IC50 values from multiple IC50 values for the RC millimole regression(Halle 1998, 2003). The log geometric mean IC50 values (in mM) of the 11 RC substancestested during Phases Ib and II (see Table 3-8) were used with the log RC LD50 values, after
transformation to log mmol/kg units (see Appendices J1 and J2), to calculate least squares
linear regressions for the data from each test method and laboratory. Each of thesemethod/laboratory regressions was compared to the RC millimole regression using an F testwith SAS PROC REG (SAS Institute 1999; see Appendix D2 for example SAS code). An F
test with a significance level of p <0.01 was used to determine whether the joint comparisonof slope and intercept indicated that the method/laboratory regressions were significantly
different from the RC millimole regression.
As an alternate analysis, a least squares linear regression using IC50 and LD50 values from the
RC was constructed for the 11 RC substances (the RC-11 regression) tested in Phases Ib andII. Each of these method/laboratory regressions was compared to the RC-11 regression using
an F test with SAS PROC GLM (SAS Institute 1999; see Appendix D2 for example SAScode) at a significance level of p <0.01. This was used to determine whether the comparisonsof slope and intercept indicated that the laboratory regressions were significantly different
from the RC-11 regression.
5.5.4

Statistical Analyses of Phase III Data

5.5.4.1 Outlier Determination for Replicate Well Concentration Data
The laboratories used the Dixon and Massey (1981) outlier test at the 99% level that wasincorporated into the EXCEL® templates to test for outlier values among replicate well dataat the different reference substance concentrations. The Study Director had the option of
excluding the outliers from the data set, which were highlighted by the template, andsubsequent calculations. All data are available in the data files provided by the laboratories,
including the outlier OD values.
5.5.4.2 Reproducibility Analyses of the PC IC50 Data
A number of analyses were performed to determine whether the SLS IC50 values werereproducible across study phases. The SLS IC50 values used to access variability weredifferent from those shown in Table 5-3. To get an assessment of the true variation of SLS
IC50 values, the reproducibility analyses included additional IC50 values from SLS tests that
did not meet the IC50 acceptance limits (see Table 5-3) for each laboratory and study phase if
they passed all other test acceptance criteria. If more than one SLS test was performed on asingle day (for any test method and laboratory), the IC50 values were averaged to determine asingle IC50 for the day. This prevented multiple data values from a single day from overly
influencing the mean for each phase. CV analyses were performed as described in Section
5.5.1 using the arithmetic mean SLS IC50 values for each method, laboratory, and study
phase.

5-29

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

For the remaining analyses of reproducibility, the IC50 values were first log-transformed toobtain normal distributions. One-way ANOVAs were performed with SAS PROC GLM(SAS Institute 1999; see Appendix D1 for example SAS code) for each method using study
phase and laboratory as individual variables. A significance level of p <0.01 was used to test
for a statistical difference among the laboratory and/or phase results.
To determine whether there was a linear time trend for the SLS IC50 data, linear regression
analyses using a least squares method were performed for each laboratory and method using
SAS PROC REG (SAS Institute 1999). Time was expressed as an index for each test. The
index number of each SLS test reflected its order of testing without respect to the time
lapsing between tests. For example, the first SLS test was assigned a time index of 1 and thesecond SLS test was assigned a time index of 2 whether it occurred the day after the first testor one week after the first test. The slopes of the linear regressions were judged to bestatistically significant at p <0.05, which indicated that the IC50 had changed significantly
over time.
5.5.4.3 Reproducibility Analyses of the Reference Substance IC50 Values
CV, one-way ANOVA analyses, and maximum:minimum IC50 ratios were performed toassess the intra- and/or inter-laboratory reproducibility of the Phase III reference substancedata, as described in Section 5.5.2.2. An additional evaluation to determine whether
normalizing the reference substance IC50 to the SLS IC50 would reduce interlaboratory
variability was performed using five substances (for each test method) for which the
ANOVAs indicated significant interlaboratory differences. The reference substance IC50
values were normalized to the SLS IC50 by calculating the reference substance IC50:SLS IC50
ratio. CVs were calculated for each substance using the mean ratios from each laboratory. To
determine whether this normalization reduced variability among the laboratories, the CVs forthe substance IC50:SLS IC50 ratios were compared to the CVs for the substance IC50.
In addition, the geometric mean IC50 values were used to calculate least squares linear
regression models after log transforming the data. Linear regressions were fit for eachmethod and laboratory using the log-transformed reference LD50 values from Table 4-2 (inmmol/kg), with log IC50 in mM. To detect differences among the linear regressions in eachlaboratory, two models were fit for each method. The first was a full model that included
effects for laboratory and interactions, and generated a regression line for each substance ineach laboratory, by test method. The second model, which was considered to be a reduced
model, assumed that one model fit all the laboratories. A goodness of fit F test was
performed to compare the full and reduced models for each method. A significance level of p
<0.01 was used to test whether the regressions among laboratories were significantly
different from one another. The following criteria were established for selection of data foruse in the regression analyses for each test method:• The substance was included in the RC• All three laboratories reported IC50 values• There was an associated rat oral reference LD50 value (see Table 4-2)
There were 47 reference substances that fit these criteria for the 3T3 and 51 test substancesthat fit the criteria for the NHK test methods.

5-30

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

5.5.4.4 Comparison of 3T3 and NHK Results with the RC Millimole Regression
To determine whether the IC50 values determined in the validation study were significantly
different from the RC values, the laboratory-specific regression values for each method werecombined using the geometric means of the laboratory-specific geometric mean IC50 values
in mM and the reference LD50 in mmol/kg. Thus, there was one regression analysis withpooled laboratory data for the 3T3 NRU test method and another regression analysis (alsowith pooled data) for the NHK NRU test method. A third linear regression was calculatedusing the IC50 and LD50 values from the RC. The IC50 values and LD50 values were logtransformed for the regression calculations. The following criteria were established for theselection of substances to be used for the regression analyses:• The substance was included in the RC• All three laboratories reported IC50 values for both the 3T3 and NHK NRU
test methods• There was an associated rat oral reference LD50 value (see Table 4-2)
Forty-seven substances met these criteria. Two models were fit for each test method to detect
differences between the NRU regression and the 47 RC substance regression. The firstregression model was a full model that included effects for the RC and the NRU regression,and generated one regression line each for the RC and the NRU test method. The second(reduced) model assumed that a single model fit the combined RC and NRU IC50 data. The
RC regression for the 47 reference substances was compared to the combined laboratory
regression for each NRU test method using an F test to simultaneously compare slopes andintercepts. The NRU regressions were statistically different from the RC regressions if
p <0.01.
To assess the accuracy of the NRU methods and the associated IC50-LD50 regressions, apredicted LD50 was calculated for each reference substance using its laboratory geometricmean IC50 in two analyses:• The RC rat-only millimole regression calculated from the 282 RC substanceswith rat LD50 values, using units of mM for the IC50 and mmol/kg for the LD50
(see Section 6.4.2)• The RC rat-only weight regression calculated from the 282 RC substanceswith rat LD50 values, using units of µg/mL for the IC50 and mg/kg for the
LD50 (see Section 6.4.3)
The LD50 values predicted from the regression analyses were used to predict GHS acute oral
toxicity categories (see Section 6.4). The accuracy of the predictions was determined by
calculating the proportion of substances for which the predicted GHS toxicity category
matched the GHS toxicity category. The LD50 predictions from these regression models werealso used to determine starting doses for acute systemic toxicity test simulations for the
purpose calculating animal use and savings that would be achieved using the NRU test
methods. The simulation modeling methods, and results from the UDP and ATC methods,
are described in Section 10.

5-31

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

5.5.5
Summary of the Data Used for Statistical Analyses
Table 5-7 summarizes the number of substances that were tested and the number ofsubstances used for the various analyses performed to determine the accuracy and reliabilityof the in vitro NRU test methods.
Table 5-7

Datasets Used for Validation Study Analyses1
3T3 NRU
Test
Method1
72

NHK NRU
Test
Method1
72

47

51

47

47

67

68

Reproducibility of acceptable rat
oral LD50 values

NA

NA

Reproducibility of IC50 values

64

68Comparison of reproducibility of
IC50 values with reproducibility ofLD50 values

53

57

Use
TestingComparison of laboratory IC50LD50 regressions to one another
Comparison of combinedlaboratory IC50-LD50 regressions
to a regression calculated withRC data
Prediction of GHS accuracy using
IC50 values in IC50-LD50
regressions; prediction of starting
doses for acute oral toxicity test
(UDP and ATC) simulations

Characteristics of Dataset
Substances tested
RC substances with IC50 values
from all laboratories andreference rat oral LD50 values
RC substances with IC50 values
for both test methods from alllaboratories and rat oralreference LD50 values
Substances with IC50 values
from at least one laboratory
62 substances with more than
one acceptable rat oral LD50
value
Substances with IC50 values
from all laboratories
Substances with IC50 values
from all laboratories and morethan one acceptable rat oral LD50
value

Abbreviations: RC=Registry of Cytotoxicity; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal
keratinocytes; NRU=Neutral red uptake; NA=Not applicable.
1
Number of substances.

5.6

Summary of NRU Test Results

Table 5-8 shows the 3T3 and NHK IC50 values as geometric means of the geometric mean
laboratory values, as a basis to compare the 3T3 and NHK NRU IC50 values for eachreference substance. The substances in Table 5-8 are organized by ascending 3T3 NRU IC50
values (as was done for Figures 5-1 and 5-2). For each method, the table provides thegeometric mean IC50 (combined across laboratories) in µg/mL, the ratio of the geometricmean IC50 to the SLS IC50, and the 3T3 IC50:NHK IC50 ratios. Geometric means were used
for this comparison because they were used for both the IC50 and LD50 regression analyses(see Sections 5.5.3.3, 5.5.4.3, and 5.5.4.4). The 3T3 and NHK NRU IC50 values werecompared using the ratios of their geometric means. The IC50 values for each reference
substance were also compared to the IC50 for SLS using the ratio of reference substancegeometric mean IC50 to SLS geometric mean IC50.
5-32

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-8

November 2006

Comparison of 3T3 and NHK NRU IC50 Geometric Means
3T3 NRU

NHK NRU

Reference SubstanceGeometric
Mean1 IC50(µg/mL)

Ratio
Geometric
Mean IC50
to SLS IC50

Geometric
Mean1 IC50(µg/mL)

Ratio
Geometric
Mean IC50
to SLS IC50

IC50 Ratios
3T3:NHK

Carbon tetrachloride
MethanolAminopterin
Triphenyltin hydroxide
Colchicine
Cycloheximide
Triethylenemelamine
Cadmium II chloride
Sodium dichromate dihydrate
Sodium arsenite
Arsenic trioxide
Mercury II chloride
Hexachlorophene
Thallium I sulfate
Haloperidol
Endosulfan
Amitriptyline HCl
Diquat dibromide monohydrate
Propranolol
Dichlorvos
Paraquat
Fenpropathrin
Physostigmine
Propylparaben
Sodium selenate
Potassium cyanide
Verapamil HCl
Parathion
Sodium oxalate
Sodium lauryl sulfate (SLS)*
Cupric sulfate pentahydrate
Acetaminophen
Dibutyl phthalate
Epinephrine bitartrate
PhenolAtropine sulfate
BusulfanSodium I fluoride
Phenylthiourea
Carbamazepine

NA
NA
0.006
0.017
0.034
0.187
0.272
0.518
0.587
0.759
1.96
4.12
4.19
5.74
6.13
6.35
7.05
8.04
13.9
17.7
20.1
24.2
25.8
26.1
29
34.6
34.9
37.4
37.7
41.7
42.1
47.7
49.7
59
66.3
76
77.7
78
79
103

NA
NA
0.00010.0004
0.001
0.004
0.007
0.01
0.01
0.02
0.05
0.1
0.1
0.1
0.1
0.2
0.2
0.2
0.3
0.4
0.5
0.6
0.6
0.6
0.7
0.8
0.8
0.9
0.9
1.0
1.0
1.1
1.2
1.4
1.6
1.8
1.9
1.9
1.9
2.5

NA
15293
669
0.01
0.007
0.073
1.85
1.84
0.721
0.477
5.26
5.8
0.029
0.152
3.36
2.13
8.96
4.48
35.3
10.7
61.6
2.43
88.5
16.6
10.2
29
66.5
30.3
337
3.99
197
518
28.7
87.4
75
81.8
260
49.8
336
83.2

NA
383.2
167.7
0.003
0.002
0.02
0.5
0.5
0.2
0.1
1.3
1.5
0.01
0.04
0.8
0.5
2.2
1.1
8.8
2.7
15.4
0.6
22.2
4.2
2.6
7.3
16.7
7.6
84.5
1.0
49.4
129.8
7.2
21.9
18.8
20.5
65.2
12.5
84.2
20.9

NA
NA
0.00001
1.7
4.9
2.6
0.1
0.3
0.8
1.6
0.4
0.7
144.5
37.8
1.8
3.0
0.8
1.8
0.4
1.7
0.3
10.0
0.3
1.6
2.8
1.2
0.5
1.2
0.1
10.5
0.2
0.1
1.7
0.7
0.9
0.9
0.3
1.6
0.2
1.2

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In Vitro Cytotoxicity Methods BRD Section 5

Table 5-8

November 2006

Comparison of 3T3 and NHK NRU IC50 Geometric Means
3T3 NRU

Reference Substance
Diethyl phthalate
Lindane
Chloramphenicol
Disulfoton
Caffeine
Strychnine
Glutethimide
Chloral hydrate
Nicotine
Procainamide HCl
Digoxin
Meprobamate
Lithium I carbonate
Phenobarbital
Acetylsalicylic acid
XyleneCitric acid
Trichloroacetic acid
Valproic acid
Sodium hypochlorite
5-Aminosalicylic acid
Boric acid
Lactic acid
Potassium I chloride
2-Propanol
Sodium chloride
Dimethylformamide
Ethanol
Gibberellic acid
Acetonitrile
1,1,1-Trichloroethane
Ethylene glycol
GlycerolNHK NRU

Geometric
Mean1 IC50(µg/mL)

Ratio
Geometric
Mean IC50
to SLS IC50

Geometric
Mean1 IC50(µg/mL)

Ratio
Geometric
Mean IC50
to SLS IC50

IC50 Ratios
3T3:NHK

107
108
128
133
153
158
174
183
361
441
466
519
5622
573
676
7212
796
902
916
1103
1667
1850
3044
3551
3618
4730
5224
6523
78103
7951
17248
24317
24655

2.6
2.6
3.1
3.2
3.7
3.8
4.2
4.4
8.7
10.6
11.2
12.4
13.5
13.7
16.2
17.3
19.1
21.6
22.0
26.5
40.0
44.4
73.0
85.2
86.8
113.4
125.3
156.4
187.3
190.7
413.6
583.1
591.2

120
18.7
348
270
638
62.5
174
133
107
1741
0.001
357
468
448
605
4662
400
413
512
1502
46.7
421
1304
2237
5364
1997
7760
10018
2856
9528
81222
41852
24730

30.1
4.7
87.2
67.7
159.9
15.7
43.6
33.3
26.8
436.3
0.0003
89.5
117.3
112.3
151.6
116.8
100.3
103.5
128.3
376.4
11.7
105.5
326.8
560.7
1344.4
500.5
1944.92510.8715.82388.02035.610489.2
6198.0

0.9
5.8
0.4
0.5
0.2
2.5
1.0
1.4
3.4
0.3
466000.0
1.5
1.2
1.3
1.1
1.5
2.0
2.2
1.8
0.7
35.7
4.4
2.3
1.6
0.7
2.4
0.7
0.7
2.7
0.8
2.1
0.6
1.0

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake;
SLS=Sodium lauryl sulfate; NA=Not available.
Reference substances are ordered by 3T3 NRU IC50 values.
1
Geometric mean IC50 of the laboratory geometric mean values.
2
Data available from only one laboratory.
3
Data available from only two laboratories.
*Acceptable positive control (SLS) values from all study phases: N=293 for the 3T3 NRU and N=281 for the NHK NRU.

5-34

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

Table 5-8 shows that there are nine reference substances for which the 3T3 and NHK NRU
IC50 values differ by at least one order of magnitude (i.e., 3T3 IC50:NHK IC50 ≤0.1 or ≥10):
aminopterin, triethylenemelamine, hexachlorophene, thallium sulfate, fenpropathrin, sodium
oxalate, acetaminophen, digoxin, and 5-aminosalicylic acid. The IC50 values for SLS, also
differed by slightly more than one order of magnitude in the two NRU test methods (41.7µg/mL for 3T3 and 3.99 µg/mL for NHK). One test method was not more consistently
sensitive (i.e., produced lower IC50 values) than the other for these nine reference substances.
The 3T3 NRU test method was more sensitive than the NHK NRU test method for four of
the nine substances: aminopterin, triethylenemelamine, sodium oxalate, and acetaminophen.
The NHK NRU test method was more sensitive than the 3T3 NRU test method for five
substances: hexachlorophene, thallium sulfate, fenpropathrin, digoxin, and 5-aminosalicylic
acid. Despite the normalization procedure, the reference substance IC50:SLS IC50 ratios forthe two methods were still greater by at least one order of magnitude for six of the nine
substances (aminopterin, triethylenemelamine, hexachlorophene, sodium oxalate,acetaminophen, and digoxin) and the order of magnitude difference increased for all six
substances. A number of factors could potentially be responsible for these differences
between the 3T3 and NHK NRU IC50 values:• Cell culture conditions (i. e., the 3T3 treatment medium contains serum while
the NHK treatment medium does not; differences in cell density in thetreatment medium)• Differences in sensitivity between the fibroblast cell line and primary
keratinocytes• Differences in sensitivity between human and mouse cells• Differences in metabolic activity between the cell types
These factors may affect the results for some substances more than others. For example, asubstance that binds to serum proteins would be less available to the 3T3 cells (which haveserum in their growth medium) than to NHK cells (which are grown without serum). No
additional testing was performed to investigate the differences between the 3T3 and NHK
NRU IC50 values.
Two substances, digoxin and aminopterin, have IC50 values that differ by five orders ofmagnitude between the two NRU test methods. Digoxin was much more toxic to the NHK
cells and aminopterin was more toxic to the 3T3 cells. Both substances are known substrates
for organic anionic transporters (OAT) (ICCVAM 2006). Such transporters are important forin vivo toxicity responses in terms of the ability of challenge substances to be absorbed, reachtarget tissues, accumulate, or be excreted. The differential susceptibilities of the 3T3 andNHK cells may be explained by differential functioning of OAT between the cell types.
Although species and tissue differences in OAT have been reported (Sekine et al. 2000;
Miyazaki et al. 2004), the reason for these differential sensitivities is not known.
The 3T3 IC50:NHK IC50 ratios shown in Table 5-8 were used to determine the frequency
distributions shown in Table 5-9. These distributions indicate that the 3T3 and NHK NRU
IC50 values were within one order of magnitude of each other for 85% of the reference
substances (obtained by adding 38.9% and 45.8% for the 0.1 < IC50 ratio ≤1 and 1 < IC50
ratio <10 ranges). Ninety-three percent of the reference substances have 3T3 and NHK NRU
5-35

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

IC50 values within two orders of magnitude of each other (obtained by adding 4.2% each forthe 10 ≤ IC50 ratio ≤100 and 0 < IC50 ratio ≤0.1 ranges to the 85% above).
Table 5-9

Frequency of 3T3:NHK IC50 Ratios1 for Reference Substances

IC50 Ratio <0.00001

Number of
Substances
1% ofSubstances
1.4

0 < IC50 Ratio ≤0.1

3

4.2

0.1 < IC50 Ratio ≤1

28

38.9

1 < IC50 Ratio <10

33

45.8

10 ≤ IC50 Ratio <1003

4.2

100 ≤ IC50 Ratio <1000

1

1.4

IC50 Ratio ≥1000

1

1.4

Not Available

2

2.8

3T3:NHK IC50 Ratio Range

Abbreviations: 3T3=Neutral red uptake using BALB/c 3T3 fibroblasts; NHK= Neutral
red uptake using normal human epidermal keratinocytes.
Note: Compiled using reference substance data from Table 5-7.

Correlations of the mean IC50 values for the reference substances common to the RC
database with the IC50 values (i.e., geometric mean of IC50 values obtained from the literature
for various basal cytotoxicity endpoints and cell types) from the RC (Halle 1998, 2003) are
shown in Figure 5-3 (3T3 values) and Figure 5-4 (NHK values). Although the validation
study tested 58 RC substances in common with the RC, IC50 values were obtained for 56
substances using the 3T3 NRU test method and 57 substances using the NHK NRU test
method. Spearman correlation analyses of the log-transformed IC50 data (in mM) indicatedthat the NRU IC50 values were significantly correlated with the RC IC50x values (p<0.001, forboth the 3T3 and NHK NRU test methods). The Spearman correlation coefficient, rs, was
0.93 for the 3T3 values and 0.86 for the NHK values.

5-36

In Vitro Cytotoxicity Methods BRD Section 5

Figure 5-3
1000
100

November 2006

RC IC50 Values vs 3T3 NRU IC50 Values for 56 Substances in Common
rs = 0.93
n = 56

10
1
0.1
0.01
0.001
0.00010.00001
0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

Registry of Cytotoxicity IC50 (mM)

Abbreviations: RC=Registry of Cytotoxicity; 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red uptake;rs=Spearman correlation coefficient; n=Number of substances; mM=Millimolar.
The diagonal line indicates the predicted values for a 1:1 correspondence. No IC50 values were obtained forcarbon tetrachloride or methanol because of insufficient toxicity. The Registry of Cytotoxicity IC50 values aregeometric means of IC50 values obtained from the literature for various basal cytotoxicity endpoints and cell
types.

5-37

In Vitro Cytotoxicity Methods BRD Section 5

Figure 5-4
1000
100

November 2006

RC IC50 Values vs NHK NRU IC50 Values for 57 Substances in Common
rs = 0.86
n = 57

10
1
0.1
0.01
0.001
0.00010.00001
0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

Registry of Cytotoxicity IC50 (mM)

Abbreviations: RC=Registry of Cytotoxicity; NHK=Normal human epidermal keratinocytes; NRU=Neutral red
uptake; rs=Spearman correlation coefficient; n=Number of substances; mM=Millimolar.
The diagonal line indicates the predicted values for a 1:1 correspondence. No IC50 values were obtained formethanol because of insufficient toxicity. The Registry of Cytotoxicity IC50 values are geometric means of IC50
values obtained from the literature for various basal cytotoxicity endpoints and cell types.

5.7

Availability of Data

All data were provided to the SMT as electronic files and paper copies. The laboratories also
maintained copies of all raw data and the electronic files. The individual test data and IC50
results for both passing and failing tests are provided in Appendix I for the reference
substances and the PC.
5.8

Solubility Test Results

A solubility protocol (see Section 2-8 and Appendix B3) designed to identify the solventthat would provide the highest concentration of a reference substance for in vitro testing wasevaluated. Each laboratory performed solubility tests on all reference substances. However,to avoid the use of different solvents by the laboratories when testing the same substance,
which might increase the variability of the IC50 results among the laboratories, the SMT
assigned the solvents to be used (see Table 5-10). The objectives of the solubility testing
were to evaluate the utility and appropriateness of the solubility protocol, and to evaluate theconcordance among laboratories in selecting the solvents for each of the 72 reference
substances.

5-38

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-10

November 2006

Solubility Test Results (mg/mL)
BioReliance1

Reference Substance

ECBC3

FAL3

IIVS3

2

3T34
NHK5
DMSO
Medium Medium

ETOH

SMT
Selection

3T34
NHK5
3T34
NHK5
3T34
NHK5
DMSO ETOHDMSO ETOHDMSO ETOH
Medium Medium
Medium Medium
Medium Medium

Phase I
Arsenic III trioxide

0.25

0.05

<2

<2

Medium

0.02560.0256<0.2<0.2

0.13560.1356<0.2<0.2<0.026<0.026<0.2<0.2

Ethylene glycol

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Propranolol HCl

<2

10

200

20

DMSO

0.2

2

200

NT

20

20

200

NT

20

2

NT

NT

Aminopterin

2

2

NT

NT

DMSO

2.0

<2

200

NT

<2

2

200

NT

0.2

0.2

200

NT

Cadmium II chloride

<2

<2

200

<200

DMSO

<2

<2

200

NT

<2

<2

200

NT

<0.2

<0.2

20

<20

Chloramphenicol

2

2

400

<200

DMSO

2.0

<2

200

NT

<2

<2

200

NT

0.2

0.2

20

20

Colchicine

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Lithium I carbonate

0.25

10

<2

NT

Medium

0.2

2.0

<20

<20

0.2

2

<200

<200

0.2

2

<2

<2

Potassium I chloride

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

2-Propanol

400

400

400

400

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Sodium I fluoride

20

20

<200

<200

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Sodium selenate

200

200

<200

<200

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Acetaminophen

10

10

400

<200

DMSO

2

2

NT

NT

2

2

NT

NT

<2

<2

200

NT

Acetonitrile

400

400

400

400

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Acetylsalicylic acid

10

10

400

200

DMSO

2

2

NT

NT

<2

<2

200

NT

2

2

NT

NT

5-Aminosalicylic acid

2

2

<200

<200

Medium

2

2

NT

NT

2

2

NT

NT

2

2

NT

NT

Amitriptyline HCl

200

200

NT

NT

DMSO

<2

<2

200

NT

<2

<2

200

NT

0.2

0.2

200

NT

Phase II

Phase III

5-39

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-10

November 2006

Solubility Test Results (mg/mL)
BioReliance1

Reference Substance

ECBC3

FAL3

IIVS3

2

3T34
NHK5
DMSO
Medium Medium

ETOH

SMT
Selection

3T34
NHK5
3T34
NHK5
3T34
NHK5
DMSO ETOHDMSO ETOHDMSO ETOH
Medium Medium
Medium Medium
Medium Medium

Atropine sulfate

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Boric aid

40

40

200

<200

Medium

20

20

NT

NT

20

20

NT

NT

2

2

NT

NT

Busulfan

<2

<2

40

<200

DMSO

<2

<2

200

NT

<2

<2

506

<200

<0.2

<0.2

20

<200

Caffeine

10

10

20

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Carbamazepine

<2

<2

40

<200

DMSO

0.2

0.2

20

20

<2

<2

200

NT

<0.2

<0.2

2

<20

Carbon tetrachloride

2

10

NT

NT

DMSO

20

20

NT

NT

<0.2

<0.2

2

NT

20

20

NT

NT

Chloral hydrate

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Citric acid

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Cupric sulfate
pentahydrate

1

0.5

<2

2

Medium

2

0.2

<200

<200

2

2

NT

NT

0.2

0.2

<200

NT

Cycloheximide

20

20

400

<200

Medium

20

20

NT

NT

20

20

NT

NT

2

2

NT

NT

Dibutyl phthalate

<2

<2

400

400

DMSO

<2

<2

200

NT

<2

<2

200

NT

<2

<2

200

NT

Dichlorvos

10

10

NT

NT

DMSO

2

2

NT

NT

<2

<2

200

NT

2

2

NT

NT

Diethyl phthalate

<2

<2

400

400

DMSO

<2

<2

200

NT

0.2

<0.2

200

NT

<2

<2

200

NT

Digoxin

0.05

0.05

200

< 200

DMSO

<2

<2

200

NT

<0.2

<0.2

200

NT

<2

<2

200

NT

Dimethylformamide

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Diquat dibromide
monohydrate

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Disulfoton

<2

<2

500

NT

DMSO

<2

<2

200

NT

<2

<2

200

NT

<2

<2

200

NT

Endosulfan

<0.05

<0.05

40

NT

DMSO

<0.2

<0.2

20

<200

<0.2

<0.2

2

<200

<0.2

<0.2

20

<200

Epinephrine bitartrate

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

2

2

NT

NT

Ethanol

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

5-40

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-10

November 2006

Solubility Test Results (mg/mL)
BioReliance1

Reference Substance

ECBC3

FAL3

IIVS3

2

3T34
NHK5
DMSO
Medium Medium

ETOH

SMT
Selection

3T34
NHK5
3T34
NHK5
3T34
NHK5
DMSO ETOHDMSO ETOHDMSO ETOH
Medium Medium
Medium Medium
Medium Medium

Fenpropathrin

<20

<20

500

NT

DMSO

<2

<2

200

NT

<0.2

<0.2

200

NT

<2

<2

200

NT

Gibberellic acid

10

10

NT

NT

Medium

2

2

NT

NT

2

2

NT

NT

2

2

NT

NT

Glutethimide

<2

<2

500

NT

DMSO

<2

<2

200

NT

<2

<2

200

NT

<2

<2

200

NT

Glycerol

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Haloperidol

<20

<20

40

NT

DMSO

<0.2

<0.2

20

<20

<0.2

<0.2

20

<20

<2

<2

20

<20

Hexachlorophene

0.05

<0.05

400

400

DMSO

<2

<2

200

NT

<2

<2

200

NT

<2

<2

200

NT

Lactic acid

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Lindane

<0.05

<0.05

400

<200

DMSO

<2

<2

200

NT

<2

<2

200

NT

<0.2

<0.2

20

<200

1

1

200

NT

DMSO

2

2

200

NT

2

2

200

NT

<0.2

<0.2

200

NT

Mercury II chloride

0.125

0.125

400

<200

DMSO

<2

<2

200

NT

<2

<2

200

NT

<0.2

<0.2

200

NT

Methanol

40

40

400

400

DMSO

20

20

NT

NT

20

20

NT

NT

<2

<2

200

NT

Nicotine

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Paraquat

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Parathion

0.05

<0.05

400

400

DMSO

<2

<2

200

NT

<2

<2

200

NT

<2

<2

200

NT

Phenobarbital

2

2

200

<200

DMSO

2

2

NT

NT

<2

<2

200

NT

<2

<2

200

NT

Phenol

40

40

400

400

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Phenylthiourea

2

2

400

<200

DMSO

2

<2

200

NT

20

20

NT

NT

<2

<2

200

NT

Physostigmine

2

2

400

200

DMSO

2

2

NT

NT

<2

<2

200

NT

<2

<2

200

NT

Potassium cyanide

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Procainamide HCl

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Meprobamate

5-41

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-10

November 2006

Solubility Test Results (mg/mL)
BioReliance1

Reference Substance

ECBC3

FAL3

IIVS3

2

3T34
NHK5
DMSO
Medium Medium

ETOH

SMT
Selection

3T34
NHK5
3T34
NHK5
3T34
NHK5
DMSO ETOHDMSO ETOHDMSO ETOH
Medium Medium
Medium Medium
Medium Medium

Propylparaben

0.25

0.25

400

400

DMSO

<2

<2

200

NT

<2

<2

200

NT

<2

<2

200

NT

Sodium arsenite

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Sodium chloride

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Sodium dichromate
dihydrate

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Sodium hypochlorite

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Sodium oxalate

<0.05

20

0.125

<0.05

Medium

<0.2

20

0.2

<2

20

20

NT

NT

<0.2

<0.2

<0.2

<0.2

Strychnine

<2

<2

2

2

Medium

0.2

<0.2

2

2

0.2

0.2

<200

<200

<0.2

<0.2

<0.2

<0.2

Thallium I sulfate

1

0.5

<2

<2

Medium

0.2

0.2

<200

<200

<0.2

<0.2

<0.2

<0.2

0.2

0.2

<20

<200

Trichloroacetic acid

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

1,1,1-Trichloroethane

10

10

400

400

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Triethylenemelamine

<2

<2

2

<20

DMSO

0.2

0.2

<200

<200

<0.2

<0.2

2

<2

<0.2

<0.2

<0.2

<0.2

Triphenyltin hydroxide <0.05

<0.05

10

<20

DMSO

<0.2

<0.2

2

<20

<0.2

<0.2

2

<200

<2

<2

2

<20

Valproic acid

10

2

NT

NT

DMSO

2

2

NT

NT

<2

<2

200

NT

2

<2

200

NT

Verapamil HCl

<0.05

0.25

200

NT

DMSO

<2

<2

200

NT

<2

<2

200

NT

<0.2

<0.2

20

NT

1

1

500

NT

DMSO

<2

<2

200

NT

2

<2

200

NT

<2

<2

200

NT

XyleneAbbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; SMT=Study Management Team; ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; DMSO=Dimethyl sulfoxide; ETOH=ethanol; NT=Not tested.
Note: Table sorted by study phase and alphabetical by substance.
1
The solubility protocol used was different from that used by the testing laboratories.
2
Solvents selected by the SMT for cytotoxicity testing. The BioReliance results were used to determine solvents for Phases I and II. Results from all laboratories were used to determine solvents for Phase III. 3T3 and NHK media
were treated as a single solvent. If a substance insoluble in one medium, and not the other, and soluble in DMSO, then DMSO was selected for use with both cell types.
3
Used protocol in Figure 2-7.
4Dulbecco’s Modification of Eagle’s Medium.
5
Keratinocyte Growth Medium (KGM from CAMBREX Clonetics®).
6
The results were obtained using a deviation from the standard protocol.
Laboratories agreed on solvent.
Laboratories did not agree on solvent. boldProtocol did not provide enough guideline information to select a single solvent.

5-42

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

5.8.1
Solubility Data
BioReliance evaluated the solubility of the reference substances, first in media, then in
DMSO, and then in ETOH, at 400 and 200 mg/mL. Based on their experience, a solubility
protocol was developed for the testing laboratories. This revised protocol required testing at
lower concentrations, and use of the various solvents at concentrations that would beequivalent when applied to the cell cultures (see Table 2-5). The solubility flow chart(Figure 2-7) illustrates the tests for solubility in 3T3 and NHK medium, DMSO, and ETOH.
Table 5-10 provides the solubility test results.
5.8.2
Solubility and Volatility Effects in the Cytotoxicity Tests
The laboratories reported solubility results for the stock solutions of reference substance foreach 3T3 and NHK test. Prior to the addition of the NR dye medium, the laboratories visuallyobserved the test cultures and documented noticeable precipitate. Table 5-11 illustrates theexistence of solubility issues (in both the 3T3 and NHK NRU test methods) as evidenced by
the observation of precipitates with some reference substances. Sections 3.2.6 and 5.4.2
provide additional information on ability of the laboratories to achieve sufficient toxicity forthe calculation of an IC50 in the presence of limited solubility. Table 5-11 also notes thepresence of volatility, as indicated by the use of film plate sealers during incubation.
Table 5-11

Reference Substances with Precipitate (PPT) and Volatility Issues1
3T3 NRU Test Method

Reference Substances

Acetonitrile
Aminopterin
5-Aminosalicylic acidArsenic III trioxide
Cadmium II chloride
Carbamazepine
Carbon tetrachloride
Citric acid
Cupric sulfate pentahydrate
Dibutyl phthalate
Dichlorvos
Diethyl phthalate
DigoxinDimethylformamide
Disulfoton
Endosulfan
Ethanol
Fenpropathrin
Gibberellic acid
Glutethimide
Lindane
Lithium I carbonate
Nicotine
Parathion
PhenolPotassium I chloride
Potassium cyanide

PPT in2X Stock
Dilutions

PPT in1X Plate
Dilutions

PPT in
Stock
and
Plate
Dilutions

NHK NRU Test Method

Volatility

PPT in2X Stock
Dilutions

PPT in1X Plate
Dilutions

PPT in
Stock
and
Plate
Dilutions

X
X

Volatility

X
X

X
X

X
X

X
X
X

X
X
X

X

X
X

X

X

X
X
X
X

X

X
X
X

X
X

X

X

X

X
X
X

X

X

X
X

X
X

X
X

X
X

5-43

X

X

X

X

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

Reference Substances with Precipitate (PPT) and Volatility Issues1

Table 5-11

3T3 NRU Test Method
Reference Substances

2-Propanol
Sodium arsenite
Sodium chloride
Sodium I fluoride
Sodium hypochlorite
Sodium oxalate
Strychnine
Trichloroacetic acid
1,1,1-Trichloroethane
Valproic acid
Verapamil HCl
Xylene

PPT in2X Stock
Dilutions

PPT in1X Plate
Dilutions

PPT in
Stock
and
Plate
Dilutions

NHK NRU Test Method

Volatility

PPT in2X Stock
Dilutions

PPT in1X Plate
Dilutions

PPT in
Stock
and
Plate
DilutionsX

Volatility

X
X

X
X
X

X
X
X
X

X
X
X

X
X

X
X
X

X

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; PPT=Precipitate.
Note: Table sorted alphabetical by reference substance.
1
Results are based on at least one laboratory having precipitate or volatility issues with a substance. Volatility was denoted by the use ofplate sealers during testing. 2X stock dilutions are prepared for each of 8 test substance concentrations. 1X plate dilutions are the result of
diluting the 2X stock solutions with medium in the 96-well plates.

5.9

Summary
•
•
•

•

•

•

The BioReliance, ECBC, and IIVS laboratories performed the 3T3 and NHK
NRU tests in compliance with GLP guidelines.
The quality and consistency of the reference substances was maintained
during the study by the central purchase and distribution of individual lots ofreference substances to the testing laboratories.
Modifications and revisions made to the protocols during Phases I and II
contributed to the optimization of the final protocols used in Phase III of thestudy. As a general rule, the protocol changes enhanced the performance ofthe methods and allowed more tests to meet the acceptance criteria.
FAL improved the quality of its NHK data prior to Phase II testing by
modifying the methods used to propagate the cells. Positive control IC50 datain Phases II and III from FAL more closely resemble the data from the otherlaboratories.
Summary test data and IC50 results are presented in tabular and graphic
formats. Comparisons of 3T3 NRU IC50 values to NHK NRU IC50 values
show that the values for 85% of the reference substances are within one orderof magnitude of each other. Digoxin and aminopterin yielded differences of
up to five orders of magnitude when the IC50 values of the 3T3 and NHK
NRU test methods were compared.
Although each laboratory followed the same solubility protocol, theysometimes obtained different results. This may have been due to thesubjective judgment of whether or not solubility was achieved. Additionally,
the laboratories may have used solubility procedures that were beyond thelevel of detail in the solubility protocol.

5-44In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

6.0

ACCURACY OF THE 3T3 AND NHK NRU TEST METHODS....................... 6-3

6.1

Accuracy of the 3T3 and NHK NRU Test Methods for Predicting Rodent
Acute Oral Toxicity ................................................................................................. 6-5
6.1.1 Linear Regression Analyses for the Prediction of Rat Acute Oral LD50
Values from In Vitro IC50 Values .................................................................. 6-5
6.1.2 Comparison of Combined-Laboratory 3T3 and NHK Regressions
to the RC Millimole Regression .................................................................... 6-8

6.2

Analysis of Outlier Substances for the RC Millimole Regression ....................... 6-9
6.2.1 Identification of Outlier Substances............................................................. 6-10
6.2.2 Evaluation of Outlier Substances................................................................. 6-12

6.3

Improving the Prediction of In Vivo Rat Oral LD50 Values from In Vitro
IC50 Data ................................................................................................................. 6-17
6.3.1 The RC Rat-Only Millimole Regression ..................................................... 6-17
6.3.2 The RC Rat-Only Weight Regression.......................................................... 6-17

6.4

Accuracy of the 3T3 and NHK NRU Test Methods for Predicting GHS
Acute Oral Toxicity Categories ............................................................................ 6-18
6.4.1 Prediction of GHS Acute Oral Toxicity Category by the RC IC50
Values Using the RC Rat-Only Millimole Regression................................ 6-20
6.4.2 Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK
NRU Test Methods Using the RC Rat-Only Millimole Regression............ 6-22
6.4.3 Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK
NRU Test Methods Using the RC Rat-Only Weight Regression................ 6-24
6.4.4 Summary of the Regressions Evaluated ...................................................... 6-27

6.5

Correlation of NRU Concentration-Response Slope with Rat Lethality
Dose-Response Slope ............................................................................................. 6-28

6.6

Strengths and Limitations of the Use of In Vitro Cytotoxicity Test
Methods with the IC50–LD50 Regressions for Predictions of Rodent Acute
Oral Toxicity .......................................................................................................... 6-31
6.6.1 In Vitro Cytotoxicity Methods ..................................................................... 6-31
6.6.2 Use of Mole-Based vs. Weight-Based Regressions for the Predictionof Toxicity for Low and High Molecular Weight Substances..................... 6-31

6.7

Salient Issues of Data Interpretation.................................................................... 6-34

6.8

Comparison of NRU Test Results to Established Performance Standards...... 6-34

6.9

Summary................................................................................................................. 6-40

6-1

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

[This Page Intentionally Left Blank]

6-2

In Vitro Cytotoxicity Test Methods BRD Section 6

6.0

November 2006

ACCURACY OF THE 3T3 AND NHK NRU TEST METHODSThis section discusses the accuracy of the 3T3 and NHK NRU test methods for predicting therodent acute oral toxicity (the LD50) of chemicals. Accuracy, the agreement between a test
result and an accepted reference value, is a critical component of the evaluation of thevalidation status of a method (ICCVAM 2003). Although the 3T3 and NHK NRU test
methods are not suitable as replacements for acute oral toxicity assays, the rationale for
evaluating the accuracy of LD50 predictions from the in vitro IC50 values is that the animalsavings produced by using these in vitro test methods to predict starting doses for acute oral
toxicity assays will be greatest when the starting dose is as close as possible to the “true”
LD50 value (see Section 10 for the evaluation of the potential reduction of animal use).
The ability of the 3T3 and NHK NRU test methods to correctly predict rodent acute oral
toxicity is based on the validity of the in vivo – in vitro (i.e., IC50-LD50) regression model.
The IC50-LD50 regression establishes the relationship between the in vitro IC50 values and theLD50 values that will be used to set the starting doses for the computer-simulated acute oral
toxicity assays in this study (see Section 10). The regressions generated by the threelaboratories for each NRU test method were not statistically different, and the data from the
3T3 and NHK NRU test methods were combined (using a geometric mean IC50 of the three
individual laboratory geometric mean IC50 values) into single regressions (see Section 6.1).
Only rat LD50 data were used for these regressions to reduce the variation that would be
produced by combining data from multiple species. Table 6-1 describes the datasets used forthe analyses in Sections 6.1 through 6.4.
To test the assumption in the Guidance Document that the RC millimole regression can beobtained using a basal cytotoxicity method with a single cell type and cytotoxicity endpoint(ICCVAM 2001b), the regressions for each NRU test method (3T3 and NHK) werecompared with regressions for the same substances that were calculated using the RC IC50
and LD50 values (see Section 6.1). Because the 3T3 and NHK regressions were notstatistically different from the RC regressions for the same chemicals, the RC data were used
to develop a regression to predict LD50 values from the NRU-generated IC50 values because
this regression was based on a larger number of substances than the NICEATM/ECVAM
regressions (see Section 6.3).
The RC millimole regression was used to identify outlier substances (i.e., those that did not
fit the regression within the established acceptance limits; see Section 6.2) tested in thevalidation study because:• Acceptance limits for the RC millimole regression had been established• The 3T3 and NHK NRU IC50 – rat oral LD50 regressions were notsignificantly different from the RC regressions calculated for the same
substances• Use of the RC regressions allow a comparison of the outlier substances
determined using RC data to those determined using the 3T3 and NHK data

6-3

In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-1

November 2006

Datasets Used for Accuracy Analyses1
3T3
NRU1

NHK
NRU1

Testing with NRU test methods

72

72

Comparison of laboratory IC50-LD50
regressions to one another47

51

Comparison of combined-laboratory
IC50-LD50 regressions to a regression
calculated with RC data

47

47

RC millimole regression

NANA

RC rat-only millimole regression

NANA

RC rat-only weight regression

NA

NA

Analysis of outliers for the RC
millimole regression

70

71

Substances with IC50 values from atleast one laboratory

67

68

Substances with IC50 values from atleast one laboratory and rat oral LD50
referene values

Use

Prediction of GHS accuracy using
IC50 values in RC rat-only regressions

Characteristics of Dataset
Substances tested; 58 substanceswere common to the RC
RC substances with IC50 values fromall laboratories and reference rat oralreference LD50 values
RC substances with IC50 values forboth test methods from alllaboratories and reference rat oral
LD50 values
RC IC50 (mM) and RC oral LD50
(mmol/kg) values for 347 substances(282 rat and 65 mouse LD50 values)
RC IC50 (mM) and RC oral LD50
values (mmol/kg) for 282 substanceswith rat oral LD50 data
RC IC50 (µg/mL) and RC oral LD50
values (mg/kg) for 282 substanceswith rat oral LD50 data

Abbreviations: RC=Registry of Cytotoxicity; 3T3=BALB/c 3T3 fibroblasts; NA=Not applicable; NHK=Normal
human epidermal keratinocytes; NRU=Neutral red uptake.
1
Number of substances.

To improve upon the RC millimole regression’s1 ability to accurately predict LD50 values
from IC50 values, and to also make this approach relevant to the testing of mixtures andsubstances without known molecular weights, two regressions were calculated (see Section
6.3). The first regression – the RC rat-only millimole regression – uses the 282 (of 347)
substances in the RC dataset that had reported rat LD50 values. The LD50 data for the
regression were limited to one species to decrease the variability in LD50 values that would
occur if the data from more than one species were combined. Rats were selected because they
are the preferred species for acute oral toxicity testing (EPA 2002b; OECD 2001a; OECD
2001d) (see Section 6.3.1). The RC rat-only millimole regression was transformed to one
based on weight units (mg/kg body weight for LD50 and µg/mL for IC50) in order to make theregression equation more generally applicable to the testing of mixtures and substances of
unknown molecular weights.
1

The RC millimole regression was created using rat and mouse oral LD50 values from RTECS® and IC50 values
from in vitro cytotoxicity assays using multiple cell lines and cytotoxicity endpoints for 347 substances withknown molecular weights (Halle 1998, 2003)
6-4

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

The ability of the 3T3 and NHK NRU IC50 data to correctly predict rat acute oral LD50 values
based on using the RC rat-only millimole regression and the RC rat-only weight regression,
was evaluated by determining the extent to which the appropriate GHS acute oral toxicity
category was identified for each reference substance (see Section 6.4). The rationale for
evaluating the accuracy of LD50 predictions is that the acute oral toxicity test methods (i.e.,
UDP, FDP, and ATC) call for starting doses to be placed as close as possible and just below
the true LD50. When the starting dose is close to the true LD50 for a test substance, fewer
animals are needed. When the starting dose is below the true LD50, there is reduced pain andsuffering because doses tend to be lower, and the test bias is more conservative. Thisapproach permits an assessment of accuracy that is specific to each GHS hazard
classification category. The discordant reference substances from the predictions of GHS
category are presented in Appendix L2.
The remainder of Section 6 discusses physical, chemical, and biological, characteristics ofsubstances that may have an impact on the accuracy of the 3T3 and NHK methods.
6.1

Accuracy of the 3T3 and NHK NRU Test Methods for Predicting Rodent
Acute Oral Toxicity

The rat LD50 values provided in Section 4.2 are used as the reference values for assessing theability of the 3T3 and NHK test methods to accurately predict acute oral toxicity2. The
accuracy of the two in vitro cytotoxicity test methods is assessed in two ways: (1) by the
goodness of fit of the in vitro IC50 data to the rat LD50 data in linear regression analyses, and(2) by the concordance (i.e., extent of agreement) between the GHS acute oral toxicity
categories (UN 2005) assigned based on rat LD50 data and those predicted using in vitro IC50
values.
6.1.1

Linear Regression Analyses for the Prediction of Rat Acute Oral LD50 Values
from In Vitro IC50 Values
As described in Section 5.5.4.3, linear regressions for each laboratory and in vitro method
were calculated using log IC50 values (mM) versus the corresponding reference log LD50
values (mmol/kg) identified in Table 4-2. The reference substances used to calculate each of
the laboratory regressions met the following criteria for each test method:• The substance was included in the RC• All three laboratories reported IC50 values• There was an associated rat acute oral LD50 reference value (see Table 4-2).
There were 47 and 51 reference substances that fit these criteria for the 3T3 and NHK test
methods, respectively. The slopes for the all of the laboratory-specific regressions were
statistically significantly different from zero (p <0.0001), which indicates a significant
correlation between in vitro IC50 values and the corresponding rat acute oral LD50 values.
Comparison of the individual laboratory regressions to one another using the goodness of fit
2

Toxicity is inversely proportional to LD50. High LD50 values reflect low toxicity and low LD50 values reflect
high toxicity

6-5

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

F-test for regression slopes and intercepts described in Section 5.5.4.3 indicated that thelaboratory-specific regressions for either NRU method were not significantly different fromone another. For the 3T3 method, p=0.605 for the slope comparisons and p=0.947 for theintercept comparisons. For the NHK method, p=0.792 for the slope comparisons and p=0.999
for the intercept comparisons.
Because the individual laboratory regressions were not significantly different, the laboratory
data were combined into a single regression for each method using the geometric mean of themean IC50 values determined by each laboratory for each substance (see the “Combinedlaboratory” regressions in Table 6-2 and Figure 6-1). The combined-laboratory 3T3
regression yielded a better fit to the reference LD50 data (R2=0.579) than the NHK regression(R2=0.463).
Table 6-2

Linear Regression Analyses of the 3T3 and NHK NRU and Rat Acute
Oral LD50 Test Results1

Laboratory

N

ECBC2
FAL2
IIVS2
Combined-laboratory3

47
47
47
47

ECBC2
FAL2
IIVS2
Combined-laboratory3

51
51
51
51

Slope
3T3 NRU
0.573
0.539
0.552
0.561
NHK NRU
0.491
0.428
0.483
0.470

Intercept

R2

0.541
0.373
0.507
0.475

0.613
0.519
0.586
0.579

0.412
0.407
0.416
0.413

0.480
0.422
0.478
0.463

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; 3T3=BALB/c 3T3
fibroblasts; N=Number of substances used to calculate the regression; NHK=Normal human epidermal
keratinocytes; NRU=Neutral red uptake; R2=Coefficient of determination.
1
Log IC50 in mM; log LD50 in mmol/kg.
2
Regression based on a single point per substance (i.e., the geometric mean of the within laboratory replicate
IC50 values and the reference rat acute oral LD50 from Table 4-2).
3
Regression based on a single point per substance (i.e., the geometric mean of the geometric mean IC50 values
obtained for each laboratory and the reference rat acute oral LD50 from Table 4-2).

6-6

In Vitro Cytotoxicity Test Methods BRD Section 6

Figure 6-1

November 2006

Combined-Laboratory 3T3 and NHK NRU Regressions
3T3 NRU

1000

log LD50 = 0.561 log IC50 + 0.475
R2 = 0.579

100

10

1

0.1

0.01

0.001

0.00010.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)

NHK NRU
1000

log LD50 = 0.470 log IC50 + 0.413
R2 = 0.463
100

10

1

0.1

0.01

0.001

0.00010.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Netural red
2
uptake; R =Coefficient of determination.
Points show the geometric means of the laboratory geometric mean IC50 values and the reference rat acute oral
LD50 values (from Table 4-2) for 47 reference substances for the 3T3 and 51 reference substances for NHK
test methods. Solid lines show the combined-laboratory regressions for each method (see Table 6-2).

6-7

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

6.1.2

Comparison of the Combined-Laboratory 3T3 and NHK Regressions to the RC
Millimole Regression
The validation study tested 58 RC substances using the 3T3 and NHK NRU test methods(see Figure 3-1). The resulting method regressions for each cell type were compared to the
RC regressions for the same substances to test the assumption in the Guidance Document that
the RC millimole regression can be obtained with a basal cytotoxicity test method using asingle cell type and endpoint (ICCVAM 2001b). The 47 substances used to calculate these
regressions met the following criteria:• The substance was included in the RC• All three laboratories reported IC50 values for both the 3T3 and NHK NRU
test methods• There was an associated rat oral reference LD50 value (see Table 4-2)
The regression calculated for the 47 substances using the RC IC50 and LD50 data is shown in
Figure 6-2. A graphic comparison of the RC regressions and the 3T3 and NHK combinedlaboratory regressions is in Figure 6-3. A statistical comparison of slope and intercept(simultaneously) using an F test showed that neither the 3T3 regression (p=0.612) nor the
NHK regression (p=0.759) was significantly different from the 47 RC substance regression.
Figure 6-2
1000

Regression for 47 RC Substances Using RC Data
log LD 50 = 0.640 log IC 50 + 0.262
R2 = 0.694

100

10

1

0.1

0.01

0.001

0.00010.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)
Abbreviations: RC=Registry of Cytotoxicity; R2=Coefficient of determination.
Points show the IC50 values and the reference rodent (rat and mouse) acute oral LD50 values from the RC for
47 reference substances. The dashed line shows the calculated regression.

6-8

In Vitro Cytotoxicity Test Methods BRD Section 6

Figure 6-3

November 2006

Regression for 47 RC Substances with the 3T3 and NHK Regressions

1000

RC Regression for 47 Substances

100

3T3 NRU Regression
NHK NRU Regression

10

1

0.1

0.01

0.001

0.00010.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)
Abbreviations: RC=Registry of Cytotoxicity; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal
keratinocytes; NRU=Neutral red uptake.
The regression for 47 RC substances using RC data is log LD50 = 0.640 log IC50 + 0.262 (R2=0.694). The
combined-laboratory 3T3 regression for the same 47 substances, is log LD50 = 0.561 log IC50 + 0.475 (R2 =0.579) (from Table 6-2). The combined-laboratory NHK regression for the same 47 substances, is log LD50 =0.471 log IC50 + 0.445 (R2 = 0.487).

6.2

Analysis of Outlier Substances for the RC Millimole Regression

The RC millimole regression and each in vitro NRU test method were used to identify
outliers among the reference substances tested in the validation study (i.e., those for which
the rodent LD50 was not accurately predicted by the in vitro IC50). The outlier substanceswere then evaluated to determine if they had common characteristics that could assist in
identifying the types of substances that are not suited for use in the 3T3 or NHK NRU test
methods for determining starting doses for acute oral toxicity assays.
The RC millimole regression was used to determine the outlier status of reference substances
because:• The RC millimole regression had associated acceptance limits (Halle 1998,
2003): a difference greater than 0.699 (or log 5) for log-observed LD50 (inmmol/kg) from the log-predicted LD50 identifies a substance as an outlier• The 3T3 and NHK IC50 – rat oral LD50 regressions were not significantly
different from the RC regressions calculated for the same substances

6-9

In Vitro Cytotoxicity Test Methods BRD Section 6•

November 2006

Use of the RC millimole regression allows a comparison of the outlier
substances determined using RC IC50 values to those determined using the
3T3 and NHK NRU IC50 values.

6.2.1
Identification of Outlier Substances
For each in vitro NRU test method, the predicted LD50 values for the reference substanceswere determined using the geometric mean IC50 values of the three geometric mean
laboratory values in the RC millimole regression. Outliers were identified using the RC
method (Halle 1998): a difference greater than 0.699 (or log 5) for log-observed LD50 (inmmol/kg) minus the log-predicted LD50 identifies a substance as an outlier (see Appendix J1for the 3T3 NRU test method and Appendix J2 for the NHK NRU test method for thepredicted LD50 values). For the best comparison with the RC outlier results, the outlier
evaluation for the 3T3 and NHK NRU test methods used same observed LD50 values as thoseused in the RC database for the 58 reference substances that were included in the RC
database (see Table 3-2). For the non-RC substances, the observed values (in Table 3-2)
were obtained from other databases such as RTECS or Hazardous Substances Database(NLM 2002). The outlier analysis included all the reference substances that yielded IC50
values from at least one laboratory in the validation study whether the in vivo LD50 values
were from rats or mice. Thus, 70 substances were used for the 3T3 NRU outlier analysis and71 substances were used for the NHK NRU outlier analysis. Table 6-3 lists the outlier
substances for the RC millimole regression when using the RC IC50 values and the 3T3 andNHK NRU IC50 values.
Table 6-3

Outlier Substances for the RC and the 3T3 and NHK NRU Methods
When the RC Millimole Regression is Used1
Substances Included in the RC Identified as Outliers in:
RC

2

3T33

NHK4

Acetaminophen (+)
Arsenic III trioxide (–)

Arsenic III trioxide (–)
Aminopterin (–)

5-Aminosalicylic acid (+)
Busulfan (–)

5-Aminosalicylic acid (+)
Busulfan (–)

Caffeine (–)

Busulfan (–)
Caffeine (–)

Cycloheximide (–)

Cycloheximide (–)

Cycloheximide (–)

Dibutyl phthalate (+)

Dibutyl phthalate (+)

Dibutyl phthalate (+)

Diethyl phthalate (+)

Diethyl phthalate (+)

Digoxin (–)

Digoxin (–)

Disulfoton (–)

Disulfoton (–)

Disulfoton (–)

Epinephrine bitartrate (–)

Epinephrine bitartrate (–)

Epinephrine bitartrate (–)

Ethanol (+)

Ethanol (+)

Ethanol (+)

Lindane (–)

Lindane (–)

Mercury II chloride (–)

Mercury II chloride (–)

Mercury II chloride (–)
Methanol (+)

6-10

In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-3

November 2006

Outlier Substances for the RC and the 3T3 and NHK NRU Methods
When the RC Millimole Regression is Used1
Substances Included in the RC Identified as Outliers in:RC

2

Nicotine (–)

3T33
Nicotine (–)

Paraquat (–)

NHK4
Nicotine (–)
Paraquat (–)

Parathion (–)

Parathion (–)

Parathion (–)

Phenobarbital (–)

Phenobarbital (–)

Phenobarbital (–)

Phenylthiourea (–)

Phenylthiourea (–)

Phenylthiourea (–)

Potassium cyanide (–)

Potassium cyanide (–)

Potassium cyanide (–)

Propylparaben (+)

Propylparaben (+)

Propylparaben (+)
Sodium oxalate (–)

Thallium I sulfate (–)

Thallium I sulfate (–)

Triethylenemelamine (–)

Triethylenemelamine (–)

Triethylenemelamine (–)

Verapamil HCl (–)

Verapamil HCl (–)

1,1,1-Trichloroethane (+)
Verapamil HCl (–)

Xylene (+)
Outliers That Were Not Included in the RC
Dichlorvos (–)

Dichlorvos (–)

Endosulfan (–)

Endosulfan (–)

Fenpropathrin (–)

Fenpropathrin (–)

Physostigmine (–)

Physostigmine (–)

Sodium hypochlorite (+)

Sodium hypochlorite (+)

Sodium selenate (–)

Sodium selenate (–)

Strychnine (–)

Strychnine (–)

Abbreviations: RC=Registry of Cytotoxicity; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes;
NRU=Neutral red uptake; (–)=Toxicity was underpredicted by the IC50 and RC millimole regression (i.e., the LD50 value
predicted by the IC50 was higher than the in vivo LD50 value); (+)=Toxicity was overpredicted by the IC50 and RC millimole
regression (i.e., the LD50 value predicted by the IC50 was lower than the in vivo rodent LD50 value).
[Note: Empty cells indicate that the substance was not an outlier for that particular IC50 value.]1
Log LD50 (mmol/kg) = 0.435 log IC50 (mM) + 0.625. Log LD50 (mmol/kg) values for outlier substances were >0.699 fromthe RC millimole regression.
2
Using RC IC50 in the RC millimole regression for the 58 RC substances tested in the validation study.
3
Using the 3T3 NRU IC50 in the RC millimole regression for the 70 reference substances that yielded IC50 values from any
laboratory in the validation study.
4
Using the NHK NRU IC50 in the RC millimole regression the RC for the 71 reference substances that yielded IC50 values
from any laboratory in the validation study.
Bolded substances have active metabolites in vivo (see Table 3-7).
Substances that showed evidence of insolubility (i.e., precipitates) during testing (see Table 5-11) are identified by italics.

When the RC millimole regression and the RC method of identifying outlier substances wereused (Halle 1998, 2003), there were 28 outliers for the 3T3 NRU test method and 31 for the
NHK NRU test method. The top part of Table 6-3 shows a comparison of the 22 RC
substances that were identified by the RC as outliers (see Table 3-2) and the RC reference
substances that were identified as outliers using either the 3T3 or NHK NRU IC50 values
with the RC millimole regression. For the 58 RC substances that were tested in the validation
6-11

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

study, 18 of the 22 RC outliers also responded as outliers in both NRU test methods, butsome of the substances were outliers only in one of the two NRU test methods. The RC
regression outliers, 5-aminosalicylic acid, caffeine, paraquat, and 1,1,1-trichloroethane were
not outliers when 3T3 data were used, and the RC outliers, digoxin, lindane, thallium sulfate,and 1,1,1-trichloroethane, were not outliers when the NHK NRU test method was used. In
contrast the 3T3 NRU test method identified three substances as outliers that were not
identified by the RC: acetaminophen, arsenic trioxide, and diethyl phthalate, and the NHK
NRU test method identified six: aminopterin, arsenic trioxide, diethyl phthalate, methanol,
sodium oxalate, and xylene. Seven additional substances, that were not included in the RC
database, were identified as outliers using the NRU IC50 values in the RC millimole
regression: dichlorvos, endosulfan, fenpropathrin, physostigmine, sodium hypochlorite,
sodium selenate, and strychnine.
6.2.2
Evaluation of Outlier Substances
A number of physico-chemical and toxicologic characteristics were evaluated for theirfrequency of occurrence among the 28 and 31 outlier substances in the 3T3 and NHK NRU
test methods, respectively, to identify attributes that may have contributed their outlier status.
This section provides a summary of these analyses based on the RC millimole regression andoutlier criteria. The frequency of outliers versus the total number of reference substances foreach physico-chemical and toxicologic category examined is shown in Appendix L1.
6.2.2.1 Physical Characteristics
A number of physical characteristics were evaluated for their frequency of occurrence in theset of outlier substances versus the complete set of reference substances. The characteristics
chosen were those that were assumed to be readily available, or relatively easy to measure,for new substances that may be tested in these NRU assays. The characteristics examined
included chemical class, molecular weight, boiling point, IC50, pH, and log Kow (i.e., log
octanol:water partition coefficient). Unfortunately, these attributes were not available for all
substances. For example, log Kow was available for 50 of the 70 (71%) substances evaluatedfor the 3T3 NRU test method and for 51 of the 71 (72%) substances evaluated for the NHK
NRU test method. Boiling point was available for only 24 of 70 (34%) substances evaluatedfor the 3T3 NRU test method and for 25 of the 71 (35%) substances evaluated for the NHK
NRU test method. For substances with log Kow >3.00, 8/13 (62%) were outliers for both the
3T3 and NHK test methods. For molecular weights >400 g/mole, 4/7 (57%) substances were
outliers using the 3T3 NRU test method and 3/7 (43%) were outliers using the NHK NRU
test method. For substances with boiling points >200oC, 9/13 (69%) were outliers using the
3T3 NRU test method and 8/13 (62%) were outliers using the NHK NRU test method.
6.2.2.2 Chemical Class
Examination of outliers by chemical class for the RC millimole regression showed that all of
the chemical classes that contained at least three reference substances also contained at least
one outlier for one test method. Two classes contained 100% outliers for both test methods:
organophosphates (3/3) and organic sulfur compounds (5/5). The remaining classes withhigher frequencies of outliers included: 2/3 (67%) amines were outliers for both test methods,
7/14 (50%) heterocylics were outliers for the 3T3 NRU and 10/14 (71%) heterocyclics were
outliers for the NHK NRU, 2/5 (40%) chlorine compounds were outliers for both test
methods, 2/6 (33%) sodium compounds were outliers for both test methods, 3/9 (33%)
alcohols were outliers for the 3T3 NRU and 4/10 (40%) alcohols were outliers for the NHK
6-12

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

NRU, and 4/14 (29%) carboxylic acids were outliers for the 3T3 NRU and 6/14 (43%)
carboxylic acids were outliers for the NHK NRU.
6.2.2.3 Solubility
Another attribute that may cause a substance to be an outlier is the lack of solubility in thetest system. Because the SMT expected the toxicity of insoluble substances to beunderpredicted in the in vitro assays, substances that formed precipitates in the tests werenoted and compared with the outlier substances. However, insolubility was not consistently
associated with the outlier substances for which toxicity was underpredicted. For example,
eight of the 22 (36%) underpredicted substances identified by applying the 3T3 results to the
RC millimole regression exhibited signs of insolubility in at least one laboratory. NHK
results showed that seven of 23 (30%) underpredicted substances exhibited signs ofinsolubility in at least one laboratory (see Table 5-11 for substances that had precipitates in
the assays). Additionally, there was evidence of insolubility in the 3T3 and NHK NRU test
methods of dibutyl phthalate and diethyl phthalate, but toxicity was overpredicted for bothsubstances, rather than underpredicted. This overprediction may be a characteristic of thephthalates, but more substances would have to be tested before a general rule could be
adopted.
There were 25 substances that showed evidence of insolubility in the 3T3 test method in atleast one laboratory, and 11 (44%) of these were outliers. Of the 24 substances showed
evidence of insolubility in at least one NHK laboratory, 11 (46%) were outliers.
6.2.2.4 Metabolism
It was anticipated that the toxicity of substances metabolized in vivo to active compounds(see Section 3.3.4.3 and Table 3-7) would be underpredicted in vitro by 3T3 and NHK cells,
which have little or no metabolic capability (Babich 1991; INVITTOX 1991). Of the 72
reference substances, 19 (26%) are known to have active metabolites in vivo, and 10 (45%)
of these were classified as outliers for 3T3. Of these 10 substances, which accounted for 36%
of the 28 outlier substances, the toxicity of six (60%) was underpredicted, while the toxicity
of four (40%) was overpredicted. Among the 31 outliers in the NHK NRU test method, nine(29%) are metabolized to active metabolites. Nine of the 19 substances known to produce
active metabolites in vivo were discordant for the NHK NRU test method. NHK cells
underpredicted the toxicity of five (56%) of these nine substances and overpredicted theother four (44%). These nine outlier substances accounted for 29% of the 31 outliers in the
NHK NRU test method. Thus, the fact that a substance has active metabolites that are notexpected to be produced in the in vitro tests does not necessarily indicate that its toxicity will
be underpredicted by in vitro basal cytotoxicity test methods.
Similarly, Halle (1998, 2003) noted that the RC substances that required metabolic activation
to produce in vivo toxicity were not necessarily outliers with respect to their fit to the RC
millimole regression. They found that eight (50%) of the 16 substances that required
metabolic activation to product toxicity were outliers (see Table L3-3 in Appendix L3).
6.2.2.5 Mechanism of Toxicity
Substances whose mechanisms of toxicity would not be detected in the 3T3 or NHK cells
would be expected to fit the RC millimole regression poorly. In particular, toxic mechanismsthat include, for example, specific actions on the central nervous system (CNS) or the heart
6-13

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

are not expected to be active in the 3T3 or NHK cells. Neurotoxic mechanisms would
include, for example, cholinesterase inhibition, CNS nicotinic receptor blockade or
activation, or any activity other than membrane destabilization such as that produced by asolvent, or disturbance of energy utilization such as interruption of oxidative
phosphorylation. Representative cardiotoxic mechanisms would include calcium channel
blockage and beta-adrenergic receptor activation or blockage.
The 72 reference substances used to validate the 3T3 and NHK NRU test methods included
16 (22%) that had specific CNS toxicity (see Table 6-4). Of these 16 substances, 10 (63%)
were outliers in both in vitro NRU test methods. Three of the six (50%) reference substancesthat are cardiotoxic were outliers in the 3T3 NRU test method and two (33%) were outliers in
the NHK NRU test method. When all the reference substances with mechanisms that are notexpected to be active in the 3T3 and NHK cells (i.e., in Table 6-4) are summed, 13/22 (59%)
are outliers for the 3T3 NRU and 12/22 (55%) are outliers for the NHK NRU. These
substances represented 13/28 (46%) and 12/31 (39%) of the total outlier substances for the
3T3 and NHK NRU test methods, respectively. Halle (1998, 2003) reported similar findings
for the RC database (i.e., approximately half of the substances expected to be outliers basedon their mechanisms of toxicity were outliers) (see Appendix L3).

6-14

In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-4

November 2006

Substances With Mechanisms of Toxicity Not Expected to Be Active in the 3T3 or NHK Cells in Culture

Substance

Mechanism of Toxicity1

3T3 Outlier2

NHK Outlier2

No

No

No

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Endosulfan

Neurotoxic
Antimuscarinic; anticholinergic action; competitive antagonism of anticholinesterase at cardiac
and CNS receptor sites.
Inhibition of phosphodiesterase leading to AMP accumulation; translocation of intracellular
Ca++; adenosine receptor antagonism; neurotoxic.
Therapeutically decreases firing of noradrenergic neurons.
Potentiation of GABAA receptor activity; inhibition of N-methyl-D-aspartate activity;
modulation of 5-hydroxytryptamine3 receptor-mediated depolarization of the vagas nerve3.
Inhibition of acetylcholinesterase resulting in acetylcholine accumulation in CNS and effector
organs.
Inhibition of acetylcholinesterase resulting in acetylcholine accumulation in CNS and effector
organs.
Affects brain neurotransmitter levels4.

Yes

Yes

Fenpropathrin

Delays closure of sodium channel causing persistent depolarization of membrane.

Yes

Yes

Glutethimide

CNS depression; anticholinergic activity.

No

No

Haloperidol

Blocks dopamine receptors.
CNS depression through inhibition of GABA receptor linked chloride channel at the picrotoxin
binding site, leading to blockade of chloride influx into neurons.
Cholinergic block causing polarization of CNS and PNS synapses.
Inhibition of acetylcholinesterase resulting in acetylcholine accumulation in CNS and effector
organs.
CNS depression through inhibition of GABA synapses; inhibits hepatic NADH cytochromeoxidoreductase.
Inhibition of acetylcholinesterase resulting in acetylcholine accumulation in CNS and effector
organs.
Increases glutamic acid in the CNS.

No

No

Yes

No

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

No

Yes

No

Atropine sulfate
Caffeine
Carbamazepine
Chloral hydrate
Dichlorvos
Disulfoton

LindaneNicotine
Parathion
Phenobarbital
Physostigmine
Strychnine

Amitriptyline HCl
Digoxin

Cardiotoxic
Blocks norepinephrine, 5-hydroxytryptamine, and dopamine presynaptic uptake; prevents
reuptake of heart norepinephrine.
Impairs ion transport and increases sarcoplasmic calcium by binding to Na+/K+ ATPase,
increasing automaticity of cardiac cells.
6-15

In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-4

November 2006

Substances With Mechanisms of Toxicity Not Expected to Be Active in the 3T3 or NHK Cells in Culture
Mechanism of Toxicity1

Substance
Epinephrine bitartrate

Adrenergic receptor stimulation.

Potassium chloride

Disturbs cardiac membrane potential and electrical activity.

Procainamide HCl
Verapamil HCl

5

Slows impulse conduction in the heart .
++Inhibition of transmembrane Ca flux in excitatory tissues; alpha-adrenergic blockade.

Abbreviations: NA=Not available or information not found; CNS=Central nervous system; GABA=Gamma aminobutyricacid; PNS=Peripheral nervous system; NADH=Nicotine adenine dinucleotide (reduced).
1
From Ekwall et al. (1998) or Hazardous Substances Data Bank (NLM 2001, 2002) unless otherwise noted.
2
As shown in Table 6-3.
3
EPA (2000b).
4
ATSDR (2000a).
5
Hardman et al. (1996).

6-16

3T3 Outlier2

NHK Outlier2

Yes

Yes

No

No

No

No

Yes

Yes

In Vitro Cytotoxicity Test Methods BRD Section 6

6.3

November 2006

Improving the Prediction of In Vivo Rat Oral LD50 Values from In Vitro IC50
Data

Because the 3T3 and NHK IC50 – rat oral LD50 regressions were not significantly different
from the RC regression for the same substances, the next step was an attempt to improve the
RC millimole regression for the prediction of LD50 values from IC50 values. Because thevalidation study provided results similar to the RC, and because the RC database has morethan 3.5 times the number of substances tested in the validation study, the RC rat data (282substances) were used to determine the relationship between IC50 and LD50. The RC data
were used to develop two new regressions, the RC rat-only millimole regression and the RC
rat-only weight regression. For reference, the original RC millimole regression, log LD50
(mmol/kg) = 0.435 x log IC50 (mM) + 0.625 (Halle 1998, 2003), is shown in Table 6-5.
6.3.1
The RC Rat-Only Millimole Regression
The first regression used the RC data for the 282 substances with rat LD50 data and theoriginal units of mM for IC50 and mmol/kg for LD50 (see Table 6-5 and Figure 6-9). Only rat
data were used because:• Rats and mice are not always equally sensitive to all substances• The majority of acute oral LD50 data used in the RC millimole regression werefrom studies using rats (282 rat data points versus 65 mouse data points)(Halle 1998, 2003)• Most acute oral toxicity testing is performed with rats.
The RC rat-only millimole regression is applicable to substances of known molecular weightthat are relatively pure.
Table 6-5

Linear Regression Analyses to Improve the Prediction of Rodent Acute
Oral LD50 Values from In Vitro NRU IC50 Using the RC Database1

Data Used
347 RC substances (282 rat and 65 mouse LD50
values) – millimole units2
282 RC substances with rat LD50 data – millimoleunits2282 RC substances with rat LD50 data – weight units4

Slope

Intercept

R2

0.435

0.625

0.4523

0.439

0.621

0.452

0.372

2.024

0.325

Abbreviations: NRU=Neutral red uptake; RC=Registry of Cytotoxicity; R2=Coefficient of determination.
1
Slopes of all regressions were significantly different (p <0.05) from zero at p <0.0001.
2
IC50 in mM; LD50 in mmol/kg.
3
Calculated from RC data (i.e., not reported by Halle [1998, 2003]).
4
IC50 in µg/mL; LD50 in mg/kg.

Table 6-5 shows that the RC millimole regression using only rat acute oral LD50 data wasessentially identical to the original regression that used both rat and mouse data. The slopechanged from 0.435 to 0.439 and the intercept changed from 0.625 to 0.621; these changes
were not statistically significantly different.
6.3.2
The RC Rat-Only Weight Regression
The second regression used the same RC rat acute oral LD50 data for the 282 substances but
was calculated using weight units rather than millimolar units (see Table 6-5 and Figure 66-17

In Vitro Cytotoxicity Test Methods BRD Section 6

November 20064b). Weight units (i.e., mg/kg for the LD50 and µg/mL for the IC50) were selected for theunits of measurement because• Millimole units are not applicable to mixtures and substances with unknown
structures or molecular weights.
• They are the most practical, i.e., hazard classification in all regulatory systems
is based on LD50 values expressed in mg/kg (see Table 1-2).
The RC rat-only weight regression is applicable for use with complex mixtures, substances
whose structures or molecular weights are unknown, and substances that are relatively
impure (i.e., mixtures that are primarily composed of a named substance).
6.4

Accuracy of the 3T3 and NHK NRU Test Methods for Predicting GHS Acute
Oral Toxicity Categories

Based on the correlations/regressions obtained between the 3T3 and NHK NRU IC50 values
and the rat LD50 values, it is clear that these in vitro methods are not suitable as replacements
for rodent acute oral toxicity tests. The use of in vitro methods to reduce animal use forrodent acute oral toxicity assays (i.e., to assist in determining the starting doses for in vivo
assays) also depends upon their accuracy for the prediction of LD50 values. However, thislatter (adjunct) use does not require the same precision in LD50 prediction as complete
replacement would.
The NRU-predicted LD50 values were determined using the in vitro NRU IC50 values in the
RC rat-only regressions presented in Table 6-5. The predicted LD50 values were used toassign each substance to a predicted GHS acute oral toxicity category (UN 2005). The
accuracy of the 3T3 and NHK NRU test methods for predicting GHS acute oral toxicity
categories was determined by comparison with categorization based on rat acute oral LD50
data. The rationale for evaluating the accuracy of LD50 predictions was that the animalsavings produced by using these in vitro NRU test methods to predict starting doses forrodent acute oral toxicity assays would be greatest when the starting dose is as close as
possible to the LD50. This approach was used because regulatory authorities use rodent acuteoral toxicity test results for hazard classification and labelling of products to protect handlers
and consumers.
The in vitro NRU test methods were evaluated for their ability to predict GHS acute oral
toxicity categories using the two regressions presented in Section 6.3, the RC rat-only
millimole regression and the RC rat-only weight regression. The same reference substanceswere evaluated for each regression. Sixty-seven and 68 substances were evaluated using the
3T3 and NHK NRU test methods, respectively. Of the original 72 reference substancestested, epinephrine bitartrate, colchicine, and propylparaben were excluded because they had
no rat acute oral LD50 reference data (see Table 4-2). Carbon tetrachloride and methanol
were excluded from the 3T3 evaluations because no laboratory attained sufficient toxicity inany test for the calculation of an IC50 (see Table 5-4). Carbon tetrachloride was excluded
from the NHK evaluations because no laboratory attained sufficient toxicity in any test forthe calculation of an IC50 (see Table 5-5).

6-18

In Vitro Cytotoxicity Test Methods BRD Section 6

Figure 6-4

November 2006

RC Rat-Only Millimole Regression (a) and RC Rat-Only Weight
Regression (b)

a.
1000

log LD 50 = 0.439 log IC 50 + 0.621
R2 = 0.452

100

10

1

0.1

0.01

0.001

0.00010.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

100

1000

10000

100000IC50 (mM)b.
100000log LD 50 = 0.372 log IC 50 + 2.024
R2 = 0.325

10000

1000

100

10

1

0.1
0.001

0.01

0.1

1

10

IC50 (µg/mL)

Abbreviations: RC=Registry of Cytotoxicity; R2=Coefficient of determination.
Regressions calculated using IC50 and rat oral LD50 datapoints for 282 substances from the RC (see Table 6-5).

6-19

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

For comparison with the NRU test method results and RC rat-only regressions, Section 6.4.1
provides the accuracy analysis for the RC database used with the RC millimole regression.
Sections 6.4.2 and 6.4.3 provide the accuracy information for the 3T3 and NHK NRU test
methods for the RC rat-only millimole regression and RC-rat only weight regression,respectively. A summary of predictivity3 is provided for each predicted toxicity category,along with the percentage of substances whose toxicity was underpredicted or overpredicted.
6.4.1

Prediction of GHS Acute Oral Toxicity Category by the RC IC50 Values Using the
RC Millimole Regression
Table 6-6 shows the concordance of the observed (i.e., in vivo) and predicted GHS acute oral
toxicity categories (UN 2005) for the 347 RC IC50 values in the RC millimole regression, log
LD50 (mmol/kg) = 0.435 x log IC50 (mM) + 0.625 (Halle 1998, 2003). Accuracy is theagreement of the in vitro category predictions with those based on the 347 rodent (282 rat
and 65 mouse) oral LD50 values used in the RC database (Halle 1998, 2003). Substances forwhich the in vitro toxicity category prediction did not match the in vivo category wereconsidered discordant for the GHS acute oral toxicity category predictions.
The overall accuracy of the RC IC50 values for correctly predicting GHS acute oral toxicity
classification category using the RC millimole regression was 40% (140/347substances)(Table 6-6). Rodent acute oral toxicity was overpredicted for 34% (118/347) andunderpredicted for 26% (89/347) of the substances. For this analysis, with respect to thepredictions of each GHS category:• None (0%) of the 12 substances with LD50 <5 mg/kg (GHS Category I) was
correctly predicted.
•
•
•

•
•

Four (15%) of 26 substances in the 5 < LD50 ≤50 mg/kg category (GHS
Category II) were correctly predicted.
Twenty (29%) of 69 substances in the 50 < LD50 ≤300 mg/kg category (GHS
Category III) were correctly predicted.
Ninety-seven (69%) of 140 substances in the 300 < LD50 ≤2000 mg/kg
category (GHS Category IV) were correctly predicted. This toxicity category
was also predicted for 106 other substances (52%; 106/203) that did not fall inthis category. Thus, the overall predictivity for this category was 48% (97/203
substances predicted for this category matched the in vivo category).
Fourteen (25%) of the 56 substances in the 2000 < LD50 ≤5000 mg/kg
category (GHS Category V) were correctly predicted.
Five (11%) of the 44 substances with LD50 >5000 mg/kg (GHS Unclassified)were correctly predicted.

.

3

Proportion of correct in vivo category matches for all substances with in vitro predictions for a particular
category. Predictivity is one of the measures of test accuracy (ICCVAM 2003).
6-20

In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-6

November 2006

Prediction of GHS Acute Oral Toxicity Category by the RC IC50 Values and the RC
Millimole Regression1
Total

Accuracy

Toxicity
Overpredicted

Toxicity
Underpredicted

0

12

0%

0%

100%

IC50-Predicted GHS Category (mg/kg)3

In Vivo Rodent Oral
LD502 (mg/kg)

LD50 <5

5 < LD50 ≤50

50 < LD50 ≤300

300 < LD50 ≤2000

2000 < LD50 ≤5000

LD50 >5000

LD50 < 5

0

5

3

4

0

5 < LD50 ≤50

0

4

13

9

0

0

26

15%

0%

85%

50 < LD50 ≤300

0

9

20

38

2

0

69

29%

13%

58%

300 < LD50 ≤2000

0

4

24

97

14

1

140

69%

20%

11%

2000 < LD50 ≤5000

0

1

5

36

14

0

56

25%

75%

0%

LD50 >5000

0

0

1

19

19

5

44

11%

89%

0%

347

40%

34%

26%

Total

0

23

66

203

49

6

Predictivity

0%

17%

30%

48%

29%

83%

Category Overpredicted

0%

61%

45%

27%

39%

0%

Category Underpredicted

0%

22%

24%

25%

33%

17%

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); RC=Registry of Cytotoxicity. Shaded cells are those containing the correct
predictions; RTECS®=Registry of Toxic Effects for Chemical Substances®.
1
The RC millimole regression is log LD50 (mmol/kg) = log IC50 (mM) x 0.435 + 0.625. Numbers in table represent numbers of substances.
2
Rat (282 values) and mouse (65 values) oral LD50 values, mostly from the 1983/84 RTECS® that were converted to mmol/kg for used in the RC (Halle 1998, 2003).
3
IC50 values from the RC are geometric mean IC50 values from in vitro cytotoxicity assays using multiple cell lines and cytotoxicity endpoints (Halle 1998,
2003). GHS categories were predicted by using the IC50 values to calculate predicted LD50 values with the RC millimole regression equation. Predicted LD50
values in mmol/kg for each substance were converted to mg/kg and used to classify the substance in the appropriate predicted GHS acute oral toxicity category.

6-21

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

The highest accuracy, 69%, for the RC IC50 values in the RC millimole regression wereobtained for substances in the 300 < LD50 ≤2000 mg/kg category (GHS Category IV). The
lowest accuracy, 0%, was obtained for substances with LD50 <5 mg/kg (GHS Category I).
Although the 11% accuracy was low for substances with LD50 >5000 mg/kg (GHS
Unclassified), the highest predictivity, 83%, was obtained for substances in this group. The
RC millimole regression generally underpredicted toxicity for substances in the highest
toxicity (i.e., lowest LD50) categories and overpredicted for substances in the lowest toxicity(i.e., highest LD50) categories (see Table 6-6).
Rodent acute oral toxicity was overpredicted for 34% (118) and underpredicted for 26% (89)of the 347 RC substances. Thus, there was a total of were 207 discordant substances. GHS
category was overpredicted for 57% (118/207) of the discordant substances andunderpredicted for 43% (89/207) of the discordant substances.
6.4.2

Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK NRU Test
Methods Using the RC Rat-Only Millimole Regression
Table 6-7 shows the concordance of the observed (i.e., in vivo) and predicted GHS acute oral
toxicity categories (UN 2005) for each in vitro test method using the geometric mean IC50
values (of the three laboratories) in the RC rat-only millimole regression, log LD50
(mmol/kg) = 0.439 x log IC50 (mM) + 0.621. Accuracy is the agreement of the in vitro
category predictions with those based on the rat acute oral LD50 reference values in Table 42. Substances for which the in vitro toxicity category prediction did not match the in vivo
category were considered discordant for the GHS acute oral toxicity category predictions.
6.4.2.1 In Vitro – In Vivo Concordance Using the RC Rat-Only Millimole Regression
The overall accuracy of the 3T3 NRU test method for correctly predicting GHS acute oral
toxicity classification category using the RC rat-only millimole regression was 31% (21/67
substances) (Table 6-7). Rat acute oral toxicity was overpredicted for 34% (23) andunderpredicted for 34% (23) of the substances. For this analysis, with respect to thepredictions of each GHS category:• None (0%) of the six substances with LD50 <5 mg/kg (GHS Category I) was
correctly predicted.
• One (9%) of 11 substances in the 5 < LD50 ≤50 mg/kg category (GHS
Category II) was correctly predicted.
• Five (42%) of 12 substances in the 50 < LD50 ≤300 mg/kg category (GHS
Category III) were correctly predicted.
• Thirteen (81%) of 16 substances in the 300 < LD50 ≤2000 mg/kg category(GHS Category IV) were correctly predicted. This toxicity category was also
predicted for 32 other substances (71%; 32/45) that did not fall in thiscategory. Thus, the overall predictivity for this category was 29% (13/45
substances predicted for this category matched the in vivo category).
• None (0%) of the 10 substances in the 2000 < LD50 ≤5000 mg/kg category(GHS Category V) were correctly predicted.
• Two (17%) of the 12 substances with LD50 >5000 mg/kg (GHS Unclassified)were correctly predicted.

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In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-7

November 2006

Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK NRU Test
Methods and the RC Rat-Only Millimole Regression1
Total

Accuracy

Toxicity
Overpredicted

Toxicity
Underpredicted

0

63

0%

0%

100%

4

3T3 -Predicted GHS Category (mg/kg)

Reference Rat Oral
LD502 (mg/kg)

LD50 <5

5 < LD50 ≤50

50 < LD50 ≤300

300 < LD50 ≤2000

2000 < LD50 ≤5000

LD50 >5000

LD50 < 5

0

2

0

4

0

5 < LD50 ≤50

0

1

6

3

1

0

11

9%

0%

91%

50 < LD50 ≤300

0

0

5

7

0

0

12

42%

0%

58%

300 < LD50 ≤2000

0

1

2

13

0

0

16

81%

19%

0%

2000 < LD50 ≤5000

0

0

0

10

0

0

105

0%

100%

0%

LD50 >5000

0

0

0

8

2

2

126,7

17%

83%

0%

67

31%

34%

34%

Total

Accuracy

Toxicity
Overpredicted

Toxicity
Underpredicted

Total

0

4

13

45

3

2

Predictivity

0%

25%

38%

29%

0%

100%

Category Overpredicted

0%

25%

15%

40%

67%

0%

Category Underpredicted

0%

50%

46%

31%

33%

0%

NHK -Predicted Toxicity Category (mg/kg)

Reference Rat Oral
LD502

LD50 <5

5 < LD50 ≤50

50 < LD50 ≤300

300 < LD50 ≤2000

2000 < LD50 ≤5000

LD50 >5000

LD50 <5

0

1

2

3

0

0

63

0%

0%

100%

5 < LD50 ≤50

0

2

5

3

1

0

114

18%

0%

82%

50 < LD50 ≤300

0

1

6

5

0

0

12

50%

8%

42%

300 < LD50 ≤2000

0

1

2

12

1

0

16

75%

19%

6%

2000 < LD50 ≤5000

0

0

0

10

0

0

105

0%

100%

0%

LD50 >5000

0

0

0

7

6

0

137

0%

100%

0%

Total

0

5

15

40

8

0

68

29%

40%

31%

Predictivity

0%

40%

40%

30%

0%

0%

Category Overpredicted

0%

40%

13%

43%

75%

0%

Category Underpredicted

0%

20%

47%

28%

25%

0%

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal
keratinocytes; NRU=Neutral red uptake; RC=Registry of Cytotoxicity. Shaded cells are those containing the correct predictions.
1
The RC rat-only millimole regression is log LD50 (mmol/kg) = log IC50 (mM) x 0.439 + 0.621. Numbers in table represent numbers of substances.
2
Reference rat oral LD50 values in mg/kg from Table 4-2.
3
Epinephrine bitartrate excluded because no rat reference acute oral LD50 was identified (see Table 4-2).
4Colchine excluded because no rat acute oral LD50 was identified (see Table 4-2).
5
Carbon tetrachloride excluded because no laboratory attained sufficient toxicity for the calculation of an IC50.
6Methanol excluded because no laboratory attained sufficient toxicity for the calculation of an IC50.
7Propylparaben excluded because no rat acute oral LD50 was identified (see Table 4-2).

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In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

The overall accuracy of the NHK NRU test method for correctly predicting the GHS acuteoral toxicity classification, when the prediction was based on the RC rat-only millimole
regression, was 29% (20/68 substances) (see Table 6-7). Toxicity was overpredicted for 40%(27) and underpredicted for 31% (21) of the 68 substances. The pattern of concordance
between in vitro and in vivo results for the NHK NRU test method with the RC rat-only
millimole regression was similar to that for the 3T3 NRU test method with the exception thatnone of the substances with a toxicity of LD50 >5000 mg/kg were correctly predicted. For
this analysis, with respect to the predictions of each GHS category:• None (0%) of the six substances with LD50 <5 mg/kg (GHS Category I) werecorrectly predicted.
• Two (18%) of 11 substances in the 5< LD50 ≤50 mg/kg category (GHS
Category II) were correctly predicted.
• Six (50%) of 12 substances in the 50< LD50 ≤300 mg/kg category (GHS
Category III) were correctly predicted.
• 12 (75%) of 16 substances in the 300< LD50 ≤2000 mg/kg category (GHS
Category IV) were correctly predicted; however, this category was also
predicted for 28 (70%; 28/40) substances that did not match the category.
Thus, the overall predictivity for this category was 30% (12/40).
• None (0%) of the 10 substances in the 2000< LD50 ≤5000 mg/kg category(GHS Category V) were correctly predicted.
• None (0%) of the 13 substances with LD50 >5000 mg/kg (GHS Unclassified)were correctly predicted.
The RC rat-only millimole regression generally underpredicted toxicity for substances in thehighest toxicity (i.e., lowest LD50) categories and overpredicted toxicity for substances in thelowest toxicity (i.e., highest LD50) categories (see Table 6-7). Although substances at thevery low and high ends of the toxicity range were poorly predicted, those in the middle range(i.e., 300 < LD50 ≤2000 mg/kg) were predicted much better, with 75 to 81% accuracy. The
pattern of accuracy for the GHS categories was similar to the pattern seen with the RC IC50
and LD50 values and the RC millimole regression (see Table 6-6) (i.e., lowest accuracy forvery toxic and very nontoxic substances and highest accuracy for substances with 300 < LD50
≤2000 mg/kg).
6.4.2.2

Discordant Substances in the Prediction of GHS Acute Oral Toxicity Category by
the 3T3 and NHK NRU Test Methods and the RC Rat-Only Millimole Regression
Appendix L2 identifies the discordant substances, that is, those for which the in vitro
predicted GHS acute oral toxicity category did not match the GHS acute oral toxicity
category assigned based on the reference rat acute oral LD50 data in Table 4-2. Of the total
number of substances used for this evaluation (67 for 3T3, 68 for NHK), the 3T3 test method
underpredicted the GHS category for 23 (50%) and overpredicted for 23 (50%) of the 46
discordant substances. The NHK test method underpredicted toxicity for 21 (44%) andoverpredicted for 27 (56%) of the 48 discordant substances.
6.4.3

Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK NRU Test
Methods Using the RC Rat-Only Weight Regression
Table 6-8 shows the concordances of the observed and predicted GHS acute oral toxicity
categories for each in vitro NRU method using the geometric mean IC50 values from the

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In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

three laboratories and the RC rat-only weight regression (Table 6-5). The regression formulafor the RC rat-only weight regression was log LD50 (mg/kg) = log IC50 (µg/mL) x 0.372 +2.024. Accuracy is the agreement of the GHS acute oral toxicity category predictions madeusing the in vitro NRU data with those based on the reference rat acute oral LD50 values(Table 4-2).
6.4.3.1 In Vitro – In Vivo Concordance Using the RC Rat-Only Weight Regression
The overall accuracy of the 3T3 NRU test method with the RC rat-only weight regression
was 31% (21/67) (Table 6-8). The toxicity was overpredicted for 33% (24) andunderpredicted for 36% (22) of the substances. For this analysis, with respect to thepredictions of the GHS category:• None (0%) of the six substances with LD50 <5 mg/kg (GHS Category I) werecorrectly predicted.
• One (9%) of 11 substances in the 5< LD50 ≤50 mg/kg category (GHS
Category II) was correctly predicted.
• Four (33%) of 12 substances in the 50< LD50 ≤300 mg/kg category (GHS
Category II) were correctly predicted; however, because 10 other substanceswere also predicted to be in this category, the overall predictivity was 29%(4/14).
• Twelve (75%) of 16 substances in the 300< LD50 ≤2000 mg/kg category(GHS Category IV) were predicted correctly. Because a total of 40 substanceswere predicted to be in this category, the overall predictivity was 30% (12/40).
• Four (40%) of 10 substances in the 2000< LD50 ≤5000 mg/kg category (GHS
Category V) were correctly predicted; however, because a total of 11
substances were predicted to be in this category, the overall predictivity was
36% (4/11).
• None (0%) of the 12 substances with LD50 >5000 mg/kg (GHS Unclassified)were correctly predicted.
The overall accuracy of the NHK predictions using the RC rat-only weight regression was
31% (21/68) (see Table 6-8). The in vivo GHS toxicity categories were overpredicted for
37% (22) and underpredicted for 32% (25) of the substances. For this analysis, with respect
to the predictions of the GHS category:• None (0%) of the six substances with LD50 <5 mg/kg (GHS Category I) werecorrectly predicted.
• One (9%) of 11 substances in the 5 < LD50 ≤ 50 mg/kg category (GHS
Category II) was correctly predicted.
• Five (42%) of 12 substances in the 50 < LD50 ≤300 mg/kg category (GHS
Category III) were correctly predicted; however, because six other substanceswere also predicted to be in this category, the overall predictivity was 33%(3/9).
• Thirteen (81%) of 16 substances in the 300 < LD50 ≤2000 mg/kg category(GHS Category IV) were predicted correctly; however, because 29 othersubstances were also predicted to be in this category, the overall predictivity
was 31% (13/42).

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In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-8

November 2006

Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK NRU
Test Methods and the RC Rat-Only Weight Regression1
3T3 -Predicted Toxicity Category (mg/kg)
Total

Accuracy

Toxicity
Overpredicted

Toxicity
Underpredicted

0

63

0%

0%

100%

0

0

114

9%

0%

91%

8

0

0

12

33%

0%

67%

3

12

0

0

16

75%

25%

0%

0

0

6

4

0

105

40%

60%

0%

0

0

0

5

7

0

126,7

0%

100%

0%

0

2

14

40

11

0

67

31%

33%

36%

Toxicity
Underpredicted
100%

Reference Rat Oral
LD502 (mg/kg)

LD50 <5

5 < LD50 ≤50

50 < LD50 ≤300

300 < LD50 ≤2000

2000 < LD50 ≤5000

LD50 >5000

LD50 <5

0

0

2

4

0

5 < LD50 ≤50

0

1

5

5

50 < LD50 ≤300

0

0

4

300 < LD50 ≤2000

0

1

2000 < LD50 ≤5000

0LD50 >5000
Total
Predictivity

0%

50%

29%

30%

36%

0%

Category Overpredicted

0%

50%

21%

28%

64%

0%

Category Underpredicted

0%

0%

50%

43%

0%

0%

NHK -Predicted Toxicity Category (mg/kg)
Reference Rat Oral
LD502 (mg/kg)

LD50 <5

5 < LD50 ≤50

50 < LD50 ≤300

300 < LD50 ≤2000

2000 < LD50 ≤5000

LD50 >5000

Total

Accuracy

LD50 <5

0

1

2

3

0

0

63

0%

Toxicity
Overpredicted
0%

5 < LD50 ≤50

0

1

5

5

0

0

114

9%

0%

91%

50 < LD50 ≤300

0

1

5

6

0

0

12

42%

8%

50%

300 < LD50 ≤2000

0

1

2

13

0

0

16

81%

19%

0%

2000 < LD50 ≤5000

0

0

0

9

1

0

105

10%

90%

0%

LD50 >5000

0

0

0

6

6

1

137

8%

92%

0%

68

31%

37%

32%

Total

0

4

14

42

7

1

Predictivity

0%

25%

36%

31%

14%

100%

Category Overpredicted

0%

50%

14%

36%

86%

0%

Category Underpredicted

0%

25%

50%

33%

0%

0%

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal
keratinocytes; NRU=Neutral red uptake; RC=Registry of Cytotoxicity.
1
The RC rat-only weight regression is log LD50 (mg/kg) = log IC50 (µg/mL) x 0.372 + 2.024.
2
Reference rat oral LD50 values in mg/kg from Table 4-2.
3
Epinephrine bitartrate excluded because no rat acute oral LD50 was identified (see Table 4-2).
4Colchine excluded because no rat acute oral LD50 was identified (see Table 4-2).
5
Carbon tetrachloride excluded because no laboratory attained sufficient toxicity for the calculation of an IC50.
6Methanol excluded because no laboratory attained sufficient toxicity for the calculation of an IC50.
7Propylparaben excluded because no rat acute oral LD50 was identified (see Table 4-2).

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In Vitro Cytotoxicity Test Methods BRD Section 6••

November 2006

One (10%) of 10 substances in the 2000 < LD50 ≤5000 mg/kg category (GHS
Category V) was correctly predicted.
One (8%) of 13 substances with LD50 >5000 mg/kg (GHS Unclassified) was
correctly predicted.

The RC rat-only weight regression generally underpredicted toxicity for substances in thehighest toxicity (i.e., lowest LD50) categories and overpredicted toxicity for substances in thelowest toxicity (i.e., highest LD50) categories (see Table 6-8). Although substances at thevery low and high ends of the toxicity range were poorly predicted, those in the middle range(i.e., 300 < LD50 ≤2000 mg/kg) were predicted much better, with 75 to 81% accuracy. The
pattern of accuracy for the GHS categories was similar to the pattern seen with the RC IC50
and LD50 values and the RC millimole regression (see Table 6-6) and with the NRU IC50 and
rat oral LD50 values and the RC rat-only millimole regression (see Table 6-7) (i.e., lowest
accuracy for very toxic and very nontoxic substances and highest accuracy for substanceswith 300 < LD50 ≤2000 mg/kg).
6.4.3.2

Discordant Substances in the Prediction of GHS Acute Oral Toxicity Category by
the 3T3 and NHK NRU Test Methods and the RC Rat-Only Weight Regression
Appendix L2 shows the substances for which the in vitro predicted GHS acute oral toxicity
category using the RC rat-only weight regression did not match those that were based on the
rat acute oral LD50 reference data. The two in vitro NRU test methods over- and underpredicted the GHS acute oral toxicity category for similar numbers of substances, comparedwith the GHS acute oral toxicity categories for the rat acute oral LD50 reference values inTable 4-2. The 3T3 NRU test method overpredicted the GHS acute oral toxicity category for
22 (48%) of 46 discordant substances, and underpredicted of 24 (52%) substances. The NHK
NRU test method overpredicted the GHS acute oral toxicity category for 25 (53%) of 47
discordant substances, and underpredicted 22 (47%) substances.
6.4.4
Summary of the Regressions Evaluated
Table 6-9 summarizes the regressions evaluated in Section 6.4 for accuracy in predicting the
GHS acute oral toxicity categories (UN 2005), and the proportion of over- or underpredictions. Prediction accuracy using the RC IC50 and LD50 values and the RC millimole
regression was higher that that for the NRU test methods with the RC rat-only regressions(i.e., 40% for the RC vs. 29% to 31% for the NRU test methods). Prediction accuracy wasslightly higher for the 3T3 NRU test method compared with the NHK NRU (i.e., 31% for
3T3 vs. 29% for NHK) using the RC rat-only millimole regression, and the same as the NHK
NRU test method (i.e., 31%) using the RC rat-only weight regression. The proportion of
discordant substances using the RC IC50 values and the RC millimole regression (60%) was
lower than that using the in vitro NRU test methods and the RC rat-only regressions (69% to71%). The proportion of discordant substances from the 3T3 test method, 69%, was the same
whether it was determined with the RC rat-only millimole regression or the RC rat-only
weight regression. The proportion of discordant substances for the NHK test method wasslightly lower with RC rat-only weight regression than with the RC rat-only millimole
regression (69% vs. 71%). The RC IC50 values and the RC millmole regression were
expected to perform better than the in vitro NRU methods and the RC rat-only regressions
since the IC50 and LD50 values used to evaluate the performance of the RC millimole
regression were exactly the same as those used to calculate the linear regression formula. The

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In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

NRU IC50 values and the reference oral LD50 values used to evaluate the RC rat-onlyregressions were different from those used to calculate the RC rat-only regressions.
Table 6-9

Comparison of Regressions and In Vitro NRU Test Methods for Their
Performance in Predicting GHS Acute Oral Toxicity Categories
N1

R2 Statistic

Accuracy

Discordant Substances2

RC millimole3

347

0.452

RC IC50 – 40%

RC IC50 – 207/347 (60%)

RC rat-only millimole3

282

0.452

RC rat-only weight3

282

0.325

3T3– 31%
NHK– 29%
3T3– 31%
NHK– 31%

3T3– 46/67(69%)
NHK– 48/68 (71%)
3T3– 46/67 (69%)
NHK– 47/68 (69%)

Regression

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005);
3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake;
RC=Registry of Cytotoxicity; R2=Coefficient of determination.
1
Number of substances used in regression.
2
Proportion of discordant substances.
3
From Table 6-5.

The accuracy of the GHS category predictions using the in vitro NRU test methods with the
RC rat-only regressions obtained for the reference substances from this validations study may
or may not be applicable to other substances. A number of reasons may explain the low
accuracy for the reference substances. One is the skewness of the substances selected fortesting with respect to fit to the RC millimole regression (see Figure 3-1). Table 3-4 showsthat 22 (38%) of the 58 RC substances selected for testing were known to poorly fit the RC
millimole regression (i.e., the predicted LD50 was outside the RC acceptance interval).
Toxicity was underpredicted for 17 (77%) of these outlier substances and overpredicted (i.e.,
predicted LD50 was lower than measured in vivo LD50) for the remaining five (23%). Table
6-3 shows that 40% (28/70 for 3T3) and 44% (31/71 for NHK) of the reference substancesthat yielded IC50 values were outliers. Other reasons for the low accuracy for GHS acute oral
toxicity prediction, such as those discussed in Section 1.2.3, include the major differences
between cell cultures and whole animals regarding the absorption, distribution (including
binding to serum proteins), availability, metabolism, and excretion of reference substances.
6.5

Correlation of NRU Concentration-Response Slope with Rat Lethality DoseResponse Slope

Because the slope calculations available for the NRU concentration-response curve analyses
were based on the Hill function, the SMT determined whether the Hill Slope correlated withthe rodent dose-mortality slope. If the two were correlated, the Hill Slope from the NRU test
methods could be used to estimate the dose-mortality slope, which could, in turn, be used toestimate the most appropriate dose progression for UDP testing in rodents. A more
immediate use for the validation study results, however, would be for the computer
simulation modeling of animal testing for the UDP and ATC acute oral toxicity methods(described in Sections 10.2 and 10.3).
Dose-mortality slope information was available for 22 of the 72 reference substances, as
shown in Table 6-10. Hill function slopes were available for 20 and 21 of the 22 substances
6-28

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

for the 3T3 and the NHK NRU test methods, respectively. The Hill function slopes weretransformed to absolute values because geometric means cannot be calculated for negative
numbers, and geometric mean Hill function slopes were calculated for the acceptable NRU
tests for each reference substance. When there was more than one dose-mortality slope
available for a substance, a geometric mean was calculated from the available values. The
absolute values of the geometric mean Hill function slopes are plotted against the geometricmean dose-mortality slopes in Figure 6-5. To determine whether there was a relationshipbetween the absolute value of the Hill Slope and the dose-mortality slope, Spearman
correlation analyses and least squares linear regression analyses were performed for eachmethod. Both analyses showed that the absolute value of the in vitro Hill function slope was
not related to the dose-mortality slope. The Spearman correlation analysis yielded
nonsignificant correlations for both in vitro NRU test methods (3T3 rs=-0.051 with p=0.831,
and NHK rs=-0.142 with p=0.541). Linear regression analyses for the prediction of dosemortality slope by the absolute value of the Hill function slope also showed that the slopes of
the regressions were not significantly different from zero (3T3 p=0.774, and NHK p=0.994).
Because there was no relationship between Hill function slope and dose-mortality slope, theHill function slope was not used to predict the dose-mortality slope for the simulation
modeling of animal testing for the UDP and ATC acute oral toxicity methods in Sections
10.2 and 10.3.
Table 6-10

Reference Substances with Dose-Mortality and NRU Hill Slopes

Reference Substance
Acetylsalicylic acid
Boric acid
Caffeine
Carbon tetrachloride
Dichlorvos
Dimethylformamide
Diquat dibromide
Ethanol
Ethylene glycol
GlycerolHexachorophene
Lactic acid
MethanolNicotine
Parathion
Potassium cyanide
Sodium arsenite
Sodium I fluoride
Trichloroacetic acid
Triethylene melamine
Valproic acid
XyleneCarbon tetrachloride

Dose-Mortality Slope1
1.45
7.70
6.27
2.06
1.24
1.11
16.57
4.57
38.38
8.90
12.84
4.04
8.53
3.00
1.31
14.50
7.60
1.26
20.97
2.10
1.20
9.60
2.06

3T3 Hill Slope2
1.658
1.511
1.069
NA
2.240
1.875
4.273
1.725
2.016
1.941
1.466
4.541
NA
11.019
1.551
1.931
2.317
3.952
1.883
0.963
2.467
1.871
NA

NHK Hill Slope2
1.906
1.083
1.215
NA
1.383
3.157
1.289
2.049
2.904
2.398
2.470
2.934
1.173
0.682
1.467
1.207
1.717
2.569
1.369
1.355
1.440
2.452
NA

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake;
NA=Not available.
1
Geometric mean if there was more than one value for each substance (from Appendix H2).
2
Geometric mean of absolute values from acceptable in vitro NRU tests.

6-29

In Vitro Cytotoxicity Test Methods BRD Section 6

Figure 6-5

November 2006

Correlation of Dose-Mortality Slope to Hill Function Slope

a.
45
40
35
30
25
20
15
10
5
0

0

1

2

3

4

5

6

7

8

9

10

11

12

3T3 NRU Hill Slope

b.
45
40
35
30
25
20
15
10
5
0
0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

NHK NRU Hill Slope
Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake.
Hill function slopes and dose-mortality slopes for the reference substances shown in Table 6-10 for (a) the 3T3 data and (b)the NHK data. The solid line indicates the theoretical, one-to-one correspondence of Hill function slope with dose-mortality
slope. Spearman’s correlation coefficients were rs=-0.051 (p=0.831) for the 3T3 and rs=-0.142 (p=0.541) for the NHK data.

6-30

In Vitro Cytotoxicity Test Methods BRD Section 6

6.6

November 2006

Strengths and Limitations of the Use of In Vitro Cytotoxicity Test Methods
with the IC50-LD50 Regressions for Prediction of Rodent Acute Oral Toxicity

6.6.1
In Vitro Cytotoxicity Methods
The NRU basal cytotoxicity methods tended to underpredict the toxicity of the most toxic
substances and to overpredict the toxicity of the least toxic substances for each regression
evaluated. The 3T3 and NHK NRU test methods were best at predicting the toxicity of
substances with 300 < LD50 ≤2000 mg/kg. The accuracy of the in vitro prediction of thisGHS category using the RC rat-only millimole regression and the RC rat-only weight
regression was 75-81%. GHS toxicity categories of substances with higher or lower LD50
values were correctly predicted with less than 50% accuracy. The worst accuracy, 0%, wasobserved for:• Substances with LD50 ≤5 mg/kg in both in vitro test methods and regressions• Substances with 2000< LD50 ≤5000 mg/kg using 3T3 with the RC rat-only
millimole regression• Substances with 2000< LD50 ≤5000 mg/kg or LD50 >5000 mg/kg using NHKwith RC rat-only millimole regression• Substances with LD50 >5000 mg/kg using 3T3 with RC rat-only weight
regression
Some substances with low toxicity and low solubility could not be tested in the in vitro NRU
test methods because the concentration of dissolved substance was inadequate to obtain an
IC50 value. None of the laboratories obtained adequate toxicity in any of the 3T3 tests ofcarbon tetrachloride or methanol, and at least one laboratory failed to achieve adequate
toxicity with gibberellic acid or xylene. No laboratory achieved adequate toxicity in any of
the NHK experiments with carbon tetrachloride, and at least one laboratory could not achieveadequate toxicity with methanol, 1,1,1-trichloroethane, or xylene. Another limitation of useof the in vitro test methods is in the testing of substances that come out of solution byforming a film on the medium surface or plastic well wall (i.e., “film out”), and for
substances that etch the laboratory ware plastics (ICCVAM 2006). Substances that etch
plastics can be detected by looking for the presence of etched rings in the 96-well plates after
exposure. Some substances that produce films in medium also etch plastic.
The prediction of rodent acute oral toxicity (and the starting doses for acute oral toxicity
tests) by the in vitro NRU methods is expected to be poor for substances with mechanisms of
toxicity that are not effective in the 3T3 and NHK cells. Such toxic mechanisms include
specific, receptor-mediated actions on the CNS or the heart.
The evaluation of the 3T3 and NHK NRU test methods for predicting starting doses forrodent acute oral toxicity testing with its potential to reduce and refine animal use is provided
in Section 10.
6.6.2

Use of Mole-Based vs. Weight-Based Regressions for the Prediction of Toxicity
for Low and High Molecular Weight Substances
The ICCVAM ATWG expressed concern that the RC rat-only weight regression may lessaccurately predict the toxicity of low and high molecular weight substances than the RC ratonly millimole regression. Using the RC IC50 and LD50 values for the 282 RC substanceswith rat oral LD50 data, analyses were performed to:
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November 2006

Determine the difference in the over and under-prediction of rodent acute oral
toxicity (i.e., LD50) from IC50 values between low molecular weightsubstances (i.e., ≤100 g/mole) and substances with molecular weights >100g/moleDetermine the difference in the over and under-prediction of rodent acute oral
toxicity from IC50 values between high molecular weight substances (i.e.,≥400 g/mole) vs. substances with molecular weights <400 g/mole.
Compare the RC rat-only millimole regression with the RC rat-only weight
regression with respect to the over- and under-prediction of the toxicity of low
and high molecular weight substancesThis analysis used the RC data rather than the validation studies data because the RC
contains data for many more substances. The analysis assumes that the regressions either
underpredicted or overpredicted the toxicity of all of the substances evaluated. In otherwords, there was a difference between the LD50 predicted by the regression and the in vivo
LD50 used to calculate the regression even if it was a tiny fraction (i.e., no substances fit the
regression exactly). The complete analysis and discussion are presented in Appendix J7. Of
the 282 RC substances with rat acute oral LD50 values, there were 51 with molecular weights≤100 g/mole and 231 with molecular weights >100 g/mole. For the 51 substances withmolecular weight ≤100 g/mole, the RC rat-only millimole regression underestimated the
toxicity of 20/51 (39%) substances and overestimated the toxicity of 31/51 (61%) substances.
The RC rat-only weight regression underestimated the toxicity of 24/51 (47%) substancesand overestimated the toxicity of 27/51 (53%) substances. Fisher’s exact test indicated that
there was no difference between the millimole and weight regressions with respect to theunder or over-prediction of toxicity for the low molecular weight substances (two-tailed
p=0.549) (see Table 6-11).
For the 231 substances with molecular weights >100 g/mole, the RC rat-only millimole
regression underestimated the toxicity of 108/231 (47%) substances and overestimated the
toxicity of 123/231 (53%). The RC rat-only weight regression underestimated the toxicity of
101/231 (44%) substances and overestimated the toxicity of 130/231 (57%). Fisher’s exact
test indicated that there were no significant differences between the millimole and weight
regressions for the under- and over-prediction of toxicity for the 231 substances withmolecular weight >100 g/mole (two-tailed p=0.575). Fisher’s exact test also showed that
there were no significant differences in the under- and over-prediction of the toxicity of the
51 substances with molecular weight ≤100 g/mole compared to the under- and overprediction of the toxicity of the 231 with molecular weight >100 g/mole (two-tailed p=0.756
for the RC rat-only weight regression, and two-tailed p=0.355 for the RC rat-only millimole
regression).

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In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-11

November 2006

Over- and Under- Prediction of Toxicity for Low and High Molecular
Weight Substances Using RC Rat-Only Weight and Millimole
Regressions
Comparison

RC rat-only millimole vs. RC rat-only weight
regression
RC rat-only millimole vs. RC rat-only weight
regression
51 Low molecular weight (≤100 g/mole)substances vs. 231 other substances (>100g/mole)51 Low molecular weight (≤100 g/mole)substances vs. 231 other substances (>100g/mole)RC rat-only millimole vs. RC rat-only weight
regression
RC rat-only millimole vs. RC rat-only weight
regression
20 High molecular weight substances (≥400
g/mole) vs. 262 other substances (<400 g/mole)
20 High molecular weight substances (≥400
g/mole) vs. 262 other substances (<400 g/mole)For
Under- and over-prediction of toxicity
for 51 substances with molecularweight ≤100 g/mole
Under- and over-prediction of toxicity
for 231 substances with molecularweight >100 g/mole

Fisher’s Exact
Test1
0.549
0.575

RC rat-only millimole regression

0.355

RC rat-only weight regression

0.756

Under- and over-prediction of toxicity
for 20 substances with molecularweight ≥400 g/mole
Under- and over-prediction of toxicity
for 262 substances with molecularweight <400 g/mole

0.480
NT

RC rat-only millimole regression

0.362

RC rat-only weight regression

0.033

Abbreviations: RC=Registry of Cytotoxicity; NT=Not tested because the proportions were the same. Toxicity was
underpredicted for 121/262 (46%) substances and overpredicted for 141/262 (54%) substances.
1
P-values.

Of the 282 RC substances with rat acute oral LD50 values, there were 20 with molecularweights ≥400 g/mole and 262 with molecular weights <400 g/mole. The RC rat-only
millimole regression underestimated the toxicity of 7/20 (35%) of the ≥400 g/mole
substances and overestimated 13/20 (65%). The RC rat-only weight regression
underestimated the toxicity of 4/20 (20%) of the substances and overestimated 16/20 (80%).
Fisher’s exact test indicated that there were no differences between the millimole and weight
regressions for the under- and over-prediction of toxicity for the 20 high molecular weightsubstances (two-tailed p=0.4801).
For the remaining 262 substances with molecular weights <400 g/mole, both the RC rat-only
millimole and the RC rat-only weight regressions underestimated the toxicity of 121/262(46%) substances and overestimated 141/262 (54%). Thus, there were no statisticaldifferences in the under- and over-esimation of toxicity for the 262 substances withmolecular weights <400 g/mole regardless of which regression was used. Fisher’s exact test
also showed that there was no statistical difference in the under- and over-prediction of the
toxicity of substances with high molecular weight (≥400 g/mole) compared with the underand over-prediction the lower molecular weight substances using the RC rat-only millimole
regression (two-tailed p=0.362). In contrast the use of the RC rat-only weight regression,
resulted in a small but statistically significant difference in the under- and over-prediction of
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In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

the toxicity of substances with high molecular weight (>400 g/mole) compared with theunder- and over-prediction of the toxicity of substances with lower molecular weight (twotailed p=0.033). The weight-based regression significantly overestimated the toxicity of thehigh molecular weight substances (compared with substances with lower molecular weight)
while the millimole regression did not.
6.7

Salient Issues of Data Interpretation

One of the most important considerations for the 3T3 and NHK NRU test methods, as forany test method, is the ability to generate good concentration-response results. In addition totechnical difficulties with these test methods, such as occasional poor cell growth and theformation of NRU crystals, this validation study yielded non-monotonic concentrationresponse curves for certain substances.
A number of substances produced non-monotonic concentration-response curves in the 3T3
and/or the NHK NRU range finding or definitive tests. Because the in vitro NRU test
methods, and the calculation of IC50 values from the resulting concentration curves, presume
that the toxic response is linear, the data from non-linear responses (e.g., biphasic curves), as
seen with aminopterin, do not always permit an IC50 determination by the standard Hillfunction analysis. In such cases, the lowest concentration that killed approximately 50% of
the cells in the range finding test was used to set the concentration range for the definitive
test. The definitive test used more closely spaced concentrations in an attempt to obtain amonotonic concentration-response curve. However, 100% toxicity (or 0%) viability wasoften unattainable in such definitive tests that exhibited a plateau of toxicity well over 0%
viability (e.g., 20%). Care must be used in the calculation of the IC50 for curves for which
toxicity plateaus to assure that the value reflects the concentration at 50% inhibition of the
VC value rather than simply the midpoint of the highest and lowest response.
Because of low toxicity and/or low solubility, some substances did not produce sufficient
toxicity for the calculation of an IC50 value. Carbon tetrachloride, methanol, xylene,
gibberellic acid, lithium carbonate, and 1,1,1-trichloroethane failed to yield acceptable IC50
results in at least one laboratory because of insufficient toxicity. All of these substances, withthe exception of methanol, produced precipitate in the cell culture medium.
6.8

Comparison of NRU Test Results to Established Performance Standards

The Guidance Document method of evaluating in vitro basal cytotoxicity assays forpredicting starting doses for rodent acute oral toxicity assays provides the existing
performance standard (ICCVAM 2001b) for the 3T3 and NHK NRU test methods. The
Guidance Document recommends testing 10 to 20 reference substances from the RC in an invitro basal cytotoxicity assay for predicting starting doses for rodent acute oral toxicity
testing (ICCVAM 2001b). These substances should cover a wide range of toxicity and fit the
RC millimole regression as closely as possible. The Guidance Document recommends using
the IC50 results for the selected reference substances from the candidate method to calculate a
new regression line with the LD50 values used by the RC. If the resulting regression isparallel to the RC millimole regression and within the ± log 5 (i.e., ± 0.699) prediction
interval for the RC, candidate assay may be considered effective for predicting starting doses
for substances in rodent acute oral toxicity assays.

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In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

One goal of the testing in Phases Ib and II of this study was to establish whether the resultsfrom the 3T3 and NHK NRU test methods were consistent with the RC millimole regression.
As discussed in Section 3.3.5, two of the major criteria for selecting the 12 coded substancestested from the 72 reference substances were:(a) Two substances must be included from each of the unclassified and classified
GHS acute oral toxicity categories, and(b) The substances must fit as closely to the RC millimole regression as possible.
Unfortunately, the SMT could not identify 12 substances that fit both criteria because there
was only one substance, aminopterin, in the LD50 <5 mg/kg category that fit the RC
millimole regression. The other substance chosen from that toxicity category was sodium
selenate. Because sodium selenate was not included in the RC, there was no indication of
how closely it would fit the RC millimole regression, and it was therefore not included in the
Phases Ib and II regression analyses. The other 10 substances selected for testing in Phases Ib
and II were colchicine, arsenic trioxide, cadmium chloride, sodium fluoride, propranolol,
lithium carbonate, potassium chloride, chloramphenicol, 2-propanol, and ethylene glycol.
The geometric mean log IC50 (mM) values from the 3T3 and NHK NRU test methods fromeach laboratory were used with the oral log rodent LD50 (mmol/kg) values from the RC (seeAppendices J1 and J2) for the least squares linear regression analyses (see Section 5.5.3.3)
for the substances tested in Phases Ib and II. The slopes for all regressions were significantly
different from zero at p <0.0001, which indicated that there was a significant relationshipbetween IC50 and LD50. The R2 values for the regressions from each laboratory, shown in
Table 6-12, show that the 3T3 NRU test method produced better-fitting regressions than thecorresponding NHK NRU test method (R2 = 0.940 to 0.953 vs. 0.577 to 0.621). The
relatively low R2 values for the NHK NRU test method were attributed to the much lower
toxicity of aminopterin in those cells (see Figures 6-6 to 6-8 and Tables 5-3 and 5-4). All
test method and laboratory-specific regressions were consistent with the RC millimole
regression. Table 6-12 shows that all joint comparisons of slopes and intercepts with the RC
millimole regression were not significant (i.e., p >0.01). The RC millimole regression slope
and intercept were used as constants for this comparison.
A graphic comparison of the IC50 regressions with the RC millimole regression as suggestedby the Guidance Document (ICCVAM 2001b) demonstrated that they were generally within
the RC millimole regression acceptance limits (see Figures 6-6, 6-7, and 6-8). According to
the Guidance Document (ICCVAM 2001b), in vitro basal cytotoxicity assays providing suchconsistency with the RC millimole regression are acceptable for predicting starting doses forrodent acute oral toxicity assays.
As an additional analysis, a regression for the 11 substances tested in Phases Ib and II (theRC-11 millimole regression), was calculated using the log RC IC50 (mM) and log LD50
(mmol/kg) values (see Table 6-12). Each of the laboratory regressions for each test method
was then compared to the RC-11 regression using an F test for a joint comparison of slope
and intercept. None of the regressions were significantly different from the RC-11 regression(p values ranged from 0.755 to 0.933).

6-35

In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-12

November 2006

Linear Regressions for 11 Substances Tested in Phases Ib and II
3T3 Regression1

Laboratory
ECBC
FAL
IIVS
Laboratory
ECBC
FAL
IIVS

Intercept
0.793
0.709
0.710

Slope

R2 Statistic

0.584
0.940
0.598
0.953
0.584
0.949
NHK Regression1

Intercept

Slope

R2 Statistic

0.401
0.429
0.373

0.530
0.548
0.549

0.577
0.621
0.590

Test Against RC
Regression2
0.040
0.024
0.041

Test Against RC-11
Regression3
0.829
0.909
0.933

Test Against RC
Regression2
0.620
0.569
0.538

Test Against RC-11
Regression3
0.805
0.853
0.755

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in Medical
Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; RC=Registry of Cytotoxicity; 3T3=BALB/c 3T3
fibroblasts; NHK=Normal human epidermal keratinocytes; R2=Coefficient of determination.
1
Laboratory and test method regressions were calculated after log transforming the NRU IC50 in mM and the RC LD50 in
mmol/kg for the 11 RC substances tested in study Phases Ib and II (shown in Figures 6-6 through 6-8).
2
Simultaneous comparison of slope and intercept with RC millimole regression: log LD50 (mmol/kg) = 0.435 x log IC50(mM) + 0.625; R2=0.452; the reported values are p values of the statistic.
3
Simultaneous comparison of slope and intercept with RC-11 regression (defined as a regression on the 11 substances): log
LD50 (mmol/kg) = 0.552 x log IC50 (mM) + 0.602; R2=0.971; the reported values are p values of the statistic.

6-36

In Vitro Cytotoxicity Test Methods BRD Section 6

Figure 6-6

November 2006

In Vitro – In Vivo Regressions1 for Phases Ib and II for ECBC
3T3 ECBC

1000

log LD50 = 0.584 log IC50 + 0.793
R2 = 0.940

Potassium chloride

100

Chloramphenicol

Lithium carbonate
2-Propanol

10
Propranolol HCl

1

EthyleneglycolSodium fluoride

Cadmium chloride

0.1
Arsenic trioxide

0.01

Colchicine
Aminopterin

0.001
0.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)

NHK ECBC

1000

log LD50 = 0.530 log IC50 + 0.401
R2 = 0.577

2-Propanol

100

EthyleneglycolPotassium chloride
Lithium carbonate
Chloramphenicol

10

Sodium fluoride
Propranolol HCl

1
Cadmium chloride

0.1
Arsenic trioxide

0.01

Colchicine
Aminopterin

0.001
0.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)

Abbreviations: ECBC=Edgewood Chemical Biological Center; RC=Registry of Cytotoxicity; 3T3=Neutral red
uptake using BALB/c 3T3 fibroblasts; NHK= Neutral red uptake using normal human epidermal keratinocytes;
R2=Coefficient of determination.
1
Regressions of substances tested in study Phases Ib and II do not include sodium selenate because it was notincluded in the RC. Regressions were calculated using the NRU IC50 values and the RC LD50 values.
The solid lines show RC millimole regression (bold) and acceptance limits (lighter). The dashed shows the
ECBC regressions.

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In Vitro Cytotoxicity Test Methods BRD Section 6

Figure 6-7
1000

November 2006

In Vitro – In Vivo Regressions1 for Phases Ib and II for FAL
3T3 FALlog LD50 = 0.598 log IC50 + 0.709
R2 = 0.953

2-Propanol

100

EthyleneglycolPotassium chloride

10

Chloramphenicol
Sodium fluoride

Propranolol HCl

1
Cadmium chloride

0.1
Arsenic trioxide

0.01

Colchicine
Aminopterin

0.001
0.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)
1000

NHK FALlog LD50 = 0.548 log IC50 + 0.429
R2 = 0.621

2-Propanol

100
Chloramphenicol

EthyleneglycolPotassium chloride

10
Sodium fluoride Lithium carbonate
Propranolol HCl

1

Cadmium chloride

0.1
Arsenic trioxide

0.01

Colchicine

0.001
0.000001 0.00001

Aminopterin

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)

Abbreviations: FAL=Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory
RC=Registry of Cytotoxicity; 3T3=Neutral red uptake using BALB/c 3T3 fibroblasts; NHK=Neutral red uptake
using normal human epidermal keratinocytes; R2=Coefficient of determination.
1
Regressions of substances tested in study Phases Ib and II do not include sodium selenate because it was notincluded in the RC. Regressions were calculated using the NRU IC50 values and the RC LD50 values.
The solid lines show RC millimole regression (bold) and acceptance limits (lighter). The dashed shows the FAL
regressions.
6-38

In Vitro Cytotoxicity Test Methods BRD Section 6

Figure 6-8
1000

November 2006

In Vitro – In Vivo Regressions1 for Phases Ib and II for IIVS
3T3 IIVS

log LD50 = 0.584 log IC50 + 0.710
R2 = 0.949

2-Propanol

100

EthyleneglycolChloramphenicol
Potassium chloride

10
Propranolol HCl

1

Sodium fluoride

Cadmium chloride

0.1

Arsenic trioxide

0.01

Colchicine
Aminopterin

0.001
0.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)
1000

NHK IIVS

log LD50 = 0.549 log IC50 + 0.373
R2 = 0.590

EthyleneglycolPotassium chloride

100

2-Propanol
Chloramphenicol

10
Sodium fluoride

Lithium carbonate

Propranolol HCl

1

Cadmium chloride

0.1

0.01

Arsenic trioxide

Colchicine
Aminopterin

0.001
0.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)

Abbreviations: IIVS=Institute for In Vitro Sciences; RC=Registry of Cytotoxicity; 3T3=Neutral red uptake
using BALB/c 3T3 fibroblasts; NHK=Neutral red uptake using normal human epidermal keratinocytes;
R2=Coefficient of determination.
1
Regressions of substances tested in study Phases Ib and II do not include sodium selenate because it was notincluded in the RC. Regressions were calculated using the NRU IC50 values and the RC LD50 values.
The solid lines show RC millimole regression (bold) and acceptance limits (lighter). The dashed shows the IIVS
regressions.
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In Vitro Cytotoxicity Test Methods BRD Section 6

6.9

November 2006

Summary

The millimole regressions developed using the validation study IC50 and LD50 values were
not significantly different from the regressions for the same 47 RC substances using the RCdata (F test; p=0.612 for the 3T3 regression and p=0.759 for the NHK regression). Because
this validation study provided results similar to the RC, which has more than 3.5 times thenumber of substances, the 282 RC substances with rat LD50 values were used to determine
the relationship between the IC50 and LD50 data. One linear regression was developed using
millimole units for the measurement of substances, the RC rat-only millimole regression, andone was developed using weight units (which are more practical in a routine testing
situation), the RC rat-only weight regression. The RC rat-only millimole regression isapplicable to substances of known molecular weight while the RC rat-only weight regression
is applicable for use with complex mixtures, substances whose molecular weight is unknown.
Characteristics that seemed promising for characterizing the RC millimole regression outliers
were chemical class, boiling point, molecular weight, and log Kow. Different chemical classes
behaved differently with respect to being outliers; ranging from 5/5 (100%) for the organic
sulfur compounds for both test methods to 4/14 (29%) for carboxylic acids for the 3T3 NRU.
Of the reference substances with boiling points >200°C, 9/13 (69%) were outliers for the 3T3
NRU and 8/13 (62%) were outliers for the NHK NRU. With respect to molecular weights,
4/7 (57%) substances with molecular weight >400 g/mole were outliers using the 3T3 data,and 3/7 (43%) were outliers using the NHK data. When log Kow was used, 8/13 (62%)
substances with a log Kow >3 were outliers for both test methods.
The lack of fit of individual substances to the RC millimole regression was not consistently
related to insolubility or to the fact that the test method systems had little to no metabolic
capability. Of the substances that exhibited precipitation, 11/25 (44%) were outliers in the
3T3 NRU assays and 11/24 (46%) were outliers in the NHK NRU assays. However, althoughthe 3T3 and NHK cells have little to no metabolic capability, the toxicity of substances
known to produce active metabolites in vivo was not underpredicted by these assays. Of the
19 substances known to produce active metabolites in vivo, 10 (53%) were outliers in the 3T3
NRU test method; the toxicity of six (60%) was underpredicted while the toxicity of four(40%) overpredicted. These 10 substances accounted for 36% of the 28 outliers identified by
the 3T3 NRU test method. Similarly, nine (47%) of the 19 substances known to produce
active metabolites in vivo were outliers in the NHK NRU test method. Of these nine, the
NHK NRU test method underpredicted the toxicity of five (56%) and overpredicted four(44%). These nine outliers accounted for 29% of the 31 outliers identified by the NHK NRU
test method.
The examination of outliers based on mechanisms of toxicity showed that 10/16 (63%)
substances with specific neurotoxic mechanisms were outliers in both the 3T3 and NHK
NRU test methods. Three of the six (50%) cardiotoxic substances were outliers in the 3T3
NRU test method and two (33%) were outliers in the NHK NRU test method. When all thereference substances with mechanisms of toxicity that are not expected to be active in the
3T3 and NHK systems (i.e., in Table 6-3) were summed, 13/22 (59%) were outliers for the
3T3 NRU and 12/22 (55%) were outliers for the NHK NRU.

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In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

The accuracy of the 3T3 and NHK NRU test methods for predicting the GHS acute oral
toxicity categories was 31% (21/67) and 29% (20/68), respectively, when used with the RC
rat-only millimole regression. The corresponding accuracy with the RC rat-only weight
regression was 31% for both methods (21/67 for 3T3, and 21/68 for NHK). Accuracy was
highest for substances in the 300< LD50 ≤2000 mg/kg range. The accuracies of theregressions, with respect to the GHS categories, were similar for both regressions (millimole
and weight) and all three laboratories.
• 0% for substances with LD50 ≤5 mg/kg (GHS Category I)• 9% to 18% for substances with 5< LD50 ≤50 mg/kg (GHS Category II)• 33% to 50% for substances with 50< LD50 ≤300 mg/kg (GHS Category III)
• 75% to 81% for substances with 300< LD50 ≤2000 mg/kg (GHS Category IV)• 0% to 40% for substances with 2000< LD50 ≤5000 mg/kg (GHS Category V)• 0% to 17% for substances with LD50 >5000 mg/kg (GHS Unclassified)
The overall accuracy for prediction of GHS category prediction using the RC IC50 and LD50
values and the RC millimole regression was higher that that for the NRU test methods with
the RC rat-only regressions (i.e., 40% for the RC vs. 29% to 31% for the NRU test methods
and RC rat-only regressions). However, the pattern of accuracy for the GHS categories was
similar. For all the accuracy analyses, the lowest accuracy was obtained for very toxic andvery nontoxic substances and highest accuracy was obtained for substances with 300 < LD50
≤2000 mg/kg.
The accuracy of GHS acute oral toxicity category predictions using the in vitro NRU test
methods with the RC rat-only regressions obtained for the reference substances may or may
not be broadly applicable to substances that might require acute oral toxicity testing. The
reasons for the low accuracy obtained in this validation study include: the differences
between cell cultures and whole animals regarding the absorption, distribution, availability,
metabolism, and excretion of reference substances, and the presence or absence of toxicity
targets; the skewness of the selection of substances for testing (with respect to fit to theregression); and the structure of the GHS acute oral toxicity categories.

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In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

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6-42

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

7.0

RELIABILITY OF THE 3T3 AND NHK NRU TEST METHODS ...................7-3

7.1

Reference Substances Used to Determine the Reliability of the 3T3 andNHK NRU Test Methods......................................................................................7-3

7.2

Reproducibility Analyses for the 3T3 and NHK NRU Test Methods.................7-5
7.2.1 Comparison of Laboratory-Specific IC50-LD50 Linear Regression
Analyses to the Mean Laboratory Regression..............................................7-5
7.2.2 ANOVA Results for the 3T3 and NHK NRU Test Methods ........................7-5
7.2.3 CV Results for the 3T3 and NHK NRU Test Methods...............................7-21
7.2.4 Comparison of Maximum to Minimum IC50 Values Using Laboratory
Means .......................................................................................................7-39
7.2.5 Comparison of Maximum:Minimum IC50 Ratios with the
Maximum:Minimum LD50 Ratios .............................................................7-40
7.2.6 Normalization of Reference Substance IC50 Values Using SLS
Values ......................................................................................................7-42

7.3

Historical Positive Control (PC) Data................................................................7-44
7.3.1 ANOVA and Linear Regression Results for the 3T3 NRU Test Method....7-46
7.3.2 ANOVA and Linear Regression Results for the NHK NRU Test
Method .....................................................................................................7-48

7.4

Laboratory Concordance for Solvent Selection.................................................7-50

7.5

Summary.............................................................................................................7-52

7-1

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

[This Page Intentionally Left Blank]

7-2

In Vitro Cytotoxicity Test Methods BRD Section 7

7.0

November 2006

RELIABILITY OF THE 3T3 AND NHK NRU TEST METHODS

The reliability of the 3T3 and NHK NRU test methods was assessed by determining intraand inter-laboratory reproducibility. Intralaboratory reproducibility is the agreement of
results produced when people in the same laboratory perform the method using the same test
protocol at different times (ICCVAM 2003). Interlaboratory reproducibility is the agreementof results among different laboratories using the same protocol and reference substances.
Interlaboratory reproducibility indicates the extent to which a method can be successfullytransferred among laboratories. Repeatability, usually applied to results within a laboratory,
is the closeness of agreement between test results obtained when the procedure is performedon the same substance under identical conditions within a given time. This study was notdesigned to assess intralaboratory repeatability.
The interlaboratory reproducibility of the test results was assessed by comparing thelaboratory-specific IC50-LD50 regressions for the 3T3 and NHK NRU test methods to themean (i.e., across-laboratory mean) laboratory regressions (see Section 7.2.1). This
comparison is relevant because the 3T3 and NHK NRU test methods are intended for use
with IC50-LD50 regressions to determine starting doses for acute oral toxicity tests.
Interlaboratory reproducibility of the 3T3 and NHK NRU test methods was also determinedusing ANOVA, CV analysis, and comparison of maximum:minimum IC50 ratios calculatedusing laboratory mean values (see Sections 7.2.2, 7.2.3, and 7.2.4, respectively), as discussed
in Section 5.5.2.2. Inter- and intra-laboratory reproducibility of the PC (SLS) was determinedusing ANOVA, CV analysis, and/or linear regression over time (see Section 7.3). The extent
of laboratory concordance in selecting the solvent to be used for each test substance(described in Section 2.10) is provided in Section 7.4.
7.1

Reference Substances Used to Determine the Reliability of the 3T3 and NHK
NRU Test Methods

The validation study was designed for the purpose of using the IC50 results of 72 reference
substances (see Table 3-2) to determine the reliability of the IC50 values from the 3T3 andNHK NRU test methods. The number of reference substances used for the reproducibility
analysis was not the same as the number of reference substances used for the accuracy
analyses in Section 6.4. In the former case, only reference substances for which all threelaboratories reported replicate IC50 values were used, while in the latter case, substances with
rat acute oral LD50 data only and at least one laboratory reporting replicate IC50 values wereused. Table 7-1 lists the reference substances that failed to yield sufficient toxicity for the
calculation of an IC50 in each laboratory, and the number of remaining reference substanceswith replicate IC50 values. The laboratories obtained acceptable IC50 values for 66 to 68
reference substances using the 3T3 NRU test method, and for 69 to 70 substances using the
NHK NRU test method. When only reference substances with IC50 values from all threelaboratories are considered, 64 and 68 substances were available to evaluate the reliability of
the 3T3 and NHK NRU test methods, respectively. The substances that were excluded fromthe 3T3 reliability analysis were carbon tetrachloride, disulfoton, gibberellic acid, lithium
carbonate, methanol, 1,1,1-trichloroethane, valproic acid, and xylene. The substances thatwere excluded from the NHK reliability analysis were carbon tetrachloride, methanol, 1,1,1trichloroethane, and xylene.

7-3

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-1

Laboratory

ECBC

FAL

IIVS

November 2006

Reference Substances Excluded from Reproducibility Analyses Becauseof Insufficient Cytotoxicity
3T3 NRU Test Method
Reference Substances
Lacking IC50 Results
Carbon tetrachloride
Methanol
1,1,1-Trichloroethane
XyleneCarbon tetrachloride
Disulfoton
Gibberellic acid
Lithium carbonate
Methanol
XyleneCarbon tetrachloride
Lithium carbonate
Methanol
Valproic acid

1

N

NHK NRU Test Method
Reference Substances
Lacking IC50 Results

N1

68

Carbon tetrachloride
Methanol
Xylene69

66

1,1,1-Trichloroethane
Carbon tetrachloride
Xylene69

68

Carbon tetrachloride
1,1,1-Trichloroethane

70

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU= Neutral
red uptake; ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; N=Number of substances.
2
Number of substances with replicate IC50 values.

Despite the fact that IC50 values were not obtained by all the laboratories for all reference
substances, Table 7-2 shows that the complete range of LD50 responses, as defined by the
GHS classification for acute oral toxicity in Table 3-1, was covered by the reference
substances for which replicate IC50 values were obtained. The 3T3 NRU IC50 values ranged
from 0.005 to 38,878 µg/mL, while the NHK values covered a larger range, from 0.00005 to
49,800 µg/mL (see Tables 5-4 and 5-5).
Table 7-2

Number of Reference Substances Tested vs Number of Reference
Substances Yielding IC50 Values from Each Laboratory, by GHS Acute
Oral Toxicity Category

GHS Category1(mg/kg)
LD50 ≤55 < LD50 ≤50
50 < LD50 ≤300
300 < LD50 ≤2000
2000 < LD50 ≤5000
LD50 >5000

Reference Oral
LD502
7
12
12
16
11
14

3T3 NRU Test
Method3
6
12
12
14
9
11

NHK NRU Test
Method3
7
12
12
16
9
12

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU= Neutral
red uptake; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005).
1
GHS category for acute oral toxicity.
2
Number of reference substances tested in each category. Reference acute oral LD50 values from rats and mice
were generated after evaluating LD50 values located through literature searches and references from toxicity
databases such as RTECS® (from Table 4-2).
3
Number of reference substances with IC50 values from all three laboratories.
7-4

In Vitro Cytotoxicity Test Methods BRD Section 7

7.2

November 2006

Reproducibility Analyses for the 3T3 and NHK NRU Test Methods

The interlaboratory reproducibility of the 3T3 and NHK NRU IC50 values was assessed bycomparing the laboratory-specific IC50-LD50 linear regressions for each method to aregression calculated using the mean IC50 values of the laboratories. The interlaboratory
reproducibility of the 3T3 and NHK NRU test methods was also assessed using ANOVA,
CV analysis, and analysis of the laboratory mean maximum:minimum IC50 ratios, as
described in Section 5.5.2.2. Intralaboratory reproducibility was assessed using a CV
analysis.
7.2.1

Comparison of Laboratory-Specific IC50-LD50 Linear Regression Analyses to the
Mean Laboratory Regression
The comparisons of laboratory-specific IC50-LD50 linear regressions to the mean laboratory
regression for each method were made because the 3T3 and NHK NRU test methods areintended for use with IC50-LD50 regressions to determine starting doses for acute oral toxicity
tests. Laboratory-specific IC50-LD50 linear regressions were generated and displayed
graphically for each method using the 64 and 68 reference substances for the 3T3 and NHK
NRU test methods, respectively, as indicated in Section 7.1. The regressions used thegeometric mean IC50 values for each substance with the rodent acute oral LD50 reference
value (Table 4-2). To determine whether the laboratory-specific regressions weresignificantly different from one another, they were compared against the mean laboratory
regression for each NRU test method that was calculated using the geometric mean of thelaboratory mean IC50 values and the rodent acute oral LD50 reference values. The mean
laboratory regression for each NRU test method is in Figure 7-1 with 95% confidence limits,and shows that the laboratory-specific regressions were all within the 95% confidence limitsof the mean laboratory regression.
7.2.2
ANOVA Results for the 3T3 and NHK NRU Test Methods
The ANOVA was performed as discussed in Section 5.5.2.2. Because the sample sizes fromthis study were small, usually three observations per laboratory, there may be differences thatwere statistically significant only because there were too few observations within thelaboratories to adequately characterize variability or because the within-laboratory variability
was small.
7.2.2.1

Differences Among the IC50 Values in Laboratories Using the 3T3 NRU Test
MethodThe ANOVA results in Table 7-3 show that there were statistically significant (p <0.01)
differences among the laboratories for 23 of the 64 (36%) reference substances evaluated.
The p values from the contrast analyses, post-hoc tests to determine which laboratory wassignificantly different from the others at p <0.01 (see Section 5.5.2.2), are also provided inTable 7-3. The substances for which statistically significant ANOVA and contrast results
were obtained are listed in Table 7-4 along with columns showing the laboratory withsignificantly differing values from the other two laboratories. Because significant laboratory
differences may have resulted from the insolubility or volatility of the test substance, Table
7-4 also indicates whether any laboratory reported insolubility or volatility during conduct of
the test. Insolubility was suggested by the presence of precipitates in either the stock
solutions or in cell culture. Volatility was identified by the need for plate sealers to containvolatile contamination of lower concentration wells by higher concentrations. Insolubility
and volatility were reported for only six of the 23 chemicals showing significant
7-5

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

interlaboratory variability. In contrast, 22 of the 41 substances that were classified as
generating interlaboratory reproducible data exhibited precipitates and/or volatility.
For the 23 substances that yielded significantly different results among laboratories, contrast
analyses indicated that the IC50 values produced by ECBC and FAL were frequently different
from the other laboratories. ECBC tended to report the lowest IC50 values (i.e., highest
toxicity) among the laboratories while FAL tended to report the highest values of the threelaboratories. ECBC reported significantly different results from the other two laboratories for
15 of the 23 substances; for 13 of the 15, ECBC’s mean value IC50 was the lowest among thelaboratories. FAL reported significantly different results from the other two laboratories for
20 of the 23 substances; for 18 of the 20, FAL’s IC50 value was the highest among thelaboratories. IIVS reported significantly different values for 11 of the 26 substances, with notendency toward highest or lowest IC50 values.

7-6

In Vitro Cytotoxicity Test Methods BRD Section 7

Figure 7-1

November 2006

Mean Laboratory and Laboratory-Specific 3T3 and NHK NRU
Regressions

a.
1000
100

Mean laboratory;: Log LD 50 = 0.582logIC 50 + 0.436
ECBC: Log LD50 = 0.543logIC 50 + 0.477
FAL: Log LD 50 = 0.547logIC 50 + 0.329
IIVS: Log LD50 = 0.532logIC 50 + 0.449

10
1
0.1
0.01
0.001
0.00010.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

1

10

100

1000

IC50 (mM)b.
1000

Mean laboratory: Log LD 50 = 0.488logIC 50 + 0.403
ECBC: Log LD50 = 0.466logIC 50 + 0.377

100

FAL: Log LD 50 = 0.429logIC 50 + 0.388
IIVS: Log LD50 = 0.463logIC 50 + 0.386

10
1
0.1
0.01
0.001
0.00010.000001 0.00001

0.0001

0.001

0.01

0.1

IC50 (mM)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red uptake; NHK=Normal human epidermal keratinocytes.
Solid lines show the mean laboratory linear regressions for the 3T3 NRU (a) and the NHK NRU (b) test methods withdashed curved lines to show the 95% confidence limits of the regression. The regressions were calculated using 64 and 68
reference substances for the 3T3 and NHK NRU test methods, respectively, as described in Section 7.1. Regressions used
geometric mean IC50 values and reference acute oral LD50 values from Table 4-2.

7-7

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
Acetaminophen
ECBC
FAL
IIVS
Acetonitrile
ECBC
FAL
IIVS
Acetylsalicylic acid
ECBC
FAL
IIVS
5-Aminosalicylic acid
ECBC
FAL
IIVS
Aminopterin
ECBC
FAL
IIVS
Amitriptyline HCl
ECBC
FAL
IIVS
Arsenic trioxide

Arithmetic
Mean IC50(mg/mL)1
50.1
40.8
66.2
43.4
8484
6433
9690
9330
760
646
1234
401
1698
1467
2070
1557
0.007
0.005
0.012
0.005
7.23
6.03
7.86
7.81
2.51

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

1.6

Arithmetic
InterLab
%CV
28

22
35
26
1.5

21
2
58
13

3.1

56
10
24
16

1.4

19
14
16
12

2.4

54
20
46
23

1.3

14
23
28
18

3.9

61

7-8

Log
Arithmetic
Mean IC50(mg/mL)1
1.7
1.61
1.82
1.64
3.93
3.81
3.99
3.97
2.88
2.81
3.09
2.6
3.23
3.17
3.32
3.19
-2.14
-2.28
-1.93
-2.33
0.86
0.78
0.9
0.89
0.4

ANOVA
P3

Contrast
P4

0.171
NA
NA
NA
0.553
NA
NA
NA
<0.001
0.581<0.001<0.001
0.054
NA
NA
NA
0.036
NA
NA
NA
0.348
0.163
0.469
0.445
0.004

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
ECBC
FAL
IIVS
Atropine sulfate
ECBC
FAL
IIVS
Boric acid
ECBC
FAL
IIVS
Busulfan
ECBC
FAL
IIVS
Cadmium chloride
ECBC
FAL
IIVS
Caffeine
ECBC
FAL
IIVS
Carbamazepine
ECBC

Arithmetic
Mean IC50(mg/mL)1
2.41
1.04
4.09
85.6
54.1
133
70
2228
1497
3987
1202
135
40
321
43.7
0.565
0.48
0.4
0.817
161
133
157
191
109
83

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

33
7
52
2.5

49
55
31
8

3.3

69
32
17
48

8.0

119
48
56
4

1.4

39
14
32
53

1.4

18
10
52
7.5

1.8

35
14

7-9

Log
Arithmetic
Mean IC50(mg/mL)1
0.38
0.02
0.61
1.93
1.73
2.12
1.85
3.35
3.18
3.6
3.08
2.13
1.6
2.51
1.64
-0.25
-0.32
-0.4
-0.09
2.21
2.12
2.2
2.28
2.04
1.92

ANOVA
P3

Contrast
P4
0.527
0.002
0.006

0.049
NA
NA
NA
0.01
NA
NA
NA
0.002
0.012
<0.001
0.033
0.124
NA
NA
NA
0.481
NA
NA
NA
0.049
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
FAL
IIVS
Carbon tetrachloride
ECBC
FAL
IIVS
Chloral hydrate
ECBC
FAL
IIVS
Chloramphenicol
ECBC
FAL
IIVS
Citric acid
ECBC
FAL
IIVS
Colchicine
ECBC
FAL
IIVS
Cupric sulfate pentahydrate
ECBC
FAL

Arithmetic
Mean IC50(mg/mL)1
152
91.8
NA
NA
NA
NA
187
151
241
170
161
55.3
273
156
829
473
1148
865
0.047
0.02
0.093
0.028
70.6
82.7
123

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

37
12
NA

NA
NA
NA
NA

1.6

25
10
10
12

4.9

67
22
30
18

2.4

41
29
13
19

4.7

85
11
45
1

21.6

85
4
44

7-10

Log
Arithmetic
Mean IC50(mg/mL)1
2.18
1.96
NA
NA
NA
NA
2.27
2.18
2.38
2.23
2.21
1.74
2.44
2.19
2.92
2.68
3.06
2.94
-1.33
-1.70
-1.03
-1.55
1.85
1.92
2.09

ANOVA
P3

Contrast
P4
NA
NA

NA
NA
NA
NA
0.004
0.008
0.002
0.181
<0.001<0.001
0.001
0.165
0.002
0.001
0.003
0.298
0.001
0.00280.00050.0914<0.0010.001<0.001In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
IIVS
Cycloheximide
ECBC
FAL
IIVS
Dibutyl phthalate
ECBC
FAL
IIVS
Dichlorvos
ECBC
FAL
IIVS
Diethyl phthalate
ECBC
FAL
IIVS
Digoxin
ECBC
FAL
IIVS
Dimethylformamide
ECBC
FAL
IIVS

Arithmetic
Mean IC50(mg/mL)1
5.7
0.293
0.125
0.647
0.109
78.3
23.5
191
20.7
20.3
9.8
32.8
18.3
113
85.5
147
106
520
351
892
317
5242
5343
5483
4900

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

31
5.9

104
45
70
23

9.2

124
17
50
7

3.3

57
35
6
11

1.7

28
34
26
24

2.8

62
39
36
21

1.1

6
10
9
4

7-11

Log
Arithmetic
Mean IC50(mg/mL)1
0.76
-0.53
-0.9
-0.19
-0.96
1.89
1.37
2.28
1.32
1.31
0.99
1.52
1.26
2.05
1.93
2.17
2.03
2.72
2.54
2.95
2.5
3.72
3.73
3.74
3.69

ANOVA
P3

Contrast
P4<0.001

0.021
NA
NA
NA
<0.001
0.012<0.001
0.005
0.002
0.001
0.002
0.823
0.127
0.092
0.07
0.846
0.043
NA
NA
NA
0.296
NA
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory

Arithmetic
Mean IC50(mg/mL)1

Diquat dibromide monohydrate
ECBC
FAL
IIVS
Disulfoton
ECBC
FAL
IIVS
Endosulfan
ECBC
FAL
IIVS
Epinephrine bitartrate
ECBC
FAL
IIVS
Ethanol
ECBC
FAL
IIVS
Ethylene glycol
ECBC
FAL
IIVS
Fenpropathrin

15.1
3.9
36.1
5.4
98.6
137
NA
60.4
8.02
5.3
15.2
3.6
59.4
51.5
63.4
63.4
6731
5360
8420
6413
25292
18325
31650
25900
27.2

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

9.3

Arithmetic
InterLab
%CV
120

23
98
25
2.3

55
55
NA
87

4.2

78
57
78
42

1.2

12
12
11
3

1.6

23
33
14
5

1.7

26
9
24
12

2.5

49

7-12

Log
Arithmetic
Mean IC50(mg/mL)1
1.18
0.59
1.56
0.73
1.99
2.14
NA
1.78
0.9
0.72
1.18
0.56
1.77
1.71
1.8
1.8
3.83
3.73
3.93
3.81
4.4
4.26
4.50
4.41
1.43

ANOVA
P3

Contrast
P4

0.017
NA
NA
NA
0.003
NA
NA
NA
0.046
NA
NA
NA
0.048
NA
NA
NA
0.075
NA
NA
NA
0.007
0.004
0.01
0.505
0.301

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
ECBC
FAL
IIVS
Gibberellic Acid
ECBC
FAL
IIVS
Glutethimide
ECBC
FAL
IIVS
Glycerol
ECBC
FAL
IIVS
Haloperidol
ECBC
FAL
IIVS
Hexachlorophene
ECBC
FAL
IIVS
Lactic acid
ECBC

Arithmetic
Mean IC50(mg/mL)1
22.6
42.4
16.7
7842
8027
NA
7657
192
167
284.3
125.3
28904
20000
38878
27833
6.26
5.3
8
5.5
4.48
5
5.3
3.1
3073
2943

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

11
63
12
1.0

3
11
NA
10

2.3

43
4
7
7

1.9

33
15
73
39

1.5

24
12
8
12

1.7

27
48
33
9

1.2

12
11

7-13

Log
Arithmetic
Mean IC50(mg/mL)1
1.35
1.63
1.22
3.89
3.9
NA
3.88
2.28
2.22
2.45
2.1
4.46
4.3
4.59
4.44
0.8
0.72
0.9
0.74
0.65
0.7
0.72
0.49
3.49
3.47

ANOVA
P3

Contrast
P4
NA
NA
NA

0.621
NA
NA
NA
<0.001
0.029<0.001<0.001
0.846
NA
NA
NA
0.006
0.03
0.002
0.061
0.174
NA
NA
NA
0.16
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
FAL
IIVS
Lindane
ECBC
FAL
IIVS
Lithium carbonate
ECBC
FAL
IIVS
Meprobamate
ECBC
FAL
IIVS
Mercury chloride
ECBC
FAL
IIVS
Methanol
ECBC
FAL
IIVS
Nicotine
ECBC
FAL

Arithmetic
Mean IC50(mg/mL)1
3487
2790
161
125
266
90.4
NA
564
NA
NA
539
353
877
386
4.32
3.5
6
3.5
NA
NA
NA
NA
378
272
412

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

16
9
2.9

58
95
36
122

NA

NA
12
NA
NA

2.5

54
14
15
2

1.7

33
5
31
3

NA

NA
NA

1.7

25
24
33

7-14

Log
Arithmetic
Mean IC50(mg/mL)1
3.54
3.45
2.21
2.1
2.43
1.96
NA
2.75
NA
NA
2.73
2.55
2.94
2.59
0.64
0.54
0.78
0.54
NA
NA
NA
NA
2.58
2.43
2.61

ANOVA
P3

Contrast
P4
NA
NA

0.066

NA

NA
NA
NA
NA
NA
NA
NA

<0.001
NA
NA
NA
0.021

NA

NA
NA
NA
NA
NA
NA
NA

0.128
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
IIVS
Paraquat
ECBC
FAL
IIVS
Parathion
ECBC
FAL
IIVS
Phenobarbital
ECBC
FAL
IIVS
Phenol
ECBC
FAL
IIVS
Phenylthiourea
ECBC
FAL
IIVS
Physostigmine
ECBC
FAL
IIVS

Arithmetic
Mean IC50(mg/mL)1
450
23.3
21.3
24.9
23.7
61.8
22.7
141
22
612
634
726
476
70.9
50.2
104
58.1
119
30.1
239
89
28.8
28.2
37.8
20.4

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

12
1.2

8
34
67
64

6.4

111
53
70
22

1.5

21
21
35
23

2.1

Log
Arithmetic
Mean IC50(mg/mL)1
2.65
1.37
1.33
1.4
1.37
1.79
1.36
2.15
1.34
2.79
2.8
2.86
2.68

41
22
24
12

7.9

90
66
28
25

1.9

30
53
5
33

7-15

ANOVA
P3

Contrast
P4
NA

1
NA
NA
NA
0.014
NA
NA
NA
0.232
NA
NA
NA
0.011

1.7
2.02
1.76
2.08
1.48
2.38
1.95
1.46
1.45
1.58
1.31

NA
NA
NA
0.007
0.004
0.006
0.718
0.149
NA
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
Potassium chloride
ECBC
FAL
IIVS
Potassium cyanide
ECBC
FAL
IIVS
Procainamide HCl
ECBC
FAL
IIVS
2-Propanol
ECBC
FAL
IIVS
Propranolol HCl
ECBC
FAL
IIVS
Propylparaben
ECBC
FAL
IIVS
Sodium arsenite

Arithmetic
Mean IC50(mg/mL)1
3635
3352
3842
3710
64.3
15.3
159
18.9
443
400
431
497
3563
2610
3970
4110
14.9
13.6
13.5
17.6
29.9
20.9
51.8
17.1
0.873

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

1.1

Arithmetic
InterLab
%CV
7

14
31
11
10.4

127
25
52
5

1.2

11
4
1
8

1.6

23
9
4
4

1.3

16
32
51
21

3.0

64
16
29
12

2.8

55

7-16

Log
Arithmetic
Mean IC50(mg/mL)1
3.56
3.53
3.58
3.57
1.81
1.18
2.2
1.28
2.65
2.6
2.63
2.7
3.55
3.42
3.6
3.61
1.17
1.13
1.13
1.25
1.48
1.32
1.71
1.23
-0.06

ANOVA
P3

Contrast
P4

0.846
NA
NA
NA
<0.0010.001<0.001
0.006
0.007
0.008
0.396
0.003
0.001
<0.001
0.004
0.002
0.488
NA
NA
NA
0.001
0.045
<0.0010.003
0.028

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
ECBC
FAL
IIVS
Sodium chloride
ECBC
FAL
IIVS
Sodium dichromate dihydrate
ECBC
FAL
IIVS
Sodium fluoride
ECBC
FAL
IIVS
Sodium hypochlorite
ECBC
FAL
IIVS
Sodium oxalate
ECBC
FAL
IIVS
Sodium selenate
ECBC

Arithmetic
Mean IC50(mg/mL)1
0.5
1.4
0.7
4764
4790
4625
4877
0.602
0.603
0.657
0.547
79.8
61.3
96.1
82
1211
823
805
2005
40.8
42
31
49.5
34.5
12.7

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

6
57
17
1.1

3
5
13
9

1.2

9
14
37
17

1.6

22
9
18
7

2.5

57
13
46
44

1.6

23
41
28
53

4.3

60
13

7-17

Log
Arithmetic
Mean IC50(mg/mL)1
-0.3
0.15
-0.15
3.68
3.68
3.67
3.69
-0.22
-0.22
-0.18
-0.26
1.9
1.79
1.98
1.91
3.08
2.92
2.91
3.3
1.61
1.62
1.49
1.69
1.54
1.1

ANOVA
P3

Contrast
P4
NA
NA
NA

0.759
NA
NA
NA
0.822
NA
NA
NA
0.016
NA
NA
NA
0.04
NA
NA
NA
0.643
NA
NA
NA
<0.001<0.001In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
FAL
IIVS
Strychnine
ECBC
FAL
IIVS
Thallium Sulfate
ECBC
FAL
IIVS
Trichloroacetic acid
ECBC
FAL
IIVS
1,1,1-Trichloroethane
ECBC
FAL
IIVS
Triethylenemelamine
ECBC
FAL
IIVS
Triphenyltin hydroxide
ECBC
FAL

Arithmetic
Mean IC50(mg/mL)1
54.2
36.5
199
389
124
83.5
7.5
2.8
13.4
6.3
928
762
1220
801
15538
NA
21250
9827
0.568
0.086
1.45
0.169
0.022
0.026
0.026

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

19
14
4.7

83
21
16
6

4.9

72
24
78
28

1.6

27
13
6
14

2.2

52
NA
11
2

16.9

135
11
18
29

1.7

29
17
81

7-18

Log
Arithmetic
Mean IC50(mg/mL)1
1.73
1.56
2.3
2.59
2.09
1.92
0.88
0.45
1.13
0.8
2.97
2.88
3.09
2.9
4.19
NA
4.33
3.99
-0.25
-1.07
0.16
-0.77
-1.66
-1.59
-1.59

ANOVA
P3

Contrast
P4<0.001
0.026<0.001<0.0010.018<0.001
0.165
NA
NA
NA
0.005
0.022
0.002
0.069
<0.001NA
NA
NA<0.001<0.001<0.001
0.002
0.688
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
IIVS
Valproic acid
ECBC
FAL
IIVS
Verapamil HCl
ECBC
FAL
IIVS
Xylene
ECBC
FAL
IIVS

Arithmetic
Mean IC50(mg/mL)1
0.015
1177
547
1807
NA
35.2
32
34.6
38.9
NA
NA
NA
724

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

55
3.3

76
12
10
NA

1.2

10
18
5
11

NA

NA
NA
NA
12

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red uptake; ECBC=Edgewood Chemical Biological Center;
FAL= Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro
Sciences; NA=No acceptable IC50 results reported or calculation was not performed (e.g., for contrast results);
CV=Coefficient of variation.
1
Results reported on the same row with chemical names are the means of all the laboratories. Results reported on the samerow as laboratories are the laboratory means.
2
Maximum laboratory mean IC50 divided by minimum laboratory mean IC50.
3p <0.01 indicated statistical significance.
4
Contrasts were performed if ANOVA was significant (p <0.01) to determine which laboratory was different from the othertwo laboratories. Significant contrasts were denoted by p <0.01. No contrast tests were performed if only two laboratories
reported IC50 values.

7-19

Log
Arithmetic
Mean IC50(mg/mL)1
-1.83
3.07
2.74
3.26
NA
1.55
1.51
1.54
1.59
NA
NA
NA
2.86

ANOVA
P3

Contrast
P4NA<0.001
NA
NA
NA
0.23

NA

NA
NA
NA
NA
NA
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-4

November 2006

Reference Substances with Significant ANOVA Differences Among
Laboratories for the 3T3 NRU Test Method
Significant Contrast Results1
FAL

IIVS

Insoluble/
Volatile2

H
L
H
H
H
H
H
H
H
H

L
H

Precipitate

L

L
L

H
H
H
H

Potassium cyanide

L

H

Procainamide HCl
2-Propanol
Propylparaben
Sodium selenate
Strychnine
Trichloroacetic acid
Triethylenemelamine

L
L

Reference Substance
Acetylsalicylic acidArsenic trioxide
Busulfan
Chloral hydrate
Chloramphenicol
Citric acidColchicine
Cupric sulfate pentahydrate
Dibutyl phthalate
Dichlorvos
Ethylene glycol
Glutethimide
Haloperidol
Meprobamate
Phenylthiourea

ECBC

L
L
L
L
M
L
L

M
H
H

L
H

L
L

Precipitate
Precipitate

M
M

Precipitate/Volatile

H
H
L

Volatile

L

Precipitate

H
H

L

Abbreviations: ANOVA=Analysis of variance; 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red uptake;
H=Laboratory reported the highest mean IC50; L=Laboratory reported the lowest mean IC50; M=Laboratory reported
a mean IC50 between the values of the other two laboratories; ECBC=Edgewood Chemical Biological Center; FAL=
Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro
Sciences.
1
Laboratories significantly different from the other two at p <0.01.
2
From Table 5-11. Precipitate reported by at least one laboratory is indicated by “Precipitate”. Use of plate sealers
by at least one laboratory to prevent volatile contamination of control wells indicated by “Volatility”.

7-20

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

7.2.2.2 Differences Among the IC50 Values in Laboratories Using the NHK NRU Test Method
The ANOVA results in Table 7-5 indicate that there were statistically significant (p <0.01)
laboratory differences for six of the 68 (9%) reference substances evaluated. These substances
are listed in Table 7-6 along with columns showing which laboratory’s IC50 values were
statistically significantly different from the other two (as indicated by the contrast results), andindications of insolubility or volatility during conduct of the assay. Insolubility was reported forthree of the six substances, but none of the six substances were volatile.
For the six substances that yielded significantly different IC50 values among the laboratories,
ECBC reported the highest IC50 value for four substances and the lowest for one, FAL reported
the lowest values for three substances and the highest for two, and IIVS reported the highest IC50
value for one substance and the lowest for two.
7.2.3
CV Results for the 3T3 and NHK NRU Test Methods
CV values were calculated as described in Section 5.5.2.2. Tables 7-3 and 7-5 provide the intraand inter-laboratory CV values for the individual reference substances. Table 7-7 summarizes
the CV values for each method and shows that median and mean values were often similar.
Median CV values were frequently lower than the corresponding means, which indicated thatlarge individual CV values skewed the CV distributions.
7.2.3.1 Reproducibility of Intralaboratory CV Values
Table 7-7 shows that the intralaboratory CV values and mean intralaboratory CV values were
the same, 26%, for both NRU test methods. The median intralaboratory CV values were alsosimilar: 23% and 24% for the 3T3 and the NHK NRU test method, respectively. Of the threelaboratories, FAL had the highest mean and median CV values and IIVS had the lowest for bothmethods.

7-21

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
Acetaminophen
ECBC
FAL
IIVS
Acetonitrile
ECBC
FAL
IIVS
Acetylsalicylic acid
ECBC
FAL
IIVS
5-Aminosalicylic acid
ECBC
FAL
IIVS
Aminopterin
ECBC
FAL
IIVS
Amitriptyline HCl
ECBC
FAL
IIVS
Arsenic trioxide

Arithmetic
Mean IC50(mg/mL)1
526
558
447
571
10104
10868
10153
9290
613
631
694
514
52.3
29.9
78.2
48.8
682
889
545
611
9.76
10.8
7.57
10.9
10.4

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

1.3

Arithmetic
InterLab
%CV
13

15
19
14
1.2

8
72
19
4

1.4

15
3
14
15

2.6

47
22
54
16

1.6

27
20
8
12

1.4

19
31
72
10

8.2

91

7-22

Log
Arithmetic
Mean IC50(mg/mL)1
2.72
2.75
2.65
2.76
4
4.04
4.01
3.97
2.79
2.8
2.84
2.71
1.72
1.48
1.89
1.69
2.83
2.95
2.74
2.79
0.99
1.03
0.88
1.04
1.02

ANOVA
P3

Contrast
P4

0.181
NA
NA
NA
0.964
NA
NA
NA
0.060
NA
NA
NA
0.044
NA
NA
NA
0.025
NA
NA
NA
0.365
NA
NA
NA
<0.001In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
ECBC
FAL
IIVS
Atropine sulfate
ECBC
FAL
IIVS
Boric acid
ECBC
FAL
IIVS
Busulfan
ECBC
FAL
IIVS
Cadmium chloride
ECBC
FAL
IIVS
Caffeine
ECBC
FAL
IIVS
Carbamazepine
ECBC

Arithmetic
Mean IC50(mg/mL)1
7.77
2.55
20.9
91.9
85.4
104
83.2
473
440
517
464
278
253
268
313
1.98
2.2
1.88
1.86
661
817
591
574
128
66.1

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

33
75
31
1.3

13
12
85
25

1.2

8
31
73
2

1.2

11
27
72
12

1.2

10
37
65
8

1.4

21
31
32
1

4.0

85
13

7-23

Log
Arithmetic
Mean IC50(mg/mL)1
0.89
0.41
1.32
1.96
1.93
2.02
1.92
2.67
2.64
2.71
2.67
2.44
2.4
2.43
2.5
0.3
0.34
0.27
0.27
2.82
2.91
2.77
2.76
2.11
1.82

ANOVA
P3

Contrast
P4
0.694<0.0010.00060.988
0.89030.90690.98320.931
0.96920.7391
0.768
0.659
NA
NA
NA
0.733
NA
NA
NA
0.296
NA
NA
NA
0.432
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
FAL
IIVS
Carbon tetrachloride
ECBC
FAL
IIVS
Chloral hydrate
ECBC
FAL
IIVS
Chloramphenicol
ECBC
FAL
IIVS
Citric acid
ECBC
FAL
IIVS
Colchicine
ECBC
FAL
IIVS
Cupric sulfate pentahydrate
ECBC
FAL

Arithmetic
Mean IC50(mg/mL)1
253
63.9
NA
NA
NA
NA
137
140
159
112
366
318
414
367
424
526
312
433
0.007
0.005
0.008
0.008
197
190
195

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

129
8
NA

NA
NA
NA
NA

1.4

17
24
32
2

1.3

13
45
44
22

1.7

25
16
17
5

1.6

22
46
10
21

1.1

4
10
6

7-24

Log
Arithmetic
Mean IC50(mg/mL)1
2.4
1.81
NA
NA
NA
NA
2.14
2.15
2.2
2.05
2.56
2.5
2.62
2.56
2.63
2.72
2.49
2.64
-2.16
-2.28
-2.12
-2.09
2.29
2.28
2.29

ANOVA
P3

Contrast
P4
NA
NA

NA
NA
NA
NA
0.302
NA
NA
NA
0.750
NA
NA
NA
0.006
0.009
0.002
0.483
0.174
NA
NA
NA
0.374
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
IIVS
Cycloheximide
ECBC
FAL
IIVS
Dibutyl phthalate
ECBC
FAL
IIVS
Dichlorvos
ECBC
FAL
IIVS
Diethyl phthalate
ECBC
FAL
IIVS
Digoxin
ECBC
FAL
IIVS
Dimethylformamide
ECBC
FAL
IIVS

Arithmetic
Mean IC50(mg/mL)1
207
0.082
0.053
0.12
0.071
32.6
28.3
47.4
22
11.1
8.56
12.4
12.2
145
174
71.5
189
0.003140.005380.00005
0.00398
7856
9353
7817
6397

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

3
2.3

43
22
78
19

2.2

41
27
73
6

1.4

20
27
30
3

2.6

44
8
94
18

107.6

88
13
36
7

1.5

19
2
1
3

7-25

Log
Arithmetic
Mean IC50(mg/mL)1
2.32
-1.09
-1.28
-0.92
-1.15
1.51
1.45
1.68
1.34
1.05
0.93
1.09
1.09
2.16
2.24
1.85
2.28
-2.5
-2.27
-4.29
-2.4
3.9
3.97
3.89
3.81

ANOVA
P3

Contrast
P4
NA

0.302
NA
NA
NA
0.408
NA
NA
NA
0.181
NA
NA
NA
0.049
NA
NA
NA
<0.001<0.001<0.001<0.001<0.001<0.0010.508<0.001In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory

Arithmetic
Mean IC50(mg/mL)1

Diquat dibromide monohydrate
ECBC
FAL
IIVS
Disulfoton
ECBC
FAL
IIVS
Endosulfan
ECBC
FAL
IIVS
Epinephrine bitartrate
ECBC
FAL
IIVS
Ethanol
ECBC
FAL
IIVS
Ethylene glycol
ECBC
FAL
IIVS
Fenpropathrin

4.73
3.59
6.77
3.84
378
140
808
186
2.35
3.44
1.42
2.19
90.6
115
81.7
75
10184
8290
12013
10250
42600
38000
49800
40000
2.6

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

1.9

Arithmetic
InterLab
%CV
37

23
55
8
5.8

99
19
26
32

2.4

43
17
50
20

1.5

24
9
35
16

1.4

18
5
19
9

1.3

15
12
9
13

2.0

39

7-26

Log
Arithmetic
Mean IC50(mg/mL)1
0.67
0.56
0.83
0.58
2.58
2.15
2.91
2.27
0.37
0.54
0.15
0.34
1.96
2.06
1.91
1.88
4.01
3.92
4.08
4.01
4.63
4.58
4.7
4.6
0.41

ANOVA
P3

Contrast
P4

0.217
NA
NA
NA
<0.001
0.002<0.001
0.018
0.029
NA
NA
NA
0.119
NA
NA
NA
0.035
NA
NA
NA
0.063
NA
NA
NA
0.031

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
ECBC
FAL
IIVS
Gibberellic Acid
ECBC
FAL
IIVS
Glutethimide
ECBC
FAL
IIVS
Glycerol
ECBC
FAL
IIVS
Haloperidol
ECBC
FAL
IIVS
Hexachlorophene
ECBC
FAL
IIVS
Lactic acid
ECBC

Arithmetic
Mean IC50(mg/mL)1
3.73
2.23
1.82
2866
2850
2940
2807
177
187
170
176
27108
34267
18023
29033
3.57
3.69
3.72
3.29
0.031
0.027
0.046
0.021
1308
1290

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

27
28
17
1.0

2
14
9
4

1.1

5
34
14
16

1.9

31
45
46
16

1.1

7
27
49
35

2.2

41
16
44
11

1.0

1
4

7-27

Log
Arithmetic
Mean IC50(mg/mL)1
0.57
0.35
0.26
3.46
3.45
3.47
3.45
2.25
2.27
2.23
2.24
4.43
4.53
4.26
4.46
0.55
0.57
0.57
0.52
-1.5
-1.57
-1.34
-1.67
3.12
3.11

ANOVA
P3

Contrast
P4
NA
NA
NA

0.862
NA
NA
NA
0.968
NA
NA
NA
0.200
NA
NA
NA
0.935
NA
NA
NA
0.097
NA
NA
NA
0.904
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
FAL
IIVS
Lindane
ECBC
FAL
IIVS
Lithium carbonate
ECBC
FAL
IIVS
Meprobamate
ECBC
FAL
IIVS
Mercury chloride
ECBC
FAL
IIVS
Methanol
ECBC
FAL
IIVS
Nicotine
ECBC
FAL

Arithmetic
Mean IC50(mg/mL)1
1320
1313
19.3
19.1
23.2
15.6
477
411
486
535
516
761
163
624
5.87
6.87
5.4
5.35
1616
NA
1133
2100
113
94.3
134

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

5
11
1.5

20
17
31
15

1.3

13
29
20
6

4.7

61
15
116
14

1.3

15
15
19
2

1.9

42
NA
19
11

1.4

17
26
59

7-28

Log
Arithmetic
Mean IC50(mg/mL)1
3.12
3.12
1.29
1.28
1.37
1.19
2.68
2.61
2.69
2.73
2.71
2.88
2.21
2.8
0.77
0.84
0.73
0.73
3.21
NA
3.05
3.32
2.05
1.97
2.13

ANOVA
P3

Contrast
P4
NA
NA

0.203
NA
NA
NA
0.295
NA
NA
NA
0.027
NA
NA
NA
0.120
NA
NA
NA
0.007
NA
NA
NA
0.700
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
IIVS
Paraquat
ECBC
FAL
IIVS
Parathion
ECBC
FAL
IIVS
Phenobarbital
ECBC
FAL
IIVS
Phenol
ECBC
FAL
IIVS
Phenylthiourea
ECBC
FAL
IIVS
Physostigmine
ECBC
FAL
IIVS

Arithmetic
Mean IC50(mg/mL)1
112
66.1
48.3
96.6
53.4
31.4
34
31.2
29
478
693
360
381
77.7
59.1
93.2
80.8
346
363
401
272
172
164
213
139

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

25
2.0

40
13
39
10

1.2

8
30
38
29

1.9

39
26
27
18

1.6

22
36
6
6

1.5

19
16
21
26

1.5

22
3
112
6

7-29

Log
Arithmetic
Mean IC50(mg/mL)1
2.05
1.82
1.68
1.98
1.73
1.5
1.53
1.49
1.46
2.68
2.84
2.56
2.58
1.89
1.77
1.97
1.91
2.54
2.56
2.6
2.44
2.24
2.21
2.33
2.14

ANOVA
P3

Contrast
P4
NA

0.047
NA
NA
NA
0.845
NA
NA
NA
0.027
NA
NA
NA
0.094
NA
NA
NA
0.133
NA
NA
NA
0.623
NA
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
Potassium chloride
ECBC
FAL
IIVS
Potassium cyanide
ECBC
FAL
IIVS
Procainamide HCl
ECBC
FAL
IIVS
2-Propanol
ECBC
FAL
IIVS
Propranolol HCl
ECBC
FAL
IIVS
Propylparaben
ECBC
FAL
IIVS
Sodium arsenite

Arithmetic
Mean IC50(mg/mL)1
2279
2560
2287
1990
45.1
29.3
89
16.9
1764
1480
1787
2027
5541
5263
4273
7087
36.9
38.27
43.8
28.6
16.8
18.1
18.6
13.8
0.532

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

1.3

Arithmetic
InterLab
%CV
13

17
28
8
5.3

86
24
112
13

1.4

16
14
12
11

1.7

26
11
27
7

1.5

21
12
6
11

1.3

16
13
15
9

2.4

44

7-30

Log
Arithmetic
Mean IC50(mg/mL)1
3.36
3.41
3.36
3.3
1.65
1.47
1.95
1.23
3.25
3.17
3.25
3.31
3.74
3.72
3.63
3.85
1.57
1.58
1.64
1.46
1.23
1.26
1.27
1.14
-0.27

ANOVA
P3

Contrast
P4

0.396
NA
NA
NA
0.340
NA
NA
NA
0.053
NA
NA
NA
0.033
NA
NA
NA
0.003
0.325
0.006
0.001
0.066
NA
NA
NA
0.061

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
ECBC
FAL
IIVS
Sodium chloride
ECBC
FAL
IIVS
Sodium dichromate dihydrate
ECBC
FAL
IIVS
Sodium fluoride
ECBC
FAL
IIVS
Sodium hypochlorite
ECBC
FAL
IIVS
Sodium oxalate
ECBC
FAL
IIVS
Sodium selenate
ECBC

Arithmetic
Mean IC50(mg/mL)1
0.79
0.336
0.47
2724
3583
1118
3470
0.737
0.784
0.851
0.576
47.4
48.7
39.7
53.7
1580
1863
1243
1633
355
355
350
360
11.2
7.47

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

32
56
14
3.2

51
7
124
9

1.5

19
14
36
17

1.4

15
14
24
13

1.5

20
31
46
11

1.0

1
15
42
26

2.2

40
12

7-31

Log
Arithmetic
Mean IC50(mg/mL)1
-0.1
-0.47
-0.33
3.44
3.55
3.05
3.54
-0.13
-0.11
-0.07
-0.24
1.68
1.69
1.6
1.73
3.2
3.27
3.09
3.21
2.55
2.55
2.54
2.56
1.05
0.87

ANOVA
P3

Contrast
P4
NA
NA
NA

0.045
NA
NA
NA
0.258
NA
NA
NA
0.313
NA
NA
NA
0.313
NA
NA
NA
0.926
NA
NA
NA
0.134
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
FAL
IIVS
Strychnine
ECBC
FAL
IIVS
Thallium Sulfate
ECBC
FAL
IIVS
Trichloroacetic acid
ECBC
FAL
IIVS
1,1,1-Trichloroethane
ECBC
FAL
IIVS
Triethylenemelamine
ECBC
FAL
IIVS
Triphenyltin hydroxide
ECBC
FAL

Arithmetic
Mean IC50(mg/mL)1
16.1
10
69.3
100
52.5
55.1
0.16
0.198
0.153
0.127
427
348
541
394
NA
8137
NA
NA
1.95
1.69
2.03
2.13
0.013
0.021
0.007

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

59
13
1.9

39
76
53
6

1.6

23
51
20
16

1.6

24
18
28
13

NA

NA
7
NA
NA

1.3

12
57
23
23

3.0

55
32
106

7-32

Log
Arithmetic
Mean IC50(mg/mL)1
1.21
1
1.84
2
1.72
1.74
-0.8
-0.7
-0.82
-0.9
2.63
2.54
2.73
2.6
NA
3.91
NA
NA
0.29
0.23
0.31
0.33
-1.89
-1.68
-2.15

ANOVA
P3

Contrast
P4
NA
NA

0.364
NA
NA
NA
0.405
NA
NA
NA
0.134
NA
NA
NA
NA
NA
NA
NA
0.562
NA
NA
NA
0.088
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
IIVS
Valproic acid
ECBC
FAL
IIVS
Verapamil HCl
ECBC
FAL
IIVS
Xylene
ECBC
FAL
IIVS

Arithmetic
Mean IC50(mg/mL)1
0.011
533
468
702
430
68.7
60.5
79.4
66.2
NA
NA
NA
486

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

32
1.6

28
25
23
17

1.3

14
22
42
8

NA

NA
NA
NA
38

Log
Arithmetic
Mean IC50(mg/mL)1
-1.96
2.73
2.67
2.85
2.63
1.84
1.78
1.9
1.82
NA
NA
NA
2.69

Abbreviations: NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; ECBC=Edgewood Chemical Biological Center; FAL=
Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; NA=No acceptable
IC50 results reported or calculation was not performed (e.g., for contrast results); CV=Coefficient of variation.
1
Results reported on the same row with chemical names are the means of all the laboratories. Results reported on the same row as laboratories
are the laboratory means.
2
Maximum laboratory mean IC50 divided by minimum laboratory mean IC50.
3p <0.01 indicated statistical significance.
4
Contrasts were performed if ANOVA was significant (p <0.01) to determine which laboratory was different from the other two laboratories.
Significant contrasts were denoted by p <0.01. No contrast tests were performed if only two laboratories reported IC50 values.

7-33

ANOVA
P3

Contrast
P4
NA

0.081
0.331
0.032
0.135
0.624
NA
NA
NA
NA
NA
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-6

November 2006

Reference Substances with Significant ANOVA Differences Among
Laboratories for the NHK NRU Test Method
Significant Contrast Results1

Reference Substance

ECBC

Arsenic trioxide
Citric acid
DigoxinDimethylformamide
Disulfoton
Propranolol HCl

FAL

IIVS

L
L
L

H

H
H
H
L

Solubility/
Volatility2
Precipitate
Precipitate

L
H
H

PrecipitateL

Abbreviations: ANOVA=Analysis of variance; NHK=Normal human epidermal keratinocytes; NRU=Neutral
red uptake; H=Laboratory reported the highest mean IC50; L=Laboratory reported the lowest mean IC50;
ECBC=Edgewood Chemical Biological Center; FAL= Fund for the Replacement of Animals in Medical
Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences.
1
Laboratories significantly different from the other two at p <0.01
2
From Table 5-11. Precipitate reported by at least one laboratory.

7.2.3.2 Reproducibility of Interlaboratory CV Values
The mean and median interlaboratory CV for the reference substances were lower in the
NHK NRU test method (mean=28%; median=21% vs. mean=47%; median=37% for 3T3(see Table 7-7).
Table 7-7

Summary of CV Results for the 3T3 and NHK NRU Test Methods

CV
Intralaboratory CV
ECBC
FAL
IIVS
Interlaboratory CV

3T3 NRU Test Method

NHK NRU Test Method

N

Mean

Median

Range

N

Mean

Median

Range

198
64
64
64
64

26%
23%
33%
21%
47%

23%
17%
31%
14%
37%

1-122%
2-95%
1-98%
1-122%
3-135%

204
68
68
68
68

26%
23%
43%
13%
28%

24%
20%
34%
13%
21%

1-129%
2-76%
1-129%
1-35%
1-91%

Abbreviations: CV=Coefficient of variation; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes;
NRU=Neutral red uptake; N=number of values; ECBC=Edgewood Chemical Biological Center; FAL= Fund for the
Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences.
Note: For the 3T3 method, the following substances were excluded because all laboratories did not obtain sufficient IC50
data: carbon tetrachloride; disulfoton; gibberellic acid; lithium carbonate; methanol; 1,1,1-trichloroethane; valproic acid; andxylene. For the NHK method, the following substances were excluded because all laboratories did not obtain sufficient IC50
data: carbon tetrachloride; methanol; 1,1,1-trichloroethane; and xylene.

7.2.3.3 Variation of CV with Chemical Property
To identify chemical characteristics that may be associated with high or low CV values, theirassociations were assessed for chemical class along with the following chemical attributes:
physical state (i.e., solid or liquid), solubility, volatility, molecular weight, log Kow, IC50, andboiling point. The CVs were also examined with respect to their association with the GHS
acute oral toxicity class (UN 2005). For categorical characteristics such as physical form,
solubility (i.e., precipitate/no precipitate), volatile/not volatile, and chemical class, the mean
7-34

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

CV values and ranges for the groups were compared to one another and to the overall mean
CV and CV range for each method. No statistical analyses were performed for thesecomparisons. Spearman correlation analyses were performed for chemical characteristics
measured by continuous variables, such as molecular weight, log Kow, and IC50, and boilingpoint.
7.2.3.4 Results of Intralaboratory CV Analysis
The intralaboratory CV analysis (see Table 7-8) uses one mean intralaboratory CV for eachreference substance that was calculated from the intralaboratory CV values from eachlaboratory. There seemed to be little difference in CV values among the categoricalphysical/chemical/toxicological attributes. The mean intralaboratory CV values for solids andliquids were similar (26 vs. 23% for 3T3; 27 vs. 24% for NHK). The mean intralaboratory
CV values for reference substances for which precipitates were observed were similar tovalues for substances with no precipitates were observed (32 vs. 23% for 3T3; 24 vs. 27% forNHK). The mean intralaboratory CV values for substances that exhibited volatility weresimilar to those that did not (31 vs. 25% for 3T3; 27 vs. 26% for NHK). Similarly, thesubstances grouped by GHS acute oral toxicity category (UN 2005) had mean intralaboratory
CV values that were similar (20-33% for 3T3; 19-31% for NHK) to the overall mean CV
values (26% for both test methods). However, the mean intralaboratory CV values for both
NRU test methods tended to increase with decreasing LD50.
Mean intralaboratory CV values were calculated for the chemical classes that contained atleast three of the reference substances included in the reproducibility analyses (i.e., 64
substances for 3T3 and 68 substances for NHK). Reference substances in the amide chemicalclass had unusually low mean intralaboratory CV values for both the 3T3 (13%) and theNHK (10%) NRU test method compared with the overall mean CV (26% for both test
methods), but there were only three substances in this chemical class (acetaminophen,dimethylformamide, procainamide HCl). Organic sulfur compounds had a high mean
intralaboratory CV for the 3T3 test method (46%), but not for the NHK NRU test method(29%) compared with the overall mean intralaboratory CV for both test methods (26%). The
intralaboratory CV values for the remaining chemical classes were unremarkable comparedwith the overall mean intralaboratory CV values.
For the characteristics amenable to correlation analysis, none of the Spearman correlation
coefficients were large (absolute value of rs <0.6), but several were statistically significantly
different from zero (p <0.05). Molecular weight (p=0.016), IC50 (p=0.002), and boiling point(p=0.009) exhibited statistically significant correlations to intralaboratory CV for the 3T3 test
NRU method. The higher molecular weight substances had higher intralaboratory CV values
and the substances with lower IC50 values had higher intralaboratory CV values. The findingthat substances with higher boiling points had higher CV values was consistent with thecategorical analysis of volatility. The substances that exhibited volatile characteristics (i.e.,cross contamination of VC wells) in the 3T3 NRU test method had slightly higher mean
intralaboratory CV values (31%) than the substances that did not exhibit volatile
characteristics (25%).

7-35

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-8

November 2006

Intralaboratory CV Values by Chemical Characteristics for the 3T3 andNHK NRU Test Methods

Class/Attribute
All chemicals
Chemical form
Solid
LiquidSolubility
Precipitate2
No precipitate
Volatility3
Volatile
Nonvolatile
Chemical Class
Alcohol
Amide
Amine
Carboxylic acid
Heterocyclic
Organophosphorous
Organic sulfur
PhenolPolycyclic
Inorganic
Inorganic chlorine
Inorganic sodium
GHS Acute Oral
Toxicity Class
LD50 ≤5 mg/kg
5 < LD50 ≤50
50 < LD50 ≤300
300 < LD50 ≤2000
2000 < LD50 ≤5000
LD50 >5000
Correlations
Molecular weight
Log Kow
IC50
Boiling point

N164

3T3 NRU Test Method
Range
Mean
1-122%
26%

N1
68

NHK NRU Test Method
Range
Mean
1-129%
26%

51
13

4-84
6-48

26
23

53
15

6-57
2-40

27
24

18
46

11-84
4-55

32
23

19
49

2-47
7-57

24
27

10
54

6-84
4-55

31
25

9
592

11-50
2-57

27
26

9
3
3
13
14
2
4
5
4
14
5
6

6-42
4-28
9-35
4-41
6-59
NA
36-59
14-30
19-35
9-43
9-33
9-34

22
13
18
18
31
NA
46
20
27
25
19
20

9
3
3
14
14
3
5
5
5
15
5
6

10-37
2-16
10-24
2-48
13-50
20-32
21-27
11-31
9-38
6-50
12-50
17-47

22
10
18
23
32
26
29
19
20
29
32
30

6
12
12
14
9
11
N
64
454
64
245

9-46
13-59
11-84
4-51
9-32
6-42
rs
0.301
0.121
-0.382
0.520

27
32
33
22
20
20
P value
0.016
0.430
0.002
0.009

7
12
12
16
9
12
N
68
484
68
245

20-40
12-50
17-37
6-57
7-50
2-40
rs
0.181
0.310
-0.346
0.226

30
31
25
25
30
19
P value
0.140
0.032
0.004
0.289

Abbreviations: CV=Coefficient of variation; 3T3=BALB/c 3T3 fibroblasts; GHS=Globally Harmonized System ofClassification and Labelling of Chemicals (UN 2005); NA=Not applicable because class had less than three observations;
NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; N=Number of values; rs=Spearman correlation
coefficient; Kow=Octanol:water partition coefficient.
1
One intralaboratory CV for each chemical was calculated by averaging the CV values for each reference substance.
2
Identified by laboratory reports of precipitate in the stock reference substance solutions or in cell culture (see Table 5-11).
3
Identified by laboratory reports of using plate sealers to avoid contamination of the VC wells (see Table 5-11).
4
Number of reference substances with CV values and log Kow data.
5
Number of reference substances with CV values and boiling point data.

Among the IC50 values obtained using the NHK NRU test method, two of the characteristics
amenable to correlation analysis were statistically significantly different from zero, although
the correlation coefficients did not have large magnitudes (absolute value of rs <0.4). The log
7-36

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

Kow (p=0.032) and IC50 (p=0.004) exhibited statistically significant correlations (p <0.05) tothe intralaboratory CV. Log Kow was positively correlated (i.e., higher log Kow values wereassociated with higher mean intralaboratory CV), but the IC50 was negatively correlated (i.e.,higher log IC50 values were associated with lower mean intralaboratory CV) to mean
intralaboratory CV.
7.2.3.5 Results of the Interlaboratory CV Analysis
Table 7-9 shows the analysis of the interlaboratory CV values. There seemed to be little
difference in interlaboratory CV values for most of the categorical physical/chemical
characteristics. The mean interlaboratory CV values for solids and liquids were similar (48%for solids vs. 42% for liquids for 3T3, and 28% for solids vs. 21% for liquids for NHK), as
were the values for substances for which precipitates were observed versus no precipitates(58% vs. 43% for 3T3, and 24% vs. 28% for NHK), and the values for substances thatexhibited volatile characteristics (51% for volatile substances vs. 46% for nonvolatile
substances for 3T3, and 32% for volatile substances vs. 26% for nonvolatile substances forNHK).
Mean interlaboratory CV values were calculated for the chemical classes that contained atleast three of the reference substances included in the reproducibility analyses (i.e., 64
substances for 3T3 and 68 substances for NHK). Reference substances in the amide chemicalclass had low mean interlaboratory CV values for both the 3T3 (15%) and the NHK (16%)
NRU test methods compared with the overall mean interlaboratory CV (47% and 28%,
respectively). Substances in the amine class also had low mean interlaboratory CV values forthe 3T3 NRU (13%), but not for the NHK NRU (20%). Organic sulfur compounds had
unusually high mean interlaboratory CV values for the 3T3 test method (100%), but not for
the NHK NRU (36%) compared with the overall mean interlaboratory CV (47% and 28%,
respectively). Because of the low number of reference substances in these classes, theseresults were deemed to not be significant.
Mean interlaboratory CV values tended to be large for chemicals in the most toxic GHS
acute oral toxicity categories, especially with the 3T3 NRU test method. The meaninterlaboratory CV for reference substances in the LD50 ≤5 mg/kg (72%) and 5 < LD50 ≤50
mg/kg (78%) classes were larger than the mean overall interlaboratory CV (47%,). For the
NHK NRU test method, the mean interlaboratory CV for chemicals in the 5 < LD50 ≤5 mg/kg(37%) and 5 < LD50 ≤50 mg/kg (41%) classes were larger than the mean overall
interlaboratory CV (28%).
For the characteristics amenable to correlation analysis, none of the correlation coefficientswere large (absolute value of rs <0.6), but IC50 (p=0.015) and boiling point (p=0.007)
exhibited statistically significant correlations (p <0.05) to interlaboratory CV in the 3T3 test
NRU method. There was a negative correlation between interlaboratory CV and IC50, but thecorrelation between boiling point and interlaboratory CV was positive. The positive
correlation of CV with boiling point was largely consistent with the categorical analysis ofvolatility. The substances that exhibited volatile characteristics in the 3T3 NRU test method
had slightly higher mean CV values than substances that did not exhibit volatile
characteristics (51% vs. 46%). Only the IC50 was significantly correlated (p=0.014) to7-37

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

interlaboratory CV with a negative correlation (rs=-0.271) when the NHK NRU test method
was used.
Table 7-9

Interlaboratory 3T3 and NHK NRU Test Method CV Values Sorted by
Chemical Characteristics

Class/Attribute
All chemicals
Chemical Form
SolidsLiquids
Solubility
Precipitate2
No precipitate
Volatility
Volatile3
Nonvolatile
Chemical Class
Alcohol
Amide
Amine
Carboxylic acid
Heterocyclic
Organic sulfur
Organophosphorous
PhenolPolycyclic
Inorganic
Inorganic chlorine
Inorganic sodium
GHS Acute Oral
Toxicity Class
LD50 ≤5 mg/kg
5 < LD50 ≤50
50 < LD50 ≤300
300 < LD50 ≤2000
2000 < LD50 ≤5000
LD50 >5000
Correlations
Molecular weight
Log Kow
IC50
Boiling point

3T3 NRU Test Method

NHK NRU Test Method

N

Range

Mean

N

Range

Mean

641

3-135%

47%

681

1-91%

28%

51
13

3-135
6-124

48
42

53
15

1-91
1-44

28
21

18
46

7-127
3-135

58
43

19
49

1-91
1-88

24
28

10
54

21-127
3-135

51
46

9
59

8-86
1-91

32
26

9
3
3
13
14
4
2
5
4
14
5
6

12-119
6-28
10-16
6-124
8-135
78-119
NA
19-64
14-85
3-127
3-127
3-60

38
15
13
38
57
100
NA
41
44
50
45
34

9
3
3
14
14
5
3
5
5
15
5
6

11-31
13-19
14-24
1-61
5-85
8-99
8-99
15-47
2-88
4-91
10-86
15-51

20
16
20
26
32
36
42
28
30
30
35
32

6
12
12
14
9
11
N
64
454
64
225

12-13533-127
8-120
11-85
3-69
6-124
rs
0.245
0.151
-0.304
0.563

72
78
37
35
29
41
P value
0.051
0.324
0.015
0.007

7
12
12
16
9
12
N
68
484
68
255

12-99
8-91
10-41
1-61
1-85
2-44
rs
0.169
0.210
-0.297-0.051

37
41
26
20
27
23
P value
0.168
0.151
0.014
0.809

Abbreviations: CV=Coefficient of variation; 3T3=BALB/c 3T3 fibroblasts; GHS=Globally Harmonized System ofClassification and Labelling of Chemicals (UN 2005); NA=Not applicable because class had less than three observations;
NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; N=Number of values; rs=Spearman correlation
coefficient; Kow=Octanol:water partition coefficient.
1
One intralaboratory CV for each chemical was calculated by averaging the CV values for each reference substance.
2
Identified by laboratory reports of precipitate in the stock reference substance solutions or in cell culture (see Table 5-11).
3
Identified by laboratory reports of using plate sealers to avoid contamination of the VC wells (see Table 5-11).
4
Number of reference substances with CV values and log Kow data.
5
Number of reference substances with CV values and boiling point data.

7-38

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

7.2.4
Comparison of Maximum to Minimum IC50 Values Using Laboratory MeansInterlaboratory reproducibility was also compared by calculating maximum to minimum
mean IC50 values using the laboratory means from each method, so that the reproducibility of
the IC50 values could be compared with the reproducibility of the reference LD50 values
derived in Section 4.2. The Figure 7-2 frequency histogram for the 3T3 NRU test method
maximum:minimum mean IC50 values shows that approximately half (37) of the 64 reference
substances produced ratios less than 2.5-fold of each other, and only nine chemicals hadratios greater than 5.5-fold, including one substance (cupric sulfate pentahydrate) that had aratio of 22.
Figure 7-2

Frequency of Maximum:Minimum 3T3 NRU IC50 Ratios

25
20
15
10
5
0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Maximum:Minimum 3T3 NRU IC50
Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red uptake.
Bars show the number of substances with maximum:minimum 3T3 NRU IC50 ratios within ±0.5 units of the barlabel (e.g., the first bar indicates that there were 14 reference substances for which the laboratory mean
maximum:minimum 3T3 NRU IC50 ratios were 0.5 to1.4). The analysis includes 64 reference substances.
Carbon tetrachloride, disulfoton, gibberellic acid, lithium carbonate, methanol, 1,1,1-trichloroethane, valproic
acid, and xylene were excluded because not all laboratories obtained IC50 values.

The Figure 7-3 frequency histogram for the maximum:minimum mean IC50 values for the
NHK NRU test method shows that ratios of 58 of the 68 chemicals were less than 2.5-fold of
one another. The highest ratio of 108 for digoxin is not shown in the figure. Comparison of
Figures 7-2 and 7-3 shows that the interlaboratory reproducibility of the NHK NRU test
method was better than that for the 3T3 NRU test method based on the distribution of the low
maximum:minimum IC50 ratios.

7-39

In Vitro Cytotoxicity Test Methods BRD Section 7

Figure 7-3

November 2006

Frequency of Maximum:Minimum NHK NRU IC50 Ratios

30

20

10

0

1

2

3

4

5

6

7

8

9

Maximum:Minimum NHK NRU IC50
Abbreviations: NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake.
Bars show the number of substances with maximum:minimum NHK NRU IC50 ratios within ±0.5 units of thebar label (e.g., the first bar indicates that there were 30 reference substances for which the laboratory mean
maximim:minimum NHK NRU IC50 ratios were 0.5 to 1.4). The analysis includes 68 reference substances.
Carbon tetrachloride, methanol, 1,1,1-trichloroethane, and xylene were excluded because not all laboratories
obtained IC50 values. The maximum:minimum IC50 for digoxin of 108 was excluded from this figure.

7.2.5

Comparison of the Maximum:Minimum IC50 Ratios with the Maximum:Minimum
LD50 Ratios
To compare the reproducibility of the NRU IC50 values with that of the LD50 values, themaximum:minimum IC50 ratios for each method (shown in Tables 7-3 and 7-5) werecompared with the maximum:minimum LD50 ratios reported in Table 4-2. This analysis
excluded reference substances for which fewer than three laboratories reported IC50 values,
and reference substances for which fewer than two acceptable acute oral LD50 values wereidentified. As a result, there were 53 substances analysed for the 3T3 NRU test method and
57 for the NHK NRU test method. The following substances were excluded from bothanalyses because fewer than two acceptable LD50 values could be identified: aminopterin;colchicine; digoxin; epinephrine bitartrate; gluthethimide; phenylthiourea; physostigmine;
procainamide HCl, propranolol HCl; propylparaben; and thallium sulfate. Carbon
tetrachloride, disulfoton, gibberellic acid, lithium carbonate, methanol, 1,1,1-trichloroethane,
valproic acid, and xylene, were excluded from the 3T3 analysis, and carbon tetrachloride,methanol, 1,1,1-trichloroethane, and xylene, were excluded from the NHK analysis, because
fewer than three laboratories reported IC50 values.
Figure 7-4 shows that the maximum:minimum LD50 ratios tend to be larger than either the
3T3 NRU IC50 or NHK NRU IC50 ratios because there are more points below the theoretical
one-to-one correspondence line than above the line. The difference between the LD50
maximum:minimum values and the NRU IC50 maximum:minimum values is more striking
for the NHK since there are fewer points above the line for the NHK graph (Figure 7-4b)
than for the 3T3 graph (Figure 7-4a).
7-40

In Vitro Cytotoxicity Test Methods BRD Section 7

Figure 7-4

November 2006

Comparison of Maximum:Minimum NRU IC50 Ratios to
Maximum:Minimum LD50 Ratios

a.
30
25
20
15
10
5
0

0

5

10

15

20

25

30

Maximum:Minimum LD 50

b.
30
25
20
15
10
5
0

0

5

10

15

20

25

30

Maximum:Minimum LD50
Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red uptake; NHK=Normal human epidermal
keratinocytes. Comparison of maximum:minimum ratios of IC50 and LD50 for 53 reference substances for the
3T3 NRU test method (a) and 57 reference substances for the NHK NRU test method (b). Solid lines show thetheoretical one to one correspondence of maximum:minimum IC50 to maximum:minimum LD50.

7-41

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

7.2.6
Normalization of Reference Substance IC50 Values Using SLS IC50 Values
As an alternate analysis for reproducibility, IC50 values for reference substances werenormalized to those of the corresponding SLS IC50values. This approach was tested using
five reference substances for each test method to determine whether such normalization
would reduce the variability, measured using CV values, of the results. The reference
substances selected for this evaluation were those for which the ANOVA indicatedstatistically significant differences among the laboratories. Because there were a number ofreference substances that met this criterion for the 3T3 NRU test method, the substanceswere selected so as to cover a wide range of rodent acute oral toxicity. One reference
substance was selected from each GHS category with the exception of the 50 ≤ LD50 <300
mg/kg category. There was no substance represented by this category because there were six
acute oral toxicity categories and only five substances were used for this assessment. The
reference substances, shown in Table 7-10, were busulfan, chloramphenicol, meprobamate,propylparaben, and triethylenemelamine. Because there were only six reference substanceswith significant ANOVAs in the NHK NRU test method, the last five reference substances inTable 7-5 (citric acid, digoxin, dimethylformamide, disulfoton, and propranolol HCl) wereselected for this analysis.
Millimolar units were used for the IC50 values in this analysis since the mole is the mostappropriate unit for measuring and comparing biological activity. The IC50 value (in mM) for
each reference substance was normalized to the corresponding SLS IC50 value (in mM) by
dividing the SLS IC50 by the reference substance IC50. Intra- and inter-laboratory CV values
were calculated for both the IC50 values and for the SLS IC50:reference substance IC50 ratios
to determine whether this type of normalization would reduce the interlaboratory CV values.
Table 7-10 shows that the mean intralaboratory CV of the IC50 values for the five substancesused in the 3T3 evaluation was 22% and the interlaboratory CV was 88%. Normalizing thereference substance IC50 values to the SLS IC50 yielded a slightly higher intralaboratory CV
of 25% and a lower interlaboratory CV of 65%. The mean intralaboratory CV of the IC50
values for the five substances used in the NHK evaluation was 14% and the interlaboratory
CV was 50%. Normalizing the reference substance IC50 values to the SLS IC50 yielded aslightly higher intralaboratory CV of 16% and a higher interlaboratory CV of 61%. Whenthe normalization ratios are examined for each chemical-by-laboratory combination (Table
7-10), nine CVs increased, five decreased, and one remained the same for the 3T3 NRU test
method, and eight increased, six decreased, and one remained the same for the NHK NRU
test method. Thus, for the reference substances used in this analysis, normalizing thereference substance IC50 to the concurrent SLS IC50 did not reduce the overall variability ofthe measurements, as measured by the CV values.

7-42

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-10

CV Values for 3T3 and NHK NRU Test Method IC50 Values andNormalized IC50 Values

Reference
Substance

IC50
(mM)1

Busulfan
ECBC
FAL
IIVS
Chloramphenicol
ECBC
FAL
IIVS
Meprobamate
ECBC
FAL
IIVS
Propylparaben
ECBC
FAL
IIVS
Triethylenemelamine
ECBC
FAL
IIVS
Mean

0.548
0.163
1.30
0.177
0.498
0.171
0.845
0.483
2.47
1.62
4.02
1.77
0.166
0.116
0.287
0.0949Citric Acid
ECBC
FAL
IIVS
Digoxin
ECBC
FAL
IIVS
Dimethylformamide
ECBC
FAL
IIVS
Disulfoton
ECBC
FAL
IIVS
Propranolol HCl
ECBC

November 2006

0.002780.000421
0.007100.000827

2.21
2.74
1.62
2.25
4.02E-06
6.89E-06
6.53E-08
5.10E-06

IntraLab
CV2 (%)

3T3 NRU Test Method
119
0.677
48
1.05
56
0.109
4
0.877
67
0.725
22
0.847
30
0.844
18
0.483
54
0.071
14
0.085
15
0.039
2
0.088
64
1.16
16
1.29
29
0.535
12
1.65
135
191
11
354
18
21.4
29
197
22
88
NHK NRU Test Method
25
0.00587
160.005317
0.007650.0047
88
62378
13
1264
36
183479
7
2389

107
128
107
87.5
1.38
0.509
2.94
0.679
0.125
0.129

InterLab
CV3 (%)SLS IC50:
Substance
IC504

19
2
1
3
99
19
26
32
21
12

7-43

0.000110.000070.000130.000130.0140
0.022
0.005
0.015
0.09470.081

IntraLab CV
SLS IC50:
Substance
IC505 (%)

InterLabCVSLS IC50:
Substance
IC506 (%)
74

70
53
9
29
30
22
21
39
23
29
3
49
20
22
9
87
11
24
23
25

65
26

14
28
16
168
10
44
26
31
7
1
19
61
6
5
20
20
15

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-10

November 2006

CV Values for 3T3 and NHK NRU Test Method IC50 Values andNormalized IC50 Values

Reference
Substance

IC50
(mM)1

IntraLab
CV2 (%)

FAL
IIVS
Mean

0.148
0.09676
11
14InterLab
CV3 (%)SLS IC50:
Substance
IC504
0.087
0.116

50

IntraLab CV
SLS IC50:
Substance
IC505 (%)
25
9
16

InterLabCVSLS IC50:
Substance
IC506 (%)

61

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; ECBC=Edgewood Chemical Biological Center; FAL= Fund for the
Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; NA=No
acceptable IC50 results reported or calculation was not performed (e.g., for contrast results); CV=Coefficient of variation.
1
Results reported on the same row with reference substance names are the arithmetic means of all the laboratories. Results
reported on the same row as laboratories are the arithmetic laboratory means.
2
CV for IC50 values from the acceptable tests within each laboratory.
3
CV calculated using the arithmetic mean IC50 values from each laboratory.
4
Concurrent SLS IC50 in mM divided by the reference substance IC 50. Results reported on the same row with reference
substance names are the arithmetic means of all the laboratories. Results reported on the same row as laboratories are thearithmetic laboratory means.
5
CV for SLS IC50:reference substance IC50 values within each laboratory.
3
CV calculated using the mean SLS IC50:reference substance IC50 values from each laboratory.

7.3

Historical Positive Control (PC) Data

The reproducibility of the PC (SLS) data was assessed by CV analysis, ANOVA, and linear
regression over time, as described in Section 5.5.4.2. To obtain an assessment of the truevariation of SLS IC50 values, the reproducibility analyses also included IC50 values from SLS
tests that failed the test acceptance criterion for the IC50 acceptance limits determined foreach study phase. Therefore, the values used for this analysis included some that were notincluded in Table 5-3. These additional SLS tests, however, passed all other test acceptancecriteria. If more than one SLS test was performed in a single day (for each method andlaboratory), the IC50 values were averaged to determine a single IC50 for the day so that themultiple results from that day would not overly influence the average.
Figure 7-5 shows the average SLS IC50 values for each method, laboratory, and study phase.
The SLS IC50 for the 3T3 test method (Figure 7-5a) was relatively consistent over the entire
period of the study (approximately 2.5 years). The intralaboratory CV values for theindividual study phases ranged from 5% to 24% (Figure 7-5a). With the exception of thePhase Ib CV at FAL, the CV values for each laboratory and phase were less than 20%. Theinterlaboratory CV values were even smaller, 6% in Phases Ia and Ib, 10% in Phase II, and
2% in Phase III.
Figure 7-5b shows that the SLS IC50 for the NHK NRU test method tended to vary withtime, but, with the exception of the values from FAL, there appeared to be no consistent
trend. The IC50 values from FAL, which changed their cell culture methods after Phase Ib(see Section 5.3.3.1), tended to decrease over time. Although the change in cell culture
methods reduced the magnitude of the IC50, the variability (as evidenced by the
intralaboratory CV values shown in Figure 7-5b) remained relatively high (CV ≥34% for all
FAL study phases).
7-44

In Vitro Cytotoxicity Test Methods BRD Section 7

Figure 7-5

November 2006

SLS IC50 for Each Laboratory and Study Phasea

3T3 NRU Test Methodb

NHK NRU Test Method

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red
uptake; ECBC=Edgewood Chemical Biological Center; FAL= Fund for the Replacement of Animals in Medical
Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; N=Number of values.
Note: Bars show mean SLS IC50 values. Error bars show standard deviation. Percent values above error bars are
intralaboratory CVs.

7-45

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

The CV values for all the laboratories and study phases show that the SLS IC50 values in the
NHK NRU test method are more variable within laboratories than the corresponding 3T3
SLS IC50 values. The CV values for the SLS IC50 for the NHK NRU test method ranged from11 to 51%, with nine of the 12 values greater than 20%. The interlaboratory CV values,
which were also greater than those for the 3T3 NRU test method, were 39% in Phase Ia, 21%
in Phase Ib, 31% in Phase II, and 8% in Phase III.
7.3.1

ANOVA and Linear Regression Results for the 3T3 NRU Test Method

7.3.1.1 Variation of SLS IC50 Values with Time
Table 7-11 shows the SLS ANOVA results from the 3T3 test method. When the IC50 values
in each laboratory were compared, there were no statistically significant differences (p <0.01)
among study phases for any laboratory. Table 7-12 shows that the slopes of the linear
regressions of the IC50 values over time (expressed as index values) were significantly
different from zero for ECBC and FAL (p=0.001 and 0.012, respectively), but, because theslopes were so small (0.000204 and -0.000324), and in different directions, these differenceswere considered to be unimportant, regardless of the statistical conclusions. The slope of the
IIVS regression of SLS IC50 over time was not significantly different from zero (p=0.651;
Table 7-12), which was consistent with the ANOVA analysis (Table 7-11), and showed thatSLS IC50 from IIVS did not vary with study phase (p=0.854). The ANOVA analysis, withstudy phase as the factor (with laboratories combined), showed that the 3T3 NRU IC50 values
from all the laboratories were consistent over time (p=0.304).
7.3.1.2 Comparison of SLS IC50 Values Among the Laboratories
When all study phases from each laboratory were combined, ANOVA, with laboratory as thefactor, showed that the SLS IC50 values in the 3T3 NRU test method differed significantly
among the laboratories (p <0.006) (Table 7-11). However, as can be seen in Figure 7-5a, theindividual laboratory SDs overlap one another.

7-46

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-11

November 2006

ANOVA Results for the SLS IC50 Values in the 3T3 NRU Test Method

ECBC
Log Mean IC50
SD
N
P1
(mM)
Test for differences between phases within each laboratory
Study Phase/
Laboratory

0.031

FAL

IIVS

Log Mean IC50
(mM)

SD

N

-0.811

0.046

9

P

Log Mean IC50
(mM)

SD

N

P1

0.015

-0.850

0.034

7

0.854

1

Phase Ia

-0.8760.042

6Phase Ib

-0.8640.066

6-0.8460.065

8-0.8380.025

5Phase II

-0.8480.027

16-0.7960.057

19-0.8540.025

8Phase III-0.8420.036

36-0.8510.066

27-0.8440.041

23-0.8260.062

63-0.8470.035

44

Test for differences between laboratories (phases combined)
All Phases-0.849

0.039

64

0.006

Test for differences between phases (laboratories combined)
Phase Ia

-0.8390.049

22Phase Ib

-0.8500.056

19Phase II

-0.8310.047

34Phase III

0.845

0.045

86

0.304

Abbreviations: ANOVA=Analysis of variance; SLS=Sodium lauryl sulfate; 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral
red uptake; N=Number of values; SD=Standard deviation; ECBC=Edgewood Chemical Biological Center; FAL=Fund for
the Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences.
1
Statistically significant at p <0.01.

7-47

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-12
Laboratory
ECBC
FAL
IIVS
ECBC
FAL
IIVS

November 2006

Linear Regression Analysis of SLS IC50 Values Over Time1
Slope
P-value (Slope)2
3T3 NRU Test Method
0.000204
0.001-0.000324
0.012
0.0000304
0.651
NHK NRU Test Method-0.000559
0.002-0.00112<0.001-0.000445
0.002Intercept-0.874-0.796-0.850-1.901-1.737-1.885Abbreviations: SLS=Sodium lauryl sulfate; 3T3=BALB/c 3T3 fibroblasts;
NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; N=Numberof values; ECBC=Edgewood Chemical Biological Center; FAL=Fund for the
Replacement of Animals in Medical Experiments Alternatives Laboratory;
IIVS=Institute for In Vitro Sciences.
1
Time was expressed as index values. The index value of each test reflected the orderof testing without respect to the time lapsing between tests.
2
Statistically significant from zero at p <0.05.

7.3.2

ANOVA and Linear Regression Results for the NHK NRU Test Method

7.3.2.1 Variation of SLS IC50 Values with Time
Table 7-13 shows the ANOVA results for the NHK NRU test method. When the IC50 values
within each laboratory were compared by study phase, the values were statistically different(p <0.01) at each laboratory. The IC50 values from the various study phases were alsosignificantly different from one another when the laboratory data were combined (p <0.001).
The change in cell culture methods at FAL after Phase Ib (see Section 5.3.3.1) contributed tothis difference. Table 7-13 shows that FAL had clearly the lowest log mean SLS IC50 for
Phases Ia and Ib. Linear regression analyses showed that the IC50 slopes over time (expressedas an index values) were statistically significantly less than zero for each laboratory (seeTable 7-12). Because the slopes were so small (-0.000559, -0.00112, and -0.000445), andnegative, their statistical significance was considered to be irrelevant.
7.3.2.2 Comparison of SLS IC50 Values Among the Laboratories
The ANOVA results, with laboratory as a factor (Table 7-13), showed that the SLS IC50 was
statistically significantly different among the laboratories when the data from the study
phases were pooled (p <0.001). Figure 7-5b shows that the SLS data from ECBC and IIVS
were rather similar to one another for Phases Ia, Ib, and III. The SLS IC50 data from FAL are
different from the other two laboratories for Phases Ia, Ib, and II, but the SDs for Phase III
show that the data from all laboratories produced similar values.

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In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-13

November 2006

ANOVA Results for the SLS IC50 Values in the NHK NRU Test Method

ECBC
Log MeanSD
N
P1
IC50 (mM)
Test for differences between phases within each laboratory
Study Phase/
Laboratory

0.001

FAL

IIVS

Log Mean
IC50 (mM)SD

N-1.656

0.125

5

P

Log Mean
IC50 (mM)

SD

N

P1

<0.001-1.9040.060

12<0.0011

Phase Ia

-1.8670.135

5Phase Ib

-1.9360.092

6-1.8290.141

10-1.9650.046

5Phase II

-2.0070.109

11-1.9820.173

15-1.8630.058

12Phase III-1.9900.098

31-1.9410.113

34-1.9720.070

19-1.8790.175

64-1.9240.073

48

Test for differences between laboratories (phases combined)
All Phases-1.9710.113

53<0.001

Test for differences between phases (laboratories combined)
Phase Ia

-1.8330.143

22

Phase Ib

-1.8910.125

21Phase II

-1.9640.139

38

Phase III-1.9710.100

84<0.001Abbreviations: ANOVA=Analysis of variance; SLS=Sodium lauryl sulfate; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; N=Number of values;
SD=Standard deviation; ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory;
IIVS=Institute for In Vitro Sciences.
1
Statistically significant at p <0.01.

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In Vitro Cytotoxicity Test Methods BRD Section 7

7.4

November 2006

Laboratory Concordance for Solvent Selection

The solvents used for the reference substances are shown in Table 7-14. For Phases Ib and II,
the SMT based their selection of solvents on the results provided by BioReliance (see Table
5-9) using the solubility protocol in Appendix G2. Despite the fact that the solubility of anindividual substance might be different in 3T3 and NHK growth media, the SMT selected thesame solvent (i.e., medium or DMSO) for both test methods, rather than having different
solvents for each method.
BioReliance occasionally achieved higher solubility values for the Phase I and II substances
than the three cytotoxicity laboratories (e.g., see the results for arsenic trioxide, aminopterin,
and chloramphenicol in Table 5-10). The laboratories were using the solubility protocols inAppendices C3 through C6 (for Phases Ib and II), which were somewhat different from theprotocol used by BioReliance. Although all the laboratories used the same protocols, they did
not always obtain similar results with respect to the solvent to be used (e.g., see the results
for aminopterin, cadmium chloride, and chloramphenicol in Table 5-10). In an attempt to
avoid the selection of a solvent for which one or more laboratories could not achieve thedesired solubility, the SMT used the solubility data from all the laboratories to determine thesolvents to be used for each chemical tested in Phase III. Table 7-14 shows that cell culture
medium was used as the solvent for 38 substances and DMSO was used for 34 substances.
Five of the substances were insoluble in medium and DMSO in at least one testinglaboratory. Arsenic trioxide was insoluble at all laboratories. IIVS also found sodium oxalate,strychnine, and triethylenemelamine insoluble in media and DMSO, and FAL found thallium
sulfate insoluble in both solvents. Therefore, the SMT used the results from the laboratories
that did achieve solubility to select the solvents to be used for testing these substances.
The testing laboratories selected the same solvent for 55 of the 72 reference substances(76%). Excluding the five substances that were found to be insoluble in both solvents by atleast one laboratory, there were 12 substances on which the laboratories disagreed:
acetaminophen, acetylsalicylic acid, carbamazepine, carbon tetrachloride, chloramphenicol,
dichlorvos, meprobamate, methanol, phenobarbital, phenylthiourea, physostigmine, andvalproic acid. Each laboratory reported relatively low solubility, ≤2 mg/mL, in medium forthese substances. Because 2 mg/mL in medium is the departure point for the selection of
medium or DMSO, small variations in solubility lead the laboratories to select different
solvents. The solubility of acetaminophen, for example was reported as 2 mg/mL in culture
media by ECBC and FAL, but <2 mg/mL by IIVS. IIVS found it soluble in 200 mg/mL
DMSO and selected DMSO as the solvent. ECBC and FAL selected culture media as thesolvent. The SMT selected DMSO as the solvent for acetaminophen to be used by alllaboratories so that they would all be assured of obtaining usable test results.

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In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-14

November 2006

Solvent Determinations by Laboratory

Reference SubstanceAcetaminophen
Acetonitrile
Acetylsalicylic acid
Aminopterin
5-Aminosalicylic acid
Amitriptyline HCl
Arsenic III trioxide
Atropine sulfate
Boric aid
BusulfanCadmium II chloride
Caffeine
Carbamazepine
Carbon tetrachloride
Chloral hydrate
Chloramphenicol
Citric acidColchicine
Cupric sulfate pentahydrate
Cycloheximide
Dibutyl phthalate
Dichlorvos
Diethyl phthalate
DigoxinDimethylformamide
Diquat dibromide
monohydrate
Disulfoton
Endosulfan
Epinephrine bitartrate
Ethanol
Ethylene glycol
Fenpropathrin
Gibberellic acid
Glutethimide
GlycerolHaloperidol
Hexachlorophene
Lactic acid
Lindane
Lithium I carbonate
Meprobamate
Mercury II chloride
MethanolNicotine
Paraquat
Parathion
Phenobarbital
PhenolPhenylthiourea

Solvent Used for
Testing1
DMSO
MediumDMSO
DMSO
MediumDMSO
MediumMediumMediumDMSO
DMSO
MediumDMSO
DMSO
MediumDMSO
MediumMediumMediumMedium
DMSO
DMSO
DMSO
DMSO
Medium

ECBC

FAL

IIVS

MediumMediumMediumDMSO
MediumDMSO
ID
MediumMediumDMSO
DMSO
MediumMediumMediumMediumDMSO
MediumMediumMediumMediumDMSO
MediumDMSO
DMSO
MediumMediumMediumDMSO
DMSO
MediumDMSO
ID
MediumMediumDMSO
DMSO
MediumDMSO
DMSO
MediumDMSO
MediumMediumMediumMedium
DMSO
DMSO
DMSO
DMSO
MediumDMSO
MediumMediumDMSO
MediumDMSO
ID
MediumMediumDMSO
DMSO
MediumDMSO
MediumMediumMediumMediumMediumMediumMediumDMSO
MediumDMSO
DMSO
MediumMediumMediumMediumMediumDMSO
DMSO
MediumMediumMediumDMSO
MediumDMSO
MediumDMSO
DMSO
MediumDMSO
Medium
DMSO
DMSO
DMSO
MediumMediumDMSO
DMSO
Medium
DMSO

DMSO
DMSO
MediumMediumMediumDMSO
MediumDMSO
MediumDMSO
DMSO
MediumDMSO
MediumMediumDMSO
MediumMediumMediumDMSO
MediumMedium
DMSO

DMSO
DMSO
MediumMediumMediumDMSO
MediumDMSO
MediumDMSO
DMSO
MediumDMSO
MediumMediumDMSO
MediumMediumMediumDMSO
DMSO
MediumMediumDMSO
DMSO
MediumMediumMediumDMSO
MediumDMSO
MediumDMSO
DMSO
MediumDMSO
Medium
DMSO
DMSO
DMSO
MediumMediumDMSO
DMSO
MediumDMSO

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In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-14

November 2006

Solvent Determinations by Laboratory

Reference SubstancePhysostigmine
Potassium I chloride
Potassium cyanide
Procainamide HCl
2-Propanol
Propranolol HCl
Propylparaben
Sodium arsenite
Sodium chloride
Sodium dichromate dihydrate
Sodium fluoride
Sodium hypochlorite
Sodium oxalate
Sodium selenate
Strychnine
Thallium I sulfate
Trichloroacetic acid
1,1,1-Trichloroethane
Triethylenemelamine
Triphenyltin hydroxide
Valproic acid
Verapamil HCl
XyleneDMSO Total
Medium Total

Solvent Used for
Testing1
DMSO
MediumMediumMediumMediumDMSO
DMSO
MediumMediumMediumMediumMediumMediumMediumMediumMediumMediumMedium
DMSO
DMSO
DMSO
DMSO
DMSO
34
38

ECBC

FAL

IIVS

MediumMediumMediumMediumMediumMediumDMSO
MediumMediumMediumMediumMediumMediumMediumMediumMediumMediumMediumMediumDMSO
Medium
DMSO
DMSO
22
49

DMSO
MediumMediumMediumMediumMediumDMSO
MediumMediumMediumMediumMediumMediumMediumMediumID
MediumMedium
DMSO
DMSO
DMSO
DMSO
DMSO
29
41

DMSO
MediumMediumMediumMediumMediumDMSO
MediumMediumMediumMediumMediumID
MediumID
MediumMediumMedium
ID
DMSO
DMSO
DMSO
DMSO
28
40

Abbreviations: DMSO=Dimethyl sulfoxide; ECBC=Edgewood Chemical Biological Center; FAL= Fund for the
Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences;
ID=Insufficient data to select solvent; Medium=Cell culture medium.
1
Solvents selected by the SMT for use by all laboratories.

7.5

Summary

Intra- and inter-laboratory reproducibility were assessed by comparing the laboratoryspecific IC50-LD50 regressions to the mean, across-laboratory regression for each method,
ANOVA, CV analysis, and comparison of maximum:minimum mean laboratory IC50 values.
ANOVA permitted statistical comparisons of laboratories and experimental averages, while
controlling for other factors. CV analysis compared the relative magnitudes of variability ona standardized scale. Reproducibility was evaluated using the results from the reference
substances that yielded IC50 values from all three laboratories: 64 and 68 reference
substances in the 3T3 and the NHK NRU test methods, respectively. The analysis of
intralaboratory reproducibility, by evaluating the similarity of the laboratory specific IC50LD50 regressions, showed that the laboratory regressions for both NRU test methods were
within the 95% confidence limits of the laboratory mean regressions.
The ANOVA showed significant interlaboratory differences for 23 substances in the 3T3
NRU test method and six in the NHK NRU test method. Intralaboratory CV values ranged
from 1-122% in the 3T3 test method and 1-129% in the NHK NRU test method. Meaninterlaboratory CV values were 26% for both NRU test methods, but NHK had a lower mean
7-52

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

interlaboratory CV (28% vs 47% for 3T3 NRU). Interlaboratory CV values ranged from 3135% in the 3T3 NRU test method and 1-91% in the NHK NRU test method. FAL had thehighest mean intralaboratory CV in both NRU test methods (33% in 3T3, 43% in NHK).
An analysis to determine the relationship between the chemical attributes and interlaboratory
CV indicated that chemical structure, physical form, solubility, and volatility had little effecton CV. The CV seemed to be related, however, to GHS acute toxicity category, IC50, andboiling point. Mean interlaboratory CV values were larger for substances in the most toxic
GHS categories than for substances in the other toxicity categories, especially with the 3T3
NRU test method. The mean interlaboratory CV for substances in the LD50 ≤5 mg/kg (72%)
and 5< LD50 ≤50 mg/kg (78%) classes were larger than the mean overall interlaboratory CV(47%) with the 3T3 NRU test method. The mean interlaboratory NHK CV was 37% forsubstances with LD50 ≤5 mg/kg, and 41% for substances with 5< LD50 ≤50 mg/kg, while themean overall interlaboratory CV was 28%. A Spearman correlation analysis showed that theIC50 was inversely correlated to interlaboratory CV for both the 3T3 (p=0.015) and NHK(p=0.014) test methods, and that boiling point was positively correlated to interlaboratory CV(p=0.007) (i.e., higher boiling points were associated with higher CV values) for the 3T3 but
not the NHK NRU test method (p=0.809).
The ANOVA results for the PC IC50 in the 3T3 NRU test method showed that there weresignificant differences among laboratories (p=0.006) but not among study phases withinlaboratories (p >0.01). However, interlaboratory CV values, which ranged from 2% to 10%
for the different study phases, were small and the intralaboratory CV values ranged from 5%
to 24%. The SLS IC50 values from the NHK NRU test method were more variable than thosefrom the 3T3 NRU test method. The ANOVA results for SLS in the NHK NRU test method
indicated that there were significant differences among laboratories (p <0.001) and among
study phases within laboratories (p ≤0.001). A change in cell culture methods at FAL after
Phase Ib decreased the SLS IC50 in subsequent phases, but FAL’s CV values still tended tobe higher than in the other laboratories. Intralaboratory CV values for the NHK SLS IC50
during the various study phases ranged from 11% to 51% and interlaboratory CV values for
SLS in the NHK NRU test method ranged from 8% in Phase III to 39% in Phase Ia.
Cell culture medium was used as the solvent for 38 substances and DMSO was used for 34
substances. Concordance among all three laboratories in selecting the solvent for thereference substances was 76% (55/72).

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In Vitro Cytotoxicity Methods BRD Section 8

November 2006

8.0

3T3 AND NHK NRU TEST METHOD DATA QUALITY ................................. 8-3

8.1

Compliance with Good Laboratory Practice Regulations ................................... 8-3
8.1.1 Guidelines Followed for Cytotoxicity Testing .............................................. 8-3
8.1.2 Quality Assurance (QA) for NRU Cytotoxicity Test Data............................ 8-4
8.1.3 Guidelines Followed for Rodent Acute Oral LD50 Data Collection .............. 8-6

8.2

Results of Data Quality Audits ............................................................................... 8-6
8.2.1 QA Statements ............................................................................................... 8-6
8.2.2 QA Statements from the Laboratories ........................................................... 8-7

8.3

Effect of Deviations or Non-compliance with GLPs ............................................. 8-9
8.3.1 Laboratory Error Rates .................................................................................. 8-9
8.3.2 Failure Rates for Definitive and PC Tests ................................................... 8-10
8.3.3 Intralaboratory Reproducibility ................................................................... 8-11
8.3.4 Prediction of GHS Acute Oral Toxicity Categories .................................... 8-12

8.4

Availability of Laboratory Notebooks ................................................................. 8-12

8.5

Summary................................................................................................................. 8-13

8-1

In Vitro Cytotoxicity Methods BRD Section 8

November 2006

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8-2

In Vitro Cytotoxicity Methods BRD Section 8

8.0

November 2006

3T3 AND NHK NRU TEST METHOD DATA QUALITY

This section of the BRD presents the extent of adherence to GLP regulations for generationof the validation study data. Data quality is described, along with deviations from theregulations and their effect (if any) on the quality of the data. Statistical analyses areprovided to compare the data generation, collection, and reporting by the two GLP compliant
laboratories and the one non-GLP compliant laboratory, as well as for the GLP-compliant
laboratory that distributed the reference substances and performed solubility studies.
Discussions of various quality assurance aspects of the study are included.
8.1

Compliance With Good Laboratory Practice Regulations

8.1.1

Guidelines Followed for Cytotoxicity Testing

8.1.1.1 Good Laboratory Practices
The SOW provided the following definition of U.S. Regulatory agency GLPs to eachlaboratory:“Regulations governing the conduct, procedures, and operations of toxicology
laboratories; regulations to assure the quality and integrity of the data and to address suchmatters as organization and personnel, facilities, equipment, facility operations, test andcontrol articles, and validation study protocol, and conduct (U.S. Food and Drug
Administration, Title 21 CFR Part 58; U.S. Environmental Protection Agency, Title 40
CFR Part 160).”
IIVS, ECBC, and BioReliance performed testing under GLP guidelines. The details of GLP
compliance and training are addressed in Section 11.2.
8.1.1.2 Spirit of GLPThe SMT determined a definition for “spirit of GLP” and provided the following to thelaboratories:“Laboratories that are non GLP-compliant shall adhere to GLP principles and othermethod parameters as put forth in this Statement of Work and the Test Method Protocols(provided by NIEHS/NICEATM); documentation and accountability shall be equal to
GLP requirements; laboratories must make assurances that they are equal in performance
criteria and that there is parity amongst the laboratories.”
FAL performed testing in the “spirit of GLP” (see Section 11.2.2.1) by following theinternational GLP standards referenced in the ECVAM Workshop 37 Report (CooperHannan 1999) and the OECD Principles of GLP (OECD 1998). The laboratory did not havetheir data and test procedures reviewed by an independent, quality assurance (QA) auditor.
The SOW directed FAL to, at a minimum, routinely document their equipment monitoring
and record keeping (see Table 8-1), and to archive all documents. The FAL already had mostof the requested procedures and guidelines in place for routine laboratory procedures before
initiation of this study. The various general laboratory-related activities were documented inworkbooks and logbooks, and the information was made available to the SMT.

8-3

In Vitro Cytotoxicity Methods BRD Section 8

Table 8-1

November 2006

SMT-Recommended Documentation for FAL
Daily

Per UseTemperatures
Laboratory (ambient), incubators,
water baths, refrigerators, freezers

Cryogenic Storage Unit
Liquid N2 volume

Humidity/CO2
Cell culture incubators

Equipment Calibration
Balances, pH meters, cell counters

Visual Observations
Cell Culture Growth

Reagents
Lot numbers and expiration datesof medium/supplements

Periodic
Laboratory Supplies1
Lot numbers and expiration dates
for stock media formulations andcomponents, NRU reagents, tissue
culture plasticware
Cells
Quantity, and cryogenic storage
conditions, for 3T3 and NHK cells
Equipment Calibration
Incubators, laminar flow hoods,
autoclaves, micropipettors,
spectrophotometer plate readers,
computers (software)

Abbreviations: SMT=Study Management Team; FAL=Fund for the Replacement of Animals in Medical Experiments
Alternatives Laboratory; NRU=Neutral red uptake; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal
keratinocytes.
1
Documentation for laboratory supplies begins when supplies are purchased and received by the laboratory

8.1.1.3 Good Cell Culture Practices (GCCP)The SMT provided guidance in the SOW for implementing GLPs in a cell culture laboratoryenvironment. The initial assumption by the SMT was that each laboratory had the basic cell
culture skills and knowledge (e.g., as described in Freshney 2000) to reliably perform the invitro NRU cytotoxicity test methods. Reviews of historical laboratory documents, andscientific and professional exchanges with the laboratory personnel, assured the SMT thateach laboratory had demonstrated, through previous validation studies and other experience,that the personnel were capable of providing quality scientific data through the use of goodcell culture practices. A comparison of the SOW and the in vitro NRU cytotoxicity protocols
showed that the guidelines developed for the NICEATM/ECVAM study were harmonious
with the guidelines in the ECVAM Good Cell Culture Practices Reports (Hartung 2002;
Coecke et al. 2005), and the OECD document on GLPs and in vitro studies (OECD 2004a).
8.1.2

Quality Assurance (QA) for NRU Cytotoxicity Test Data

8.1.2.1 Coded Reference Substances
BioReliance acquired 73 high purity chemicals (72 reference substances and one positive
control substance) from reputable commercial sources. Sixty-four of the reference substanceswere ≥99% pure, and seven were between 90 and 99% pure. Lactic acid had the lowest
purity, 89% (See Appendix F1). The substances were coded with unique identification
numbers and provided to the testing laboratories in a blinded fashion. Procurement ofchemicals and their preparation for distribution was performed under GLP guidelines and the
SOW provided by the SMT (see Appendix G). Section 3.4 provides detailed information on
the acquisition and distribution of reference substances.

8-4

In Vitro Cytotoxicity Methods BRD Section 8

November 2006

8.1.2.2 Solubility Testing and Data Review
All laboratories performed solubility tests on all reference substances using the solvents andprocedures specified in the protocols provided by the SMT, and submitted solubility data to
the SMT in the form of hard copy printouts and electronic worksheets. The Study Directors
reviewed all laboratory procedures and all data produced at their respective laboratories, andthe QA designee in each GLP-compliant laboratory reviewed all data in their laboratory. The
SMT Project Coordinators served as informal QA reviewers for FAL (i.e., reviewed all theraw data sheets). The errors and omissions detected were reported to FAL, and corrections
were requested. The SMT reviewed all solubility data and NRU assay data produced by all of
the laboratories.
The SMT reviews of the submitted data in Phases Ia and Ib revealed that, even after data
review by the Study Directors, the data files contained an unacceptably high frequency oferrors (see Section 2.6.2.5). The laboratories were alerted to the problem and personnel fromall laboratories attended a weeklong training session at the IIVS laboratories in Gaithersburg,
Maryland to enhance harmonization among the laboratories. Errors continued to be found indata files submitted for Phase III after the training, albeit less frequently; however, such
errors generally resulted from the rush to rapidly complete the data files for submission to the
SMT shortly after the conclusion of each test. The formal QA reviews of the files occurred
later in each phase of the study.
The most common errors included typographical mistakes, transcriptional and data entry
errors in the Microsoft® EXCEL® and the GraphPad PRISM® 3.0 templates, and incorrectlabeling of files. The SMT reviewed every electronic file and hard copy printout throughout
the study and alerted the Study Directors of the affected laboratories when errors were found.
All data files were checked for consistency within the documents, and for compliance withthe protocols. The SMT also documented errors on the hard copy printouts in the form ofhandwritten notations to the files (at NICEATM) and added these notations to the electronic
data summary files compiled for data management. Files that were revised and/or correctedby the Study Director were resubmitted to the SMT and identified as corrected files.
8.1.2.3 NRU Cytotoxicity Test Tallies
The Study Directors periodically received individualized test tallies specific to theirlaboratories from NICEATM that detailed:• The number of range finder tests performed by the laboratory• The number of definitive tests performed, and the pass/fail status of each
• The number of PC tests performed, and the pass/fail status of each
• The number of acceptable tests completed• The test completion status for each chemical (i.e., whether one range finder
test had been completed, and the number of acceptable definitive tests had
been completed)
The laboratories compared the NICEATM tallies to their own records to verify theirconsistency and accuracy. Discrepancies were resolved through direct communication
between the Study Director and the SMT.

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In Vitro Cytotoxicity Methods BRD Section 8

November 2006

8.1.3
Guidelines Followed for Rodent Acute Oral LD50 Data Collection
For the purposes of this validation study, the in vitro NRU test methods were proposed forpredicting starting doses for rodent acute oral toxicity test methods, rather than as
replacement tests for the in vivo test method. No in vivo tests were performed for thisvalidation study. All in vivo data (i.e., rat and mouse LD50 values) were collected by
NICEATM through reviews of the literature and from publicly available databases. All
relevant data and pertinent information were gathered and stored in an Excel® spreadsheet.
8.1.3.1 Rodent Acute Oral LD50 Values Used in the Registry of Cytotoxicity (RC)The RC is a database of acute oral LD50 values for rats and mice obtained primarily from the
1983/84 RTECS database compiled by NIOSH, and IC50 values from in vitro cytotoxicity
assays using multiple cell lines and cytotoxicity endpoints for chemicals with known
molecular weights (Halle 1998, 2003). Collection and reporting methods used for generating
the data in RTECS® were not a part of the data collection hierarchy employed by NIOSH,
and the data in this database were not evaluated for quality and accuracy. Many of the values
come from secondary sources with no citation to the original report. GLP guidelines were notused to determine acceptable data for the database. The only criterion used by NIOSH forreporting acute oral toxicity data in RTECS® was that the LD50 value was the most toxic
LD50 value for a chemical that could be found in the literature, regardless of the number ofother values available, or their distribution.
8.1.3.2 Rodent Acute Oral LD50 Values Collected by NICEATM from Other Sources
One critical aspect of the validation study design was the establishment of a rat acute oral
LD50 reference value for each of the 72 reference substances (see Section 4). These reference
values were used to evaluate the extent to which the two in vitro NRU test methods could
predict rat acute oral LD50 values. Primary rat acute oral LD50 studies were located throughsearching electronic databases, published articles, and secondary references. Rat data were
not available for three of the reference substances and mouse acute oral LD50 values wereused. Only seven of the 455 LD50 values collected from the literature were produced under
GLP guidelines.
8.2

Results of Data Quality Audits

The QA unit or designee in each GLP laboratory provided a systematic and criticalcomparison of the data provided in the laboratory’s study reports to the raw data in thelaboratory records. The SOW provided to each laboratory contained the following guidance
regarding QA statements:“The Final Reports for all phases of the Validation Study shall be audited by the Quality
Assurance unit of the Testing Facility for GLP compliance and a QA Statement shall be
provided by the Testing Facility. Each Final Report shall identify: 1) the phases and datainspected, 2) dates of inspection, and 3) dates findings were reported to the Study
Director and Testing Facility management. The QA Statement shall identify whether the
methods and results described in the Final Report accurately reflect the raw data
produced during the Validation Study.”
8.2.1
QA Statements
The QA statements from the GLP-compliant laboratories addressed the reviews of:• Protocols• Laboratory standard operating procedures (SOPs)
8-6

In Vitro Cytotoxicity Methods BRD Section 8•••

November 2006

Laboratory operations, in general
3T3 and NHK NRU experiment data
The report submitted to the SMT

The QA statements from the GLP laboratories affirm that the methods described in the
protocols are the methods that the laboratory personnel used, and that the data reported to the
SMT accurately reflect the raw data obtained by the laboratory. See Section 8.2.2 for
information about the QA statements for the non-GLP laboratory.
8.2.2

QA Statements from the Laboratories

8.2.2.1 BioReliance QA Statements
The Study Director/Laboratory Director provided the following statement in all of the final
reports:“The solubility studies, acquisition, preparation, and distribution of the test chemicals
were conducted in compliance with GLP. Although not audited (per SOW), the work
described in this report for Phase X (i.e., Ia, Ib, and II) fully and accurately reflects to thebest of my knowledge the raw data generated in the study.”
8.2.2.2 FAL QA Statements
The Study Director for FAL performed the final review of all data and reports before sending
them to the SMT, and provided the following two statements in the final reports provided to
the SMT.
• “The laboratory worked under the principles of GLP whilst not being a GLPcompliant laboratory.”• “The report accurately reflects the work undertaken and the results obtained at
the FRAME Alternatives Laboratory.”
Formal QA statements were not provided to FAL because the SMT performed informal QA
reviews.
8.2.2.3 ECBC QA Statements
The QA statements reported the particular study phase and laboratory procedures that were
examined for GLP compliance. In addition, the laboratory’s statement noted that the scope ofwork, associated protocols, and quality control (QC) acceptance criteria were updated orchanged during the study, which made the assessment of the procedures and data for
conformance to the SOPs more difficult. However, compliance with the requirements andintent of GLP guidelines was continually assessed during the review of the SOPs and theobservance of operations. The QA reviews found the ECBC protocols to be in compliancewith the NICEATM/ECVAM study protocols. The aspects of the studies inspected by the
QA designee were:• Review of protocols and laboratory SOPs
• Review of waste handling procedures• Review of laboratory operations• Certification of new personnel• Review of data
• Review of the final report for each testing phase

8-7

In Vitro Cytotoxicity Methods BRD Section 8

November 2006

The QA designee also observed the preparation of reference substances, 96-well plate
configuration, application of reference substance, annotation to the workbook, andappropriate sterile technique while performing the testing. The number of inspections oflaboratory operations was reduced in the latter phases of the study because the same
personnel conducted the testing throughout the entire study.
ECBC Review Dates of the Study Phases• Phase Ia: July 2002 through May 2003
• Phase Ib: July 2002 through January 2003
• Phase II: May 2003 through February 2004
• Phase III: November 2003 through March 2005
8.2.2.4 IIVS QA Statements
Because the IIVS QA unit is small, it carried out reviews of different aspects of theprocedures at different times. The IIVS QA Statement reads:“This study has been divided into a series of in-process phases. Using a random sampling
approach, Quality Assurance monitors each of these phases over a series of studies.
Procedures, documentation, equipment records, etc., are examined to assure that the study
is performed in accordance with the U.S. FDA Good Laboratory Practice regulations (21CFR 58), the U.S. EPA GLP Standards (40 CFR 792 and 40 CFR 160) and the OECD
Principles of Good Laboratory Practice and to assure that the study is conductedaccording to the protocol and relevant Standard Operating Procedures.”
The aspects of the studies inspected by the QA designee were as follows:• Protocol and initial paperwork• Reading of the plates (definitive test)• Dilution of the test articles (definitive test)• Treatment of the cells• Termination of treatment and addition of the NR dye (definitive test)• Cell concentration determination and seeding of the plates (third definitivetest)• Termination of treatment and addition of the NR dye• Washing the cells• Draft report and data
• Final report
IIVS Review Dates of Various Aspects of the Test Phases• Phase Ia: August 2002
Final Report Review: October 2005
• Phase Ib: January 2003
Final Report Review: October 2005
• Phase II: July-August 2003
Final Report Review: October 2005
• Phase III: January-November 2004 Final Report Review: October 2005
8.2.2.5 Other QA Information
Data generated by the laboratories and reviewed by their respective Study Directors were
submitted to the SMT. Often, the data were provided electronically within days of the end of
testing. The SMT was active as a secondary QA reviewer of all information provided by the

8-8In Vitro Cytotoxicity Methods BRD Section 8

November 2006

Study Directors. If the SMT found discrepancies, the Project Coordinators corresponded withthe appropriate Study Director to identify and rectify the error. The Study Director madecorrections/adjustments to the discrepancies in data reporting and presented the changes to
the SMT. The SMT did not initiate any external data quality audits.
The quality of the reference substances was assured in the form of certificates of analysis
provided by the chemical manufacturer to BioReliance at the time of purchase. The SMT andthe laboratories obtained certificates of analysis from CAMBREX for Clonetics® NHK
culture medium and supplements. In addition, the SMT obtained QC data directly fromCAMBREX technical departments concerning the NHK medium’s ability to support
keratinocyte growth.
8.3

Effect of Deviations or Non-compliance with GLPs

Rates for several types of errors (i.e., documentation, testing methods, and data management)
were determined by the SMT. Many of the errors (particularly in Phases Ia and Ib) were theresult of minor mistakes (e.g., typographical, mislabeling) and did not affect the quality ofthe data.
8.3.1
Laboratory Error Rates
The SMT was concerned about the number of errors that were seen in documentation andtesting methods during Phases Ia and Ib, and compiled the detected errors from eachlaboratory. The types of errors found included errors in documentation (e.g., reference
substance identification did not match on all associated data sheets; IC20 and IC80 values weretransposed in the EXCEL® template; a test acceptance criterion flag in a data sheet wasincorrect) and in testing (e.g., wrong dilution scheme was used for the PC; wrong SLS IC50
was used as the PC IC50). Error rates were compiled as the number of tests with errors per
total number of tests. As shown in Table 2-3, FAL had the highest error rates: 93% for the
3T3 NRU test method and 41% for the NHK NRU test method. The highest error rates in theother laboratories were 10% for the 3T3 NRU test method and 23% for the NHK NRU test
method (both ECBC).
There were relatively few errors detected in the Phase III data files. The SMT did notcompile the typographical and transcriptional errors found, but reported them directly to theappropriate Study Director so that the data sheets could be immediately corrected. The SMT
did not detect errors in the raw optical density data from the 96-well plates provided in eachdata file. The laboratories and the SMT corrected typographical and transcriptional errors(e.g., incorrect logIC50 value entered) in the EXCEL® templates. The EXCEL® template
formulas were used for the statistical analyses.
An assessment of error rates was performed specifically for Phase III for one particularclerical error – the transfer of the final results (e.g., ICx values) from the GraphPad PRISM®
3.0 template to the Microsoft® EXCEL® template. It was often necessary for the SMT torevise the EXCEL® data files provided by the laboratories because the incorrect values had
been transferred to EXCEL®. Table 8-2 summarizes the Phase III error rates resulting fromthe transfer of data from PRISM® to EXCEL®.

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Phase III Error Rates in the Transfer of Data to the EXCEL® Template

Table 8-2

Laboratory

Number of Errors
Detected

Number of Definitive
Tests

Percentage of Tests
with Detected Errors

ECBC

49

402

12

FAL

171

513

33

IIVS

25

419

6

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences.

8.3.2
Failure Rates for Definitive and PC Tests
Table 8-3 presents the test failure (i.e., did not meet test acceptance criteria) rates
experienced in Phase III. Approximately 25% of all 3T3 definitive tests and 18% of all NHK
definitive tests failed. If a definitive test (see Section 2.3.2.2 for the definition of a definitivetest) failed, the laboratory repeated the test and attempted to obtain three acceptabledefinitive tests for each reference substance in each cell type (see Section 2.5 for criteria for
repeating tests). The PC tests failed 0 to 18% of the time with a combined average failure rateof 8% for both cell types. FAL had the highest individual laboratory test failure rates for 3T3
definitive tests (30%), NHK definitive tests (32%), and NHK PC tests (18%). ECBC had thehighest failure rate for 3T3 PC tests (11%). IIVS had no PC test failures.
Table 8-3

Definitive Test and Positive Control (PC) Test Failure Rates in Phase III
Test Type

3T3 NRU Test Method

NHK NRU Test Method

ECBC FAL IIVS Total ECBC FAL IIVS Total

Total

Definitive Tests - Acceptable

169

177

176

522

173

175

174

522

1044

Definitive Tests - Total

215

257

225

697

187

256

194

637

1334

% Failed Definitive Tests

21

30

22

25

8

32

10

18

22

PC Tests - Acceptable

66

40

16

122

58

37

20

115

237

PC Tests - Total

74

42

17

133

59

45

20

124

257

% Failed PC Tests

11

5

6

8

2

18

0

7

8

14

6

14

34

0

22

0

22

56

7

2

6

5

0

9

0

4

4

Definitive Tests Failed Only
Because PC Tests Failed% Definitive Tests Failed Only
Because PC Tests Failed

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; NRU=Neutral red uptake;
3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes.
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The Phase III guidelines required each laboratory to provide three acceptable definitive tests
for each substance for both cell types (3 x 60 x 2 = 360 definitive tests). PC tests were runconcurrently with the definitive tests, and more than one reference substance was usuallytested in conjunction with each PC test. Because of test failures, each laboratory performed
additional testing to obtain the three acceptable definitive tests required for each substance.
Table 8-4 presents the success rates for each laboratory for Phase III testing and a total for
all the laboratories combined.
Table 8-4

Combined Definitive and Positive Control (PC) Test Success Rates for the
3T3 and NHK Methods in Phase III

Test TypeAcceptable Definitive Tests/
Total Definitive Tests% Acceptable Definitive
Tests
Acceptable PC Tests/Total
PC Tests% Acceptable PC Tests

ECBC

FAL

IIVS

Total

342/402352/513350/4191044/1334

85%

69%

84%

78%

124/133

77/87

36/37

237/257

93%

89%

97%

92%

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; NRU=Neutral red uptake;
3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes.

8.3.3
Intralaboratory Reproducibility
CV values for each method were determined for each reference substance in each laboratory
using the IC50 values from the acceptable definitive tests, as described in Section 5.5.2.
Table 8-5 presents the average CV values for the substances tested in each of the study
phases, and for the entire study.
Table 8-5

CV Values for Definitive Tests

Cell
Type

Labs

3T3

ECBC
FAL
IIVS

NHK

ECBC
FAL
IIVS

Phases I & II
Number of
Average
Reference% CV
Substances
12
17
11
28
11
20
12
12
12

Phase IIIAll Phases
Number of
Number of
Average
Average
Reference
Reference% CV% CV
Substances
Substances
57
24
69
23
55
33
66
33
56
22
68
21

24
31
14

57
57
58

22
45
14

69
69
70

23
42
14

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; 3T3=BALB/c 3T3
fibroblasts; NHK=Normal human epidermal keratinocytes; CV=Coefficient of variation.
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8.3.4
Prediction of GHS Acute Oral Toxicity Categories
Predicted LD50 values were determined using the in vitro NRU IC50 values in the IC50-LD50
regressions presented in Table 6-5. The predicted LD50 values were used to assign each
substance to a predicted GHS acute oral toxicity category (UN 2005). The accuracy of the
3T3 and NHK NRU test methods for predicting GHS categories was determined bycomparison with categorization based on in vivo rat oral LD50 values (in mg/kg) in Table 42. Using the RC rat-only millimole regression, the accuracy of the predictions and the extent
of underprediction or overprediction are shown for each laboratory in Table 8-6. The
laboratories generally agreed with each other in their predictions. Although FAL had thehighest error rates and CV values, their predictions of GHS categories were consistent withthe other laboratories. The laboratories determined category matches for 25 to 30% of thereference substances for the 3T3 NRU test method and 29 to 31% of the reference substances
for the NHK NRU test method. For the 3T3 NRU test method, toxicity was overpredicted for
38% of the reference substances and underpredicted for 33 to 38% of them. For the NHK
NRU test method, toxicity was overpredicted for 35 to 38% of the reference substances andunderpredicted for 32 to 34% of them. (See Appendix J for additional laboratorycomparisons for the other in vitro – in vivo regressions evaluated in Section 6.)
8.4

Availability of Laboratory Notebooks

All laboratories maintained laboratory notebooks using a template provided by IIVS, andprovided copies of the notebooks to the SMT (archived at NICEATM) after completion ofeach testing phase. The notebooks contained information from all aspects of testing
including, but not limited to:• Environmental conditions• Reagent identification• Preparation of 96-well plates• Preparation of reference substances• Treatment of cell cultures• Visual observations of cell cultures• NRU assays• Data analysis

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GHS Acute Oral Toxicity Category Predictions by Laboratory1

Table 8-6

ECBC

Total
Reference
Substances
64

FAL

Labs

3T3

NHK

November 2006

Category
Match

Toxicity
Toxicity
Overpredicted Underpredicted

30%

38%

33%

64

25%

38%

38%

IIVS

64

27%

38%

36%

ECBC

68

31%

35%

34%

FAL

68

29%

38%

32%

IIVS

68

31%

37%

32%

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; 3T3=BALB/c 3T3
fibroblasts; NHK=Normal human epidermal keratinocytes; GHS=Globally Harmonized System forClassification and Labelling of Chemicals (UN 2005).
1
3T3 and NHK NRU test method IC50 data (geometric mean of within laboratory replicates) used with the RC
rat-only millimole regression, log LD50 (mmol/kg) = 0.439 x log IC50 (mM) + 0.621, to assign GHS category. In
vivo category was based on reference rodent oral LD50 values (mg/kg) in Table 4-2. For each method, thereference substances evaluated were those for which all three laboratories obtained IC50 values.

8.5

Summary••••

The determinations of test method and data collection errors showed that FAL
consistently had the highest error levels; however, the laboratory’s GHS acuteoral toxicity category predictions were comparable to the other laboratories’
results.
The laboratories reported no significant deviations from the protocols, anddeviations that did occur during the testing phases were generally quickly
acknowledged and addressed by the Study Directors. If a deviation occurredthat would affect the data (e.g., improper concentration of DMSO solvent), theStudy Director would reject the test, notify the SMT, and perform an
additional test. Improper transfer of data to either the EXCEL® or PRISM®
templates, which would affect the data summaries and analyses, wererecognized, documented, and rectified by the Study Director and/or the SMT.
The SMT reviewed all data sheets to ensure that data were not inadvertently
attributed to the incorrect data summary files, and that the correct data wereused in all statistical analyses.
An electronic copy of all data for this validation study can be obtained fromNICEATM upon request by mail, fax, or e-mail to Dr. William S. Stokes,
NICEATM, NIEHS, P. O. Box 12233, MD EC-17, Research Triangle Park,NC, 27709, (phone) 919-541-2384, (fax) 919-541-0947, (e-mail)niceatm@niehs.nih.gov.

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9.0

OTHER SCIENTIFIC REPORTS AND REVIEWS OF IN VITROCYTOTOXICITY TEST METHODS AND THEIR ABILITY TO
PREDICT IN VIVO ACUTE TOXICITY AND OTHER TOXICEFFECTS.................................................................................................................. 9-3

9.1

Relevant Studies ....................................................................................................... 9-3
9.1.1 Correlation of NRU Cytotoxicity Values with Rodent Lethality .................. 9-3
9.1.2 Use of In Vitro Cytotoxicity Data to Reduce the Use of Animals in
Acute Oral Toxicity Testing .......................................................................... 9-7
9.1.3 Other Evaluations of 3T3 or NHK NRU Methods ...................................... 9-10

9.2

Independent Scientific Reviews ............................................................................ 9-13
9.2.1 In Vitro Acute Toxicity Testing for the Classification and Labeling ofChemicals..................................................................................................... 9-13
9.2.2 Use of In Vitro Cytotoxicity Data for Estimation of Starting Doses for
Acute Oral Toxicity Testing ........................................................................ 9-14
9.2.3 Validation of 3T3 NRU Assay for Phototoxicity ........................................ 9-17

9.3

Studies Using In Vitro Cytotoxicity Methods with Established
Performance Standards......................................................................................... 9-18
9.3.1 Guidance Document (ICCVAM 2001b)...................................................... 9-18
9.3.2 King and Jones (2003) ................................................................................. 9-19
9.3.3 A-Cute-Tox Project: Optimization and Pre-Validation of an In Vitro
Test Strategy for Predicting Human Acute Toxicity (Clemedson 2005)..... 9-19

9.4

Summary................................................................................................................. 9-20

9-1

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In Vitro Cytotoxicity Test Methods BRD Section 9

9.0

November 2006

OTHER SCIENTIFIC REPORTS AND REVIEWS OF IN VITROCYTOTOXICITY TEST METHODS AND THEIR ABILITY TO PREDICTIN VIVO ACUTE TOXICITY AND OTHER TOXIC EFFECTS

In vitro cytotoxicity methods based on NRU have been evaluated for a number of uses. Thissection reviews studies that used in vitro NRU cytotoxicity methods to:• Predict acute rodent oral toxicity• Predict starting doses for acute systemic toxicity tests• Predict other in vivo toxicity endpoints, including phototoxicity and eye
irritation.
Section 9.1 describes studies that evaluated in vitro cytotoxicity test methods that measured
NRU for its ability to predict acute systemic toxicity in rodents, and other in vivo endpoints.
Also reviewed are studies that evaluated the use of in vitro cytotoxicity results to reduceanimal use in acute toxicity testing. Section 9.2 reviews independent evaluations of the useof in vitro cytotoxicity methods to predict acute oral toxicity, and to determine starting doses
for acute systemic toxicity assays. Also discussed is a 3T3 NRU test method that has beenvalidated and accepted for regulatory use for detecting phototoxic potential using a protocol
similar to that used in the NICEATM/ECVAM validation study. The conclusions of thesereports will be compared to the conclusions reached in this study, wherever possible. Section
9.3 reviews published studies that used the Guidance Document approach (ICCVAM 2001b),
which established the current test method performance standard.
9.1

Relevant Studies

9.1.1
Correlation of NRU Cytotoxicity Values with Rodent Lethality
This section reviews five published in vitro cytotoxicity studies that correlated cytotoxicity
values (i.e., IC20 or IC50) from NRU cytotoxicity test methods that used various cell types, torat and/or mouse acute LD50 values from various exposure routes. In these sections, italics
are used to identify reference substances tested in the reviewed studies that were also tested
in the NICEATM/ECVAM validation study. Table 9-1 characterizes the substances tested in
the reviewed studies by providing the ranges of their rat oral LD50 values. Also shown forcomparison are the mouse and/or rat oral LD50 ranges for the NICEATM/ECVAM validation
study and the RC. The table shows that the substances tested by Peloux et al. (1992), Fautrel
et al. (1993), and Rasmussen (1999), covered a wide range of rat acute LD50 values. The
substances used by Roguet et al. (1993) and Creppy et al. (2004) covered a much smaller
range. Table 9-2 characterizes the test substances by chemical class based on NLM Medical
Subject Heading (MeSH®) descriptors.

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In Vitro Cytotoxicity Test Methods BRD Section 9

Table 9-1

November 2006

Rat Acute Oral LD50 Ranges for Test Substances Used in Previous In
Vitro NRU Cytotoxicity Studies and the NICEATM/ECVAM Study1

Study/Database
Peloux et al. (1992)
Fautrel et al. (1993)
Roguet et al. (1993)
Rasmussen (1999)
Creppy et al. (2004)
NICEATM/ECVAM Validation3
RC4

Rat Acute Oral LD50 Range (mg/kg)2
2 – 14500
2 – 14500
0.04 – 176
1 – 10298
48 – 9245
2 – 19770
1 – 31015

N
30
31
28
20
2
72
347

Abbreviations: N=Number of substances in the study/database; RC=Registry of Cytotoxicity.
1
Studies reviewed in Section 9.1.1.
2
Values cited in the studies or from references provided by the studies.
3
Current study summarized in this BRD.
4The RC includes both rat and mouse LD50 values.
5Upper limit of range is an LD50 calculated from the in vitro NRU IC50 because there was no in vivo value
available for that substance.

Table 9-2

Chemical Classes Represented by the Substances Used in Published
Studies for Correlation of In Vitro NRU Cytotoxicity with Rodent Acute
Lethality

Chemical Class1

Study2

Chemical Class1

Alcohols
AmidesAminesArsenicals
Carboxylic Acids
Chlorine
CopperEthers

1, 2, 3, 4
1, 2, 3
1, 2
3, 4
1, 2, 3, 4
3, 4
3, 4
1, 2

Fluorine
Heterocyclics
Hydrocarbons
Iron
Lactones
Lithium
Mercury
MetalsStudy2

Chemical Class1

Study2

3, 4
1, 2, 3, 4, 5
1, 2, 3, 4, 5
3
1, 2
1, 2, 3, 4
3, 4
3, 4

NitrilesNitrogen
Organophosphates
Phenols
Polycyclics
Potassium
SodiumSulfur

1, 2
3, 4
3, 4
3, 4
3
3, 4
3, 4
1, 2, 3, 4

Study references: 1=Peloux et al. (1992) (24/25 substances were organic compounds); 2=Fautrel et al. (1993)(30/31 substances were organic compounds); 3=Roguet et al. (1993) (22/30 substances were organic
compounds); 4=Rasmussen (1993) (13/20 substances were organic compounds); 5=Creppy et al. (2004) (2/2
substances were organic compounds).
1
Classification by NLM Medical Subject Heading (MeSH®) descriptors.
2
Studies reviewed in Section 9.1.1.

9.1.1.1 Peloux et al. (1992)
The authors used several different in vitro cytotoxicity methods with primary rat hepatocytes
to determine the correlation with rat/mouse intraperitoneal (i.p.) or intravenous (i.v.) LD50
values for the 25 substances tested. The in vitro cytotoxicity methods, which used 20-hour
test substance exposure durations, assessed the following endpoints: NRU, total protein
content, LDH release, MTT reduction. MTT is metabolized by mitochondrial succinate
dehydrogenase of viable cells to yield a purple formazan reaction product. The IC50 values

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In Vitro Cytotoxicity Test Methods BRD Section 9

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obtained using the four endpoints were highly correlated (r = 0.973 to 0.999) to each other.
When performing the IC50-LD50 regressions, Peloux et al. (1992) used the lowest reported
published LD50 value for acute rat or mouse studies that administered the test substances
using the i.p. or i.v. routes. The IC50 values obtained using NRU as the endpoint had thehighest correlation coefficient, r = 0.877, to the rat/mouse i.p./i.v. LD50 values. The total
protein assay yielded r = 0.872, the MTT reduction assay yielded r = 0.808, and the LDH
release assay yielded r = 0.789.
Peloux et al. (1992) followed the recommendations of Fry et al. (1988, 1990) and usedparenteral LD50 values rather than oral LD50 values for comparison with in vitro values. Fry
et al. (1988, 1990) recommended the use of the i.p./i.v. LD50 values for comparisons because
they proposed that cells in vivo receive a more direct test substance exposure via these routes
than through the oral route. They had posited that in vitro cell cultures would mirror this(direct) toxicity because they also receive direct exposure to test substances via the cell
culture medium. The authors also noted that the oral route of exposure presents confounding
variables such as, 1) only a fraction of a test substance would be available in the systemic
circulation due to limited absorption or pre-systemic metabolism, and 2), the level of thesubstance in the systemic circulation decreases due to elimination mechanisms (e.g.,
metabolism, excretion). Fry et al. (1990) had reported a correlation of only r = 0.49 for invivo/in vitro comparisons of oral LD50 and IC50 values (from a total protein assay) and acorrelation of r = 0.68 for i.p. LD50 and ID50 values1.
9.1.1.2 Fautrel et al. (1993)
Six laboratories tested the cytotoxicity of 31 substances in primary rat hepatocyte culturesusing a 24-hour exposure followed by measurement of NRU. The investigators performed
linear regression analyses for the prediction of rat i.v., i.p., and oral LD50 values from the
NRU IC50 values. The regressions for the various in vivo administration routes did not use thesame substances because LD50 values were not available for all of the tested substances in allof the routes. Oral, i.v., and i.p. LD50 values were available for 27, 24, and 18 substances,respectively, and IC50 values were obtained for 15, 14, and 11 of these substances,respectively. The regression for the i.v. data was statistically significant (r = 0.88, n = 11),
but the i.p. (r = 0.48, n = 14) and oral regressions (r = 0.17, n = 15) were not. The finding thatthe i.v. LD50 values corresponded more closely with the in vitro cytotoxicity data than did theoral LD50 was thought to be the result of having fewer pharmacokinetic variables (i.e.,
absorption, distribution, etc.) to consider following i.v. administration.
9.1.1.3 Roguet et al. (1993)
Roguet et al. (1993) tested the cytotoxicity of 28 MEIC substances in primary rat hepatocytes
exposed for 21 hours, followed by the measurement of NRU. A correlation of the NRU IC50
values to oral LD50 values obtained from the unpublished data of B. Ekwall et al. (personalcommunication) yielded a statistically significant linear correlation (p <0.001) with r = 0.80
when the in vivo and in vitro data were in molar units. [NOTE: The LD50 values subsequently
published by Ekwall et al. (1998) were from the 1997 edition of RTECS®.] The authors
reported that the toxicities of thioridazine, malathion, and copper sulfate were overestimated,
and the toxicity of potassium cyanide was underestimated by the correlation, but their criteria
for over- and under- estimation were not provided.
1

ID50: index of cytotoxicity; concentrations (µg/mL) producing a 50% reduction in protein value.
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In Vitro Cytotoxicity Test Methods BRD Section 9

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The in vivo toxicity of potassium cyanide was also underpredicted in the
NICEATM/ECVAM validation study. Table 6-3 shows that potassium cyanide was an
outlier for which toxicity was underpredicted when using the IC50 values from both the 3T3
and NHK NRU test methods in the RC millimole regression (log LD50 mmol/kg = 0.435 log
IC50 mM + 0.625). The GHS category predictions using both NRU test methods and the RC
rat-only millimole regression (log LD50 mmol/kg = 0.439 log IC50 mM + 0.621), and the RC
rat-only weight regression (i.e., log LD50 =0.372 log IC50 + 2.024), were also higher (i.e., lesstoxic) than the in vivo category (see Appendix L2).
9.1.1.4 Rasmussen (1999)
Twenty MEIC substances were tested for cytotoxicity using NRU release from 3T3 cells
following 24-hr exposure, with and without the addition of a Aroclor-induced rat liver
microsomal preparation (S9 mix). Similar to the present validation study, Rasmussen (1999)
observed that xylene was non-toxic to the cells, even though it was dissolved in ethanol
instead of DMSO. In the presence of S9, the cytotoxicities of malathion, 2,4dichlorophenoxyacetic acid, propranolol, thioridazine, lithium sulfate, copper sulfate, andthallium sulfate, were significantly decreased (p <0.05), while the cytotoxicities of 1,1,1trichloroethane, phenol, nicotine, and paraquat were significantly increased (p <0.05).
Because the NICEATM/ECVAM validation study used cells with little or no xenobiotic
metabolizing capability, it could be expected that these systems would overpredict the
toxicity of substances that would be inactivated by the addition of a metabolizing system, or
to underpredict the toxicity of substances that are metabolized to more toxic substances.
None of the four substances in common for which toxicity was decreased by the addition of
S9 were overpredicted in the NICEATM/ECVAM study. However, the toxicities of two of
the four substances in common for which toxicity was increased by the addition of S9, were
underpredicted in the NICEATM/ECVAM study. Table 6-3 shows that nicotine was an
outlier whose toxicity was underpredicted when using the 3T3 and NHK IC50 values in the
RC millimole regression (log LD50 mmol/kg = 0.435 log IC50 mM + 0.625). Paraquat was an
outlier whose toxicity was underpredicted when using the NHK IC50 value in the RC
millimole regression. The GHS category predictions for both substances using both NRU test
methods with the RC rat-only millimole regression (log LD50 mmol/kg = 0.439 log IC50 mM+ 0.621) and the RC rat-only weight regression (log LD50 mg/kg = 0.357 log IC50 µg/mL +2.194) were also higher than the in vivo category (see Appendix L2).
Although both the IC20 and IC50 values were determined in the Rasmussen (1999) study, only
the IC20 values were used for correlations with the rat acute oral LD50 values from RTECS®.
The units of the LD50 values were not reported, but the correlations were assumed to be inmolar units because the IC20 and IC50 values were reported in µM units. Significant
correlations (p <0.001) between IC20 and LD50 values were obtained with and without rat
liver microsomes. The correlation of IC20 with LD50 was slightly higher with the S9 mix (r =0.72 vs. 0.68 for oral LD50 values, and 0.82 vs. 0.78 for i.p. LD50 values).
Although the presence of S9 increased the cytotoxicity of some substances to the 3T3 cells, it
decreased the toxicity of others, and yielded only a small improvement in the correlation to invivo data. Rasmussen (1999) concluded that the toxicity of the S9 mix (0.32 mg protein/mL),
itself, was insignificant because it reduced cell survival by less than 10% compared with cells

9-6In Vitro Cytotoxicity Test Methods BRD Section 9

November 2006

without S9. However, others have shown that S9 microsomal mixes could produce
significant cytotoxic effects. Kohn (1993) showed that an S9 mix containing 0.07 mg
protein/mL was cytotoxic to all types of murine neurons in culture when the cells were
exposed for four days or longer. Non-neuronal cells tolerated higher concentration exposuresof S9, but exhibited cytoplasmic inclusions when exposed to S9 at 0.35 mg protein/mL. Dal
Negro et al. (2006) reported 100% cell death of human monocyte-derived U-937 cells when
the S9 fraction (1 mg protein/mL) and co-factors were applied to the cells for a 72-hour
incubation. Both of these studies used longer exposure durations, and/or higher protein
concentrations, than the Rasmussen (1999) study.
9.1.1.5 Creppy et al. (2004)
Creppy et al. (2004) used a 48-hour NRU assay to determine the cytotoxicity of ochratoxin A(OTA) and fumonisin B1 (FB1) on cultured C6 glioma (rat brain), Caco-2 (human intestinal),
and Vero (green monkey kidney) cells. The IC50 determined in the NRU assay was used in
the RC millimole regression to predict rodent acute oral LD50 values. The predicted LD50 for
OTA using the C6 glioma cells was similar to mouse LD50 values generated from four in vivo
mouse studies, but the LD50 values predicted by the other cell lines were about 50 times
greater. The authors found the relative insensitivity of the Vero cells surprising because OTA
is a kidney toxin. There were no available in vivo rodent oral LD50 values with which tocompare the predicted LD50 of FB1, which ranged from 671 to 924 mg/kg for the three cell
types tested.
9.1.2

Use of In Vitro Cytotoxicity Data to Reduce the Use of Animals in Acute Oral
Toxicity Testing

9.1.2.1 Halle et al. (1997): Animal Savings with the ATC Method Using Cytotoxicity Data
This study assessed the animal savings that would be produced by using IC50 data in an IC50LD50 regression to determine a starting dose for ATC testing. No cytotoxicity testing was
performed for this study. Instead, the authors used the IC50 values from the RC database andthe RC millimole regression to predict the LD50 for 347 RC substances. The predicted LD50
values were then used to determine the starting doses for simulated ATC testing.
At the time of the Halle et al. (1997) study, the ATC method (1996 version from OECD) was
designed to classify substances using three classes of acute oral toxicity and an unclassified
group, as defined by the acute oral toxicity classification system of the EU (see Table 9-3).
As a result, the fixed doses for the ATC testing were 25, 200, and 2000 mg/kg. The authors
used the LD50 predicted by the RC IC50 and the RC millimole regression for the 347 RC
substances as a starting point to estimate the number of ATC dose steps, and number ofanimals, that would be needed to classify the substances in the EU category associated withthe rodent oral LD50 (i.e., rat or mouse values from RTECS®). The method required thesimulated ATC testing for each substance to start at the fixed ATC dose nearest to thepredicted LD50. The outcome of the simulated testing of three animals per fixed dose wasdetermined by the in vivo LD50. If the test dose was lower than the in vivo LD50, animals
were assumed to live and, if the test dose was higher than the LD50, the animals wereassumed to die. Testing of the substance would proceed with higher (when the animals lived)or lower fixed doses (when the animals died) until the substance was placed into the EU
toxicity category indicated by the in vivo rodent oral LD50.

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Table 9-3

November 2006

EU1 Classes of Acute Oral Toxicity
Category

LD50 (mg/kg)

1

LD50 ≤252

25 < LD50 ≤200

3

200 < LD50 ≤2000

Unclassified

LD50 >2000

Abbreviations: EU=European Union
1
Anon (1993)

The method of Halle et al. (1997) can be illustrated with digoxin, which has an in vivo mouse
LD50 of 18 mg/kg (from RTECS®). The predicted LD50 of 414 mg/kg was calculated using
the RC IC50 in the RC millimole regression (log LD50 [mmol/kg] = 0.435 x log IC50 (mM) +0.625). Simulated ATC testing would start at the nearest fixed dose, 200 mg/kg. The three
animals tested were assumed to die, and then three more animals would be tested at 25
mg/kg. The animals tested at 25 mg/kg were assumed to die and digoxin would be classifiedin category 1 for LD50 ≤25 mg/kg. Thus, the classification of digoxin using the 4-categorysystem required six animals.
Using such simulations of ATC testing, Halle et al. (1997) estimated that 2139 animals
would be used to test the 347 substances:• Three hundred twenty-eight would require testing with two doses using threetest animals each.
• Nineteen would require testing with three doses using three animals each.
Halle et al. (1997) cited Schlede et al. (1995) in reporting that the average number of animals
required to classify substances using the ATC method was 9.11 animals per test. Using thisaverage, ATC testing of the 347 RC substances would require 3161 animals. Thus, Halle etal. (1997) estimated that there would be a 32% reduction ([3161-2139]/3161) in the numberof test animals used when the LD50 prediction from the RC millimole regression was usedwith the 1996 version of the ATC method, in lieu of the standardanimal classification
procedure (Halle et al. 1997).
The simulated average animal savings for the ATC in the NICEATM/ECVAM validation
study at dose-response slopes of 2.0 and 8.3 was 4.8% to 10.2% (0.51 to 1.09 animals) for
the 3T3 (67 reference substances) and NHK (68 reference substances) NRU test methods(see Section 10.3.3.2), depending on the regression evaluated. This is considerably lower
than the average savings of 32% estimated by Halle et al. (1997). However, there are anumber of differences between the evaluation performed by Halle et al. (1997) and the
NICEATM/ECVAM study that contribute to the difference in calculated animal savings:• The NICEATM/ECVAM study used six GHS acute toxicity categories forclassification whereas Halle et al. (1997) used the EU toxicity classification
scheme, which had only four toxicity categories. The accuracy of category
prediction by any method would be higher with fewer categories.

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•

•

•

•

November 2006

The NICEATM/ECVAM study used experimentally derived in vitro
cytotoxicity data from a standardized protocol to estimate starting doses(using two regressions based on the RC substances with rat LD50 data),
whereas Halle et al. (1997) used IC50 data from the RC database.
The reference substances tested in the NICEATM/ECVAM study poorly fit
the RC millimole regression. Nearly half of the reference substances evaluatedwere outliers (28/70 [40%] in the 3T3 NRU test method, and 31/71 [44%] in
the NHK NRU test method) (see Table 6-3). The RC database had 95/347(27.4%) substances outside of the prediction intervals.
The NICEATM/ECVAM study used computer simulations of ATC testing,which incorporated assumptions about mortality distributions, to determine
animals used, whereas Halle et al. (1997) used simplified assumptions (i.e., allanimals lived when test dose was less than the in vivo LD50 and all animals
died when test dose was greater than the in vivo LD50).
The NICEATM/ECVAM study determined animal savings by comparing
animal use with starting doses determined by the in vitro data, to animals usedat the default starting dose of 300 mg/kg. Halle et al. (1997) used the averageanimal use for the ATC for comparison to animal use with simulated testing.

9.1.2.2 Spielmann et al. (1999): Animal Savings Using Cytotoxicity Data with the UDP
Spielmann et al. (1999) recommended an in vitro cytotoxicity procedure as a range finding
test for the in vivo toxicity test to reduce the number of animals used in acute toxicity tests.
The authors identified nine substances in both the RC database and an evaluation of acutetoxicity methods by Lipnick et al. (1995). They then compared the LD50 values from Lipnick
et al. (1995) to LD50 predictions calculated when using the RC IC50 values in the RC
millimole regression formula (log LD50 [mmol/kg] = 0.435 x log IC50 [mM] + 0.625). For
seven of the nine substances, the LD50 prediction was within an order of magnitude of theexperimental LD50 reported by Lipnick et al. (1995). Spielmann et al. (1999) concluded that
the RC millimole regression provided an adequate prediction of LD50, and that in vitro
cytotoxicity data could be used to predict starting doses for the UDP. The authors
recommended using the IC50, with the RC millimole regression, to calculate a starting dose
(i.e., an estimated LD50) for the UDP, FDP, or ATC method whenever an IC50 was available.
If no IC50 was available, Spielmann et al. (1997) recommended determining cytotoxicity
using a standard cell line and specific cytotoxic endpoint (e.g., NRU, total protein, MTT
reduction). They recommended testing 10 to 20 RC substances to demonstrate that the invitro cytotoxicity test methods provide results that are consistent with the RC millimole
regression. The resulting IC50 values would then be used to calculate a new regression (using
the LD50 values reported in the RC), which would be compared to the RC millimole
regression. If the new regression fit into the acceptance interval (± log 5 of the fitted
regression line) of the RC millimole regression, the RC millimole regression would be used
to predict starting doses for the UDP. If the new regression is parallel to the RC millimole
regression, but outside the ± log 5 acceptance interval, then the new regression would be
used for the prediction of the starting dose.
Spielmann et al. (1999) contended that the RC millimole regression provides a sufficient
prediction of LD50 values from IC50 values for substances that do not require metabolic
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November 2006

activation and are not very toxic (i.e., LD50 > 200 mg/kg). The authors acknowledged that thefit of substances with LD50 <200 mg/kg to the RC millimole regression is not good, andattributed the poor fit of these substances to the need for metabolic activation to a more toxic
substance. They suggested that the prediction of starting doses using cytotoxicity data can beused with the UDP and ATC methods, but not with the FDP because dosing is not sequential(which contradicted a claim made earlier in the paper that the approach could be used with
the FDP). They did not estimate the number of animals that might be saved with thisapproach, but did recommend that the approach be validated experimentally using several
established cell lines with a limited number of representative substances from the RC.
9.1.2.3 EPA (2004): U.S. EPA HPV Challenge Program Submission
In response to the EPA HPV Chemical Challenge Program, PPG Industries, Inc., themanufacturer of Propanoic acid, 2-hydroxy-, compound with 3-[2-(dimethylamino)ethyl] 1(2-ethylhexyl) (4-methyl-1,3-phenylene)bis[carbamate] (1:1) [CASRN 68227-46-3], and thesponsor of this compound, submitted data to the EPA. This is an isolated intermediate used to
produce a resin component of paint products. PPG provided the following types of data intheir submission to the EPA: physical-chemical, environmental fate and pathway,
ecotoxicity, and toxicology. The acute mammalian toxicology data were generated using
both in vitro and in vivo methods.
An in vitro NRU cytotoxicity test was conducted with 3T3 cells to estimate a starting dose
for the in vivo acute UDP oral toxicity test (OECD 2001a) (see Appendix M1 for the OECD
UDP test guideline). The use of this in vitro NRU test method was intended to minimize thenumber of animals used for in vivo testing. The estimated LD50 of the compound as
determined by the NRU assay was 489 mg/kg. Therefore, the starting dose for the UDP study
was set at 175 mg/kg, which is the first default dose below the estimated LD50 value; this isalso the default starting dose for the UDP, and is used when no information on which to basea starting dose is available. A total of fifteen female rats received the compound at 175, 550,
or 2000 mg/kg. Five of nine rats treated at 2000 mg/kg died prematurely on Days 2 and 3,
and by Day 15, 2/4 surviving animals at this dose had lost up to 25% of their Day 1 bodyweights. The LD50 was estimated to be 2000 mg/kg, with a 95% confidence interval of 11235700 mg/kg. Thus, the in vitro NRU test method overpredicted the toxicity of the compoundby estimating an LD50 value that was lower than that determined in the UDP test. The reportauthors reported that a greater than predicted number of animals was used for the UDP
testing because the estimated LD50, 489 mg/kg and, consequently, the starting dose, wasmuch lower than the in vivo LD50 of 2000 mg/kg. However, because the UDP started with thedefault starting dose of 175 mg/kg, the claim that more animals were used is incorrect,
because animal use with the default starting dose is the baseline against which other animal
use should be compared.
9.1.3
Other Evaluations of 3T3 or NHK NRU Test Methods
This section briefly reviews five studies that evaluated NRU test methods for purposes otherthan the prediction of starting doses for acute oral toxicity assays. NRU test methods using
either 3T3 or NHK cells have been evaluated for use as alternatives to the Draize eyeirritation test, to measure phototoxicity, and to predict acute lethality in humans. Except forthe 3T3 NRU phototoxicity assay, NRU methods have not been scientifically validated by anindependent review for any of these purposes or accepted for regulatory use. The use of the

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November 2006validated 3T3 NRU test method to determine phototoxic potential is addressed in Section
9.2.
The in vitro NRU protocols evaluated in the five reviewed studies are similar to those used in
the NICEATM/ECVAM validation study, all of which were based on the method ofBorenfreund and Puerner (1985). The major difference is that most studies used a 24-hour
test substance exposure duration for the 3T3 NRU test method, while the
NICEATM/ECVAM 3T3 study used a 48-hour exposure duration. The major difference
between the NHK protocols used in the reviewed studies and the protocol used in the
NICEATM/ECVAM study is that the cell culture medium was changed at the time of test
substance application in the NICEATM/ECVAM study.
9.1.3.1 Draize Eye Irritation
Triglia et al. (1989)
Four laboratories collaborated in an interlaboratory validation study to test the NHK NRU
assay marketed by Clonetics® Corporation2 for its intra- and inter-laboratory reproducibility
and ability to predict in vivo ocular irritancy. Each laboratory tested 11 blind-codedsurfactant-based substances and compared the IC50 values to in vivo Draize ocular irritancy
scores.
The test exhibited the following performance characteristics for the comparison of in vitro
and in vivo data:• Specificity (percentage of non-irritants correctly detected) = 93%• Sensitivity (percentage of true irritants correctly detected) = 80%• Predictive values (probability that an unknown agent will be properlyclassified)o Positive predictive value = 90%
o Negative predictive value = 87%
The authors reported that there was excellent correlation among the laboratories, and goodcorrelation between the in vitro IC50 values and in vivo Draize scores (Spearman Rankcorrelation coefficients between in vivo and in vitro data for the laboratories ranged from
0.67-0.76). The authors also concluded that the NRU test could not replace the Draize test,
but may be an effective screening tool for use in a battery of in vitro alternatives
Sina et al. (1995)
Sina et al (1995) evaluated the NHK NRU test method along with six other in vitro methods
to determine whether they could be used as complimentary tests in a battery approach toestimate ocular irritation. The NRU data correlated poorly with Draize ocular scores for the
33 pharmaceutical intermediates tested. The Spearman correlation coefficient for the IC50 andmaximum average Draize score (MAS) was -0.10, and the Pearson correlation coefficient
was -0.04.

2

Clonetics® Corporation sponsored this study. It was not clear in the publication if Clonetics® Corporation
participated as one of the testing laboratories.
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Brantom et al. (1997)
This study examined the potential of 10 alternative methods to predict the eye irritation
potential of cosmetic ingredients. Four laboratories tested 55 coded substances (23 single
ingredients and 32 formulations) using the 3T3 NRU test method, and used the resulting IC50
values to predict modified maximum average scores (MMAS) for the Draize test.
An endpoint was generated for each test by interpolation from a plot of percent cell survival
versus test substance concentration. A prediction model was developed from data of 30
single ingredients (29 surfactants and one substance not classified by the authors) to equate
the IC50 value to an MMAS.
The interlaboratory CV for the IC50 values was 37.3 ± 29.8% (7.5 ± 6.8, log transformed).
Most of the mean IC50 values from a single laboratory differed by plus or minus an order ofmagnitude from the means of all the laboratories for each substance, which the authorsinterpreted as “no significant outliers”. Correlations of NRU-predicted MMAS scores with invivo MMAS scores yielded Pearson’s r values ranging from 0.25 to 0.32 for the four
laboratories.
Although the authors concluded the interlaboratory reproducibility was good, the IC50 values
did not predict the MMAS. The r values for the in vitro/in vivo correlations were low (0.246
to 0.316) and the tests all underpredicted irritants and overpredicted non-irritants. Four
substances were outside of the 95% confidence intervals and the authors concluded that the
3T3 NRU test method had wide applicability to test the remaining 51 coded substances
according to the limitations in the prediction model, but that it was not effective as a standalone replacement for the Draize test across the entire irritation scale. The authors did not
identify the test substances.
Harbell et al. (1997)
This publication reported the results of the evaluation of 12 in vitro cytotoxicity assays topredict ocular irritation. Data were voluntarily submitted to the U.S. Interagency Regulatory
Alternatives Group (IRAG), composed of members from CPSC, EPA, and FDA. The NHK
NRU test method was one of the tests evaluated by six laboratories testing surfactants andsurfactant-containing formulations (the 3T3 NRU test method was not tested). Twolaboratories submitted results for the same test substances, but the other four submitted datafor various sets of substances and formulations.
The correlation of results from the two laboratories that independently tested the same
substances was r=0.99. Correlations between the IC50 data and in vivo maximum average
Draize score (MAS) ranged from -0.92 to -0.54. The IRAG concluded that the assays weresuitable as a screening and adjunct assay to assess eye irritation over the range of toxicities
found in personal care and household products, and recommended that its use be limited towater-soluble materials. Although the method was also evaluated for surfactants, IRAG
recommended that the evaluation continue for its performance in predicting eye irritation forvarious product classes (e.g., fabric softeners, shampoos). In addition, the substance’s
physical form should be considered because the in vitro toxicity of a solution of the test
substance will not necessarily predict toxicity of the parent, solid substance in vivo.

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9.1.3.2 Predicting Human Lethal Blood Concentrations (LC)Seibert et al. (1992)
This single laboratory study was designed to evaluate various aspects of cellular toxicity infour in vitro test systems for their relevance and reliability with respect to acute systemic
toxicity, in particular, human LC. The 3T3 NRU test method was one of four methods
evaluated with 10 MEIC substances.
The authors stated that final conclusions on the relevance of the in vitro systems for in vivo
data could not be determined because the variations in LC were unknown so that limits forover or underprediction of human in vivo toxicity using experimental models could not be
defined. In addition, the ability of in vitro toxicity to predict in vivo toxicity may depend on
toxicokinetic factors that were not considered in the in vitro systems.
9.2

Independent Scientific Reviews

This section summarizes independent scientific reviews of the use of in vitro cytotoxicity
methods for the prediction of rodent acute oral toxicity, and for the reduction of animal use inacute toxicity testing. The conclusions of these reviews are compared to the conclusions of
the current study. Also discussed is the 3T3 NRU phototoxicity method, because it is similar
to the 3T3 NRU test method used in the current validation study and has been validated by
ECVAM and is the subject of OECD Test Guideline 432 (OECD 2004).
9.2.1

In Vitro Acute Toxicity Testing for the Classification and Labelling of Chemicals

9.2.1.1 Seibert et al. (1996): ECVAM Workshop 16
ECVAM sponsored a workshop in 1994 to review the current status of various in vitro
methods and to determine their potential uses for reducing, refining, and/or replacing the useof laboratory animals for acute systemic toxicity testing. The workshop participants reviewed
various types of toxicity, in vitro cytotoxicity testing schemes and strategies, inclusion of
biokinetic parameters, biotransformation, biodistribution in vitro and in vivo, and a proposed
acute toxicity testing scheme for the classification of substances.
The workshop participants agreed that some studies showed good correlations between invitro cytotoxicity data and LD50 values. They also acknowledged that in vitro basal
cytotoxicity tests could not address all the different of mechanisms of acute systemic toxicity.
Additional approaches to replacing animals would have to incorporate the three main typesof cellular level toxic effects that can lead to in acute systemic toxicity (i.e., basal
cytotoxicity, selective toxicity, and cell-specific function toxicity). The participants
determined that it is also important that any alternative method take into account the active
concentration and meaningful dose of a test substance in an in vitro cell culture system.
Quantitative comparisons of test substance concentrations must be made to evaluate theeffects of the test substances regarding the three types of cytotoxicity.
The biokinetics of a test substance (determined by its absorption, distribution, metabolism,and elimination) must be considered when making predictions of in vivo toxicity using invitro toxicity data. Various methods can be used to convert in vitro effective concentrationsof a test substance to equivalent body doses. Test substance factors, such as physicochemical
characteristics (e.g., pKa, lipophilicity, volatility), estimates of protein binding, and in vitro

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November 2006

characteristics (e.g., cell concentration, cell protein concentration, ratio of cell/medium
volumes, medium albumin concentration), are needed for such conversions.
An in vitro tiered testing scheme was proposed by the workshop participants for using invitro methods to determine the acute oral toxicity of a substance:• Stage 1: Basal cytotoxicity test
• Stage 2: Hepatocyte-specific cytotoxicity test to assess the role ofbiotransformation in producing toxicity• Stage 3: Test system that evaluates non-hepatocyte-specific selective
cytotoxicity (i.e., effects on cell-specific functions)This testing scheme was proposed as an approach to classify substances by their in vitro
toxicity. The lowest IC50 value determined at any of the testing stages would be used toclassify a substance (i.e., very toxic, toxic, harmful, and no label). The workshop participants
recommended that a feasibility study be conducted to determine the practicability, relevance,and reliability of this tiered testing scheme. As noted in the NICEATM/ECVAM study (seeSection 6.4), the in vitro basal cytotoxicity tests are not suitable as replacements for rodent
acute oral toxicity tests and could only be used as an adjunct test, and not a stand-alone test,
for classifying substances for acute oral toxicity. However, in vitro tests could be used to
identify starting doses for acute toxicity testing to reduce the number of animals used.
9.2.2

Use of In Vitro Cytotoxicity Data for Estimation of Starting Doses for Acute Oral
Toxicity Testing

9.2.2.1

ICCVAM (2001a): Estimation of Animal Savings Using Cytotoxicity Data with the
ATC Method
Participants at Workshop 2000 examined the influence of starting dose on animal use in the
ATC method (ICCVAM 2001a; Section 2.2.3, pp.12-14; no testing was performed at theWorkshop). The participants made inferences from the 1996 version of the ATC method thatwas based on the EU toxicity classification system (Table 9-1). The fixed doses for testing
were 25, 200, and 2000 mg/kg. Normally, classification of a substance requires testing three
animals in two to four dosing steps (i.e., six to 12 animals). The number of dosing steps
increases with increasing distance between the true toxicity class and the starting dose. They
estimated that one to three dosing steps could be avoided (i.e., three to nine animals saved) if
the optimum starting dose could be predicted by in vitro cytotoxicity testing.
The predicted savings of one to three dosing steps was made under ideal conditions. The
Workshop 2000 report (ICCVAM 2001a) provides a biometric analysis at a dose-mortality
slope of 2.0 that shows that the greatest animal savings would occur for substances with very
high and very low toxicity. Three animals are needed to classify a substance in the <25
mg/kg class if the true LD50 is 1 mg/kg and 25 mg/kg is the starting dose, but six animals areneeded if the test starts from the default starting dose of 200 mg/kg (i.e., an animal savings of50%). For a substance with a true LD50 of 10000 mg/kg, 11.3 animals on average are needed
when the default starting dose is used, but only 7.7 animals would be needed at the 2000
mg/kg starting dose (i.e., an animal savings of 31%). For substances with a true LD50 of 2000
mg/kg, no animals would be saved by starting at the 2000 mg/kg dose (compared to startingat the default starting dose of 200 mg/kg).

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Although these analyses were performed assuming the 1996 ATC method used starting dosesof 25, 200, 2000 mg/kg, the Workshop 2000 participants noted that the animal savings thatwould be produced by improving the starting dose would not be significantly different for thecurrent ATC method that uses GHS doses of 5, 50, 300, and 2000 mg/kg (or up to 5000
mg/kg) (OECD 2001c; see Appendix M for the current ATC test guideline). The Workshop
2000 participants did not predict the animal savings when in vitro cytotoxicity methods areused to estimate starting doses for the ATC, other than the biometric analysis describedabove.
The NICEATM/ECVAM study yielded patterns of animal savings with the ATC that weresimilar to those discussed at the 2000 Workshop (i.e., animal savings were greater for
substances with a lower or higher LD50 than the default starting dose; see Section 10.3.3.3).
Depending on the NRU test method and regression evaluated, the average animal savings per
test (for the 67 or 68 reference substances evaluated) predicted by the NICEATM/ECVAM
7validation study at a dose-mortality slope of 2.0 were:• 22.6 to 30.4 % (2.21 to 2.96 animals) for substances in the LD50 ≤5 mg/kg
category• 10.2 to 13.0 % (1.17 to 1.51 animals) for substances in the 5< LD50 ≤50
mg/kg category• 3.8 to 4.3 % (0.42 to 0.47 animals) for substances in the 50< LD50 ≤300
mg/kg category• -9.5 to -6.1% (-0.93 to -0.60 animals) for substances in the 300< LD50 ≤2000
mg/kg category• -0.03 to 12.7% (-0.30 to 1.43 animals) for substances in the 2000< LD50
≤5000 mg/kg category• 17.1 to 25.5% (2.03 to 3.02 animals) for substances with LD50 >5000 mg/kg
The major differences between the evaluation reviewed by the Workshop 2000 participants
and the NICEATM/ECVAM study were:• The NICEATM/ECVAM study used the GHS toxicity categories forclassification whereas the Workshop participants used the EU classification
scheme, which has fewer toxicity categories. The accuracy of category
prediction is higher with fewer categories.
• The NICEATM/ECVAM study used in vitro cytotoxicity data to estimate
starting doses using two regressions based on the RC substances with rat LD50
data, whereas the Workshop 2000 participants used the fixed ATC doses as
starting doses.
• The NICEATM/ECVAM study used computer simulations of ATC testing forindividual substances whereas Workshop 2000 participants used an evaluationthat estimated animal use based on fixed in vivo LD50 values and the fixed
ATC doses.

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9.2.2.2

ICCVAM (2001a): Estimation of Animal Savings Using Cytotoxicity Data with the
UDP
Workshop 2000 participants examined the effect of starting dose on animal usage in the UDP
assay by making inferences from the computer simulations of animal use shown in the peerreview BRD for the UDP (ICCVAM 2000). When the rule that requires testing to stop whenfour animals have been tested after the first reversal is used, and no other stopping rules are
considered, the animal use is relatively insensitive to the slope of the dose-mortality curve.
The number of animals required when the starting dose equals the true LD50 is approximately
six. However, approximately nine animals are required when the starting dose is 1% of thetrue LD50. Thus, animal use is 30% less when the starting dose is the true LD50 compared to astarting dose that is 1% of the true LD50 (ICCVAM 2001a, section 2.2.4, pg. 16). When UDP
testing stops based on the likelihood-ratio stopping rule, the animal use depends principallyon the slope of the dose-mortality curve. The Workshop 2000 participants estimated that 25
to 40% of the animals would be saved when the starting dose is equal to the true LD50,
compared to the savings at a starting dose 1% of the true LD50.
According to the UDP BRD (ICCVAM 2000) used by the Workshop participants, UDP
simulations at a mortality-response slope of 2.0 showed that an average of 12.4 animals per
test were used when the starting dose was 1% of the true LD50, but an average of 8.7 animals
was used when the starting dose was the true LD50 (i.e., a 30% reduction). At a slope of 8.3,
an average of 11 animals per test were used when the starting dose was 1% of the true LD50,
but an average of only six animals were used when the starting dose was the true LD50 (i.e., a
46% reduction). The animal savings predicted by Workshop 2000 participants was 25 to 40%
based on starting at the true LD50 in comparison to starting at a dose that is 1% of the true
LD50.
Depending on the regression evaluated, the average animal savings predicted in the
NICEATM/ECVAM validation study at dose-response slopes of 2.0 and 8.3 were 5.8 to
7.8% (0.49 to 0.66 animals) using the 3T3 (67 reference substances) and NHK (68 reference
substances) NRU test methods (see Section 10.2.3). When averaged for the reference
substances in each GHS category, the highest mean animal savings at a mortality-response
slope of 2.0 was obtained for reference substances in the 2000 < LD50 ≤5000 mg/kg and LD50
>5000 mg/kg categories. Animal savings were 11.3 to 16.7% (1.28 to 1.65 animals) using the
3T3 and NHK NRU test methods for the two regressions evaluated. The average animalsavings for the substances in these categories at a dose-mortality slope of 8.3 were 12.1 to
21.0% (1.11 to 1.63 animals) for both methods and regressions. The major differences
between the evaluation performed by the Workshop 2000 participants and the
NICEATM/ECVAM study were that:• The default starting dose used for the NICEATM/ECVAM simulations was
175 mg/kg (see Section 10.2.2), rather than 1% of the true LD50 assumed by
the Workshop 2000 participants.
• The NRU IC50 was used in two regressions of in vitro data against in vivo data
to estimate starting doses. This estimation was not always close to the true
LD50, which was the value used by the Workshop 2000 participants. For
example, LD50 values predicted by the NICEATM/ECVAM study forphenylthiourea were approximately 540 mg/kg by the 3T3 IC50 and

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approximately 904 mg/kg by the NHK IC50 using the RC rat-only millimole
regression. The true in vivo LD50 for phenylthiourea is 3 mg/kg. Workshop
2000 participants used a best-case scenario when they assumed that in vitro
cytotoxicity precisely predicted the true LD50.
9.2.3
Validation of the 3T3 NRU Assay for Phototoxicity
An NRU assay using 3T3 cells was validated by ECVAM, and accepted for regulatory use,
to detect the phototoxic potential of test substances. The 3T3 NRU test for phototoxicity
requires a 60-minute exposure to the test substance, a 50-minute exposure to ultraviolet(UVA, 315-400 nm) light, followed by removal of test substance and incubation for another24 hours in fresh medium (Spielmann et al. 1998). NR medium is then added, and NRU is
measured after a 3-hour incubation. Phototoxic potential is assessed by comparing thedifferences in cytotoxicity between test plates containing the test substance that have notbeen exposed to UVA and comparable test plates exposed to UVA.
Two different models, employing the Photoinhibition Factor (PIF) and the Mean Photo
Effect (MPE), were validated for the prediction of in vivo phototoxic potential. The accuracyof the models for classifying the phototoxic potential of the 30 substances tested in ninelaboratories was 88% for the PIF, and 92% for the MPE, when compared with in vivo
classifications. Interlaboratory variability for classification (i.e., phototoxic vs. nonphototoxic) was assessed using a bootstrapping approach. For each substance, theclassification based on a single experiment was compared to the classification based on themean PIF or mean MPE. The interlaboratory variability for classification was 0 to 18.8%
using PIF and 0 to 20% using MPE.
The ECVAM Scientific Advisory Committee confirmed the scientific validity of the method
in 1997 (ECVAM 1997) and its regulatory acceptance was noted in Annex V of Council
Directive 67/548/EEC part B.41 on phototoxicity, in 2000. An OECD Test Guideline, 432,
was finalized in 2004 (OECD 2004). The 3T3 NRU phototoxicity test is used in a tiered
testing approach to determine the phototoxic potential of test substances.
The performance of the 3T3 NRU phototoxicity assay could not be compared with theperformance of the 3T3 NRU test method used in this validation study because different
classification schemes were used (i.e., a two-category classification for the phototoxicity vs.
a six-class scheme for acute oral toxicity). The ECVAM measurements of interlaboratory
variability also used different techniques and were not comparable to those used for the
NICEATM/ECVAM study.
9.2.3.1 NHK NRU Phototoxicity Assay
FAL participated in the European Union/European Cosmetic, Toiletry and Perfumery
Association (EU/COLIPA) study (30 substances tested using NHK and 3T3 cells) and the
ECVAM/COLIPA study (20 substances tested using NHK cells) (Clothier et al. 1999). The
studies showed that the NHK NRU test method could be used to predict phototoxic potential.
The accuracy for predicting in vivo results was similar to that of the 3T3 NRU phototoxicity
test (see Table 9-4). The NHK NRU phototoxicity test uses the same test substance exposure
duration (approximately 2 hours) as the 3T3 NRU test method, but the duration of culture
after UV exposure is 72 hours rather than 24 hours. NRU was measured after a 45-minuteincubation with NR.
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Although the NHK NRU phototoxicity method achieved good concordance with in vivo
phototoxicity, it has not yet been validated for regulatory use.
Table 9-4

Correct Identification of In Vivo Phototoxicants by the NHK NRU
Phototoxicity Assay
Study

EU/COLIPA(Spielmann et al. 1998)
ECVAM/COLIPA
Combined Study Data

3T3 NRU Phototoxicity
MethodNHK NRU Phototoxicity
Method29/30 (97%)1

28/30 (93%)1

NA
45/45 (100%)2

18/20 (90%)1
19/20 (95%)2
44/45 (98%)2

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red
uptake; EU=European Union; ECVAM=European Centre for the Validation of Alternative Methods;
COLIPA=The European Cosmetic Toiletry and Perfumery Association; NA=not available.
1
Mean Photo Effect (MPF) prediction model.
2
Photoinhibition Factor (PIF) prediction model.

9.3

Studies Using In Vitro Cytotoxicity Methods with Established PerformanceStandards

The procedure provided in the Guidance Document for evaluating basal cytotoxicity assays
for use in predicting starting doses for acute oral toxicity assays provides the existing
performance standards for the 3T3 and NHK NRU test methods (ICCVAM 2001b).
9.3.1
Guidance Document (ICCVAM 2001b)
In addition to guidance for evaluating in vitro basal cytotoxicity methods for use inpredicting starting doses for rodent acute oral toxicity assays, the Guidance Document
provided results from testing 11 reference substances using the recommended 3T3 and NHK
NRU protocols (ICCVAM 2001b). The 11 substances were chosen from the RC database so
as to have a close fit to the RC millimole regression and to cover a wide range ofcytotoxicity. The major differences between the Guidance Document protocols and theprotocols used in this validation study are the reduced NR concentrations (from 50 µg/mL to
25 µg/mL in the 3T3 NRU test method, and from 50 µg/mL to 33 µg/mL in the NHK NRU
test method), the increased duration of test substance exposure in the 3T3 NRU test method,from 24 to 48 hours, and the lack of a refeeding step in the NHK NRU test method just prior
to substance application (see Sections 2.6 and 2.7 for further detail). Despite thesedifferences, the Guidance Document shows that the test results for the 11 substances in boththe 3T3 and NHK NRU test methods were similar to the results in the RC database. The
calculated regressions for the 11 Guidance Document substances were:• log LD50 = 0.506 log IC50 + 0.475 (R2=0.985) for the 3T3 NRU test method• log LD50 = 0.498 log IC50 + 0.551 (R2=0.936) for the NHK NRU test method• log LD50 = 0.435 log IC50 + 0.625 for the RC millimole regression
The 3T3 and NHK NRU regressions were compared with the RC millimole regression (347substances) to show that the regression lines, as well as all 11 substance data points, were

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within the acceptance interval (± 0.5 log around the regression) of the RC millimole
regression (see Guidance Document Figures 3 and 4, p.13 [ICCVAM 2001b]).
9.3.2
King and Jones (2003)
This study also tested the 11 substances recommend in the Guidance Document using therecommended 3T3 NRU protocol. The IC50 - LD50 regression obtained was comparable to the
RC millimole regression and to the 11 substance regression provided in the GuidanceDocument (ICCVAM 2001b). The regression was log LD50 = 0.552 log IC50 + 0.503(R2=0.929) and the RC millimole regression was log LD50 = 0.435 log IC50 + 0.625. The 11substance regression fit within the acceptance interval (± 0.5 log) of the RC millimole
regression.
King and Jones (2003) also showed that a 3T3 NRU test method that was adapted for highthroughput testing by using three test sample concentrations yielded approximately the same
IC50 as an eight concentration-response. A regression used to compare the IC50 values using
the two different concentration-response approaches yielded R2=0.945.
9.3.3

A-Cute-Tox Project: Optimization and Pre-Validation of an In Vitro Test Strategy
for Predicting Human Acute Toxicity (Clemedson 2005)
The A-Cute-Tox Project is an Integrated Project under the EU 6th framework program thatstarted in January 2005, with a termination date of January 2010. It was initiated in responseto the REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals)
Directive and the 7th amendment of the Cosmetics Directive, which calls for the broad
replacement of animal experiments for finished products by 2003, and for ingredients by
2009. The project is an extension of the NICEATM/ECVAM validation study and the EDIT
program, which is the continuation of the MEIC program. The partnership is made up of the
EDIT Consortium, ECVAM, and 35 other European toxicity research group partners.
The aim of the project is to develop a simple and robust in vitro testing strategy forprediction of human acute oral toxicity, which could replace the animal acute oral toxicity
tests currently used for regulatory purposes. The objectives of A-Cute-Tox are:• Compilation, critical evaluation, and generation of high quality in vitro and invivo data for comparative analysis.
• Identifying factors (e.g., kinetics, metabolism, and organ specificity) that
influence the correlation between in vitro toxicity (concentration) and in vivo
toxicity (dosage), and to define an algorithm that accounts for these effects.
• Explore innovative tools and cellular systems to identify new toxicity endpoints and strategies to better anticipate animal and human toxicity.
• To design a simple, robust and reliable in vitro test strategy associated withthe prediction model for acute toxicity that is amenable to high-throughput
testing.
The project has been divided into the following workpackages that will be implemented byvarious configurations of research partners:• WP1: Generation of a “high quality” in vivo database (through literaturesearches and historical data) and establishment of a depository list ofreference substances

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•
•
•
•
•
•
•
•

November 2006

WP2: Generation of a “high quality” in vitro database (including data from the
NICEATM/ECVAM study, EDIT studies, and MEIC studies)
WP3: Iterative amendment of the testing strategy
WP4: New end-points and new cell systems
WP5: Alerts and correctors in toxicity screening (I): Role of absorption,
distribution, and excretion
WP6: Alerts and correctors in toxicity screening (II): Role of metabolism
WP7: Alerts and correctors in toxicity screening (III): Role of target organ
toxicity (i.e., neuro-, nephro-, hepato-toxicity)
WP8: Technical optimisation of the amended test strategy
WP9: Pre-validation of the test strategy

A-Cute-Tox aims to extend the NICEATM/ECVAM and MEIC/EDIT approaches toward afull replacement test strategy by improving the prediction of acute oral toxicity using invitro methods, and then validating the testing procedure.
9.4

Summary••••

In vitro NRU cytotoxicity test methods using various cell types have been
evaluated for their correlation with rodent lethality endpoints (e.g., rat/mousei.v., i.p., and oral toxicity). Peloux et al. (1992) and Fautrel et al. (1993)
showed good correlations (r=0.877 and 0.88, respectively) of in vitro
cytotoxicity with rodent i.p./i.v. and i.v. toxicity data, respectively.
3T3 and NHK NRU test methods have been evaluated for purposes other thanthe prediction of starting doses for acute toxicity studies (e.g., ocular irritancy;
human LC values, in vivo phototoxicity).
A 3T3 NRU test method has been validated by ECVAM for the identificationof in vivo phototoxic potential.
No in vitro test methods have been validated for the prediction of acute oral
toxicity. Estimations of animal savings using in vitro cytotoxicity data toestimate starting doses for the UDP did not use actual in vitro cytotoxicity
data. Instead, animal savings were estimated by assuming that the in vivo
starting dose equals the true LD50, which is an approach that assumes thatcytotoxicity data can perfectly predict in vivo lethality. These theoreticalpredictions of animal savings in the UDP ranged from 25 to 40% (ICCVAM
2001a), as compared with the average animal savings of 5.3 to 7.8% predictedusing computer simulation modeling of the UDP for the reference substancestested in the NICEATM/ECVAM study. Halle et al. (1997) used the in vitro
cytotoxicity data in the RC to determine that an animal savings of 32% can beattained for the ATC method by using the LD50 predicted by the RC
regression as the starting dose. For the reference substances tested in the
NICEATM/ECVAM validation study, most of which were a poor fit to the
RC millimole regression, the average animal savings for the ATC, as
determined by computer simulation modeling, was 4.8 to 10.2%.

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10.0

ANIMAL WELFARE CONSIDERATIONS (REFINEMENT,REDUCTION, AND REPLACEMENT)............................................................. 10-3

10.1

Use of 3T3 and NHK NRU Test Methods to Predict Starting Doses forRodent Acute Oral Toxicity Assays ..................................................................... 10-4

10.2

Reduction and Refinement of Animal Use for the UDP..................................... 10-4
10.2.1 In Vivo Testing Using the UDP ................................................................... 10-4
10.2.2 Computer Simulation Modeling of the UDP ............................................... 10-6
10.2.3 Animal Savings in the UDP When Using 3T3- and NHK-Based
Starting Doses .............................................................................................. 10-7
10.2.4 Refinement of Animal Use for the UDP When Using 3T3- and NHKBased Starting Doses ................................................................................. 10-15
10.2.5 Accuracy of UDP Outcomes Using the IC50-Based Starting Doses .......... 10-16

10.3

Reduction and Refinement of Animal Use in the ATC Method ...................... 10-16
10.3.1 In Vivo Testing Using the ATC Method .................................................... 10-16
10.3.2 Computer Simulation Modeling of the ATC Method................................ 10-17
10.3.3 Animal Savings for the ATC Method When Using 3T3- and NHKBased Starting Doses ................................................................................. 10-18
10.3.4 Refinement of Animal Use in the ATC Method When Using 3T3- andNHK-Based Starting Doses ....................................................................... 10-26
10.3.5 Accuracy of the ATC Method Outcomes Using the IC50-Based
Starting Doses ............................................................................................ 10-27

10.4

The Impact of Accuracy on Animal Savings ..................................................... 10-28

10.5

The Impact of Prevalence on Animal Savings................................................... 10-30

10.6

Summary............................................................................................................... 10-31

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10.0

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ANIMAL WELFARE CONSIDERATIONS (REFINEMENT, REDUCTION,
AND REPLACEMENT)As demonstrated in Section 6, in vitro basal cytotoxicity methods cannot be used as
replacement assays1 for rodent acute oral toxicity test methods for hazard classification.
However, as described in this section, these methods can be used to reduce2 and refine3
animal use in the UDP or ATC acute oral toxicity assays, as shown by the computer
simulations of such testing. Although the use of in vitro cytotoxicity data to determine
starting doses for the FDP may reduce the use of animals for the FDP, even though death is
not the primary endpoint, such an evaluation will not be provided in this document.
The test guidelines recommend using information on structurally-related substances and theresults of any other toxicity tests (EPA 2002b) for the test substance, including in vitro
cytotoxicity results, to approximate the LD50 and the slope of the dose-mortality curve(OECD 2001a; OECD 2001d; EPA 2002a). However, for the purposes of the reduction andrefinement evaluation conducted in this section, it was assumed that no information otherthan 3T3 and NHK NRU IC50 data would be available. To determine the extent of animal
reduction or refinement that would occur in the UDP and the ATC method when using astarting dose based on 3T3 or NHK NRU IC50 values rather than the default starting dose,computer models were used to simulate the in vivo testing of the reference substances used in
the validation study.
Section 10.1 lists the regressions that were used with IC50 data from the 3T3 and NHK NRU
test methods to determine starting doses for the UDP and the ATC. Sections 10.2.1 and10.3.1 summarize the animal testing procedures in the current test guidelines for the UDP
and the ATC, respectively. The procedures for using computer simulation of the animal
testing of the reference substances are described in Sections 10.2.2 and 10.3.2. The computer
simulations were used to determine the numbers of animals used and the numbers of animals
that “died” for each test. The modeling was performed using five different dose-mortality
slopes4 (i.e., 8.3, 4.0, 2.0, 0.8, and 0.5) because such slope information was not available forall of the reference substances used. To simplify the presentation of results, the animal use
figures provided in Sections 10.2.3, 10.2.4, 10.3.3, and 10.3.4 include the data for only twoof the slopes, 8.3 and 2.0. The slope of 2.0 is the default used for the calculation of LD50 by
the UDP method (OECD 2001a; EPA 2002a) and the slope of 8.3 is shown to represent
substances, such as pesticides, with higher slopes. The results for the other three slopes were
calculated, and are provided in Appendices N and Q. The numbers of animals used aresummarized to show the mean number of animals tested when the default starting dose isused and the mean number of animals used when the starting dose was determined from the
3T3 or NHK NRU IC50 values. The difference in animal use between the default starting
doses and the IC50-based starting doses is referred to as the animal savings. Differences were1

Replacement alternative: a new or modified test method that replaces animals with nonanimal systems or one
animal species with a phylogenetically lower one (e.g., a mammal with an invertebrate).
2
Reduction alternative: a new or modified test method that reduces the number of animals required.
3
Refinement alternative: a new or modified test method that refines procedures to lessen or eliminate pain or
distress in animals or enhances animal well-being.
4
The dose-mortality slope is the slope of the dose-response curve for mortality.
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tested for statistical significance (at p <0.05) using a one-sided Wilcoxon signed ranked test
based on the number of substances evaluated. Sections 10.2 and 10.3 summarize mean
animal use by the total number of substances tested and by the number of substances in each
GHS category. Sections 10.2.4 and 10.3.4 provide the mean number of animal deaths
compared to the mean number of animals used for each default and IC50-based starting doseto determine whether the IC50-based starting doses lead to a reduction in the number ofanimals used and the number that die (i.e., refinement). Sections 10.2.5 and 10.3.5 discussconcordance for the reference substance outcomes of simulated testing using the IC50-based
starting doses, with the outcomes of the default starting doses. Sections 10.4 and 10.5 discuss
the impact of accuracy and the impact of prevalence (i.e., the number of substances to betested in each GHS category) on animal savings.
10.1

Use of the 3T3 and NHK NRU Test Methods to Predict Starting Doses forRodent Acute Oral Toxicity Assays

The IC50 values developed from the 3T3 and NHK NRU test methods were used to predict
starting doses for rodent acute oral toxicity tests using the following linear regressions of
IC50-LD50 values (from Section 6.3):• The RC rat-only millimole regression: log LD50 (mmol/kg) = 0.439 log IC50(mM) + 0.621• The RC rat-only weight regression: log LD50 (mg/kg) = 0.372 log IC50(µg/mL) + 2.024
The IC50 values from each in vitro NRU test method were evaluated with each regression andsimulated acute oral toxicity test method,. The criteria for the use of a reference substance forthis evaluation were that it must have:• Replicate IC50 values from at least one laboratory• A rat acute oral LD50 reference value (from Table 4-2)
Sixty-seven and 68 reference substances were evaluated for the 3T3 and the NHK NRU test
methods, respectively. Of the 72 reference substances tested, epinephrine bitartrate,colchicine, and propylparaben were excluded because they did not have associated rat oral
LD50 data. Carbon tetrachloride and methanol were excluded from the 3T3 evaluations, andcarbon tetrachloride was excluded from the NHK evaluations, because none of thelaboratories achieved sufficient toxicity in any test for the calculation of an IC50.
10.2

Reduction and Refinement of Animal Use for the UDP

10.2.1In Vivo Testing Using the UDPThis section describes the general dosing procedure for the UDP (OECD 2001a; EPA 2002a).
Although doses, interval between doses, and dose progression, may be adjusted as necessary,
the procedures described reflect the default guidance. Guidance on the types of animals thatcan be used, animal housing, clinical observations, etc., are outside the scope of the current
discussion and are provided in the test guidelines (see Appendices M1 and M2).
10.2.1.1 Main Test
The UDP is based on a staircase design in which single animals are dosed, in sequence, at
48-hour intervals. The effect on the first animal determines the dose of the next animal. If thefirst animal dies or is in a moribund state within 48 hours after dosing, the dose administered

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to the next animal is lowered by dividing the original dose by one-half log (i.e., 3.2, which isthe default dose progression). If the first animal survives, the dose administered to the next
animal is increased by one-half log times the original dose. A dose progression of one-half
log unit corresponds to a dose-mortality slope of 2.0. The default dose progression can be
adjusted if the analyst has prior information upon which to estimate a slope.
The starting dose recommended by the guideline is one dose progression step below theanalyst’s best estimate of the LD50, because, in the UDP test method, the LD50 estimate tends
to move toward the starting dose. A default starting dose of 175 mg/kg is used if there is no
information on which to base a starting dose. The default dosing scheme, using the dose
progression of 3.2, is 1.75, 5.5, 17.5, 55, 175, 550, 1750, and 5000 mg/kg (EPA 2002a) or
1.75, 5.5, 17.5, 55, 175, 550, and 2000 mg/kg (OECD 2001a). The difference between thetwo reflects the different maximum doses emphasized in the different guidelines. Dosing
single animals, upward or downward, in sequence proceeds until the first of three conditions,referred to as stopping rules, is met:
• Three consecutive animals survive at the upper dose limit (2000 or 5000
mg/kg)• Five reversals5 occur in any six consecutive animals tested• Four or more animals have followed the first reversal, and the likelihoodratios specified by the guideline exceed the critical value. For a wide varietyof LD50 values and dose-mortality slopes, this rule is satisfied with four to six
animals after the first reversal. Three likelihood values are calculated: alikelihood at an LD50 point estimate (called the rough estimate or doseaveraging estimate); a likelihood at a value below the point estimate (the point
estimate divided by 2.5); and a likelihood at a value above the point estimate(the point estimate multiplied by 2.5). The ratios of the likelihoods are
examined to determine whether they exceed a critical value.
If none of these conditions is met, the dosing stops after 15 animals have been used.
10.2.1.2 Limit Test
The UDP guidelines include a limit test using three to five animals dosed sequentially at2000 mg/kg (OECD 2001a) or 5000 mg/kg (EPA 2002a). The EPA guideline for testing at alimit dose calls for proceeding to the main test if the first animal dosed at 5000 mg/kg dies
(EPA 2002a). If the first animal lives, two more animals are dosed, in sequence, with 5000
mg/kg. If both animals live, then testing is terminated, and the substance is designated ashaving an LD50 >5000 mg/kg. If one or both animals die, then two more animals are dosed insequence. As soon as a total of three animals survive, the test is terminated, with theconclusion that LD50 >5000 mg/kg. However, the main test is conducted if three animals die.

5

Reversal: a situation where a nonresponse (i.e., animal lives) is observed at some dose, and a response isobserved at the next dose tested (i.e. animal dies), or vice versa. Reversal is created by a pair of responses. (SeeAppendices M1 and M2)

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The OECD guideline for testing at a limit dose calls for proceeding to the main test if the firstanimal dosed at 2000 mg/kg dies (OECD 2001a). If the animal lives, four more animals aresequentially dosed. The main test is performed if three animals die. If three or more animals
survive, testing is terminated with the conclusion that the LD50 >2000 mg/kg.
10.2.2Computer Simulation Modeling of the UDP
Ten thousand UDP testing simulations were run for each substance, in vitro NRU test
method, and dose-mortality slope. Because the analysis assumed there was no information
upon which to estimate a dose-mortality slope, the modeling used the default dose
progression factor of 3.2, and 5000 mg/kg as the upper limit dose because this upper limit isemphasized in the EPA guideline (EPA 2002a)6. If the starting dose estimated from the invitro IC50 value was ≥4000 mg/kg, then the limit test, rather than the main test, wasperformed. If, during the dose progression, the next highest dose to be administered wasapproximately 4000 mg/kg or greater, then the limit dose of 5000 mg/kg was administered. If
a dose one step below the IC50-estimated LD50 was used as the starting dose, the other doses
administered corresponded to the default doses specified in the guidelines (OECD 2001a;
EPA 2002a). The simulation modeling procedures also used a lower limit of 1 mg/kg. Thus, adose of 1 mg/kg was administered if the dose progression fell below 1 mg/kg. To estimate
animal use by the default method, a starting dose of 175 mg/kg was used; the other doses
administered after the default starting dose corresponded to the doses specified in theguidelines (OECD 2001a; EPA 2002a).
The simulation was performed using SAS version 8 (SAS 1999) and implemented the
distributional assumptions underlying the dose-mortality relationship. The lowest dose atwhich an animal dies in response to the administration of a toxic substance varies fromanimal to animal. For an entire population of animals, mortality is assumed to have a lognormal distribution, with the mean equal to the log of the true LD50. Sigma (σ), thevariability of the simulated population, is the inverse of the slope of the dose-mortality curve.
Because of a lack of information concerning the actual dose-mortality curves, the simulations
assumed several different values of the slope, but no corresponding changes were made in
the dose progression. Dose-mortality slopes of 0.5, 0.8, 2.0, 4.0, and 8.3 were used because
these were used in the simulation modeling used to evaluate the current version of the UDP
guidelines (ICCVAM 2001c).
To model the variability of the IC50 values within and among laboratories, the values for eachreference substance were log-transformed to normalize their distribution. The mean andvariance of these log-transformed values were used to generate a log-normal distribution
from which an IC50 value was randomly selected. This IC50 value was used with theregressions to determine starting doses using two different methods. One method used theLD50 estimated from the IC50 and the regression as the starting dose, while the other used theclosest default dose that was lower than the estimated LD50. The latter method isrecommended by the EPA and OECD test guidelines (EPA 2002a; OECD 2001a), and theresults from that simulation are presented in Section 10.2. The UDP is only usable forregulatory purposes if the starting dose is set below the expected LD50. Appendix Q contains
6

The results from UDP simulations for a limit dose of 2000 mg/kg will be presented in a future addendum tothis document.
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In Vitro Cytotoxicity Test Methods BRD Section 10

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the results obtained when the LD50 that was estimated by the IC50 and the regression wasused as the starting dose.
The simulation procedure used the following steps for each reference substance:
1. The LD50 value (in mg/kg) from Table 4-2 was entered as the true LD50 value
and the choices of assumed slope were entered as the true slopes for the dosemortality curve.
2. An IC50 value was selected from a distribution identified by the mean andvariance of the IC50 values for each chemical to reflect the variation in IC50
values produced by the different laboratories (see Tables 5-4 and 5-5 formean IC50 values and standard deviations for the 3T3 and NHK NRU test
methods, respectively).
3. The IC50 value from Step 2 was used in the regression model being evaluatedto predict a LD50 value, which was used to determine the starting dose.
4. The dosing simulation was run three times: once with the default starting doseof 175 mg/kg, once at the next default dose below the LD50 estimated by the
regression being evaluated, and once at a dose equal to that of the LD50
estimated by the regression being evaluated.
5. For each simulated trial, the animals are dosed sequentially; therefore for eachanimal (i) there is a corresponding dose (i) that is administered to the animal.
For the first animal in each trial, it is the starting dose for that trial. For eachsubsequent animal, the dose is dependent on the previous dose and theprevious animal’s response, as described in Section 10.2.1. For animal (i), theprobability of a response is computed with the cumulative log-normal
distribution at the dose administered. That is,
P ( response ) = P ( x < log[ dose (i )]) where x ~ N ( µ , σ ) ,where µ is the log of the true LD50 value, and σ is the inverse of theassumed slope of the dose-mortality curve. One observation is then sampledfrom a binomial distribution with this calculated probability of success todetermine whether the animal lives or dies.
6. Dosing simulation is stopped as soon as one of the stopping rules is satisfied.
Steps 2-6 were repeated 10,000 times in order to compute an average animal use for eachmethod evaluated.
10.2.3

Animal Savings in the UDP When Using 3T3- and NHK-Based Starting Doses

10.2.3.1 The Effect of the Dose-Mortality Slope on Animal Use
As described in Section 10.2.2, the simulation modeling of animal use for the UDP assumed
five different dose-mortality slopes in order to assess animal use under various conditions of
population variability. Table 10-1 shows that the number of animals used for the UDP
decreases with increasing slope for both the default starting dose and the IC50-determined
starting dose when based on the RC rat-only millimole regression. The IC50-determined
starting dose was the next default dose lower than the regression-estimated LD50. For
example, because the LD50 predicted for cadmium chloride by the 3T3 NRU IC50 with the
RC rat-only millimole regression was 16 mg/kg, the starting dose was 1.75 mg/kg (i.e., thenext default dose below the predicted LD50). This approach is consistent with the UDP

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guidelines (OECD 2001a; EPA 2002a) as a means of reducing the number of animals thatmight experience pain and suffering from a treatment. This approach also overcomes thenonconservative bias of the UDP, which tends to yield an LD50 close to the starting dose.
Table 10-1 shows that, for each dose-mortality slope, the mean number of animals saved wasstatistically significant (p<0.05) when compared to mean number of animals needed when
the default starting dose was used. When expressed as a percentage of the number of animals
used when the default starting dose is used, animal savings also generally increased withincreasing slope of the dose-response. The animal savings is the same at all slopes tested, but
fewer animals are used at the steeper slopes, which increases the relative percentages ofanimals saved.
Table 10-1

Change in Animal Use1 with Dose-Mortality Slope for the UDP2

Dose-Mortality Slope

With Default Starting
Dose1,3With IC50-Based Starting
Dose1,4

Animals Saved5

0.5

3T3 NRU Test Method
10.01 ±0.10
9.48 ±0.11

0.53* (5.3%)

0.8

9.95 ±0.139.34 ±0.14

0.61* (6.1%)

2.0

9.35 ±0.168.80 ±0.17

0.54* (5.8%)

4.0

8.68±0.18

8.15 ±0.19

0.52* (6.0%)

8.3

7.95 ±0.18

7.42 ±0.20

0.53* (6.6%)

NHK NRU Test Method
0.5

10.01 ±0.09

9.53 ±0.12

0.49* (4.9%)

0.8

9.96 ±0.139.41 ±0.15

0.55* (5.5%)

2.0

9.36 ±0.168.86 ±0.18

0.50* (5.3%)

4.0

8.66 ±0.178.18 ±0.20

0.48* (5.6%)

8.3

7.92 ±0.187.43 ±0.20

0.49* (6.2%)

Abbreviations: UDP=Up-and-Down Procedure; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal
keratinocytes; RC=Registry of Cytotoxicity.
*Statistically significant (p<0.05) by a one-sided Wilcoxon signed rank test. Percentage difference is shown in parentheses.
1
Mean numbers of animals ±standard errors for 10,000 simulations for each of the 67 substances in the 3T3 NRU test
method and 68 substances in the NHK NRU test method. Although the simulations used whole animals, averaging theresults over a large number of simulations produced fractional numbers. Upper limit dose =5000 mg/kg.
2
OECD (2001a); EPA (2002a).
3
Default starting dose = 175 mg/kg.
4
The starting dose = next lower default dose to the predicted LD50, which was calculated from the IC50 value in the RC ratonly millimole regression: log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621. The IC50 value for each reference substance
was randomly selected from the distribution of values obtained during the testing with each method.
5
Difference between mean animal use with the default starting dose and mean animal use with the IC50-based starting dose.

To simplify the presentation of animal savings and the comparison of the various regressions
and starting doses, the results of subsequent analyses presented in Section 10.2.3 are limited
to the dose-mortality slopes of 2.0 and 8.3. The slope of 2.0 is the default used for the
calculation of LD50 by the UDP method (OECD 2001a; EPA 2002a) and the slope of 8.3 isshown to represent substances, such as pesticides, with higher slopes. Animal savings results
for the other dose-mortality slopes were calculated, and are presented in Appendices N1-N3.
Although using the next lower default dose to the in vitro-determined LD50 value overcomes

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November 2006

the bias of the UDP toward the starting dose (OECD 2001a, EPA 2002a) and is theappropriate approach for regulatory use, animal savings results using the estimated LD50 as
the starting dose were also calculated (see Appendix Q).
10.2.3.2 Mean Animal Use for UDP Simulations – Comparison of Regressions andPredictions from the 3T3 and NHK NRU Test Methods
Table 10-2 shows the mean animal use for the simulated UDP testing of the reference
substances described in Section 10.1. Mean animal use is shown for the default starting doseand for starting doses that were one default dose lower than the LD50 predicted from the invitro NRU methods and the regressions evaluated in Section 6.4 for the prediction of GHS
category. The difference in animal use between the two starting doses is the mean animalsavings produced by using the starting dose based on the in vitro NRU methods. All
differences (i.e., mean animal savings) were statistically significant (p <0.05) by a one-sided
Wilcoxon signed rank test. Mean animal savings ranged from 0.49 to 0.66 (6.2% to 7.0%)
animals per test depending upon the in vitro NRU test method, regression, and dose-mortality
slope. The lowest mean animal savings were obtained for the RC rat-only millimole
regression (0.49 [6.2%] to 0.54 [5.8%] animals for the different test methods and dosemortality slopes), and the greatest mean animal savings were obtained with the RC rat-only
weight regression (0.54 [6.8%] to 0.66 [7.0%] animals per test).
The animal savings using the in vitro NRU test methods with the RC rat-only regressions
apply only to the reference substances evaluated in this validation study, and are based on
substances pre-selected for their known in vivo toxicities and may not be broadly applicableto other substances. Table 3-4 shows that 22 (38%) of the 58 RC substances selected fortesting were known to have a poor fit to the RC millimole regression (i.e., the in vivo LD50
was outside the RC acceptance interval for the predicted LD50). Table 6-3 shows that 40%(28/70 for the 3T3) and 44% (31/71 for the NHK) of the reference substances that produced
IC50 values were outliers. The RC rat-only millimole regression evaluated here is very similar
to the RC millimole regression (see Table 6-5). Substances with better fits to the regression
are more likely to yield greater animal savings.
10.2.3.3 Animal Savings in the UDP by GHS Acute Oral Toxicity Category Using 3T3- andNHK-Based Starting Doses
Tables 10-3 and 10-4 show mean animal use and mean animal savings for the UDP when thedefault starting dose and the IC50-predicted starting doses were used, and when the reference
substances are grouped by GHS category (UN 2005). The data come from the same analyses
as the data provided in Table 10-2. The IC50-predicted starting doses were based on the:• RC rat-only millimole regression (Table 10-3)• RC rat-only weight regression (Table 10-4)

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Table 10-2

November 2006

Mean Animal Use1 in the UDP2 Using Starting Doses Based on the 3T3 and NHK NRU Test
Methods with the Different Regressions
With
With IC50Animals
Default
Based
Starting
Starting
Saved5
3
4
Dose
Dose
Dose-mortality Slope = 2.0

With
With IC50Animals
Default
Based
Starting
Starting
Saved5
3
5
Dose
Dose
Dose-mortality Slope = 8.3

RC rat-only millimole6

9.35 ±0.168.80 ±0.17

0.54* (5.8%)

7.95 ±0.18

7.42 ±0.20

0.53* (6.6%)

RC rat-only weight7

9.36 ±0.168.70 ±0.16

0.66* (7.0%)

7.94 ±0.18

7.32 ±0.19

0.62* (7.8%)

Assay/Regression
3T3 NRU Test Method

NHK NRU Test Method
6

RC rat-only millimole
7

RC rat-only weight

Dose-mortality Slope = 2.0

Dose-mortality Slope = 8.3

9.36 ±0.168.86 ±0.18

0.50* (5.3%)

7.92 ±0.187.43 ±0.20

0.49* (6.2%)

9.36 ±0.168.80 ±0.17

0.56* (6.0%)

7.92 ±0.18

7.38 ±0.20

0.54* (6.8%)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity; UDP=Up-and-Down Procedure.
*Statistically significant (p <0.05) by a one-sided Wilcoxon signed rank test. Percentage difference is shown in parentheses.
1
Mean numbers of animals ±standard errors for 10,000 simulations for each of the 67 substances in the 3T3 NRU test method and 68 substances in the NHK
NRU test method. Although the simulations used whole animals, averaging the results over a large number of simulations produced fractional numbers. Upper
limit dose =5000 mg/kg.
2
OECD (2001a); EPA (2002a).
3
Default starting dose =175 mg/kg.
4
The starting dose = one default dose lower than the predicted acute oral LD50 calculated using the IC50 value in the specified regression. The IC50 value for eachreference substance was randomly selected from the distribution of values obtained during the in vitro testing with each test method.
5
Difference between mean animal use with default starting dose and mean animal use with the IC50-based starting dose.
6
log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
7
log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024.

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November 2006

These analyses showed that:• For each in vitro NRU test method and regression, animal savings were
statistically significant for substances in the 2000 < LD50 ≤5000 mg/kg and
LD50 >5000 mg/kg toxicity categories.
• For substances with 5 < LD50 ≤50 mg/kg and 50 < LD50 ≤300 mg/kg, both invitro NRU test methods with each regression used slightly more animals thanthe default-starting dose, but the differences were not statistically significant.
Animal Savings for the UDP by GHS Acute Oral Toxicity Category Using 3T3- and NHKBased Starting Doses with the RC Rat-Only Millimole Regression
Table 10-3 shows the animal savings by GHS category when the IC50 values are used with
the RC rat-only millimole regression. Mean animal savings were statistically significant (p<0.05) by a one-tailed Wilcoxon signed rank test for the following GHS toxicity categories,
test methods, and dose-mortality slopes:• The use of the NHK NRU test method at both dose-mortality slopes for
substances with 300 < LD50 ≤2000 mg/kg that produced savings of 0.49
(6.5%) to 0.52 (6.1%) animals per test.
• The use of the 3T3 NRU test method at the 8.3 dose-mortality slope for
substances with 300 < LD50 ≤2000 mg/kg that produced a saving of 0.31
(4.1%) animals per test.
• The use of both in vitro NRU test methods at both dose-mortality slopes for
substances with 2000 < LD50 ≤5000 mg/kg that produced savings of 1.11
(12.1%) to 1.28 (11.9%) animals per test.
• The use of both in vitro NRU test methods and both dose-mortality slopes for
substances with an LD50 >5000 mg/kg that produced savings of 1.47 (14.8%)
to 1.58 (20.3%) animals per test.
The mean animal savings for the 3T3 and NHK NRU test methods were similar for most
toxicity categories at both dose-mortality slopes, with the mean savings with the 3T3 slightly
higher than with the NHK. For the dose-mortality slope of 2.0, the mean animal savings withthe 3T3 NRU test method ranged from -0.42 (-5.5%) to 1.58 (16.0%) animals per test for thevarious toxicity categories, and savings for the NHK NRU test method ranged from -0.34 (3.5%) to 1.47 (14.8%) animals per test. For the dose-mortality slope of 8.3, animal savingsfor the 3T3 NRU test method ranged from -0.29 (-4.3%) to 1.58 (20.3%) animals per test andsavings for the NHK NRU test method ranged from -0.33 (-3.9%) to 1.47 (19.2%) animals
per test. Animal savings were also obtained for highly toxic substances (LD50 ≤5 mg/kg) withboth the 3T3 (0.96 [9.9%] to 1.14 [10.0%] animals per test) and NHK (0.71 [7.3%] to 0.75
[6.7%] animals per test) NRU test methods, but the savings were not statistically significant.
No mean animal savings (≤-0.28 animal per test) were observed for substances with 50<
LD50 ≤300 mg/kg by either the 3T3 or the NHK NRU test method. This category includes thedefault starting dose of 175 mg/kg. Animal savings were not expected for this category
because savings were determined by comparing animal use with the IC50-based starting dosewith animal use at the default starting dose. No animal savings (-0.07 to -0.34 animals pertest) were observed for substances with 5< LD50 ≤50 mg/kg for either NRU test method.
None of these differences in animal use was statistically significant.

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In Vitro Cytotoxicity Test Methods BRD Section 10

Table 10-3

November 2006

Animal Use1 for the UDP2 by GHS Acute Oral Toxicity Category3 Using Starting Doses Based on the 3T3
and NHK NRU Test Methods with the RC Rat-Only Millimole Regression4
Dose-mortality Slope = 2.0

GHS Acute Oral Toxicity
Category3

Number ofReference
Substances

With Default
Starting
Dose5

With IC50Based
Starting
Dose6

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
12

11.32 ±0.20
9.68 ±0.23
7.76 ±0.108.53 ±0.2110.73 ±0.109.87 ±0.34

10.19 ±0.70
9.74 ±0.458.18 ±0.218.14 ±0.219.46 ±0.158.29 ±0.49

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
13

11.21 ±0.24
9.65 ±0.16
7.78 ±0.118.55 ±0.2210.75 ±0.08
9.87 ±0.32

10.47 ±0.71
9.99 ±0. 45
8.12 ±0.218.03 ±0.239.54 ±0.20
8.41 ±0.44

Animals
Saved7

Dose-mortality Slope = 8.3
With Default
Starting
Dose5

3T3 NRU Test Method
1.14 (10.0%)
9.70 ±0.28-0.07 (-0.7%)
8.46 ±0.28-0.42 (-5.5%)
6.61 ±0.19
0.38 (4.5%)
7.46 ±0.24
1.28* (11.9%)9.17 ±0.23
1.58* (16.0%)
7.76 ±0.59
NHK NRU Test Method0.75 (6.7%)
9.66 ±0.27-0.34 (-3.5%)
8.43 ±0.26-0.34 (-4.4%)
6.57 ±0.19
0.52* (6.1%)
7.49 ±0.25
1.21* (11.3%)
9.17 ±0.23
1.47* (14.8%)
7.66 ±0.59

With IC50Based
Starting
Dose6

Animals
Saved78.74 ±0.438.54 ±0.476.90 ±0.197.15 ±0.19
7.96 ±0.31
6.18 ±0.69

0.96 (9.9%)-0.08 (-1.0%)-0.29 (-4.3%)
0.31* (4.1%)
1.21* (13.2%)
1.58* (20.3%)

8.95 ±0.528.77 ±0.496.85 ±0.19
7.00 ±0.20
8.06 ±0.29
6.18 ±0.69

0.71 (7.3%)-0.33 (-3.9%)-0.28 (-4.2%)
0.49* (6.5%)
1.11* (12.1%)
1.47* (19.2%)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); NHK=Normal humanepidermal keratinocytes; RC=Registry of Cytotoxicity; UDP=Up-and-Down Procedure.
*Statistically significant (p<0.05) by a one-sided Wilcoxon signed rank test. Percentage difference shown in parentheses.
1
Mean numbers of animals used ±standard errors for 10,000 simulations for each substance with an upper limit dose of 5000 mg/kg. Although the simulations used wholeanimals, averaging the results over a large number of simulations produced fractional numbers. Results are provided for 67 substances in the 3T3 NRU test method and 68
substances in the NHK NRU test method. Substances were categorized using the rat acute oral LD50 reference values in mg/kg from Table 4-2.
2
OECD (2001a); EPA (2002a).
3
UN (2005).
4
The RC rat-only millimole regression is log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
5
Default starting dose = 175 mg/kg.
6
The starting dose was one default dose lower than the predicted LD50 calculated using the IC50 value for each reference substance in the RC rat-only millimole regression.
The IC50 value for each reference substance was randomly selected from the distribution of values obtained during the testing with each method.
7
Difference between mean animal use with the default starting dose and mean animal use with the predicted starting dose.

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In Vitro Cytotoxicity Test Methods BRD Section 10

November 2006

The animal savings from the future use of these in vitro NRU test methods with the RC ratonly millimole regression will depend on the proportion of test substances that will fall into
each of the GHS categories.
Animal Savings for the UDP by GHS Category Using 3T3- and NHK-Based Starting Doses
with the RC Rat-Only Weight Regression
Table 10-4 shows the mean animal savings by GHS acute oral toxicity category when the
IC50 values are used with the RC rat-only weight regression. A comparison of mean animalsavings, by category, with the RC rat-only millimole regression, indicates that, in most cases,
animal savings were slightly higher for the RC rat-only weight regression than for the
millimole regression. In the RC rat-only weight regression, the mean differences between
animal use for the default starting dose and mean animal use with the IC50-determined
starting dose were statistically significant (p <0.05) by a one-sided Wilcoxon signed rank test
for the following GHS categories, NRU test methods, and dose-mortality slopes:• The use of the 3T3 NRU test method at the 8.3 mortality-slope for substanceswith 300< LD50 ≤2000 mg/kg that produced a savings of 0.28 (3.8%) animals
per test.
• The use of both in vitro NRU test methods at both dose mortality slopes for
substances with 2000< LD50 ≤5000 mg/kg that produced savings of 1.28
(14.0%) to 1.64 (15.2%) animals per test.
• The use of both in vitro NRU test methods at both dose-mortality slopes for
substances with LD50 >5000 mg/kg that produced savings of 1.53 (20.0%) to
1.65 (16.7%) animals per test.
For the dose-mortality slope of 2.0, the mean animal savings (for the various GHS
categories) with the 3T3 NRU test method ranged from -0.25 (-3.3%) to 1.65 (16.7%)
animals per test, and from -0.24 (-3.1%) to 1.54 (15.6%) animals per test using the NHK
NRU test method. At the dose-mortality slope of 8.3, animal savings with the 3T3 NRU test
method ranged from -0.18 (-2.7%) to 1.63 (21.0%) animals per test, and savings for the NHK
NRU test method ranged from -0.18 (-2.7%) to 1.53 (20.0%) animals per test. Animalsavings were also obtained for highly toxic substances (LD50 ≤5 mg/kg) with both the 3T3(0.78 [8.0%] to 0.90 [8.0%] animals per test) and NHK (0.69 [7.1%] to 0.72 [6.4%] animals
per test) NRU test methods, but these savings were not statistically significant.
There were no mean animal savings (≤-0.18 animals per test) for substances with 50 < LD50
≤300 mg/kg with either in vitro NRU test method. This category includes the default starting
dose of 175 mg/kg. Animal savings were not expected for this category because savings were
determined by comparing animal use at the IC50-based starting dose with animal use at thedefault starting dose. For the NHK NRU test method, there were no animal savings (-0.07 to-0.13 animals per test) when used for substances with 5 < LD50 ≤50 mg/kg. None of thesesmall changes in animal use were statistically significant.
The animal savings from testing new substances with these in vitro NRU test methods using
the RC rat-only weight regression will depend on the proportion of test substances that fall
into each of the GHS categories.

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In Vitro Cytotoxicity Test Methods BRD Section 10

Table 10-4

November 2006

Animal Use1 for the UDP2 by GHS Acute Oral Toxicity Category3 Using Starting Doses Based on the 3T3
and NHK NRU Test Methods with the RC Rat-Only Weight Regression4

GHS Acute Oral Toxicity
Category3

Number ofReference
Substances

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
12

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
13

Dose-mortality Slope = 2.0
Dose-mortality Slope = 8.3
With
With IC50With Default
With IC50Animals
Animals
Default
Based
Starting
Based
7Starting
Saved
Saved7
Starting
5
Dose
Starting Dose
5
Dose
Dose
3T3 NRU Test Method
11.29 ±0. 20
10.38 ±0.62
0.90 (8.0%)
9.70 ±0.28
8.92 ±0.37
0.78 (8.0%)
9.71 ±0.229.58 ±0.42
0.13 (1.3%)
8.47 ±0.28
8.41 ±0.44
0.06 (0.8%)
7.74 ±0.10
7.99 ±0.18-0.25 (-3.3%)
6.58 ±0.19
6.76 ±0.18-0.18 (-2.7%)
8.52 ±0.218.16 ±0.19
0.35 (4.1%)
7.46 ±0.24
7.17 ±0.16
0.28* (3.8%)
10.78 ±0.11
9.14 ±0.24
1.64* (15.2%)
9.20 ±0.24
7.61 ±0.37
1.59* (17.3%)
9.87 ±0.348.23 ±0.48
1.65* (16.7%)
7.76 ±0.59
6.14 ±0.69
1.63* (21.0%)
NHK NRU Test Method
11.21 ±0.24
10.49 ±0.71
0.72 (6.4%)
9.66 ±0.27
8.97 ±0.52
0.69 (7.1%)
9.70 ±0.189.78 ±0.41-0.07 (-0.8%)
8.45 ±0.27
8.59 ±0.44-0.13 (-1.6%)
7.75 ±0.11
7.99 ±0.21-0.24 (-3.1%)
6.58 ±0.19
6.76 ±0.18-0.18 (-2.7%)
8.54 ±0.21
8.20 ±0.22
0.34 (3.9%)
7.48 ±0.23
7.17 ±0.16
0.31 (4.1%)
10.77 ±0.08
9.40 ±0.25
1.38*(12.8%)
9.18 ±0.237.90 ±0.33
1.28* (14.0%)
9.88 ±0.32
8.34 ±0.44
1.54*(15.6%)
7.66 ±0.566.12 ±0.63
1.53* (20.0%)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); NHK=Normal humanepidermal keratinocytes; RC=Registry of Cytotoxicity; UDP=Up-and-Down Procedure.
*Statistically significant (p <0.05) by a one-sided Wilcoxon signed rank test. Percent difference is shown in parentheses.
1
Mean number of animals used ±standard errors for 10,000 simulations for each substance with a limit dose of 5000 mg/kg. Although the simulations used whole animals,
averaging the results over a large number of simulations produced fractional numbers. Results are provided for 67 substances for the 3T3 NRU test method and 68
substances for the NHK NRU test method categorized using the rat acute oral LD50 reference values in mg/kg from Table 4-2.
2
OECD (2001a); EPA (2002a).
3
UN (2005).
4
The RC rat-only weight regression is log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024
5Default starting dose = 175 mg/kg.
6
The starting dose was one default dose lower than the predicted LD50 calculated using the IC50 values for each reference substance in the RC rat-only weight regression.
The IC50 value for each reference substance was randomly selected from the distribution of values obtained during the testing with each method.
7
Difference between mean animal use with the default starting dose and mean animal use with the predicted starting dose.

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In Vitro Cytotoxicity Test Methods BRD Section 10

November 2006

10.2.4

Refinement of Animal Use for the UDP When Using 3T3- and NHK-Based
Starting Doses
A procedure refines animal use when it lessens or eliminates pain or distress in animals or
enhances animal well-being (ICCVAM 2003). This section evaluates whether the use of 3T3and NHK-based starting doses refines animal use by reducing the number of animals that die
and experience accompanying pain and distress during UDP testing, compared to the numberof animals that die when the default starting dose of 175 mg/kg is used. Table 10-5 reports
the results for the UDP simulation modeling using the 5000 mg/kg limit dose. For every
regression evaluated, the mean number of deaths when using the IC50-based starting doses
were essentially equal to the mean number of deaths when using the default starting dose.
The percentage of deaths, however, was slightly higher for the IC50-based starting doses thanfor the default starting dose because the total number of animals used was lower for the IC50based starting doses. Thus, fewer animals were used when using an IC50-based starting dosecompared with use of the default starting dose, but the same numbers of animals died.
Table 10-5

Animal Deaths1 in the UDP2 Using Starting Doses Based on the 3T3 andNHK NRU Test Methods

Assay/Regression

With Default Starting Dose3

With IC50-Based Starting Dose4

Used

Used

Dead

3T3 NRU Test Method
RC rat-only millimole5
6

RC rat-only weight% DeathsDead% Deaths

Dose-Mortality Slope = 2.0
9.35

4.11

44.0%

8.80

4.09

46.5%

9.36

4.11

43.9%

8.70

4.05

46.6%

Dose-Mortality Slope = 8.3
5

RC rat-only millimole
6

RC rat-only weight

7.95

3.44

43.3%

7.42

3.43

46.2%

7.94

3.43

43.2%

7.32

3.39

46.3%

NHK NRU Test Method5

RC rat-only millimole

Dose-Mortality Slope = 2.0
9.36

4.08

43.6%

8.86

4.07

45.9%

RC rat-only weight

9.36

4.08

43.6%

8.80

4.02

45.7%

RC rat-only millimole5

7.92

3.39

42.8%

7.43

3.39

45.6%

7.92

3.39

42.8%

7.38

3.35

45.4%

6

Dose-Mortality Slope = 8.3
6

RC rat-only weight

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; RC=Registry ofCytotoxicity; UDP=Up-and-Down Procedure.
1
Numbers are mean numbers of animals used for 10,000 simulations for each substance. Although the simulations usedwhole animals, averaging the results over a large number of simulations produced fractional numbers. Upper limit dose =5000 mg/kg. Results are provided for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test
methods.
2
OECD (2001a); EPA (2002a).
3
Default starting dose = 175 mg/kg.
4
The starting dose was one default dose lower than the predicted LD50 calculated using the IC50 value in the regression
specified. The IC50 value for each reference substance was randomly selected from the distribution of values obtained during
the testing with each method.
5log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
6
log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024.

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November 2006

10.2.5
Accuracy of UDP Outcomes Using the IC50-Based Starting Doses
For each of the reference substances, the outcome of the simulated UDP testing, thesimulated LD50 was used to classify the substance into a GHS acute oral toxicity category.
The accuracy of GHS toxicity category assignments using the IC50-based starting doses wasdetermined by calculating the proportion of reference substances for which the GHS acuteoral toxicity category obtained using the IC50-based starting dose matched the categories
obtained using the default starting dose.
The concordance between the GHS categories determined using the 3T3 and NHK NRU test
methods with the RC rat-only millimole regression, and those determined using the UDP
default starting dose, was 96% for 3T3 and 97% for NHK (see Appendix N1). The
discordant reference substances were acetaminophen and sodium dichromate dihydrate in the
3T3 NRU test method, and acetaminophen, caffeine, and sodium dichromate dihydrate in the
NHK NRU test method. The use of the IC50-based starting dose from both in vitro NRU test
methods resulted in a higher GHS category (i.e., higher simulated LD50) for acetaminophen(simulated LD50 = 2047 vs. 1765 mg/kg for 3T3, and LD50 = 2174 vs. 1755 mg/kg for NHK),and a lower GHS category for sodium dichromate dihydrate (simulated LD50 = 44 vs. 52
mg/kg for 3T3 and LD50 = 45 vs. 52 mg/kg for NHK) than when using the default starting
dose. The NHK-based starting dose resulted in a lower GHS category for caffeine (simulated
LD50 = 280 vs. 357 mg/kg).
The concordance of GHS acute toxicity category predictions with those determined using thedefault starting dose was 97% for the 3T3 and NHK NRU test methods when the RC rat-only
weight regression was used (see Appendix N2). The discordant reference substances were
caffeine and sodium dichromate dihydrate. The simulated LD50 outcome for caffeine waslowered from 338 mg/kg for the default starting dose to 272 mg/kg for the 3T3-based starting
dose, and from 339 mg/kg to 270 mg/kg for the NHK-based starting dose. The simulated
LD50 outcome for sodium dichromate dihydrate was lowered from 51 mg/kg for the default
starting dose to 48 mg/kg for the 3T3-based starting dose, and from 51 mg/kg to 49 mg/kg
for the NHK-based starting dose.
Thus, the use of the IC50-based starting doses did not significantly alter the outcome of thesimulated UDP tests compared with the outcome obtained using the default starting doses.
10.3

Reduction and Refinement of Animal Use in the ATC Method

10.3.1In Vivo Testing Using the ATC Method
This section describes the general dosing procedure for the conduct of the ATC procedure(OECD 2001d). The ATC is used to assign a test substance to the appropriate GHS category
for classification and labeling. This is done by estimating the range of the LD50 values for thetest substance, rather than calculating a point estimate of the LD50. The time between
administration of test substance doses is determined by the onset, duration, and severity of
toxic signs. Guidance on the types of animals to use, animal housing, clinical observations,etc., which are outside the scope of the current discussion, are provided in the test guideline(See Appendix M3).

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10.3.1.1 Main Test
The ATC method uses a stepwise administration of test substances to three animals at a time,at one of a number of fixed doses: 5, 50, 300, and 2000 mg/kg (and 5000 mg/kg, if
necessary). The starting dose is selected so that at least some of the animals die at that dose.
If no information on which to base a starting dose is available, a default starting dose of 300
mg/kg is used. The next step is determined by the starting dose and the outcome of the three
animals tested at the starting dose and may be a decision to stop testing, test additional
animals at the same dose, test at the next higher dose, or test at the next lower dose. For
example, if two to three animals die or are in a moribund state after receiving the 300 mg/kg
starting dose, the next step is to administer 50 mg/kg to three more animals. However, if no,or one, animal dies at 300 mg/kg, three additional animals are tested at that dose. Most
substances require two to four dosing steps before they can be classified, and testing can be
stopped. See Appendix M3 for the outcome-based testing sequence for each starting dose.
10.3.1.2 Limit Test
For test substances that are likely to be nontoxic, the ATC guideline includes a limit test inwhich six animals (three animals per step [see Appendix M3]) are tested at the limit dose of2000 mg/kg or three animals are tested at a limit dose of 5000 mg/kg (OECD 2001d).
10.3.2Computer Simulation Modeling of the ATC Method
The simulation for the ATC method was performed using MATLAB® (The MathWorks, Inc.
1996-2004) computational software, which is functionally comparable with SAS® version 8.
Two thousand simulations of ATC testing were run for each substance, in vitro NRU test
method, and dose-mortality slope, using an upper limit dose of 2000 mg/kg7. The simulation
implements the distributional assumptions underlying the dose-mortality response. The
lowest dose at which an animal dies in response to the administration of a toxic substancevaries from animal to animal. For an entire population of animals, mortality is assumed tohave a log-normal distribution with the mean equal to the log of the true LD50. Sigma (σ), thevariability of the simulated population, is the inverse of the slope of the dose-mortality curve.
For any given dose, the probability that an animal will die is computed by the cumulative
log-normal distribution:

1 log dose $( t$log trueLD )Probability (death) =dt& e 2"" 2# $%
50

2

2

Because of a lack of information regarding the real dose-mortality curves, the simulations
assumed several different values of the slope (i.e., the inverse of σ). Dose-mortality slopes of! chosen, so as to be comparable to the slopes chosen for
0.5, 0.8, 2.0, 4.0, and 8.3 were
simulation modeling of the UDP (see Section 10.2.2).
To model the variability of the IC50 values within and among laboratories, the values for each
substance were log-transformed to normalize their distribution. The mean and variance of

7

The results from ATC simulations for a limit dose of 5000 mg/kg will be presented in a future addendum tothis document.

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November 2006

these log-transformed values were used to generate a log-normal distribution from which torandomly select an IC50 value.
The simulation procedure used the following steps for each substance:
1. The rodent acute oral LD50 value (in mg/kg) from Table 4-2 was entered as
the true LD50 value and the choices of assumed slope were entered as the true
slope for the dose-mortality curve.
2. An IC50 value was selected from a distribution identified by the mean andvariance of the IC50 values computed from the data to reflect that different
laboratories produce different IC50 values in different situations (see Tables 54 and 5-5 for mean IC50 values and standard deviations for the 3T3 and NHK
NRU test methods, respectively).
3. The IC50 value from Step 2 was used in the regression model being evaluatedto compute a predicted LD50 value for determining the starting dose.
4. The dosing simulation (of 2000 iterations) was run twice: once with thedefault starting dose of 300 mg/kg and once with a starting dose equal to thenext fixed dose below the predicted LD50, which was estimated by the
regression being evaluated (i.e., the IC50-based starting dose). If the IC50based starting dose was greater than the 2000 mg/kg limit dose, then testingproceeded using the 2000 mg/kg limit test rather than the main test.
5. For every dose group of three animals, one observation was sampled from abinomial distribution with the probability of death calculated by theprobability equation for a population of three. The sampled value, referred toas N1, indicates the number of animals, 0, 1, 2, or 3, in the dosing group thatdie.
6. If N1 ≤1, step 4 is repeated with the same dose. The resulting sampled value
from the binomial distribution is referred to as N2.
7. If N2 ≤1 and the dose is the highest dose tested, or the dose has already been
decreased, a toxicity category is assigned and testing is terminated. If the doseis not the highest dose tested, or if the dose has not been decreased, the next
higher fixed dose is administered and step 4 is repeated.
8. If N1 >1 or N2 >2, and the dose is the lowest dose tested, or if the dose has
already been increased, a toxicity category is assigned and testing isterminated. If the dose is not the lowest dose tested, or if the dose has notalready been increased, the next lower fixed dose is administered and step 4 isrepeated.
10.3.3

Animal Savings for the ATC Method When Using 3T3- and NHK-Based Starting
Doses

10.3.3.1 The Effect of the Dose-Mortality Slope on Animal Use
As described in Section 10.3.2, the simulation modeling of animal use for the ATC used fivedifferent dose-mortality slopes to assess animal use under various conditions of population
variability. Table 10-6 shows how mean animal use for the simulated ATC changes withdose-mortality slope for both the default starting dose of 300 mg/kg and a starting dose thatwas one fixed dose lower than that predicted by the 3T3 and NHK NRU IC50 values with the
RC rat-only millimole regression. The mean number of animals used for the ATC method

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decreased slightly with increasing slope for both the default starting dose and the IC50-based
starting dose.
The mean numbers of animals saved at all dose-mortality slopes were statistically significant(p <0.05 by one-sided Wilcoxon signed rank tests) when compared with mean animal use
with the default dose, and tended to decrease with increasing slope. To simplify thepresentation of animal savings and comparisons of the various regressions and starting doses,
subsequent results in Section 10.3.3 are shown only for dose-mortality slopes of 2.0 and 8.3.
As stated earlier, these slopes are shown here because the slope of 2.0 is the default used forthe calculation of LD50 by the UDP method (OECD 2001a; EPA 2002a) and the slope of 8.3
is shown to represent substances, such as pesticides, with higher slopes. Results for the otherdose-mortality slopes were computed, and are presented in Appendices N3 and N4.
Table 10-6

Change in Animal Use1 with Dose-Mortality Slope in the ATC Method2

Dose-Mortality Slope

With Default Starting
With IC50- Based
Dose1,3Starting Dose1,4
3T3 NRU Test Method

Animals Saved5

0.5

11.25 ±0.05

10.56 ±0.17

0.69* (6.1%)

0.8

11.10 ±0.07

10.46 ±0.19

0.64* (5.8%)

2.0

10.89 ±0.1210.27 ±0.24

0.62* (5.7%)

4.0

10.73 ±0.15

10.15 ±0.26

0.58* (5.4%)

8.3

10.64 ±0.1710.13 ±0.27

0.51* (4.8%)

NHK NRU Test Method
0.5

11.25 ±0.05

10.43 ±0.16

0.82* (7.3%)

0.8

11.10 ±0.07

10.31 ±0.18

0.79* (7.1%)

2.0

10.91 ±0.11

10.11 ±0.24

0.80* (7.3%)

4.0

10.75 ±0.15

9.98 ±0.27

0.77* (7.1%)

8.3

10.67 ±0.179.96 ±0.29

0.70* (6.6%)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; ATC=Acute Toxic Class method; NHK=Normal human epidermal
keratinocytes; RC=Registry of Cytotoxicity.
*Statistically significant (p <0.05) by a one-sided Wilcoxon rank test. Percent difference is shown in parentheses.
1
Mean numbers of animals used ± standard errors for 2000 simulations each for 67 substances in the 3T3 NRU test method
and 68 substances in the NHK NRU test method. Although the simulations used whole animals, averaging the results over alarge number of simulations produced fractional numbers. Upper limit dose = 2000 mg/kg.
2
OECD (2001d).
3
Default starting dose = 300 mg/kg.
4
Next fixed dose lower than the predicted LD50 calculated using the IC50 value for each reference substance in the RC ratonly millimole regression: log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621. The IC50 value for each reference substance
was randomly selected from the distribution of values obtained during the testing with each method.
5
Difference between mean animal use with the default starting dose and mean animal use with the IC50-based starting dose.

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November 2006

10.3.3.2 Mean Animal Use for ATC Simulations – Comparison of Regressions andPredictions from the 3T3 and NHK NRU Test Methods
Table 10-7 shows the mean animal use for testing the reference substances using thesimulated ATC method, when the starting dose was the default starting dose and when thestarting dose was one fixed dose lower than that determined by the 3T3 and NHK-predicted
LD50, and the regressions evaluated in Section 6.4 for prediction of GHS category. The meandifference in animal use between the two starting doses is the mean animal savings. All mean
animal savings were statistically significant (p <0.05 using one-sided Wilcoxon signed rank
tests), and ranged from 0.51 (4.8%) to 1.09 (10.2%) animals per test depending upon the
NRU test method, regression, and dose-mortality slope. The lowest mean animal savings
were obtained for the RC rat-only millimole regression (0.51 [4.8%] to 0.80 [7.3%] animals
per test), and the highest were obtained with the RC rat-only weight regression (0.91 [8.6%]
to 1.09 [10.2%] animals per test).
The animal savings obtained using the in vitro NRU test methods with the RC rat-onlyregressions apply only to the reference substances evaluated in this validation study, and are
based on substances pre-selected for their known in vivo toxicities and may not be broadly
applicable to other substances. Table 3-4 shows that 22 (38%) of the 58 RC substances
selected for testing were known to have a poor fit to the RC millimole regression (i.e., thepredicted LD50 was outside the RC acceptance interval). Table 6-3 shows that 40% (28/70 in
the 3T3) and 44% (31/71 in the NHK) of the reference substances that yielded IC50 values
were outliers. Substances that better fit the regression are likely to yield greater animalsavings.

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Table 10-7

November 2006

Animal Use1 for the ATC2 Method Using Starting Doses Based on the 3T3 and NHK NRU Test Methods with
the Different Regressions

Method/Regression

With Default
Starting Dose3

3T3 NRU Test Method
RC rat-only millimole6
7

RC rat-only weight7

RC rat-only weight

Animals
Saved5Dose-Mortality Slope = 2.0

With Default
Starting Dose3

With IC50- Based
Starting Dose5

Animals
Saved5Dose-Mortality Slope = 8.3

10.89 ±0.1210.27 ±240.62* (5.7%)

10.64 ±0.1710.13 ±0.27

0.51* (4.8%)

10.89 ±0.129.85 ±0.24

1.04* (9.6%)

10.64 ±0.179.55 ±0.29

1.09* (10.2%)

NHK NRU Test Method
RC rat-only millimole6

With IC50- Based
Starting Dose4

Dose-Mortality Slope = 2.0

Dose-Mortality Slope = 8.3

10.91 ±0.11

10.11 ±0.24

0.80* (7.3%)

10.67 ±0.179.96 ±0.29

0.70* (6.6%)

10.91 ±0.119.95 ±0.24

0.96* (8.8%)

10.67 ±0.179.75 ±0.30

0.91* (8.6%)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; ATC=Acute Toxic Class method; NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
*Statistically significant (p<0.05) using a one-sided Wilcoxon signed rank test. Percentage difference is shown in parentheses.
1
Mean numbers of animals used ±standard errors for 2000 simulations each for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU
test method. Upper limit dose =2000 mg/kg.
2
OECD (2001d).
3
Default starting dose =300 mg/kg.
4
Starting dose was one fixed dose lower than the predicted LD50 calculated using the IC50 value for each reference substance in the regression specified. The IC50
value for each reference substance was randomly selected from the distribution of values obtained during the testing with each test method.
5
Difference between mean animal use with the default starting dose and mean animal use with the IC50-based starting dose.
6
log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
7
log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024.

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10.3.3.3 Animal Savings in the ATC Method by GHS Acute Oral Toxicity Category Using
the 3T3- and NHK -Based Starting Doses
Tables 10-8 and 10-9 show mean animal use and mean animal savings for the ATC whenused with the in vitro NRU test methods, organized by GHS category (UN 2005), and whenbased on the:• RC rat-only millimole regression (Table 10-8)• RC rat-only weight regression (Table 10-9)
The following data come from the same analyses as the data provided in Table 10-7.
The analyses showed that:• For each in vitro NRU test method and regression, the highest mean animalsavings were generally in the LD50 ≤5 mg/kg and LD50 >5000 mg/kg toxicity
categories.
• For each NRU test method and regression, the lowest mean animal savings
were in the 300 < LD50 ≤2000 mg/kg toxicity category.
Animal Savings in the ATC Method by GHS Category Using the 3T3- and NHK-Based
Starting Doses with the RC Rat-Only Millimole Regression
Table 10-8 shows the mean animal savings in the ATC method by GHS category for the invitro NRU test methods used with the RC rat-only millimole regression. Mean differences
between animal use for the default starting dose and with the IC50-determined starting dosewere statistically significant (p <0.05) by a one-sided Wilcoxon signed rank test for thefollowing GHS toxicity categories, NRU test methods, and dose-mortality slopes:• The use of both in vitro NRU test methods at both dose-mortality slopes for
substances with 5 < LD50 ≤50 mg/kg produced savings of 1.15 (9.8%) to 1.33
(11.4%) animals per test
• The use of the 3T3 NRU test method at both dose-mortality slopes for
substances with 300 < LD50 ≤2000 mg/kg used more animals per test (i.e.,
produced savings of -0.92 [-9.5%] to -1.30 [-14.0%] animals per test)• The use of both in vitro NRU test methods at both dose-mortality slopes for
substances with LD50 >5000 mg/kg produced savings of 2.03 (17.1%) to 2.66
(22.2%) animals per test
At the dose-mortality slope of 2.0, the mean animal savings with the 3T3 NRU test method
ranged from -0.92 (-9.5%) to 2.68 (27.4%) animals per test, and the animal savings with the
NHK NRU test method ranged from -0.60 (-6.1%) to 2.96 (30.4%) animals per test. At thedose-mortality slope of 8.3, the mean animal savings with the 3T3 NRU test method ranged
from -1.30 (-14.0%) to 2.70 (29.7%) animals per test, and the animal savings with the NHK
NRU test method ranged from -0.85 (-9.2%) to 2.99 (33.0%) animals per test.

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Table 10-8

November 2006

Animal Savings1 for the ATC2 Method by GHS Acute Oral Toxicity Category3 Using Starting Doses Basedon the 3T3 and NHK NRU Test Methods with the RC Rat-Only Millimole Regression4
Dose-Mortality Slope = 2.0

GHS Acute Oral Toxicity
Category3

Number ofReference
Substances

With Default
Starting
Dose5

With IC50Based
Starting
Dose6

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
12

9.77 ±0.1711.56 ±0.2110.81 ±0.20
9.75 ±0.07
11.22 ±0.08
11.85 ±0.04

7.09 ±1.09
10.39 ±0.52
10.39 ±0.1710.67 ±0.48
11.14 ±0.08
9.82 ±0.78

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
13

9.74 ±0.16
11.56 ±0.21
10.83 ±0.219.77 ±0.06
11.22 ±0.08
11.86 ±0.03

6.78 ±1.31
10.38 ±0.35
10.39 ±0.29
10.37 ±0.49
11.25 ±0.129.43 ±0.73

Animals Saved

Dose-Mortality Slope = 8.3
7With Default
Starting Dose5

3T3 NRU Test Method
2.68 (27.4%)
9.08 ±0.08
1.17* (10.2%)
11.75 ±0.16
0.42 (3.9%)9.42 ±0.26-0.92* (-9.5%)
9.26 ±0.10
0.08 (0.7%)
11.88 ±0.10
2.03* (17.1%)
12.00 ±0.000
NHK NRU Test Method
2.96 (30.4%)
9.09 ±0.08
1.18* (10.2%)
11.76 ±0.17
0.44 (4.0%)
9.44 ±0.26-0.60 (-6.1%)
9.26 ±0.10-0.03 (-0.3%)
11.87 ±0.102.43* (20.5%)
12.00 ±0.000

WithIC50Based
Starting
Dose6
6.38 ±1.09
10.60 ±0.439.27 ±0.1110.56 ±0.62
11.77 ±0.109.81 ±0.84
6.09 ±1.2310.42 ±0.45
9.63 ±0.49
10.11 ±0.63
11.89 ±0.15
9.34 ±0.80

Animals
Saved72.70 (29.7%)
1.15* (9.8%)
0.15 (1.6%)-1.30* (-14.0%)

0.11 (0.9%)
2.19* (18.3%)
2.99 (33.0%)
1.33* (11.4%)-0.20 (-2.1%)-0.85 (-9.2%)-0.02 (-0.2%)
2.66* (22.2%)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; ATC=Acute Toxic Class method; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN2005); NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
*Statistically significant (p <0.05) by a one-sided Wilcoxon signed rank test. Percentage difference is shown in parentheses.
1
Mean number of animals used ±standard errors for 2000 simulations for each substance with an upper limit dose of 2000 mg/kg. Results are provided for 67 substances inthe 3T3 NRU test method and 68 substances in the NHK NRU test method categorized using the rat acute oral LD50 reference values in mg/kg from Table 4-2. Although
the simulations used whole animals, averaging the results over a large number of simulations produced fractional numbers.
2
OECD (2001d).
3
GHS for acute oral toxicity (UN 2005).
4
The RC rat-only millimole regression is log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
5
Default starting dose =300 mg/kg.
6
The starting dose was the next fixed dose lower than the predicted LD50 using the IC50 for each reference substance in the RC rat-only millimole regression. The IC50 value
for each reference substance was randomly selected from the distribution of values obtained during the testing with each method.
7
Difference between mean animal use with the default starting dose and mean animal use with the IC50-based starting dose.

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November 2006

At both the 2.0 and 8.3 dose-mortality slopes, the mean animal savings using the 3T3 NRU
test method were lower than the corresponding savings using the NHK NRU test method, forsubstances in at least four of the six toxicity categories: LD50 ≤5 mg/kg; 5 < LD50 ≤50 mg/kg;
300 < LD50 ≤2000 mg/kg; and LD50 >5000 mg/kg. The mean animal savings per test werehigher with the 3T3 NRU test method than the NHK NRU test method for substances in the
2000< LD50 ≤5000 mg/kg category at both dose-mortality slopes. For substances in the 50 <LD50 ≤300 mg/kg category, the mean animal savings using the 3T3 NRU test method was
greater than the savings using the NHK NRU test method, when the dose-mortality slope
equaled 8.3. When the 3T3 NRU test method was used, the highest mean animal savings
occurred when testing substances in the LD50 ≤5 mg/kg category (2.68 [27.4%] animals per
test at dose-mortality slope = 2.0, and 2.70 [29.7%] at dose-mortality slope = 8.3). When the
NHK NRU test method was used, the highest mean animal savings occurred when testing
substances in the LD50 ≤5 mg/kg category (2.96 [30.4%] animals per test at dose-mortality
slope = 2.0, and 2.99 [33.0%] animals per dose at dose-mortality slope = 8.3). However, theanimal savings were not statistically significant with either in vitro NRU test method.
The smallest mean animal savings (≤0.44) in both in vitro NRU test methods were observed
for substances with LD50 values between 50 and 5000 mg/kg. Because the default starting
dose was 300 mg/kg, little change in mean animal use was expected for substances in the 50< LD50 ≤300 mg/kg and 300 < LD50 ≤2000 mg/kg categories. The mean animal savings fromboth in vitro NRU test methods and both dose-mortality slopes for the substances in the 50 <LD50 ≤300 mg/kg category were -0.20 to 0.44 animals per test. There were no animal savingsfor substances in the 300 < LD50 ≤2000 mg/kg category using either NRU test method or
dose-mortality slope. In fact, significantlyore animals were used when the starting doses werebased on the 3T3 NRU IC50 than using the default starting dose (-0.92 to -1.30 animals pertest). More animals were also used when the starting doses were based on the NHK NRU
IC50 (-0.85 to -0.60 animals/test), but the difference was not statistically significant.
The animal savings in the various GHS acute oral toxicity categories using the in vitro NRU
test methods with the RC rat-only millimole regression applies only to the reference
substances evaluated in this validation study, and may not be broadly applicable to othersubstances. The animal savings for future testing using the in vitro NRU test methods with
the RC rat-only millimole regression will depend on the prevalence of test substances in eachof the GHS acute oral toxicity categories.
Animal Savings with the ATC Method by GHS Category Using 3T3- and NHK-Based
Starting Doses with the RC Rat-Only Weight Regression
Table 10-9 shows the animal savings for the simulated ATC method by GHS category forthe in vitro NRU methods used with the RC rat-only weight regression. Mean animal savings
were statistically significant (p <0.05) by a one-tailed Wilcoxon signed rank test for thefollowing GHS toxicity categories, NRU test methods, and dose-mortality slopes.
• The use of both in vitro NRU test methods at both dose-mortality slopes for
substances with 5 < LD50 ≤50 mg/kg produced savings of 1.25 (10.8%) to
1.51 (13.0%) animals per test.

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In Vitro Cytotoxicity Test Methods BRD Section 10

Table 10-9

November 2006

Animal Savings1 for the ATC2 Method by GHS Acute Oral Toxicity Category3 Using Starting Doses
Based on the 3T3 and NHK NRU Test Methods with the RC Rat-Only Weight Regression4

GHS Acute Oral Toxicity
Category3

Number ofReference
Substances

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
12

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
13

Dose-Mortality Slope = 2.0
Dose-Mortality Slope = 8.3
With
With IC50With
With IC50Animals
Animals
Default
Based
Default
Based
Starting
Saved7
Starting
Starting
Saved7
Starting
6
5
6
5
Dose
Dose
Dose
Dose
3T3 NRU Test Method
9.77 ±0.177.56 ±1.03
2.21 (22.6%)
9.08 ±0.08
6.85 ±0.99
2.24 (24.6%)
11.56 ±0.21
10.06 ±0.38
1.51* (13.0%) 11.75 ±0.16
10.27 ±0.33
1.48* (12.6%)
10.81 ±0.20
10.35 ±0.18
0.47* (4.3%)
9.42 ±0.26
9.20 ±0.10
0.22 (2.4%)
9.75 ±0.07
10.67 ±0.50-0.93* (-9.5%)
9.26 ±0.1010.65 ±0.66-1.39 (-15.0%)
11.22 ±0.08
9.80 ±0.51
1.43* (12.7%) 11.88 ±0.109.44 ±0.88
2.43 (20.5%)
11.85 ±0.04
8.83 ±0.83
3.02* (25.5%) 12.00 ±0.00
8.67 ±0.91
3.33* (27.7%)
NHK NRU Test Method
9.74 ±0.166.87 ±1.282.87 (29.4%)
9.09 ±0.08
6.18 ±1.20
2.91 (32.0%)
11.56 ±0.2110.31 ±0.19
1.25* (10.8%) 11.76 ±0.1710.40 ±0.33
1.36* (11.5%)
10.83 ±0.21
10.41 ±0.28
0.42 (3.8%)
9.44 ±0.26
9.63 ±0.49-0.20 (-2.1%)
9.77 ±0.62
10.46 ±0.50-0.69 (-7.1%)
9.26 ±0.1010.23 ±0.65-0.97 (-10.4%)
11.22 ±0.09
10.69 ±0.37
0.53 (4.7%)
11.87 ±0.10
11.03 ±0.60
0.84 (7.1%)
11.86 ±0.03
8.91 ±0.78
2.94* (24.8%) 12.00 ±0.00
8.75 ±0.85
3.25* (27.1%)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; ATC=Acute Toxic Class method; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN2005); NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
*Statistically significant (p <0.05) by a one-sided Wilcoxon signed rank test. Percentage difference is shown in parentheses.
1
Mean number of animals used ±standard errors for 2000 simulations for each substance with an upper limit dose of 2000 mg/kg. Although the simulations used wholeanimals, averaging the results over a large number of simulations produced fractional numbers. Results are provided for 67 substances in the 3T3 NRU test method and68 substances in the NHK NRU test method categorized using the rat acute oral reference LD50 values in mg/kg from Table 4-2.
2
OECD (2001d).
3
GHS for acute oral toxicity (UN 2005).
4
From Table 6-2; log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024
5Default starting dose = 300 mg/kg.
6
The starting dose was one fixed dose lower than the predicted LD50 calculated using the IC50 for each reference substance in the RC rat-only weight regression. The IC50
value for each reference substance was randomly selected from the distribution of values obtained during the testing with each method.
7
Difference between mean animal use with the default starting dose and mean animal use with the IC50-based starting dose.

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•
•

•
•

November 2006

The use of the 3T3 NRU test method at the 2.0 dose-mortality slope for
substances with 50 < LD50 ≤300 mg/kg produced savings of 0.47 (4.3%)
animals per test.
The use of the 3T3 NRU test method at the 2.0 dose-mortality slope for
substances with 300 < LD50 ≤2000 mg/kg produced savings of -0.93 (-9.5%)
animals per test (i.e., used more animals per test than the default starting
dose).
The use of the 3T3 NRU test method at the 2.0 dose-mortality slope for
substances with 2000 < LD50 ≤5000 mg/kg produced savings of 1.43 (12.7%)
animals per test.
The use of both in vitro NRU test methods at both dose-mortality slopes for
substances with LD50 >5000 mg/kg produced savings of 2.94 (24.8%) to 3.33
(27.7%) animals per test.

The mean animal savings with the 3T3 and NHK NRU test methods were similar for mostacute oral toxicity categories at both dose-mortality slopes; the mean savings for the 3T3
NRU test method was slightly higher than for the NHK NRU test method for most toxicity
categories. At the dose-mortality slope of 2.0, the mean animal savings for the 3T3 NRU test
method (for the various toxicity categories) ranged from -0.93 (-9.5%) to 3.02 (25.5%)
animals per test, and savings for the NHK NRU test method ranged from -0.69 (-7.1%) to
2.94 (24.8%) animals per test. At the dose-mortality slope of 8.3, animal savings with the
3T3 NRU test method ranged from -1.39 (-15.0%) to 3.33 (27.7%) animals per test, andsavings with the NHK NRU test method ranged from -0.97 (-10.4%) to 3.25 (27.1%) animals
per test.
There were no mean animal savings (≤-0.69 animals) for substances with 300 < LD50 ≤2000
when either in vitro NRU test method was used. The mean animal savings for the substancesin the 50 < LD50 ≤300 mg/kg category using both in vitro NRU test methods and dosemortality slopes were also relatively small (-0.20 to 0.47 animals per test). Because thedefault starting dose was 300 mg/kg, little change in mean animal use was expected for
substances in the 50< LD50 ≤300 mg/kg and 300 < LD50 ≤2000 mg/kg categories.The highest
mean animal savings (≤-0.69 animals) occurred for substances with LD50 >5000 mg/kg
when either in vitro NRU test method was used. For both test methods and dose-mortality
slopes, the mean animal savings for substances in this category were 2.94 (24.8%) to 3.33
(27.7%) animals per test and were statistically significant. Mean animal savings were alsohigh (2.21 [22.6%] to 2.91 [32.0%] animals per test) for substances with LD50 ≤5 mg/kg, butthese savings were not statistically significant.
The animal savings in the various GHS categories using the two in vitro NRU test methods
with the RC rat-only weight regression applies only to the reference substances evaluated inthis validation study, and may not be broadly applicable to other substances.
10.3.4

Refinement of Animal Use in the ATC Method When Using 3T3- and NHK-Based
Starting Doses
A procedure refines animal use when it lessens or eliminates pain or distress in animals, orenhances animal well-being (ICCVAM 2003). This section evaluates whether the use of 3T3and NHK-based starting doses refines animal use by reducing the number of animals that die
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when the IC50-predicted starting doses are used, compared to the number of animals that die
when using the default ATC starting dose of 300 mg/kg. Table 10-10 reports the results for
the ATC simulation modeling using the 2000 mg/kg limit dose. For every regression
evaluated, the mean number of deaths when using the 3T3- and NHK-based starting doses
was less than the mean number of deaths when using the default starting dose, byapproximately 0.4 to 0.5 deaths per test. For the RC rat-only millimole regression and the RC
rat-only weight regression, the percentage of deaths (compared with the numbers of animals
used) was also slightly lower with the in vitro-based starting dose compared with the default
starting dose. In general, fewer animals were used with the in vitro-based starting dose, andfewer animals died.
Table 10-10 Animal Deaths1 for the ATC2 Method Using Starting Doses Based on the
3T3 and NHK NRU Test Methods

Method/Regression

Default Starting Dose3
Used

Dead

3T3 NRU Test Method5

RC rat-only millimole
6

RC rat-only weight% Deaths

IC50- Based Starting Dose4
Used

Dead

% Deaths

Dose-Mortality Slope = 2.0
10.89

3.77

34.6%

10.27

3.31

32.2%

10.89

3.77

34.6%

9.85

3.27

33.2%

Dose-Mortality Slope = 8.3
5

RC rat-only millimole
RC rat-only weight

6

10.64

3.20

30.1%

10.13

2.77

27.3%

10.64

3.20

30.1%

9.55

2.73

28.6%

10.91

3.72

34.1%

10.11

3.19

31.6%

10.91

3.72

34.1%

9.95

3.21

32.3%

NHK NRU Test Method
RC rat-only millimole5
RC rat-only weight

6

Dose-Mortality Slope = 2.0

Dose-Mortality Slope = 8.3
5

RC rat-only millimole
RC rat-only weight

6

10.67

3.15

29.5%

9.96

2.67

26.8%

10.67

3.15

29.5%

9.75

2.67

27.4%

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; ATC=Acute Toxic Class method; NHK=Normal human epidermal
keratinocytes; RC=Registry of Cytotoxicity.
1
Mean numbers of animals used for 2000 simulations for each of 67 substances in the 3T3 NRU test method and 68
substances in the NHK NRU test method. Although the simulations used whole animals, averaging the results over a large
number of simulations produced fractional numbers. Upper limit dose =2000 mg/kg.
2
OECD (2001d).
3
Default starting dose =300 mg/kg.
4
The starting dose was one fixed dose lower than the predicted LD50 calculated by using the IC50 for each reference
substance in the regression evaluated. The IC50 value for each reference substance was randomly selected from the
distribution of values obtained during the testing with each method.
5log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
6
log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024.

10.3.5
Accuracy of the ATC Method Outcomes Using the IC50-Based Starting Doses
The accuracy of the outcome of the simulated ATC testing (i.e., the simulated GHS acuteoral toxicity category) using the IC50-based starting dose was determined by calculating the
proportion of reference substances for which the simulated GHS category for the IC50-based
starting dose matched the simulated GHS category for the default starting dose.
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When the RC rat-only millimole regression with the 3T3 and NHK NRU test methods wasused, the concordance of simulated GHS categories for the IC50-based starting doses withthose for the default starting dose was 99% for both in vitro NRU test methods (seeAppendix N3). The discordant reference substance in the 3T3 NRU test method wascaffeine. The simulated GHS category using the 3T3-based starting dose was 50 < LD50 ≤300
mg/kg, and the simulated GHS category using the default starting dose was 300 < LD50
≤2000 mg/kg.
The discordant reference substance in the NHK NRU test method was sodium dichromate
dihydrate. The simulated GHS acute oral toxicity category using the NHK-based starting
dose was 5 < LD50 ≤50 mg/kg and the simulated GHS category using the default starting
dose was 50 < LD50 ≤300 mg/kg. Both discordant substances were predicted to have astarting dose one category below the actual category.
When the RC rat-only weight regression was used with the 3T3 and NHK NRU test methods,
the concordance of simulated GHS acute toxicity category predictions with those determinedusing the default starting dose was 99% and 97% for the 3T3 and the NHK NRU test
methods, respectively (see Appendix N4). The discordant reference substance in the 3T3
NRU test method was caffeine. The simulated GHS acute oral toxicity category for caffeine
using the 3T3-based starting dose was 50 < LD50 ≤300 mg/kg and that using the default
starting dose was 300 < LD50 ≤2000 mg/kg. The discordant reference substances in the NHK
NRU test method were caffeine and sodium dichromate dihydrate. The simulated GHS acuteoral toxicity category for caffeine using the NHK-based starting dose was 50 < LD50 ≤300
mg/kg and the simulated GHS category using the default starting dose was 300 < LD50 ≤2000
mg/kg. The simulated GHS acute oral toxicity category for sodium dichromate dihydrateusing the NHK-based starting dose was 5 < LD50 ≤50 mg/kg while that for the default
starting dose was 50 < LD50 ≤300 mg/kg. Similar to what was seen with the RC millimole
regression, the predicted starting doses for the discordant substances were one GHS category
below the actual category.
Thus, the use of the IC50-based starting doses did not significantly alter the outcomes of thesimulated ATC tests compared with the outcome based on the default starting dose.
10.4

The Impact of Accuracy on Animal Savings

Two types of accuracy analyses were performed for the NICEATM/ECVAM validation
study. The first analyses determined the accuracy of using the NRU IC50 values with an IC50LD50 regression to predict LD50 values. It calculated the concordance for GHS acute oral
toxicity category by comparing the GHS categorization yielded by the NRU-predicted LD50
values (using the in vitro NRU IC50 values in the regressions presented in Table 6-5) with the
GHS categorization based on rat acute oral LD50 data (see Section 6.4). The second analysis
determined the accuracy of the simulation outcomes using the IC50-based starting doses (seeSections 10.2.5 and 10.3.5). It calculated the concordance for the GHS acute oral toxicity
category outcomes obtained using the IC50-based starting doses with the GHS category
outcomes obtained using the default starting dose. The magnitude of animal savings did notcorrelate with either determination of accuracy and the accuracy determinations for IC50based predictions and IC50-based outcomes for GHS category did not correlate with one
another.
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Animal savings did not correlate with the accuracy of the GHS acute oral toxicity category
predictions based on the LD50 values calculated using the IC50 values in the RC rat-onlyregressions (see Sections 6.4.2 and 6.4.3). Substances in categories with the lowest accuracy
produced the highest animal savings. For example, using the RC rat-only millimole
regression with the in vitro NRU IC50 values yielded very low accuracy (0 to 17%) for GHS
acute oral toxicity category prediction for substances with LD50 >5000 mg/kg (see Table 67), but the highest animal savings of 14.8 to 20.3% occurred in this category (see Table 103). Animal savings were small, 4.5 to 6.5%, for substances with 300 ≤ LD50 ≤2000 mg/kg,
but the accuracy of 75-81% for GHS acute oral toxicity category prediction was relatively
high. The reason that animal savings is unrelated to the accuracy of prediction of GHS acuteoral toxicity category based on the LD50 values calculated using IC50 values in the RC ratonly regressions is because two different standards are used for comparison in the twoanalyses:•
GHS acute oral toxicity category predictions using IC50 values in the RC ratonly regressions are compared with the GHS categories derived from the invivo reference LD50•
The number of animals used (to determine animal savings) was comparedwith the animal use at the default starting dose of 175 mg/kg for the UDP or
300 mg/kg for the ATC
Despite the relatively poor GHS accuracy for the low toxicity chemicals (the toxicity ofalmost all were overpredicted by one GHS category), animal savings were greatest due to thefact that testing goes to the limit dose faster.
The accuracy of the simulated GHS toxicity category assignments using the IC50-based
starting doses for UDP and ATC test simulations was determined by calculating the
proportion of reference substances for which the GHS acute oral toxicity category obtainedusing the IC50-based starting dose matched the categories obtained using the default starting
dose (see Sections 10.2.5 and 10.3.5). The accuracy of these GHS toxicity category
assignments based on the simulation outcomes does not correlate with animal savings using
the IC50 values in the RC rat-only regressions (see Sections 6.4.2 and 6.4.3). For example,
the accuracy of GHS acute oral toxicity category outcomes for the ATC test method whenusing the RC rat-only millimole regression was 100% for the 3T3 NRU test method for
substances with 300 ≤ LD50 ≤2000 mg/kg (see Appendix N3). In contrast, the animal savingsfor those substances was negative at -6.1 to -14.0% (i.e., more animals were used comparedwith the default starting dose) (see Table 10-8). The reason the outcome-based GHS acuteoral toxicity category predictions is unrelated to animal savings is that two different
parameters are being measured in the two analyses:•
The accuracy of the simulatedGHS acute oral toxicity outcomes using theIC50-based starting doses measured outcome (i.e., simulated GHS category
based on the simulated LD50 outcome for the UDP and simulated GHS
category for the ATC)•
The animal savings analysis measured the number of animals used at the IC50based starting dose and the default starting dose of 175 mg/kg for the UDP or
300 mg/kg for the ATC

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Thus, the measurements for the two analyses are different: outcome (i.e., GHS category) andnumber of animals used to achieve the outcome.
In addition, accuracy of the GHS toxicity category assignments based on the simulation
outcomes does not correlate with the accuracy of the GHS acute oral toxicity category
predictions using the IC50 values in the RC rat-only regressions (see Section 6.4.2 and 6.4.3).
For example, the overall accuracy of GHS acute oral toxicity category outcomes for the ATCtest method when using the RC rat-only millimole regression was 99% for both in vitro NRU
test methods (see Section 10.3.5 and Appendix N3). In contrast, the overall accuracy of
GHS acute oral toxicity category predictions using the using the IC50 values in the RC ratonly millimole regression was 31% for the 3T3 NRU test method and 29% for the NHK
NRU test method (see Table 6-7). The reason the simulated outcome-based GHS acute oral
toxicity category predictions differed from the accuracy of the GHS acute oral toxicity
category predictions based on the calculation of LD50 using the IC50 in the IC50-LD50
regression is because two different standards are used for comparison in the two analyses:•
Simulated GHS acute oral toxicity outcomes for the IC50-based starting doses
were compared with the simulated GHS category outcomes using the default
starting doses•
GHS acute oral toxicity category predictions using the IC50 values in the RC
rat-only regressions were compared with the GHS category derived from thein vivo reference LD50
Thus, despite that the IC50 values and IC50-LD50 regressions predicted GHS acute oral
toxicity categories poorly, the GHS acute oral toxicity category outcomes using the IC50based starting doses were practically the same as the GHS acute oral toxicity category
outcomes using the default starting dose.
10.5

The Impact of Prevalence on Animal Savings

As stated several times in this section, the animal savings for substances tested in the
futureusing the 3T3 and NHK NRU test methods to determine the staring dose for rodent
acute oral toxicity test methods will depend on the proportion of test substances that fall into
each of the GHS acute toxicity hazard categories. Although the prevalence of substancesamong the different categories will depend, to a large extent, on the mandate of a particularregulatory agency, Spielmann et al. (1999) indicated that 76% (845/1115) of the industrial
substances submitted to the Federal Institute for Health Protection of Consumers andVeterinary Medicine in Berlin, Germany, since 1982 had LD50 >2000 mg/kg. The extent towhich these substances represent the population of substances in commerce is not known.
However, if the results of the validation study are broadly applicable to substances to betested in the future, and if such substances are relatively nontoxic, the selection of starting
doses using the in vitro NRU test methods may save a considerable number of animals sinceanimal savings for the validation study were highest for the least toxic substances.

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Summary

Computer simulation modeling of UDP testing using the default dose progression shows that,for the subset of reference substances evaluated, the prediction of starting doses using the
3T3 and NHK NRU test methods with the RC rat-only millimole regression resulted in astatistically significant (p <0.05) decrease in the number of animals used by an average of
0.49 (6.2%) to 0.54 (5.8%) animals per test, depending upon the in vitro NRU test method
and the dose-mortality slope (2.0 or 8.3) used. The mean animal savings improved slightly, to0.54 (6.8%) to 0.66 (7.0%) animals per test, when the RC rat-only weight regression wasused.
When reference substances were grouped by GHS category, there were no mean animalsavings by simulated UDP testing for substances with 50< LD50 ≤300 mg/kg. The highest,
and statistically significant, animal savings were observed with both in vitro NRU test
methods when testing substances with 2000< LD50 ≤5000 mg/kg and LD50 >5000 mg/kg.
When using the RC rat-only millimole regression, animal savings for these categories ranged
from 1.28 (11.9%) to 1.58 (20.3%) animals per test. The use of the RC rat-only weight
regression improved animal savings slightly for the substances in these toxicity categories to
1.28 (14.0%) to 1.65 (16.7%) animals per test. Although the use of IC50 values to estimate
starting doses for the simulated UDP deceased the number of animals used per test, it did notchange the number of animals that would have died during the procedures.
Computer simulation modeling of ATC testing showed that, for the reference substancestested in this validation study, the prediction of starting doses using the 3T3 and NHK NRU
test methods with the RC rat-only millimole regression resulted in a statistically significant(p <0.05) decrease in the number of animals for ATC testing by an average of 0.51 (4.8%) to
0.80 (7.3%) animals per test, depending upon the in vitro NRU test method and the dosemortality slope (2.0 or 8.3) used. Animal savings improved to a mean of 0.91 (8.6%) to 1.09
(10.2%) animals per test when the RC rat-only weight regression was used.
When test substances were grouped by GHS category, the mean animal savings for ATC
testing using the RC rat-only millimole regression were statistically significant with the 3T3
NRU test method at both dose-mortality slopes for substances with 5 < LD50 ≤50 mg/kg(1.15 [9.8%] to 1.17 [10.2%] animals per test), and for substances with LD50 >5000 mg/kg(2.03 [17.1%] to 2.19 [18.3%] animals per test). Significantly more animals were needed
when the 3T3-based starting doses were used, than the default starting dose for reference
substances with 300 < LD50 ≤2000 mg/kg (i.e., the animal savings were negative: -0.92 [9.5%] to -1.30 [-14.0%] animals). The mean animal savings with the NHK NRU test method
and the RC rat-only millimole regression were statistically significant at both dose-mortality
slopes for substances with 5 < LD50 ≤50 mg/kg (1.18 [10.2%] to 1.33 [11.4%] animals pertest), and for substances with LD50 >5000 mg/kg (2.43 [20.5%] to 2.66 [22.2%] animals pertest). When the RC rat-only weight regression was used, statistically significant savings inanimals used were observed with both in vitro NRU test methods and dose-mortality slopes
for substances with 5 <LD50 ≤50 mg/kg (1.25 [10.8%] to 1.51 [13.0%] animals per test), andfor substances with LD50 >5000 mg/kg (2.94 [24.8%] to 3.33 [27.7%] animals per test). The
use of IC50 values to estimate starting doses for the ATC refined animal use by producing

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approximately 0.5 to 0.6 fewer mean animal deaths per test than when the default starting
dose of 300 mg/kg was used.
The use of the IC50-based starting doses did not significantly alter the GHS category
outcomes of the simulated UDP or ATC when compared with the outcomes based on thedefault starting dose. The concordance for GHS acute oral toxicity category for the IC50based starting dose with the default starting dose was 97 to 99% for both in vitro NRU
methods and IC50-LD50 regressions evaluated.
The magnitude of animal savings did not correlate with the accuracy of GHS categorization
yielded by the NRU-predicted LD50 values (using the in vitro NRU IC50 values in the IC50LD50 regressions) or with the accuracy of GHS category outcomes since the accuracy andanimals savings analyses used different standards for comparison.
The specific animal savings using the 3T3 and NHK NRU test methods with the RC rat-onlyregressions apply only to the reference substances evaluated in this validation study, and may
not be broadly applicable to other substances. Spielmann et al. (1999) indicated that 76%(845/1115) of the industrial substances submitted to the Federal Institute for HealthProtection of Consumers and Veterinary Medicine in Berlin, Germany, since 1982 had LD50
>2000 mg/kg. The extent to which these substances represent the population of substances incommerce is not known. However, if the results of the validation study are broadly
applicable to substances to be tested in the future, and if such substances are relatively
nontoxic, the selection of starting doses using the in vitro NRU test methods may save aconsiderable number of animals since animal savings for the validation study were highest
for the least toxic substances.

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11.0

November 2006

PRACTICAL CONSIDERATIONS .................................................................... 11-3

11.1 Transferability of the 3T3 and NHK NRU Test Methods.................................. 11-3
11.1.1 Facilities and Major Fixed Equipment..................................................................... 11-3
11.1.2 Availability of Other Necessary Equipment and Supplies ...................................... 11-4
11.1.3 Problems Specific to the NHK Test Method ........................................................... 11-5
11.2 3T3 and NHK NRU Test Method Training Considerations .............................. 11-6
11.2.1 Required Training and Expertise ............................................................................. 11-6
11.2.2 Training Requirements to Demonstrate Proficiency................................................ 11-7
11.2.3 Personnel Needed to Perform the In Vitro NRU Test Methods............................... 11-8
11.3 Cost Considerations ............................................................................................... 11-8
11.3.1 3T3 and NHK NRU Test Methods .......................................................................... 11-8
11.3.2 Rodent Acute Oral Toxicity Testing...................................................................... 11-11
11.4 Time Considerations for Performing the 3T3 and NHK NRU Tests.............. 11-12
11.4.1 The 3T3 NRU Test Method ................................................................................... 11-12
11.4.2 The NHK NRU Test Method................................................................................. 11-12
11.4.3 Prequalification of NHK Medium ......................................................................... 11-12
11.4.4 In Vivo Testing....................................................................................................... 11-12
11.4.5 The Limit Test........................................................................................................ 11-13
11.5

Summary............................................................................................................... 11-14

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In Vitro Cytotoxicity Methods BRD Section 11

11.0

November 2006

PRACTICAL CONSIDERATIONS

The 3T3 and NHK NRU test methods are proposed as adjuncts, rather than replacements for,in vivo acute oral toxicity assays. Data from these in vitro basal cytotoxicity test methods areused with a linear regression model to predict the rat acute oral LD50 of the test substance,
which is then used to determine the starting dose for subsequent rat acute oral toxicity tests,
as described in Sections 10.2.2 and 10.3.2. This section discusses practical issues involved inusing these two in vitro NRU test methods for predicting starting doses for rat acute oral
toxicity tests. Practical issues that need to be considered with respect to the implementationof these cell culture methods include the need for, and availability of, specialized equipment,
personnel training and expertise requirements, cost considerations, and time expenditures.
11.1

Transferability of the 3T3 and NHK NRU Test Methods

Transferability of a test method is defined as the ability of a method or procedure to beaccurately and reliably performed in different, competent laboratories (ICCVAM 2003).
Accuracy and reliability of these NRU test methods are discussed in Sections 6 and 7,
respectively.
Protocols for the 3T3 and NHK NRU test methods, including solubility testing, andprequalification of keratinocyte growth medium, have been optimized and are available on
the ICCVAM/NICEATM website (http://iccvam.niehs.nih.gov/methods/invitro.htm). The
protocols were designed with GLP-compliance in mind and can be easily implemented or
adapted by scientists with the appropriate technical experience.
Although the in vitro and in vivo test methods require some similar, general laboratory skills(e.g., preparation of solutions and test substance doses, record keeping), in vitro testing
requires skills specific to cell culture systems (e.g., aseptic techniques, microscopic
evaluation of cell cultures, propagation of cells in medium) but not to the maintenance,
handling, or treatment of rodents.
11.1.1
Facilities and Major Fixed Equipment
The following lists of facility requirements, equipment and supplies, and training andexpertise are common to most in vitro mammalian cell culture laboratories. Required
equipment and supplies are also described in detail in the validation study 3T3 and NHK
protocols (Appendices B and C), the Guidance Document (ICCVAM 2001b), and Hartung etal. (2002).
11.1.1.1 Facility Requirements
The testing facility should be appropriate for operating a scientific laboratory (e.g.,laboratory space, air handling procedures, access to utilities, shipping/receiving department[for appropriate receipt and handling of cell culture materials], etc.). Each facility should
provide:• Adequate facilities, equipment, and supplies• Proper health and safety guidelines• Satisfactory quality assurance procedures
Each facility should conform to all appropriate statutes (i.e., local, state, provincial, federal,national, international) concerning safety guidelines (e.g., general workplace safety
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guidelines, chemical handling and disposal guidelines, biohazard guidelines). Hartung et al.
(2002) provides recommended safety guidelines for working with potentially infectious
materials (e.g., HIV, hepatitis B, hepatitis C) and human materials (e.g., cells, tissues, fluids).
11.1.1.2 Cell Culture Laboratory
The testing facility should have a designated cell culture laboratory to ensure that in vitro
cytotoxicity assays are performed under clean and proper aseptic conditions. The dedicatedlaboratory should be located such that through traffic is minimal to reduce possible
disturbances that can lead to contamination which could compromise the cell culture assays.
The room temperature of the laboratory should be regulated, monitored, and documented.
Access to the laboratory and its supplies and test chemicals should be restricted toappropriate personnel.
11.1.1.3 Major Equipment
Each testing facility should have at a minimum the following equipment:• Incubator (37ºC ± 1ºC, 90% ± 10% humidity, 5.0% ± 1% CO2/air)• Laminar flow clean bench/cabinet (standard: "biological hazard")• Inverted phase contrast microscope• 96-well plate spectrophotometric plate reader equipped with 540 nm ± 10 nm
filter (if testing in 96-well plates)• Autoclave• Refrigerator• Freezer (-70ºC)• Cryogenic (liquid nitrogen) freezer/storage unit
• Computer
Equipment maintenance and calibration should be routinely performed and documented
according to GLP guidelines and testing facility SOPs.
11.1.2Availability of Other Necessary Equipment and Supplies

11.1.2.1 General Equipment
Each testing facility should have at a minimum the following equipment:• Low speed centrifuge• Adjustable temperature waterbath• Pipettors• Balance• pH meter• Cell counting system• Water bath sonicator• Magnetic stirrer• Vortex mixer• Antistatic bar ionizer (for reduction of static on tissue culture plates)
Equipment maintenance and calibration should be routinely performed and documented as
per GLP guidelines and testing facility SOPs. The types of equipment listed in this section
are available from scientific and laboratory supply companies (e.g., Thomas Scientific http://www.thomassci.com/index.jsp; Fisher Scientific - https://www.fishersci.com/).
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11.1.2.2 Cell Culture Materials and Supplies
The following supplies are needed for the in vitro NRU test methods. Specific product andprivate company names are provided either as an identification of actual materials/brands
used in the validation study or as examples. Mention of these names does not imply
endorsement of the product or company.
• Tissue culture plasticware (flasks [e.g., 25 cm2, 75-80 cm2], 96-well plates,
disposable pipettes)• Laboratory glassware (e.g., flasks, bottles, graduated cylinders)• Adhesive film plate sealers (e.g., Excel Scientific SealPlate)• Sterile filtration systems (e.g., vacuum filtration units with 0.22 µm and 0.45
µm sterile filters)• Culture medium and supplements (e.g., DMEM; prequalified NHK medium)• NCS (bovine)• Balanced salt solutions (e.g., HBSS, D-PBS)
Cell culture supplies are generally available through the major scientific and laboratory
supply companies and through specialty companies (e.g., GIBCO, SIGMA-Aldrich,
CAMBREX/Biowhittaker, Becton Dickinson). Compositions of culture media,
supplements/additives, salt solutions, NRU assay chemicals, and the volumes of each neededfor each test method, should be defined. All tissue culture flasks and dishes needed to assure
proper cell propagation should be identified.
11.1.2.3 Cell Cultures
3T3 Mouse Fibroblasts: BALB/c 3T3 cells, clone 31, can be obtained fromnational/international cell culture repositories (e.g., American Type Culture Collection[ATCC], Manassas, VA, product # CCL-163).
NHKs: These non-transformed keratinocyte cells from cryopreserved primary or secondary
cells can be obtained from national/international cell culture repositories (e.g., CAMBREX
Bio Science, 8830 Biggs Ford Road, Walkersville, MD), or isolated from donated tissue
using proper collection, preparation, and propagation techniques. It may be difficult, at times,
to obtain adequate supplies of keratinocytes; the preparation of a pool of cells depends on theavailability of tissue donors. It is recommended that testing laboratories procure of acommercially available stock pool of cells and store them indefinitely in a cryogenic freezer.
All cell stock and cultures used for testing must be certified as free of contamination by
mycoplasma and bacteria.
11.1.3
Problems Specific to the NHK NRU Test Method
FAL had difficulty obtaining an adequate supply of NHK medium during the validation
study. Communication between the UK distributor and the laboratory was uneven and the
SMT attempted to resolve the supply issue on several occasions. The other laboratories
periodically had difficulties in obtaining NHK medium and supplements that adequately
supported keratinocyte growth. Although the purchased medium and supplements met the
manufacturer’s QA/QC standards, certain lots of the medium and supplements did notsupport the growth of NHK cells to the extent needed in the test protocol. To deal with these
problems, an NHK medium prequalification protocol was incorporated into the study to
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avoid unnecessarily repeating studies because of medium and supplements that did notadequately support cell growth. These experiences illustrate the need for multiple sources ofkeratinocyte cell culture medium. They also suggest that the NHK results could be morevariable than the 3T3 results because of the batch-to-batch differences in NHK growth
medium and supplements.
11.2

3T3 and NHK NRU Test Method Training Considerations

The ECVAM Good Cell Culture Practice Task Force Report 1 (Hartung et al. 2002)
encouraged the establishment of practices and principles that will reduce uncertainty in thedevelopment and application of in vitro test methods. Training in good cell culture practices,in conjunction with good laboratory practices, are essential for all in vitro cytotoxicity testingand should be employed to ensure that data produced from the 3T3 and NHK NRU test
methods are reproducible, credible, and acceptable.
In vitro cytotoxicity test methods require personnel trained specifically in sterile tissue/cell
culture techniques and general laboratory procedures. Personnel should have mandatory
training in good cell culture practices, in the specialized culture procedures needed for theseassays, and in safety and handling practices appropriate to the types of substances that may
be tested in the laboratory (Hartung et al. 2002).
The facility management should establish scientific guidelines and procedures, train andsupervise professional and technical staff, and evaluate results and performance within theirdiscipline area relative to the testing requirements. Performance of the tests requires amoderate degree of technical capability and a high degree of skill in monitoring andmaintaining appropriate cell growth conditions, troubleshooting the potential and real
problems in culture systems, and analyzing and interpreting in vitro cytotoxicity data. Eachindividual engaged in the conduct of a study, or responsible for its supervision, shall haveeducation, training, and experience, or combination thereof, to enable that individual toperform the assigned duties. The NRU test methods do not require that personnel be trained
to perform in vivo testing.
11.2.1Required Training and Expertise
Personnel performing in vitro testing should have training in basic cell culture aspects suchas: sterile technique, handling culture media, feeding cultures, cell counting, subculture(trypsinization), detection and elimination of contamination, cell growth and measurement ofgrowth curves, viability assays, and storage and freezing/thawing of cells. Additionally,
training is encouraged for special culture procedures such as primary cell and tissue cultures,
toxicity testing, and viability assays. Laboratory personnel should be trained in theapplication of GLP requirements (see Section 8.1.1), and in the safe storage, handling, anddisposal of toxic substances.
11.2.1.1 Specific Training and Expertise Needed
Personnel performing the in vitro cytotoxicity test methods should be well experienced ingeneral cell culture techniques and should be able to:• Work with cryogenic freezing apparatus• Pipette solutions with large volume pipettors and multi-channel pipettors

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Establish cells in culture vessels under aseptic conditions and monitor growth;recognize normal and abnormal cell growth characteristics; and document
observations of cell cultures throughout all aspects of the procedure
Perform the in vitro assays by following the protocols to grow the cells, count,transfer, and feed the cells, treat the cells with test substances, perform
application of adhesive plate sealers to culture plates for control of volatile
substances, perform the NRU assay, perform optical density measurements,
transfer data to electronic templates
Operate equipment necessary for maintaining cell culture laboratories (e.g.,
incubators, biohazard hoods, spectrophotometric microtiter plate readers)

11.2.1.2 General Laboratory Expertise Needed
Personnel should also be able to understand and perform basic laboratory techniques andlaboratory management:• Prepare cell culture solutions (e.g., culture medium, NRU solutions), measure
pH, know proper storage conditions, and maintain proper documentation• Prepare test substances for application to cell cultures, follow solubility
protocols to adequately prepare test chemicals in solution, recognize solubility
issues (e.g., insolubility nature of chemical, precipitation), and implement
procedures for dissolving the test chemicals• Monitor and control laboratory environment (e.g., temperature, humidity,
lighting, traffic), maintain equipment to support cell cultures (e.g.,temperature, humidity, gas flow, calibrations)11.2.2Training Requirements to Demonstrate Proficiency
Laboratories establish their own criteria for proficiency but, over the course of training,laboratory personnel should be able to understand the protocol, perform the protocol withguidance from an experienced supervisor/trainer and, eventually, perform the protocol withminimal or no supervision. An experienced supervisor determines when a technician isadequately trained because there are no standardized criteria or tasks that can be used toaccurately measure competence. After the technician demonstrates competence in executing
all the aspects of the test protocols(s), it is appropriate to perform routine assessments oftechnical competence using a benchmark, coded control test substance (e.g., SLS). It isessential that the laboratory staff be certified as proficient in using the test methods to test
unknowns.
The laboratories in the validation study were selected because of their experience inperforming in vitro cytotoxicity assays but were required to develop additional skills throughPhases I and II (e.g., data collection and transfer to Excel® and PRISM® templates).
Inexperienced laboratory personnel were trained by having them perform “training” assays
using SLS. In the early phases of the validation study, the laboratories continued training bytesting coded reference substances of various toxicities, and performing solubility testing on
substances of varying solubilities. These procedures helped improve proficiency among thelaboratories for the final phase of the validation study.

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11.2.2.1 Proficiency With GLP-Compliance
Results from these test methods will be submitted to regulatory agencies that will, for themost part, require GLPs. Laboratories should work toward attaining GLP compliance. GLP
compliance in each laboratory is determined by its independent QA unit. ECBC and IIVS
conducted this validation study in compliance with GLP (see Section 8.1.1). Their respective
QA units (as per GLPs) reviewed the various aspects of the study and issued QA statementsthat addressed whether the test methods and the results described in the Final Reportaccurately followed the test protocol and reflected the raw data produced during the study,
and provided assurance that all testing was done under according to GLP. FAL (which was
non-GLP-adherent) followed the GLP standards referenced in Section 8.1.1 as guidelines for
conducting this study. FAL had no QA unit to judge GLP compliance.
11.2.3
Personnel Needed to Perform the In Vitro NRU Test Methods
The facility management will be responsible for determining which qualified personel meetthe criteria (e.g., scientific knowledge, specialized training) for the following positions
needed for adequate performance of the in vitro NRU test methods and oversight of thetesting.
• Study Director: the individual with the overall responsibility for the technicalconduct of the testing (e.g., is familiar with the test procedures, provides SOPs
and ensures GLP compliance, analyzes and interprets the data, determines test
acceptance, oversees recordkeeping procedures, and produces the test reports.
• Quality Assurance Officer: monitors the testing to assure conformance with
GLP requirements; must be independent of the Study Director.
• Laboratory Technician(s): individuals trained in sterile tissue/cell culture
techniques and general laboratory procedures and who are capable ofperforming the test methods according to GLPs.
11.3

Cost Considerations

11.3.1

3T3 and NHK NRU Test Methods

11.3.1.1 Equipment Costs
Major instruments and equipment needed to implement the in vitro cytotoxicity test methods
are described in Section 11.1.1. Ranges of costs for some of the equipment were obtained
from on-line catalogues for two major scientific equipment and supplies companies (Thomas
Scientific - http://www.thomassci.com/index.jsp; Fisher Scientific https://www.fishersci.com/). These prices are for equipment that will meet the minimum
needs of the NRU test methods (see Table 11-1). These costs were researched in August
2006.
11.3.1.2 Costs for Cell Cultures and Supplies
Supplies such as cell culture chemicals, the reagents used to measure NRU, and cell culture
plasticware are available from numerous suppliers, and are not cost prohibitive.

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Table 11-1

November 2006

Costs for Major Laboratory Equipment
Range of Costs1

Equipment
Class II Biological Safety Cabinet$7,300 – $12,200

CO2 Incubator$5,100 – $16,400

Spectrophotometer Microplate Reader$5,000 – $7,500

Freezer (capable of -70°C)$8,000 – $15,300

Refrigerator$1,300 – $9,800

Centrifuge (benchtop model)$2,100 – $8,500

Microscope (inverse phase contrast)$3,000 – $14,500

Coulter Counter2, 3$3,000 – $9,000

Autoclave (benchtop model)2$3,500 – $15,400

Cryogenic (liquid nitrogen) Storage$1,000 – $3,700

1From on-line scientific equipment catalogues (Thomas Scientific - http://www.thomassci.com/index.jsp; Fisher
Scientific - https://www.fishersci.com/). [searched August 2006]
2
May be useful, but not required for performing the tests.
3
Other automatic cell counters may be used.

The 3T3 NRU test method is generally less expensive to perform than the NHK NRU test
method. One vial of the immortalized 3T3 cells (~$200 [ATCC]) can be propagatedindefinitely by passaging cells and periodically cryopreserving batches of cells. The NHK
NRU test method requires a fresh sample of primary cells for each test run (~$380 per vial
[CAMBREX]). Because primary NHK cells are passaged only once after initiating theculture, there are no cells available to cryopreserve a stock batch of cells. The DMEM
medium used for the 3T3 cells is less expensive, more “generic”, and more readily available
than keratinocyte-specific NHK medium. (See Table 11-2.)
11.3.1.3 Commercial Testing
The following price quotes are provided as examples of test costs and were acquired fromcommercial laboratories through Internet contact or through personal communication. Use of
information from these specific laboratories does not imply endorsement of them.
A representative of MB Research Laboratories (Spinnerstown, PA,
http://www.mbresearch.com/) provided a quote (personal communication, 2005) for an invitro 24-hr cytotoxicity test (but not a 48-hour test period) of $1050 (USP standards1) or$1950 (ISO standards1) for a set of three test chemicals. The lead laboratory for the
NICEATM/ECVAM study, IIVS (Gaithersburg, MD, http://www.iivs.org/) provides
1

USP=United States Pharmacopeia; ISO=International Standards Organization. These organizations provide
international standard testing requirements for products that require high quality for public use.

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commercial laboratory GLP-compliant testing using this study’s protocols (48-hour test
period) at a cost of $1120 - $1850 per chemical/sample for one cell type (personalcommunication 2005) (see Table 11-2).
Table 11-2

Costs for Cell Culture Materials and Commercial Laboratory In Vitro
Cytotoxicity Testing

Item

Cost
(approximate)

Number ofTests Possible

Other
One vial can produce an
indefinite supply of cells by
propagating the cells in culture
and periodically freezing apool of cells.
Since cells are passaged only
once beyond cryopreservation,
new vials should be thawed asneeded to maintain continuous
testing.
Establish cells in culture (~20mL/vial of cells; 60 mL/3
vials), seed cells in 96-wellplates (12 mL/plate; 180
mL/15 plates); prepare stock
solution and eight
concentration dilutions (~20mL/chemical; 300 mL/15
plates).

3T3 Cells~$200/vial1

indefinite

NHK Cells~$380/vial2~5 (96-wellplates)Dulbeccos’ Minimum
Essential Medium (DMEM) with
supplements~$20/500mL3~15 (96-wellplates)~$80/500 mL2~15 (96-wellplates)Same as DMEM (above)$1050/$1950(USP/ISO) per 3 test
materials4

1 test/material

in vitro NRU cytotoxicity test
(24-hour test period)$1120 (GLP) per test
material (minimum of 5
materials tested
simultaneously)4

1 range finder, 2
definitive tests
per test material

in vitro NRU cytotoxicity test
(48-hour test period)$1850 (GLP) per single
test material (tested
individually)4

1 range finder, 2
definitive tests
per test material

in vitro NRU cytotoxicity test
(48-hour test period)

NHK Medium with
supplements
Commercial
Laboratory Testing(MB ResearchLaboratories [GLPcompliant])
Commercial
Laboratory Testing(Institute for In Vitro
Sciences [GLPcompliant])
Commercial
Laboratory Testing(Institute for In Vitro
Sciences)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red
uptake; USP/ISO= United States Pharmacopeia/International Standards Organization GLP=Good Laboratory
Practices
1
Catalogue price from American Type Culture Collection (ATCC) (http://www.atcc.org/)
2
Catalogue price from CAMBREX (http://www.cambrex.com/Welcome.asp)
3
Catalogue price from INVITROGEN (http://www.invitrogen.com/content.cfm?pageid=1)
4
Personal communication (Raabe 2005)
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11.3.2
Rodent Acute Oral Toxicity Testing
As stated in Section 11.3.1.3, presentation of price quotes from commercial laboratories
provides examples of test costs and does not imply an endorsement of that laboratory. Table
11-3 provides some commercial prices for acute oral systemic toxicity testing. MB ResearchLaboratories performs the UDP test at a cost of $750 for three rats and charges $250 for each
additional rat needed. In the best-case scenario, the UDP test needs only three rats ($750). In
the worst-case scenario, this test would need an additional 12 rats (15 maximum for the test);
the total cost of the test would be $3,750. In this costing strategy, $250 is saved for each rat
not used by an accurate prediction of the starting dose by the 3T3 or NHK NRU test method.
Because the in vitro cytotoxicity test costs from $350 to $1850 per chemical, there is no net
savings in animal costs if fewer than two to six animals are saved.
Table 11-3

Commercial Prices for Conducting In Vivo Acute Rat Toxicity Testing
GLP-Compliant

Non GLPCompliant

$1200

$1000

$800

$650

$2700

$2200

$950

NA

$1500

NA

Acute Oral Rat Toxicity: LD50

$3000

NA

Acute Oral Toxicity – UDP$730 for the first 3
animals; $250 each
additional animal

NA

Test
Acute Oral Toxicity UDP:
Limit Test - 2000 mg/kg
Acute Oral Toxicity UDP:
Limit Test - 5000 mg/kg
Acute Oral Toxicity UDP:
LD50
Acute Oral Rat Toxicity:
single dose2
Acute Oral Rat Toxicity: twodoses2Company
Product Safety
Laboratories
Product Safety
Laboratories
Product Safety
Laboratories1
Bio ResearchLaboratories
Bio ResearchLaboratories
Bio ResearchLaboratories
MB ResearchLaboratories1

Abbreviations: UDP=Up-and-Down Procedure; GLP=Good Laboratory Practices; NA=Not available.
1
Personal communication (Wnorowski 2005).
2
Washington State Biological Testing Methods #80-12 For the Designation of Dangerous Waste; Part B: Acute Oral Rat
Toxicity Test [http://www.ecy.wa.gov/pubs/80012.pdf]. This test method is an adaptation of the EPA Health Affects Test
Guidelines OPPTS 870.110 Acute Oral Toxicity and American Society for Testing and Materials (ASTM) methods E 116390 (Standard test method for estimating acute oral toxicity in rats) and E 1372-90 (Standard test method for conducting a 90day oral toxicity study in rats).

The President of Product Safety Laboratories, Gary Wnorowski, (Dayton, NJ,
http://www.productsafetylabs.com/), provided a cost quote of $2700 for determination of a
rat LD50 value using the UDP test; the cost is independent of the number of rats that areneeded. Each test dose is administered ~24-48 hours after the previous dose and each animal
test generally does not exceed four days. The time involved in providing the LD50 value isapproximately three months (initiation of the test to provision of the final report). Having theestimated LD50 value would not affect the cost of the in vivo test but could reduce the numberof animals needed.
Bio Research Laboratories performs the rat acute oral toxicity test using a test method that
determines lethality and signs of acute toxicity from a waste sample administered in a single
dose, by gavage, to a limited number of rats. The bioassay determines if the test sample
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produces an LD50 either greater than or less than a regulatory threshold corresponding to ahazardous waste designation (i.e., 5000, 500, 50 mg/kg). A minimum of 10 rats is used at thetested dose for the regulatory threshold value that is relevant to the test sponsor. In thistesting scenario, knowledge of the estimated LD50 would not reduce animal use or test costs
if a single predetermined dose is tested.
11.4

Time Considerations for Performing the 3T3 and NHK NRU Tests

11.4.1The 3T3 NRU Test Method
Approximately one week is needed to thaw cryopreserved 3T3 cells, propagate them, andpassage them at least two times before subculturing them into the 96-well test plates. After
subculture into 96-well plates, the cells are incubated another 24 hours to reach the proper
confluence, and then exposed to test chemical for 48 hours. The initial 3T3 NRU test (range
finder or definitive test) takes approximately 10 days. However, after the cells are establishedin culture, they can be passaged for approximately two months before having to go back to
the cryopreserved cells to start a new culture. A 3T3 NRU test can be completed in less thanfour consecutive days when started from an established stock culture. Multiple substances
can be tested at the same time, and different tests can overlap each other; thus, many
substances can be tested in a relatively short time.
11.4.2The NHK NRU Test Method
Approximately one week is needed to thaw cryopreserved NHK cells, propagate them, andpassage them into the 96-well test plates. After subculture into 96-well plates, the cells are
incubated another 48-72 hours to reach the proper confluence and then exposed to test
chemical for 48 hours. The entire NHK NRU test (range finder or definitive test) requires
approximately 11-12 days. Cells can be seeded at different densities from one starter vial in
the culture flasks so that passaging the cultures can take place on different days. Once thecells are established in culture, they are passaged once to the 96-well test plates and an NHK
NRU test can usually be completed in five to six consecutive days. Multiple substances canbe tested at the same time, and different tests can overlap each other; thus, many substances
can be tested in a relatively short time.
11.4.3Prequalification of NHK Medium
The protocol for the prequalification of NHK medium requires nearly identical steps, andsimilar time-line (i.e., 11-12 days), as required for the NHK rangefinder and definitive tests.
Table 11-2 provides an estimate of how many tests could be performed using one 500 mL
bottle of medium with supplements (~15 tests in 96-well plates).
11.4.4
In Vivo Testing
According to guidelines for acute oral toxicity testing, single animals or groups of animals
are dosed in sequence, usually at 2-4 day intervals, and observations are generally made forup to 14 days (for animals that are not moribund) for the main test and limit dose test (EPA
2002a; OECD 2001a; OECD 2001b, OECD 2001c). The addition of 3T3 or NHK NRU
testing to estimate a starting dose prior to the implementation of the UDP main test or limit
dose test would take 10-12 days, but could save up to 14 days of observation for every
animal not used.

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11.4.5
The Limit Test
The in vitro NRU test methods can provide a savings of time when used to determine if an invivo acute oral toxicity limit test can be employed as the initial test for a substance withunknown in vivo toxicity. If the IC50 value from an in vitro NRU test could accurately predict
an LD50 that is greater than, or equal to, the limit dose (i.e., 2000 mg/kg or 5000 mg/kg), thein vivo test could start at the limit test dose. This approach has the potential to eliminate theneed to do the main test and could result in a net savings of six days for the UDP test method
and about one day for the ATC test method. Table 11-4 illustrates the following:••

Time needed to perform the 3T3 and NHK NRU test
Time needed to reach the limit test starting dose when initiating the in vivo
main test using the default starting doses (UDP and ATC)

The times presented in Table 11-4 use the following assumptions:• 3T3 cells reach ≤50% confluence in approximately 24 hours• NHK cells reach >20% confluence in approximately 48 hours• Animals show no evident toxicity 48 hours post-dosing, and additional
animals are dosed at the next higher default dose
• Limit test dose = 5000 mg/kg for the UDP and 2000 mg/kg for the ATC
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Table 11-4

November 2006

Comparison of Time Needed for In Vitro and In Vivo TestingTime

3T3 NRU
Test Method

NHK NRU
Test Method

UDP(5000 mg/kg
upper limit)

ATC(2000 mg/kg
upper limit)

Day 1

Seed cells in 96-wellplate
Incubate for 24 ±2 hr

Seed cells in 96-well plate
Incubate forapproximately 48 to 72 hr

Dose 1 animal at
default dose
(175 mg/kg)
Observe for 48 hr

Dose 3 animals atdefault dose(300 mg/kg)
Observe for 48 hr

Day 2

Apply test substance
Incubate for 48 ±0.5 hr

Incubate

Observe

Observe0 – 1 animal dies
Dose 3 animals atdefault dose(300 mg/kg)
Observe 48 hr

Day 3

Incubate

Apply test substance
Incubate for 48 ±0.5 hr

No death
Dose 1 animal at next
default dose
(550 mg/kg)
Observe 48 hr

Day 4

NRU: 3 ±0.1 hr
Elute NR: 0.33 to 0.75 hr
OD540 measurement
Calculate IC50
Estimate LD50 andStarting Dose*

Incubate

Observe

Observe

NRU: 3 ±0.1 hr
Elute NR: 0.33 to 0.75 hr
OD540 measurement
Calculate IC50
Estimate LD50 andStarting Dose*No death
Dose 1 animal at next
default dose
(1750 mg/kg)
Observe 48 hr

0 – 1 animal dies
Dose 3 animals at next
default dose
(2000 mg/kg)
Starting Point for the
Limit Test

Day 5

Day 6

Observe

Day 7

No death
Dose 1 animal at next
default dose
(5000 mg/kg)
Starting Point for the
Limit Test

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; UDP=Up-andDown Procedure; ATC=Acute Toxic Class method; hr=Hour; NR=Neutral red; OD540=Optical density at 540 nm.

11.5

Summary•

All equipment and supplies should be readily commercially available. During
the validation study, direct communication with the NHK medium supplierinsured that specific lots of medium were available to the laboratories. The
test methods are expected to be transferable to laboratories experienced withmammalian cell culture methods.

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November 2006

Much of the training and expertise needed to perform the 3T3 and NHK NRU
test methods are common to mammalian cell culture procedures. Additional
technical training would not be extensive because these test methods are
similar to other in vitro mammalian cell culture assays, and no extraordinary
techniques are necessary. GLP training should be provided to technicians toensure proper adherence to protocols and documentation procedures.
Prices for commercial testing for one chemical are $1,120 to $1,850 (Table
11-2) for in vitro cytotoxicity testing in the 3T3 and NHK test methods,
respectively, to determine the IC50 (Raabe 2005, personal communication). In
contrast, the in vivo rat acute oral testing for LD50 determination could costfrom $750 - $3,750 (Table 11-3), depending on the test method used and the
toxicity of the test substance. Comparison of costs of in vitro testing to in vivo
testing is difficult because the in vitro NRU test methods are not replacements
for the animal testing, and animal testing would be performed regardless of
the responses of the 3T3 or NHK cells. The use of these in vitro NRU test
methods may not reduce the overall cost of the in vivo rat acute oral toxicity
test, but has the potential to reduce the number of animals needed for a study.

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12.0

November 2006

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GLOSSARY1

Accuracy2: (a) The closeness of agreement between a test method result and an acceptedreference value. (b) The proportion of correct outcomes of a test method. It is a measure oftest method performance and one aspect of “relevance”. Accuracy is highly dependent on theprevalence of positives in the population being examined.
Acute Toxic Class (ATC) method: An acute oral systemic toxicity test method based on
testing groups of animals at fixed doses in a sequential manner. The lethality outcomes areused to classify a test substance into the appropriate GHS acute oral toxicity category.
ANOVA: One-way (and two-way) analysis of variance. ANOVA compares themeasurements (continuous variables) of three or more groups when the data are categorized
in one way (one-way) or two ways (two-way). ANOVA assumes that the populations
compared are normally distributed and that the variances for the groups to be compared areapproximately equal.
Assay2: The experimental system used. Often used interchangeably with “test” and “testmethod.”Biphasic dose-response: Dose-response in which cytotoxicity increases (as dose increases),plateaus, and then increases again. See Section 2.6.3.
Category prediction: The acute oral GHS hazard category that includes the predicted LD50
value for a test chemical.
Coded substances: Substances labeled by code rather than name so that they can be tested
and evaluated without knowledge of their identity or anticipation of test results. Coded
substances are used to avoid intentional or unintentional bias when evaluating laboratory or
test method performance.
Coefficient of determination: In linear regression, it denotes the proportion of the variancein Y and X that is shared. Its value ranges between zero and one and it is commonly called
called “R2.” For example, R2 = 0.45, indicates that 45% of the variance in Y can be explainedby the variation in X and that 45% of the variance in X can be explained by the variation inY.

1

The definitions in this Glossary are restricted to their uses with respect to in vitro cytotoxicity testing and the
NRU test methods.
2
Definition used by the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM 2003).
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Coefficient of variation: A statistical representation of the precision of a test. It is expressed
as a percentage and is calculated as follows: standard deviation  × 100%meanConcordance2: The proportion of all substances tested that are correctly classified as
positive or negative. It is a measure of test method performance and one aspect of“relevance.” The term is often used interchangeably with “accuracy.” Concordance is highly
dependent on the prevalence of positives in the population being examined. In the
NICEATM/ECVAM study, concordance was used to describe the proportion of test
substances that were correctly classified into GHS acute oral toxicity hazard categories, or todescribe the proportion of test substances for which the laboratories obtained the sameclassification result.
Confluence: A state in which cells in culture come into contact with other cells in the same
culture to form a complete sheet of cells (monolayer). For this study, confluence isdetermined as a percentage of cell coverage of the tissue culture vessel growth surface (e.g.,cell monolayer has 80% confluency).
Cytotoxicity: The adverse effects resulting from interference with structures and/or
processes essential for cell survival, proliferation, and/or function. For most chemicals,
toxicity is a consequence of non-specific alterations in "basal cell functions" (i.e., via
mitochondria, plasma membrane integrity, etc.), which may then lead to effects on organspecific functions and/or death of the organism. These effects may involve the integrity ofmembranes and the cytoskeleton, cellular metabolism, the synthesis and degradation orrelease of cellular constituents or products, ion regulation, and cell division.
Definitive test: The main test of the cytotoxicity assay for determining the IC50. The
concentration closest to the range finder test IC50 serves as the midpoint of the concentrations
tested in a definitive test. Compared to the range finder test, the definitive test uses a smaller
dilution factor for the concentrations tested.
Discordant chemicals: Chemicals for which the LD50 is not accurately predicted by the IC50
(and the associated regression formula) or the GHS toxicity category is not accurately
predicted by the IC50 (and the associated regression formula). Also referred to as “outliers.”
EDIT: Evaluation-guided Development of New In vitro Test Batteries. An international
project initiated by Björn Ekwall in 1998 and continued by the Scandinavian Society for Cell
Toxicology to develop new in vitro tests for toxicity and toxicokinetics to be incorporated
into test batteries for predicting acute and chronic systemic toxicity.
Endpoint2: The biological process, response, or effect assessed by a test method.

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Fixed Dose Procedure (FDP): An acute oral systemic toxicity test method based on testinggroups of animals at fixed doses. Evident toxicity outcomes are used to classify a test
substance into the appropriate GHS acute oral toxicity category.
Geometric mean: The antilog of the mean of the logarithm of the values. It is less affectedby extreme values than the arithmetic mean.
Globally Harmonized System (GHS): A classification system presented by the United
Nations that provides (a) a harmonized criteria for classifying substances and mixtures
according to their health, environmental and physical hazards, and (b) a harmonized hazard
communication elements, including requirements for labeling and safety data sheets.
Good Laboratory Practices (GLP)2: Regulations promulgated by the U.S. Food and Drug
Administration and the U.S. Environmental Protection Agency, and principles andprocedures adopted by the Organization for Economic Cooperation and Development andJapanese authorities that describe record keeping and quality assurance procedures forlaboratory records that will be the basis for data submissions to national regulatory agencies.
Guidance Document: Guidance Document on Using In Vitro Data to Estimate In Vivo
Starting Doses for Acute Toxicity (ICCVAM 2001b).
Hazard2: The potential for an adverse health or ecological effect. A hazard potential results
only if an exposure occurs that leads to the possibility of an adverse effect being manifested.
Hill function: The IC50 values are determined from the concentration-response using a Hillfunction which is a four parameter logistic mathematical model relating the concentration of
the test chemical to the response (typically following a sigmoidal shape).

Y = Bottom +Top − Bottom
1+ 10 (logEC50−log X)HillSlope

where Y=response (i.e., % viability), X is the substance concentration producing the
response, Bottom is the minimum response (0% viability, maximum toxicity), Top is the
maximum response (maximum viability), EC50 is the substance concentration at the response
midway between Top and Bottom, and HillSlope describes the slope of the curve. When
Top=100% viability and Bottom=0% viability, the EC50 is the equal to the IC50.
Hill function (rearranged): Some unusual dose-responses did not fit the Hill function well.
To obtain a better model fit, the Bottom parameter was estimated without constraints (theprevious practice was to use Bottom=0). However, when Bottom≠0, the EC50 reported by the
Hill function was not the same as the IC50 since the Hill function defines EC50 as the point
midway between Top and Bottom. Thus, the Hill function calculation using the Prism®
software was rearranged to calculate the concentration corresponding to the IC50 as follows: Top − Bottom −1log Y − Bottomlog IC50 = log EC50 −HillSlope13-3

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where IC50 is the concentration producing 50% toxicity, EC50 is the concentration producing
a response midway between the Top and Bottom responses; Top is the maximum response(maximum survival), Bottom is the minimum response (0% viability, maximum toxicity),
Y=50 (i.e., 50% response), and HillSlope describes the slope of the response. The X from thestandard Hill function equation is replaced, in the rearranged Hill function equation, by the
IC50.
Hormesis: a dose-response characterized by a compound’s ability to cause an opposite effectat low doses than it causes at high doses. A stimulatory effect at low doses and an inhibitory
effect in high doses is often the observed manifestation of hormesis.
IC50: test chemical concentration producing 50% inhibition of the endpoint measured (i.e.,cell viability).
Interlaboratory reproducibility2: A measure of whether different qualified laboratories
using the same protocol and test substances can produce qualitatively and quantitatively
similar results. Interlaboratory reproducibility is determined during the prevalidation andvalidation processes and indicates the extent to which a test method can be transferred
successfully among laboratories.
Intralaboratory repeatability2: The closeness of agreement between test results obtained
within a single laboratory when the procedure is performed on the same substance underidentical conditions within a given time period.
Intralaboratory reproducibility2: The first stage of validation; a determination of whether
qualified people within the same laboratory can successfully replicate results using a specifictest protocol at different times.
In vitro: In glass. Refers to assays that are carried out in an artificial system (e.g., in a test
tube or petri dish) and typically use single-cell organisms, cultured cells, cell-free extracts, or
purified cellular components.
In vivo: In the living organism. Refers to assays performed in multicellular organisms.
Kow: Octanol:water partition coefficient.
LC50: Acute lethal serum or blood concentrations.
LD50: The calculated value of the oral dose that produces lethality in 50% of test animals(rats and mice). The LD50 values serve as reference values for the in vitro tests.
LD50 (initial): Acute oral rat and mouse LD50 values used during the chemical selection
process. For RC chemicals, LD50 values were those used in the RC database, which werelargely from the 1983/84 RTECS®. For chemicals that were not included in the RC, theinitial LD50 values came from HSDB or 2002 RTECS®.

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LD50 (reference): Acute oral rodent LD50 values from rats and mice were located throughliterature searches and references from major toxicity databases such as RTECS®. Studies
were reviewed to identify the most appropriate LD50 values for each chemical. Values
obtained using feral animals, preanesthetized animals, or animals less than 4 weeks of age
were not used. Values reported as inequalities were not used. Reference LD50 values were
determined by calculating the geometric mean of the acceptable LD50 values. Data were usedin generation of the laboratory-specific and combined-laboratory 3T3 and NHK NRU
regressions.
Maximum:minimum value: Ratio of minimum acceptable LD50 (or IC50) to maximum
acceptable LD50 (or IC50).
MEIC: Multicentre Evaluation of In Vitro Cytotoxicity. An international effort establishedby the Scandinavian Society for Cell Toxicology and initiated in 1983 to evaluate therelationship and relevance of in vitro cytotoxicity for predicting the acute toxicity ofchemicals in humans.
Millimolar regressions: Linear regressions with IC50 values in mmol/L and LD50 values in
mmol/kg.
Negative control: An untreated sample containing all components of a test system, except
the test substance solvent, which is replaced with a known non-reactive material, such as
water. This sample is processed with test substance-treated samples and other control
samples to determine whether the solvent interacts with the test system.
Neutral red (NR): A weakly cationic water-soluble dye that stains living cells by readily
diffusing through the plasma membrane and concentrating in lysosomes where it
electrostatically binds to the anionic lysosomal matrix.
Neutral red uptake (NRU): Concentration of neutral red dye in the lysosomes of living
cells. Altering the cell surface or the lysosomal membrane by a toxicological agent causes
lysosomal fragility and other adverse changes that gradually become irreversible. The NRU
test method makes it possible to distinguish between viable, damaged, or dead cells because
these changes result in decreased uptake and binding of NR measurable by optical density
absorption readings in a spectrophotometer.
NHK: Normal Human epidermal Keratinocytes (from neonatal foreskin).
Optical density (OD): The absorption (i.e., OD measurement) of the resulting coloredsolution (colorimetric endpoint) in the NRU assay measured at 540 nm ± 10 nm in a
spectrophotometric microtiter plate reader using blanks as a reference
Outlier: For any measurement, an extreme value in the NICEATM/ECVAM study wasreferred to as an “outlier” if it passes a statistical test for outliers at the 99% level. With
respect to chemicals, it refers to chemicals that do not fit (using the specified criteria) an
IC50-LD50 linear regression model. It may also refer to chemicals for which the predicted

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acute oral GHS toxicity category does not match the reference in vivo GHS acute oral
toxicity category.
Performance2: The accuracy and reliability characteristics of a test method (see “accuracy”,“reliability”).
pH: A measure of the acidity or alkalinity of a solution. pH 7.0 is neutral; higher pHs arealkaline, lower pHs are acidic.
Plate reader: A spectrophotometric device for measuring light intensity as a function ofcolor/wavelength (i.e., optical density/absorption at 540 nm ± 10 nm for NRU) in 96-well
microtiter tissue culture plates.
Positive control: A sample containing all components of a test system and treated with asubstance known to induce a positive response, which is processed with the test substancetreated and other control samples to demonstrate the sensitivity of each experiment and toallow for an assessment of variability in the conduct of the assay over time.
Predictivity2: Proportion of in vivo category matches for all substances with in vitro
predictions for a particular category. Predictivity is an indicator of test accuracy.
Protocol2: The precise, step-by-step description of a test, including the listing of allnecessary reagents, criteria and procedures for the evaluation of the test data.
Quality assurance (QA)2: A management process by which adherence to laboratory testing
standards, requirements, and record keeping procedures is assessed independently by
individuals other than those performing the testing.
Quality control (QC): A management process for ensuring products or services are designed
and produced to adhere to a defined set of quality criteria to meet or exceed customer
requirements; similar to quality assurance.
Range finder: Initial test performed to determine starting doses for the main (definitive) test.
The NRU assays test eight concentrations of the test chemical or the PC by diluting the stock
solution in log dilutions to cover a large concentration range.
RC millimole regression: log (LD50) = 0.435 x log (IC50) + 0.625; for estimating an LD50
value in mmol/kg (body weight) from an IC50 value in mM. Developed using the 347 IC50
and oral LD50 (282 rat and 65 mouse) values from the RC.
RC rat-only millimole regression: log (LD50) = 0.439 x log (IC50) + 0.621; for estimating an
LD50 value in mmol/kg (body weight) from an IC50 value in mM. Developed from the IC50
values (in mM) and acute oral LD50 values (in mmol/kg) for the 282 substances with rat LD50
values in the RC database (Halle 1998, 2003).

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RC rat-only weight regression: log (LD50) = 0.372 x log (IC50) + 2.024; for estimating an
LD50 value in mg/kg (body weight) from an IC50 value in µg/mL. Developed from the IC50
values (in µg/mL) and acute oral LD50 values (in mg/kg) for the 282 substances with rat LD50
values in the RC database (Halle 1998, 2003).
Reduction alternative2: A new or modified test method that reduces the number of animals
required.
Reference substances: Substances selected for use during the research, development,
prevalidation, and validation of a proposed test method because their response in the in vivo
reference test method or the species of interest is known (see “reference test”). Reference
substances should represent the classes of chemicals for which the proposed test method is
expected to be used and cover the range of expected responses (negative, weak to strong
positive).
Reference test method2: The accepted in vivo test method used for regulatory purposes toevaluate the potential of a test substance to be hazardous to the species of interest.
Refinement alternative2: A new or modified test method that refines procedures to lessen or
eliminate pain or distress in animals or enhances animal well-being.
Registry of Cytotoxicity (RC): Database that consists of in vivo acute oral toxicity data (i.e.,
LD50 values) from rats and mice and in vitro cytotoxicity data (i.e., IC50 values) frommultiple cell lines and cytotoxicity endpoints for 347 chemicals with known molecularweights (Halle 1998, 2003). A regression model constructed from these data was proposed
by ZEBET, as a method to reduce animal use by identifying the most appropriate starting
doses for acute oral systemic toxicity tests.
Relevance2: The extent to which a test method correctly predicts or measures the biologicaleffect of interest in humans or another species of interest. Relevance incorporates
consideration of the “accuracy” or “concordance” of a test method.
Reliability2: A measure of the degree to which a test method can be performed reproducibly
within and among laboratories over time. It is assessed by calculating intra- and interlaboratory reproducibility and intralaboratory repeatability.
Replacement alternative2: A new or modified test method that replaces animals withnonanimal systems or one animal species with a phylogenetically lower one (e.g., a mammal
with an invertebrate).
Reproducibility2: The consistency of individual test results obtained in a single laboratory(intralaboratory reproducibility) or in different laboratories (interlaboratory reproducibility)
using the same protocol and test substances (see intra- and inter-laboratory reproducibility).
RTECS®: Registry of Toxic Effects for Chemical Substances. Compendium of data
extracted from the open scientific literature. The database includes toxicity data (e.g., acute

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toxicity) and specific numeric toxicity values (e.g., LD50). Compiled by the U.S. National
Institute for Occupational Safety and Health (NIOSH) and now licensed to MDL Information
Systems, Inc.
Sensitivity2: The proportion of all positive substances that are classified correctly as positivein a test method. It is a measure of test method accuracy.
Simulation modeling: Computer simulation modeling of the acute systemic toxicity assays
to determine animal use. The simulation process uses a simulated population of animals fortesting, a reference endpoint (i.e., “true” LD50 value), and its assumed log-normal
distribution. Mortality is assumed to have a mean equal to the log of the true LD50. The SD,
which reflects the variability of the simulated population, is the inverse of the slope of thedose-mortality curve. Due to a lack of information for the real dose-mortality curve, thesimulations assumed slopes of 0.5, 0.8, 2, 4, and 8.3.
Solubility: The amount of a test substance that can be dissolved (or thoroughly mixed with)
culture medium or solvent. The solubility protocol was based on a U.S. EPA guideline (EPA
1998) that involves testing for solubility in a particular solvent, beginning at a relatively highconcentration and proceeding to successively lower concentrations by adding more solvent as
necessary for dissolution. Testing stops when, upon visual observation, the procedure
produces a clear solution with no cloudiness or precipitate.
Solvent control: An untreated sample containing all components of a test system, including
the solvent that is processed with the test substance-treated and other control samples toestablish the baseline response for the samples treated with the test substance dissolved in thesame solvent. When tested with a concurrent negative control, this sample also demonstrates
whether the solvent interacts with the test system.
Specificity2: The proportion of all negative substances that are classified correctly as
negative in a test method. It is a measure of test method accuracy.
Spirit of GLP: Guidance provided in the Statement of Work specifically for the non GLPcompliant laboratory that participated in the validation study. Based on the GLP standards
referenced in the ECVAM Workshop 37 Report (Cooper-Hannan et al. 1999) and the OECD
Principles of GLP (OECD 1998). “Laboratories that are non GLP-compliant shall adhere to
GLP principles and other method parameters. Documentation and accountability shall beequal to GLP requirements. Laboratories must make assurances that they are equal in
performance criteria and that there is parity amongst the laboratories.”
TESS: Toxic Exposure Surveillance System. A comprehensive poisoning surveillance
database maintained by the American Association of Poison Control Centers (AAPCC).
Test2: The experimental system used; used interchangeably with “test method” and “assay”.
Test method2: A process or procedure used to obtain information on the characteristics of a
substance or agent. Toxicological test methods generate information regarding the ability of a

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substance or agent to produce a specified biological effect under specified conditions. Used
interchangeably with “test” and “assay”. See also “validated test method” and “reference
test”.
Test method component: Structural, functional, and procedural elements of a test methodthat are used to develop the test method protocol. These components include uniquecharacteristics of the test method, critical procedural details, and quality control measures.
3T3: BALB/c 3T3 clone A31 mouse fibroblasts developed in 1968 from disaggregated 14- to
17-day-old BALB/c mouse embryos (American Type Culture Collection [ATCC]; # CCL163).
Tiered testing: A testing strategy where all existing information on a test substance isreviewed, in a specified order, before in vivo testing.
Toxicity underpredicted: Measured l LD50 value of a test substance is lower than thepredicted LD50 value.
Toxicity overpredicted: Measured LD50 value of a test substance is higher than thepredicted LD50 value.
Transferability2: The ability of a test method or procedure to be accurately and reliably
performed in different, competent laboratories.
Up-and-Down Procedure (UDP): An acute oral systemic toxicity test method used tominimize the number of animals required to estimate the acute oral toxicity of a chemical,
estimate the LD50 and confidence interval (CI), and observe signs of toxicity. Single animals
are tested sequentially. Subsequent doses are based on the outcome of the previous animal.
Validated test method2: An accepted test method for which validation studies have been
completed to determine the accuracy and reliability of this method for a specific proposed
use.
Validation2: The process by which the reliability and accuracy of a procedure are established
for a specific purpose.
Vehicle control (VC): The VC consists of appropriate cell culture medium for the cells in
the test (i.e., DMEM for 3T3 cells and keratinocyte growth medium for the NHK cells). For
chemicals dissolved in DMSO, the VC consists of medium with the same amount of solvent
as that used in the test chemical concentrations that are applied to the 96-well test plate. The
final DMSO concentration is ≤0.5% (v/v) in the VCs.
Volatility: Ability of a test chemical to evaporate. A general indicator of volatility issues in
the NRU test methods is the percent difference in the mean OD values for the two VC
columns on the test plate. If the difference is greater than 15%, then chemical volatility canbe suspected, especially if the VC adjacent to the highest test concentration had a

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significantly reduced OD value. Volatility may be an issue for compounds with a specificgravity of less than 1.
Weight of evidence (process): The strengths and weaknesses of a collection of information
are used as the basis for a conclusion that may not be evident from the individual data.
Weight regressions: Linear regressions with IC50 values in µg/mL and LD50 values in
mg/kg.
ZEBET: The German National Center for the Documentation and Evaluation of Alternative
Methods to Animal Experiments.

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NIH Publication No: 07-4518

BACKGROUND REVIEW DOCUMENTIn Vitro Cytotoxicity Test Methods for Estimating
Acute Oral Systemic Toxicity

Volume 2 of 2
Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)
National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)
National Institute of Environmental Health Sciences
National Institutes of Health
U. S. Public Health Service
Department of Health and Human Services

In Vitro Cytotoxicity Test Methods
for Estimating Acute Oral Systemic Toxicity
Background Review Document
Volume 2 of 2
Prepared by
The National Toxicology Program (NTP) Interagency Centerfor the Evaluation of Alternative Toxicological Methods (NICEATM)

National Institute of Environmental Health Sciences (NIEHS)
P.O. Box 12233
Mail Drop: EC-17
Research Triangle Park, NC 27709

November 2006
NIH Publication No. 07-4518
National Institute of Environmental Health Sciences
National Institutes of Health
U.S. Public Health Service
Department of Health and Human Services

This document is available electronically at:
http://iccvam.niehs.nih.gov/methods/invitro.htm

Printed: 11/30/2006

In Vitro Cytotoxicity Test Methods BRD Table of Contents

November 2006

TABLE OF CONTENTSAPPENDICES.................................................................................................................. xiii
LIST OF FIGURES .......................................................................................................... xvi
LIST OF TABLES...........................................................................................................xvii
LIST OF ACRONYMS AND ABBREVIATIONS ........................................................... xx
ACKNOWLEDGMENTS................................................................................................ xxv
PREFACE........................................................................................................................xxix
EXECUTIVE SUMMARY............................................................................................ xxxiii
VOLUME 1 OF 2
1.0

INTRODUCTION AND RATIONALE FOR THE USE OF IN VITRO
NEUTRAL RED UPTAKE CYTOTOXICITY TEST METHODS TO
PREDICT STARTING DOSES FOR IN VIVO ACUTE ORALTOXICITY TESTING .........................................................................................1-3
1.1

Historical Background and Rationale for the Use of In Vitro
Cytotoxicity Assays to Predict Starting Doses for Rodent Acute
Oral Toxicity Tests....................................................................................1-4
1.1.1
The Multicentre Evaluation of In Vitro Cytotoxicity (MEIC)
Program........................................................................................1-4
1.1.2
An International Evaluation of Selected In Vitro Toxicity
Test Systems for Predicting Acute Systemic Toxicity ...................1-6
1.1.3
The Registry of Cytotoxicity (RC) ................................................1-7
1.1.4
The ZEBET Initiative to Reduce Animal Use ...............................1-9
1.1.5
The International Workshop on In Vitro Methods forAssessing Acute Systemic Toxicity ...........................................1-10
1.1.6
The NICEATM/ECVAM In Vitro NRU Cytotoxicity Study .......1-11
1.1.6.1
Study Design .............................................................1-12

1.2

Regulatory Rationale and Applicability for the Use of In Vitro
Cytotoxicity Test Methods to Predict Starting Doses for Acute
Oral Toxicity Testing in Rodents............................................................1-14
1.2.1
Current Regulatory Testing Requirements for Acute Oral
Toxicity ......................................................................................1-14
1.2.1.1
Test Methods for Assessing Acute Oral Toxicity ........1-16
1.2.2
Intended Regulatory Uses for In Vitro Cytotoxicity Test
Methods......................................................................................1-16
1.2.3
Similarities and Differences in the Endpoints of In Vitro
Cytotoxicity Test Methods and Rodent Acute Oral Toxicity
Test Methods ..............................................................................1-17
1.2.4
Use of In Vitro Cytotoxicity Test Methods in the Overall
Strategy of Hazard Assessment ...................................................1-18i

In Vitro Cytotoxicity Test Methods BRD Table of Contents

1.3

2.0

November 2006

Scientific Basis for the In Vitro NRU Test Methods .............................. 1-18
1.3.1
Purpose and Mechanistic Basis of In Vitro NRU Test
Methods ..................................................................................... 1-19
1.3.2
Similarities and Differences in the Modes/Mechanisms of
Action for In Vitro NRU Test Methods Compared with theSpecies of Interest ...................................................................... 1-20
1.3.3
Range of Substances Amenable to the In Vitro NRU Test
Methods ..................................................................................... 1-20

TEST METHOD PROTOCOL COMPONENTS OF THE 3T3 AND NHK
IN VITRO NRU TEST METHODS .....................................................................2-3
2.1

Basis for Selection of In Vitro NRU Cytotoxicity Test Methods..............2-4
2.1.1
Guidance Document Rationale for Selection of In Vitro NRU
Cytotoxicity Test Methods............................................................2-4
2.1.2
Guidance Document Rationale for Selection of Cell Types...........2-4

2.2

Overview of the 3T3 and NHK NRU Test Methods ................................2-5
2.2.1
The 3T3 NRU Test Method ..........................................................2-6
2.2.1.1
Initiating and Subculturing 3T3 Cells..........................2-6
2.2.1.2
Preparation of Cells for 96-well Plate Assays .............2-8
2.2.1.3
Reference Substance Application.................................2-8
2.2.2
The NHK NRU Test Method .......................................................2-8
2.2.2.1
Initiating and Subculturing NHK Cells........................2-8
2.2.2.2
Preparation of Cells for 96-well Plate Assays .............2-8
2.2.2.3
Reference Substance Application.................................2-9
2.2.3
Measurement of NRU in the 3T3 and NHK Test Methods ............2-9

2.3

Descriptions and Rationales of the 3T3 and NHK NRU Test
Methods.....................................................................................................2-9
2.3.1
Materials, Equipment, and Supplies..............................................2-9
2.3.1.1
3T3 Cells.....................................................................2-9
2.3.1.2
NHK Cells................................................................. 2-10
2.3.1.3
Tissue Culture Materials and Supplies ...................... 2-10
2.3.1.4
Cell Culture Materials .............................................. 2-10
2.3.1.5
Equipment................................................................. 2-10
2.3.1.6
Culture Medium ........................................................ 2-11
2.3.2
Reference Substance Concentrations/Dose Selection .................. 2-12
2.3.2.1
Range Finder Test..................................................... 2-12
2.3.2.2
Definitive Test ........................................................... 2-12
2.3.3
NRU Endpoints Measured .......................................................... 2-13
2.3.3.1
NRU and Measurement ............................................. 2-13
2.3.3.2
Determination of IC50, IC20, and IC80 Values .............. 2-13
2.3.4
Duration of Reference Substance Exposure ................................ 2-14
2.3.5
Known Limits of Use ................................................................. 2-14
2.3.5.1
Solubility/Precipitation/Volatility.............................. 2-14
2.3.5.2
Biokinetic Determinations ......................................... 2-15
2.3.5.3
Organ-Specific Toxicity............................................. 2-15

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2.3.5.4

2.3.6
2.3.7

2.3.8
2.3.9
2.3.102.3.11
2.3.122.3.132.3.14The Role of Cytotoxicity Tests in an In Vitro
Battery Approach for Possible Replacement of In
Vivo Acute Toxicity Testing .......................................2-15
Basis of the Response Assessed ..................................................2-16
Appropriate Positive, Vehicle, and Negative Controls.................2-16
2.3.7.1
Positive Control ........................................................2-16
2.3.7.2
Vehicle Control .........................................................2-16
2.3.7.3
Negative Control .......................................................2-17
Acceptable Ranges of Control Responses ...................................2-17
2.3.8.1
Vehicle Controls as a Quality Control Tool ...............2-18
Nature of Experimental Data Collected.......................................2-18
2.3.9.1
NRU OD Measurements ............................................2-18
2.3.9.2
Information and Data Collected ................................2-18
Data Storage Media ....................................................................2-19
Measures of Variability ..............................................................2-19
Methods for Analyzing NRU Data..............................................2-20
Decision Criteria for Classification of Reference Substances ......2-20
Information and Data Included in the Test Report.......................2-20

2.4

Proprietary Components of the In Vitro NRU Test Methods................2-22

2.5

Basis for the Number of Replicate and Repeat Experiments for the
3T3 and NHK NRU Test Methods..........................................................2-22

2.6

Basis for Modifications to the 3T3 and NHK NRU Test Method
Protocols ..................................................................................................2-22
2.6.1
Phase Ia: Laboratory Evaluation Phase .......................................2-22
2.6.1.1
NR Dye Crystals........................................................2-23
2.6.1.2
3T3 Cell Growth........................................................2-23
2.6.1.3
NHK Cell Growth......................................................2-23
2.6.1.4
Vehicle Control OD Limits ........................................2-24
2.6.1.5
Precipitate Formation ...............................................2-24
2.6.1.6
Dilution Factor .........................................................2-24
2.6.1.7
Test Acceptance Criteria ...........................................2-25
2.6.2
Phase Ib: Laboratory Evaluation Phase .......................................2-25
2.6.2.1
NR Crystal Formation ...............................................2-25
2.6.2.2
Heating of Reference Substance Solutions .................2-26
2.6.2.3
Growth of Untreated Cells.........................................2-27
2.6.2.4
Correction of Reference Substance OD Values ..........2-27
2.6.2.5
Laboratory Error Rates .............................................2-28
2.6.2.6
Resultant Protocol Changes for Phase II ...................2-28
2.6.2.7
Test Acceptance Criteria ...........................................2-29
2.6.3
Phase II: Laboratory Qualification Phase ....................................2-29
2.6.3.1
Testing of Volatile Reference Substances ...................2-29
2.6.3.2
Atypical Concentration-Responses ............................2-31
2.6.3.3
Hill Function.............................................................2-33
2.6.3.4
Insoluble Reference Substances .................................2-33
2.6.3.5
Inadequate Cell Growth in NHK Medium..................2-34
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2.6.3.6

Performance Standards for Media to Support
NHK Growth............................................................. 2-34
2.6.3.7
Test Acceptance Criteria for Phase II........................ 2-35
2.6.4 Phase III: Laboratory Testing Phase .............................................. 2-35
2.6.4.1
Required Cytotoxicity Values .................................... 2-35
2.6.4.2
Revisions to Data Analysis Procedures ..................... 2-35

3.0

2.7

Differences Between the 3T3 and NHK NRU Protocols for the
Validation Study and the Guidance Document Standard Protocols...... 2-36

2.8

Overview of the Solubility Protocol........................................................ 2-37

2.9

Basis of the Solubility Protocol............................................................... 2-38
2.9.1
Initial Solubility Protocol Development...................................... 2-38
2.9.2
Basis for Modification of the Phase II Protocol........................... 2-39

2.10

Components of the Solubility Protocol................................................... 2-40
2.10.1 Medium, Supplies, and Equipment Required .............................. 2-40
2.10.1.1 Medium and Chemical Supplies ................................ 2-40
2.10.1.2 Equipment................................................................. 2-40
2.10.1.3 Procedures................................................................ 2-40
2.10.2 Data Collection........................................................................... 2-41
2.10.3 Variability in Solubility Measurements....................................... 2-41
2.10.4 Solubility Issues During the Testing of the Reference
Substances.................................................................................. 2-41
2.10.5 Analysis of Solubility Data......................................................... 2-41

2.11

Summary ................................................................................................. 2-42

REFERENCE SUBSTANCES USED FOR VALIDATION OF THE 3T3
AND NHK NRU TEST METHODS ....................................................................3-3
3.1

Rationale for the 72 Reference Substances Selected for Testing ............3-3
3.1.1
Reference Substance Selection Criteria.........................................3-3
3.1.2
Candidate Reference Substances...................................................3-4
3.1.3
Selection of Reference Substances for Testing..............................3-5

3.2

Characteristics of the Selected Reference Substances .............................3-6
3.2.1
Source Databases Represented by the Selected Reference
Substances....................................................................................3-6
3.2.2
Chemical Classes Represented by the Selected Reference
Substances.................................................................................. 3-28
3.2.3
Product/Use Classes Represented by the Selected Reference
Substances.................................................................................. 3-28
3.2.4
Toxicological Characteristics of the Selected Reference
Substances.................................................................................. 3-28
3.2.4.1
Corrosivity ................................................................ 3-28
3.2.4.2
Toxicity Targets ........................................................ 3-32
3.2.4.3
Metabolism ............................................................... 3-32
3.2.5
Selection of Reference Substances for Testing in Phases Ib
and II.......................................................................................... 3-32
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3.2.6

4.0

5.0

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Unsuitable and Challenging Reference Substances .....................3-34

3.3

Reference Substance Procurement, Coding, and Distribution..............3-34
3.3.1
Exceptions..................................................................................3-35

3.4Reference Substances Recommended by the Guidance Document........3-35

3.5

Summary .................................................................................................3-36

RODENT ACUTE ORAL LD50 REFERENCE VALUES USED TO
ASSESS THE ACCURACY OF THE 3T3 AND NHK NRU TESTMETHODS ...........................................................................................................4-3
4.1

Methods Used to Obtain Rodent Acute Oral LD50 Reference
Values ........................................................................................................4-3
4.1.1
Identification of Candidate Rodent Acute Oral LD50
Reference Data .............................................................................4-3
4.1.2
Criteria Used to Select Candidate Rodent Acute Oral Data for
Determination of LD50 Reference Values......................................4-5
4.1.2.1
Final Exclusion Criteria ..............................................4-6
4.1.2.2
Assumptions Regarding Materials, Animals, andMethods ......................................................................4-6
4.1.2.3
Calculation Reference LD50 Values .............................4-7
4.1.2.4
Use of Rat and Mouse Data.........................................4-7

4.2

Final Rodent Acute Oral LD50 Reference Values ....................................4-7

4.3

Relevant Toxicity Information for Humans.............................................4-8

4.4

Accuracy and Reliability of the Rodent Acute Oral LD50 Reference
Values ......................................................................................................4-13
4.4.1
Variability Among the Acceptable LD50 Values..........................4-13
4.4.2
Comparison of Rodent Acute Oral LD50 Reference Values
with the Corresponding RC LD50 Values ...................................4-14
4.4.3
Comparison of the Variability Among Acceptable LD50
Values to Those Obtained in Other Studies .................................4-15

4.5

Summary .................................................................................................4-16

3T3 AND NHK NRU TEST METHOD DATA AND RESULTS .......................5-3
5.1

Study Timeline and Participating Laboratories ......................................5-3
5.1.1
Statements of Work (SOW) and Protocols ....................................5-3
5.1.2
Study Timeline .............................................................................5-4
5.1.3
Participating Laboratories.............................................................5-4

5.2

Coded Reference Substances and GLP Guidelines..................................5-5
5.2.1
Coded Reference Substances ........................................................5-5
5.2.2
Lot-to-Lot Consistency of Reference Substances ..........................5-5
5.2.3
Adherence to GLP Guidelines.......................................................5-5

5.3

3T3 and NHK NRU Test Method Protocols.............................................5-5
5.3.1
Phase Ia: Laboratory Evaluation Phase .........................................5-6v

In Vitro Cytotoxicity Test Methods BRD Table of Contents

5.3.2
5.3.3
5.3.4

November 2006

5.3.1.1
Protocol Changes and the Effect on the Data ..............5-6
Phase Ib: Laboratory Evaluation Phase.........................................5-6
5.3.2.1
Protocol Changes and the Effect on the Data ..............5-7
Phase II: Laboratory Qualification Phase ......................................5-7
5.3.3.1
Protocol Changes and the Effect on the Data ..............5-8
Phase III: Main Validation Phase..................................................5-9
5.3.4.1
Protocol Changes and the Effect on the Data ..............5-9

5.4

Data Used to Evaluate Test Method Accuracy and Reliability ............. 5-10
5.4.1
PC Data...................................................................................... 5-10
5.4.1.1
Phase Ib PC Data Acceptance Limits ........................ 5-12
5.4.1.2
Phase II PC Data Acceptance Limits......................... 5-12
5.4.1.3
Phase III PC Data Acceptance Limits ....................... 5-12
5.4.2
Reference Substance Data .......................................................... 5-12

5.5

Statistical Approaches to the Evaluation of 3T3 and NHK Data.......... 5-26
5.5.1
Statistical Analyses for Phase Ia Data......................................... 5-27
5.5.1.1
Outlier Determination for Replicate Well
Concentration Data................................................... 5-27
5.5.1.2
Curve Fit Criteria ..................................................... 5-27
5.5.1.3
Reproducibility Analyses for PC IC50 Values............. 5-27
5.5.2
Statistical Analyses for Phase Ib Data......................................... 5-28
5.5.2.1
Outlier Determination for Replicate Well
Concentration Data................................................... 5-28
5.5.2.2
Reproducibility Analyses of the Reference
Substance IC50 Values ............................................... 5-28
5.5.3
Statistical Analyses for Phase II Data ......................................... 5-28
5.5.3.1
Outlier Determination for Replicate Well
Concentration Data................................................... 5-28
5.5.3.2
Reproducibility Analyses of the Reference
Substance IC50 Values ............................................... 5-28
5.5.3.3
Comparison of 3T3 and NHK Test Results with
the RC Millimole Regression ..................................... 5-29
5.5.4
Statistical Analyses for Phase III Data ........................................ 5-29
5.5.4.1
Outlier Determination for Replicate Well
Concentration Data................................................... 5-29
5.5.4.2
Reproducibility Analyses of the PC IC50 Data ........... 5-29
5.5.4.3
Reproducibility Analyses of the Reference
Substance
IC50 Values................................................................ 5-30
5.5.4.4
Comparison of 3T3 and NHK Test Results with
the RC Millimole Regression ..................................... 5-31
5.5.5
Summary of the Data Used for Statistical Analyses .................... 5-32

5.6

Summary of NRU Test Results............................................................... 5-32

5.7

Availability of Data ................................................................................. 5-38

5.8

Solubility Test Results ............................................................................ 5-38

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5.8.1
5.8.2
5.9
6.0

November 2006

Solubility Data............................................................................5-43
Solubility and Volatility Effects in the Cytotoxicity Tests...........5-43

Summary .................................................................................................5-44

ACCURACY OF THE 3T3 AND NHK NRU TEST METHODS ......................6-3
6.1

Accuracy of the 3T3 and NHK NRU Test Methods for Predicting
Rodent Acute Oral Toxicity......................................................................6-5
6.1.1
Linear Regression Analyses for the Prediction of Rat Acute
Oral LD50 Values from In Vitro IC50 Values..................................6-5
6.1.2
Comparison of Combined-Laboratory 3T3 and NHK
Regressions to the RC Millimole Regression ................................6-8

6.2

Analysis of Outlier Substances for the RC Millimole Regression ...........6-9
6.2.1
Identification of Outlier Substances ............................................6-10
6.2.2
Evaluation of Outlier Substances ................................................6-12
6.2.2.1
Physical Characteristics............................................6-12
6.2.2.2
Chemical Class .........................................................6-12
6.2.2.3
Solubility...................................................................6-13
6.2.2.4
Metabolism................................................................6-13
6.2.2.5
Mechanism of Toxicity...............................................6-13

6.3

Improving the Prediction of In Vivo Rat Oral LD50 Values from In
Vitro IC50 Data .........................................................................................6-17
6.3.1
The RC Rat-Only Millimole Regression .....................................6-17
6.3.2
The RC Rat-Only Weight Regression .........................................6-17

6.4

Accuracy of the 3T3 and NHK NRU Test Methods for Predicting
GHS Acute Oral Toxicity Categories .....................................................6-18
6.4.1
Prediction of GHS Acute Oral Toxicity Category by the RC
IC50 Values Using the RC Millimole Regression.........................6-20
6.4.2
Prediction of GHS Acute Oral Toxicity Category by the 3T3
and NHK NRU Test Methods Using the RC Rat-Only
Millimole Regression..................................................................6-22
6.4.2.1
In Vitro – In Vivo Concordance Using the RC
Rat-Only Millimole Regression..................................6-22
6.4.2.2
Discordant Substances in the Prediction of GHS
Acute Oral Toxicity Category by the 3T3 andNHK NRU Test Methods and the RC Rat-Only
Millimole Regression.................................................6-24
6.4.3
Prediction of GHS Acute Oral Toxicity Category by the 3T3
and NHK NRU Test Methods Using the RC Rat-Only
Weight Regression......................................................................6-24
6.4.3.1
In Vitro – In Vivo Concordance Using the RC
Rat-Only Weight Regression......................................6-25
6.4.3.2
Discordant Substances in the Prediction of GHS
Acute Oral Toxicity Category by the 3T3 andNHK NRU Test Methods and the RC Rat-Only
Weight Regression.....................................................6-27
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6.4.4

7.0

November 2006

Summary of the Regressions Evaluated ...................................... 6-27

6.5

Correlation of NRU Concentration-Response Slope with Rat
Lethality Dose-Response Slope............................................................... 6-28

6.6

Strengths and Limitations of the Use of In Vitro Cytotoxicity Test
Methods with the IC50–LD50 Regressions for Prediction of Rodent
Acute Oral Toxicity................................................................................. 6-31
6.6.1
In Vitro Cytotoxicity Methods .................................................... 6-31
6.6.2
Use of Mole-Based vs. Weight-Based Regressions for thePrediction of Toxicity for Low and High Molecular Weight
Substances.................................................................................. 6-31

6.7

Salient Issues of Data Interpretation...................................................... 6-34

6.8

Comparison of NRU Test Results to Established PerformanceStandards ................................................................................................ 6-34

6.9

Summary ................................................................................................. 6-40

RELIABILITY OF THE 3T3 AND NHK TEST METHODS ............................7-3
7.1

Reference Substances Used to Determine the Reliability of the 3T3
and NHK Test Methods ............................................................................7-3

7.2

Reproducibility Analyses for the 3T3 and NHK Test Methods ..............7-5
7.2.1
Comparison of Laboratory-Specific IC50-LD50 Linear
Regression Analyses to the Mean Laboratory Regression .............7-5
7.2.2
ANOVA Results for the 3T3 and NHK NRU Test Methods .........7-5
7.2.2.1
Differences Among the IC50 Values inLaboratories Using the 3T3 NRU Test Method............7-5
7.2.2.2
Differences Among the IC50 Values inLaboratories Using the NHK NRU Test Method........ 7-21
7.2.3
CV Results for the 3T3 and NHK NRU Test Methods ................ 7-21
7.2.3.1
Reproducibility of Intralaboratory CV Values ........... 7-21
7.2.3.2
Reproducibility of Interlaboratory CV Values ........... 7-34
7.2.3.3
Variation of CV with Chemical Property ................... 7-34
7.2.3.4
Results of the Intralaboratory CV Analysis................ 7-35
7.2.3.5
Results of the Interlaboratory CV Analysis ................ 7-37
7.2.4
Comparison of Maximum to Minimum IC50 Values Using
Laboratory Means ...................................................................... 7-39
7.2.5
Comparison of Maximum:Minimum IC50 Ratios with the
Maximum:Minimum LD50 Ratios............................................... 7-40
7.2.6
Normalization of Reference Substance IC50 Values Using
SLS IC50 Values ......................................................................... 7-42

7.3

Historical Positive Control (PC) Data ................................................... 7-44
7.3.1
ANOVA and Linear Regression Results for the 3T3 NRU
Test Method .............................................................................. 7-46
7.3.1.1
Variation of the SLS IC50 Values with Time ............... 7-46
7.3.1.2
Comparison of SLS IC50 Values Among theLaboratories ............................................................. 7-46
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7.3.2

8.0

9.0

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ANOVA and Linear Regression Results for the NHK NRU
Test Method ...............................................................................7-48
7.3.2.1
Variation of SLS IC50 Values with Time.....................7-48
7.3.2.2
Comparison of SLS IC50 Values Among theLaboratories..............................................................7-48

7.4

Laboratory Concordance for Solvent Selection .....................................7-50

7.5

Summary .................................................................................................7-52

3T3 AND NHK NRU TEST METHOD DATA QUALITY ................................8-3
8.1

Compliance with Good Laboratory Practice Regulations.......................8-3
8.1.1
Guidelines Followed for Cytotoxicity Testing...............................8-3
8.1.1.1
Good Laboratory Practices .........................................8-3
8.1.1.2
Spirit of GLP ...............................................................8-3
8.1.1.3
Good Cell Culture Practices (GCCP)..........................8-4
8.1.2
Quality Assurance (QA) for NRU Cytotoxicity Test Data.............8-4
8.1.2.1
Coded Reference Substances .......................................8-4
8.1.2.2
Solubility Testing and Data Review .............................8-5
8.1.2.3
NRU Cytotoxicity Test Tallies......................................8-5
8.1.3
Guidelines Followed for Rodent Acute Oral LD50 Data
Collection .....................................................................................8-6
8.1.3.1
Rodent Acute Oral LD50 Values Used in theRegistry of Cytotoxicity (RC).......................................8-6
8.1.3.2
Rodent Acute Oral LD50 Values Collected by
NICEATM from Other Sources ....................................8-6

8.2

Results of Data Quality Audits .................................................................8-6
8.2.1
QA Statements..............................................................................8-6
8.2.2
QA Statements from the Laboratories ...........................................8-7
8.2.2.1
BioReliance QA Statements .........................................8-7
8.2.2.2
FAL QA Statements .....................................................8-7
8.2.2.3
ECBC QA Statements ..................................................8-7
8.2.2.4
IIVS QA Statements .....................................................8-8
8.2.2.5
Other QA Information .................................................8-8

8.3

Effect of Deviations or Non-compliance with GLPs ................................8-9
8.3.1
Laboratory Error Rates .................................................................8-9
8.3.2
Failure Rates for Definitive and PC Tests ...................................8-10
8.3.3
Intralaboratory Reproducibility...................................................8-11
8.3.4
Prediction of GHS Acute Oral Toxicity Categories .....................8-12

8.4

Availability of Laboratory Notebooks....................................................8-12

8.5

Summary .................................................................................................8-13

OTHER SCIENTIFIC REPORTS AND REVIEWS OF IN VITROCYTOTOXICITY TEST METHODS AND THEIR ABILITY TO
PREDICT IN VIVO ACUTE TOXICITY AND OTHER TOXICEFFECTS..............................................................................................................9-3ix

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November 2006

9.1

Relevant Studies........................................................................................9-3
9.1.1
Correlation of NRU Cytotoxicity Values with Rodent
Lethality.......................................................................................9-3
9.1.1.1
Peloux et al. (1992).....................................................9-4
9.1.1.2
Fautrel et al. (1993) ....................................................9-5
9.1.1.3
Roguet et al. (1993).....................................................9-5
9.1.1.4
Rasmussen (1999) .......................................................9-6
9.1.1.5
Creppy et al. (2004) ....................................................9-7
9.1.2
Use of In Vitro Cytotoxicity Data to Reduce the Use of
Animals in Acute Oral Toxicity Testing .......................................9-7
9.1.2.1
Halle et al. (1997): Animal Savings with the ATC
Method Using Cytotoxicity Data .................................9-7
9.1.2.2
Spielmann et al. (1999): Animal Savings Using
Cytotoxicity Data with the UDP ..................................9-9
9.1.2.3
EPA (2004): U.S. EPA HPV Challenge Program
Submission ................................................................ 9-10
9.1.3 Other Evaluations of 3T3 or NHK NRU Methods.......................... 9-10
9.1.3.1
Draize Eye Irritation ................................................. 9-11
9.1.3.2
Predicting Human Lethal Blood Concentrations(LC) .......................................................................... 9-13

9.2

Independent Scientific Reviews.............................................................. 9-13
9.2.1
In Vitro Acute Toxicity Testing for the Classification andLabeling of Chemicals................................................................ 9-13
9.2.1.1
Seibert et al. (1996): ECVAM Workshop 16 .............. 9-13
9.2.2
Use of In Vitro Cytotoxicity Data for Estimation of Starting
Doses for Acute Oral Toxicity Testing ....................................... 9-14
9.2.2.1
ICCVAM (2001a): Estimation of Animal Savings
Using Cytotoxicity Data with the ATC Method .......... 9-14
9.2.2.2
ICCVAM (2001a): Estimation of Animal Savings
Using Cytotoxicity Data with the UDP ...................... 9-16
9.2.3
Validation of 3T3 NRU Assay for Phototoxicity......................... 9-17
9.2.3.1
NHK NRU Phototoxicity Assay.................................. 9-17

9.3

Studies Using In Vitro Cytotoxicity Test Methods with Established
Performance Standards .......................................................................... 9-18
9.3.1
Guidance Document (ICCVAM 2001b)....................................... 9-18
9.3.2
King and Jones (2003)................................................................ 9-19
9.3.3
A-Cute-Tox Project: Optimization and Pre-Validation of an
In Vitro Test Strategy for Predicting Human Acute Toxicity(Clemedson 2005) ...................................................................... 9-19

9.4

Summary ................................................................................................. 9-20

ANIMAL WELFARE CONSIDERATIONS (REFINEMENT,REDUCTION, AND REPLACEMENT) ........................................................... 10-3
10.1

Use of 3T3 and NHK NRU Test Methods to Predict Starting Doses
for Rodent Acute Oral Toxicity Assays.................................................. 10-4x

In Vitro Cytotoxicity Test Methods BRD Table of Contents

11.0

November 2006

10.2

Reduction and Refinement of Animal Use for the UDP ........................10-4
10.2.1 In Vivo Testing Using the UDP...................................................10-4
10.2.1.1 Main Test ..................................................................10-4
10.2.1.2 Limit Test ..................................................................10-5
10.2.2 Computer Simulation Modeling of the UDP ...............................10-6
10.2.3 Animal Savings in the UDP When Using 3T3- and NHKBased Starting Doses ..................................................................10-7
10.2.3.1 The Effect of the Dose-Mortality Slope on Animal
Use............................................................................10-7
10.2.3.2 Mean Animal Use for UDP Simulations –Comparison of Regressions and Predictions fromthe 3T3 and NHK NRU Test Methods ........................10-9
10.2.3.3 Animal Savings in the UDP by GHS Acute Oral
Toxicity Category Using 3T3- and NHK-Based
Starting Doses ...........................................................10-9
10.2.4 Refinement of Animal Use for the UDP When Using 3T3and NHK-Based Starting Doses ................................................10-15
10.2.5 Accuracy of the UDP Outcomes Using the IC50-Based
Starting Doses ..........................................................................10-16

10.3

Reduction and Refinement of Animal Use in the ATC Method ..........10-16
10.3.1 In Vivo Testing Using the ATC Method....................................10-16
10.3.1.1 Main Test ................................................................10-17
10.3.1.2 Limit Test ................................................................10-17
10.3.2 Computer Simulation Modeling of the ATC Method ................10-17
10.3.3 Animal Savings for the ATC Method When Using 3T3- andNHK-Based Starting Doses ......................................................10-18
10.3.3.1 The Effect of Dose-Mortality Slope on Animal
Use..........................................................................10-18
10.3.3.2 Mean Animal Use for ATC Simulations –Comparison of Regressions and Predictions fromthe 3T3 and NHK NRU Test Methods ......................10-20
10.3.3.3 Animal Savings in the ATC Method by GHS Acute
Oral Toxicity Category Using the 3T3- and NHKBased Starting Doses...............................................10-22
10.3.4 Refinement of Animal Use in the ATC Method When Using
3T3- and NHK-Based Starting Doses........................................10-26
10.3.5 Accuracy of the ATC Method Outcomes Using the IC50Based Starting Doses ................................................................10-27

10.4

The Impact of Accuracy on Animal Savings........................................10-28

10.5

The Impact of Prevalence on Animal Savings......................................10-30

10.6

Summary ...............................................................................................10-31

PRACTICAL CONSIDERATIONS ..................................................................11-3
11.1

Transferability of the 3T3 and NHK NRU Test Methods .....................11-3
11.1.1 Facilities and Major Fixed Equipment ........................................11-3
xiIn Vitro Cytotoxicity Test Methods BRD Table of Contents

11.1.2

11.1.3

November 2006

11.1.1.1 Facility Requirements ............................................... 11-3
11.1.1.2 Cell Culture Laboratory............................................ 11-4
11.1.1.3 Major Equipment ...................................................... 11-4
Availability of Other Necessary Equipment and Supplies ........... 11-4
11.1.2.1 General Equipment ................................................... 11-4
11.1.2.2 Cell Culture Materials and Supplies.......................... 11-5
11.1.2.3 Cell Cultures............................................................. 11-5
Problems Specific to the NHK NRU Test Method ...................... 11-5

11.2

3T3 and NHK NRU Test Method Training Considerations.................. 11-6
11.2.1 Required Training and Expertise ................................................ 11-6
11.2.1.1 Specific Training and Expertise Needed .................... 11-6
11.2.1.2 General Laboratory Expertise Needed ...................... 11-7
11.2.2 Training Requirements to Demonstrate Proficiency.................... 11-7
11.2.2.1 Proficiency with GLP-Compliance ............................ 11-8
11.2.3 Personnel Needed to Perform the In Vitro NRU Test Methods.... 11-8

11.3

Cost Considerations ................................................................................ 11-8
11.3.1 3T3 and NHK NRU Test Methods.............................................. 11-8
11.3.1.1 Equipment Costs ....................................................... 11-8
11.3.1.2 Costs for Cell Cultures and Supplies ......................... 11-8
11.3.1.3 Commercial Testing .................................................. 11-9
11.3.2 Rodent Acute Oral Toxicity Testing ......................................... 11-11

11.4

Time Considerations for Performing the 3T3 and NHK NRU Tests .. 11-12
11.4.1 The 3T3 NRU Test Method ...................................................... 11-12
11.4.2 The NHK NRU Test Method.................................................... 11-12
11.4.3 Prequalification of NHK Medium............................................. 11-12
11.4.4 In Vivo Testing ......................................................................... 11-12
11.4.5 The Limit Test.......................................................................... 11-13

11.5

Summary ............................................................................................... 11-14

12.0

REFERENCES ................................................................................................... 12-1

13.0

GLOSSARY........................................................................................................ 13-1

xii

In Vitro Cytotoxicity Test Methods BRD Table of Contents

November 2006

APPENDICESVOLUME 2 OF 2
Appendix A NICEATM/ECVAM Validation Study Management.............................A-1
Appendix B Validation Study Test Method Protocols (Phase III) .............................. B-1
B1
Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake (NRU)
Cytotoxicity Test........................................................................................ B-3
B2
Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test .......................................... B-25
B3
Test Method Protocol for Solubility Determination (Phase III)................. B-47
B4
Test Method Procedure for Prequalification of Normal Human
Epidermal Keratinocyte Growth Medium (Phase III)................................ B-59
Appendix C Validation Study Test Method Protocols (Phases Ia, Ib, and II)............C-1
C1
Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase Ia) ....................................................................... C-3
C2
Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test (Phase Ia).......................... C-23
C3
Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase Ib) ..................................................................... C-41
C4
Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test (Phase Ib) ......................... C-63
C5
Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase II)...................................................................... C-85
C6
Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test (Phase II) ........................ C-109
Appendix D SAS Codes ................................................................................................D-1
D1
SAS Code for ANOVA and Contrasts ........................................................D-3
D2
SAS Code for Regression Comparisons......................................................D-7
Appendix E Neutral Red Dye Experiments ................................................................. E-1
E1
Institute for In Vitro Sciences (IIVS) Assessment of Protocol Variablesin the NICEATM/ECVAM Evaluation of Cytotoxicity Assays................... E-5
E2
Neutral Red (NR) Dye Experiments – 3T3 Cells – IIVS........................... E-13
E3
Neutral Red (NR) Dye Experiments – NHK Cells – IIVS......................... E-19
E4
Neutral Red (NR) Dye Experiments – 3T3 Cells – ECBC ........................ E-25
Appendix F Reference Substance Information ........................................................... F-1
F1
NRU Test Information for the 72 Reference Substances ............................. F-3
F2
Chemical, Physical, and Biological Information from the Literature forthe 72 Reference Substances .................................................................... F-11
F3
Candidate Reference Substances .............................................................. F-27
Appendix G Statement of Work (SOW).......................................................................G-1
G1
A Validation Study For In Vitro Basal Cytotoxicity Testing .......................G-3
G2
Procedures for Acquisition, Preparation, Solubility Testing, andDistribution of Test Chemicals for a Validation Study for In Vitro
Basal Cytotoxicity Testing .......................................................................G-57
xiii

In Vitro Cytotoxicity Test Methods BRD Table of Contents

November 2006

Appendix H Rat and Mouse Oral LD50 Database........................................................H-1
H1
Rat and Mouse Oral LD50 Database ........................................................... H-3
H2
Evaluation of the Candidate Reference Oral LD50 Data ............................ H-39
Appendix I
I1
I2
I3
I4

In Vitro NRU Data .................................................................................... I-1
3T3 NRU Reference Substance Data........................................................... I-3
NHK NRU Reference Substance Data .......................................................I-59
3T3 NRU Positive Control (SLS) Data.....................................................I-111
NHK NRU Positive Control (SLS) Data ..................................................I-127

Appendix J
J1
J2
J3
J4
J5
J6
J7

LD50 and Toxicity Category Predictions ..................................................J-1
3T3 NRU Predictions: RC Millimole Regression ........................................ J-5
NHK NRU Predictions: RC Millimole Regression .................................... J-11
3T3 NRU Predictions: RC Rat-Only Millimole Regression....................... J-17
NHK NRU Predictions: RC Rat-Only Millimole Regression..................... J-21
3T3 NRU Predictions: RC Rat-Only Weight Regression ........................... J-25
NHK NRU Predictions: RC Rat-Only Weight Regression......................... J-29
Comparison of Millimole Regression with Weight Regression
Regarding Prediction of Toxicity (LD50) for Low or High Molecular
Weight Chemicals..................................................................................... J-34

Appendix K
K1
K2
K3
K4

IC50 and LD50 Data for Regressions ........................................................K-1
IC50 and LD50 Values Used for Laboratory-Specific Regressions ............... K-3
IC50 and LD50 Values Used for Combined-Laboratory Regressions.......... K-17
RC IC50 and LD50 Values for RC Substances with Rat Oral LD50 Data..... K-23
Individual Laboratory LD50 Predictions: RC Rat-Only Millimole
Regression ............................................................................................... K-33

Appendix L Outlier Information ................................................................................. L-1
L1
Outlier Characterization for the 3T3 and NHK NRU Test Methods
with the RC Millimole Regression ............................................................. L-3
L2
Discordant Substances for GHS Acute Oral Toxicity Category
Predictions Using the 3T3 and NHK NRU Test Methods and RC RatOnly Regressions ....................................................................................... L-9
L3
Analysis of Outliers by Halle (1998, 2003) for the RC Millimole
Regression ............................................................................................... L-17
Appendix M Acute Oral Toxicity Test Guidelines ...................................................... M-1
M1
OECD UDP Test Guideline ...................................................................... M-3
M2
EPA UDP Test Guideline........................................................................ M-31
M3
OECD ATC Method Test Guideline ....................................................... M-71
M4
OECD FDP Test Guideline ..................................................................... M-87
M5
OECD Guidance on Acute Oral Toxicity Testing...................................M-103
Appendix N UDP/ATC Simulation Modeling Results................................................. N-1
N1
UDP Simulation Results Using Starting Doses One Default Dose
Lower than the LD50 Predicted by the 3T3 and NHK NRU IC50 and the
RC Rat-Only Millimole Regression – 5000 mg/kg Upper Limit Dose ........ N-3

xiv

In Vitro Cytotoxicity Test Methods BRD Table of Contents

N2
N3
N4

November 2006

UDP Simulation Results Using Starting Doses One Default Dose
Lower than the LD50 Predicted by the 3T3 and NHK NRU IC50 and the
RC Rat-Only Weight Regression – 5000 mg/kg Upper Limit Dose ..........N-13
ATC Simulation Results Starting at the Next Fixed Dose Below theLD50 Predicted by the 3T3 and NHK NRU IC50 and the RC Rat-Only
Millimole Regression - 2000 mg/kg Upper Limit Dose ............................N-23
ATC Simulation Results Starting at the Next Fixed Dose Below theLD50 Predicted by the 3T3 and NHK IC50 and the RC Rat-Only
Weight Regression – 2000 mg/kg Upper Limit Dose................................N-33

Appendix O Federal Register Notices ...........................................................................O-1
O1
70FR14473 - Request for Nominations for an Independent Peer Review
Panel To Evaluate In Vitro Testing Methods for Estimating Acute Oral
Systemic Toxicity and Request for In Vivo and In Vitro Data .....................O-3
O2
69FR61504 - Availability of Updated Standardized In Vitro
Cytotoxicity Test Method Protocols for Estimating Acute Oral
Systemic Toxicity; Request for Existing In Vivo and In Vitro Acute
Toxicity Data ...........................................................................................O-5
O3
69FR11448 - Notice of the Availability of Agency Responses to
ICCVAM Test Recommendations for the Revised Up-and-Down
Procedure for Determining Acute Oral Toxicity and In Vitro Methods
for Assessing Acute Systemic Toxicity ......................................................O-7
O4
66FR49686 - Report of the International Workshop on In Vitro
Methods for Assessing Acute Systemic Toxicity; Guidance Document
on Using In Vitro Data to Estimate In Vivo Starting Doses for Acute
Toxicity: Notice of Availability and Request for Public Comment .............O-9
O5
65FR57203 - Notice of an International Workshop on In Vitro
Methods for Assessing Acute Systemic Toxicity, co-sponsored by
NIEHS, NTP and the U.S. Environmental Protection Agency (EPA):
Workshop Agenda and Registration Information......................................O-11
O6
65FR37400 - Notice of an International Workshop on In Vitro
Methods for Assessing Acute Systemic Toxicity, co-sponsored by
NIEHS, NTP and the U.S. Environmental Protection Agency (EPA):
Request for Data and Suggested Expert Scientists ....................................O-15
Appendix P In Vitro Cytotoxicity Test Methods and the High Production
Volume (HPV) Challenge Program......................................................... P-1
P1
Supplemental Acute Toxicity Protocol ....................................................... P-3
P2
Office of Pollution Prevention and Toxics (OPPT) Letters to
Manufacturers/Importers ............................................................................ P-9
Appendix Q Additional UDP Simulation Modeling Results........................................Q-1
Q1
UDP Simulation Results for the RC Rat-Only Millimole Regression
Starting at the LD50 Predicted by the 3T3 and NHK NRU IC50 –5000 mg/kg Upper Limit Dose .................................................................. Q-3
Q2
UDP Simulation Results for the RC Rat-Only Weight Regression
Starting at the LD50 Predicted by the 3T3 and NHK NRU IC50 LD50 –5000 mg/kg Upper Limit Dose .................................................................Q-13
xv

In Vitro Cytotoxicity Test Methods BRD List of Figures

November 2006

LIST OF FIGURES
Figure 1-1
Figure 1-2
Figure 2-1
Figure 2-2
Figure 2-3
Figure 2-4
Figure 2-5
Figure 2-6
Figure 2-7
Figure 3-1
Figure 4-1
Figure 5-1
Figure 5-2
Figure 5-3
Figure 5-4
Figure 6-1
Figure 6-2
Figure 6-3
Figure 6-4
Figure 6-5
Figure 6-6
Figure 6-7
Figure 6-8
Figure 7-1
Figure 7-2
Figure 7-3
Figure 7-4
Figure 7-5

RC Millimole Regression for In Vitro Cytotoxicity (IC50x) and Rat andMouse Acute Oral LD50 Values for 347 Chemicals .....................................1-9
NICEATM/ECVAM Validation Study Phases .......................................... 1-13
Major Steps in the Performance of the NRU Test Methods .........................2-7
3T3 NRU OD for SLS as a Function of NR Concentration andDuration.................................................................................................... 2-26
SLS IC50 Values for Each NR Concentration and Incubation Duration(3T3 NRU) ............................................................................................... 2-27
Representative Concentration-Response for 2-Propanol in a 3T3 NRU
Range Finder Test..................................................................................... 2-30
Representative Concentration-Response for Aminopterin in a NHK
NRU Range Finder Test............................................................................ 2-31
Representative Concentration-Response for Aminopterin in a NHK
NRU Definitive Test ................................................................................. 2-32
Flow Chart for Determination of Reference Substance Solubility in
Medium, DMSO, or ETOH....................................................................... 2-38
The Fifty-Eight (58) Selected RC Reference Substances on the RC
Millimole Regression................................................................................ 3-27
Correlation of LD50 Values With the Reference LD50 Values for the
58 RC Chemicals ...................................................................................... 4-14
Reference Substance IC50 Results for the 3T3 NRU Test Method byLaboratory ................................................................................................ 5-24
Reference Substance IC50 Results for the NHK NRU Test Method byLaboratory ................................................................................................ 5-25
RC IC50 Values vs 3T3 NRU IC50 Values for 56 Substances in
Common ................................................................................................... 5-37
RC IC50 Values vs NHK NRU IC50 Values for 57 Substances in
Common .................................................................................................. 5-38
Combined-Laboratory 3T3 and NHK NRU Regressions .............................6-7
Regression for 47 RC Substances Using RC Data .......................................6-8
Regression for 47 RC Substances with the 3T3 and NHK Regressions........6-9
RC Rat-Only Millimole Regression (a) and RC Rat-Only Weight
Regression (b)........................................................................................... 6-19
Correlation of Dose-Mortality Slope to Hill Function Slope...................... 6-30
In Vitro – In Vivo Regressions for Phases Ib and II for ECBC ................... 6-37
In Vitro – In Vivo Regressions for Phases Ib and II for FAL...................... 6-38
In Vitro – In Vivo Regressions for Phases Ib and II for IIVS...................... 6-39
Mean Laboratory and Laboratory-Specific 3T3 and NHK NRU
Regressions.................................................................................................7-7
Frequency of Maximum:Minimum 3T3 NRU IC50 Ratios ......................... 7-39
Frequency of Maximum:Minimum NHK NRU IC50 Ratios....................... 7-40
Comparison of Maximum:Minimum NRU IC50 Ratios to Maximum:
Minimum LD50 Ratios .............................................................................. 7-41
SLS IC50 for Each Laboratory and Study Phase......................................... 7-45
xvi

In Vitro Cytotoxicity Test Methods BRD List of Tables

November 2006

LIST OF TABLES
Table ES-1
Table 1-1
Table 1-2
Table 2-1
Table 2-2
Table 2-3
Table 2-4
Table 2-5
Table 3-1
Table 3-2
Table 3-3
Table 3-4
Table 3-5
Table 3-6
Table 3-7
Table 3-8
Table 4-1
Table 4-2
Table 4-3
Table 4-4
Table 5-1
Table 5-2
Table 5-3
Table 5-4
Table 5-5
Table 5-6
Table 5-7
Table 5-8
Table 5-9
Table 5-10
Table 5-11
Table 6-1
Table 6-2
Table 6-3

Datasets Used for Validation Study Analyses.......................................... xxxvi
Summary of Current U.S. Legislation for Using Acute Toxicity Data
for Product Labeling .................................................................................1-14
Regulatory Classification Systems for Acute Oral Toxicity .......................1-15
Measured VC OD540 Values and Targets...................................................2-17
Refeeding/No Refeeding Data for the NHK NRU Test Method.................2-24
Error Rates in Phase Ib by Laboratory and Test.........................................2-28
Cell Seeding Densities ..............................................................................2-37
Comparison of Concentrations Tested in the Various Solubility
Protocols...................................................................................................2-39
GHS Classification Scheme for Acute Oral Toxicity ...................................3-3
Reference Substances Used in the 3T3 and NHK NRU Test Methods
Validation Study - Sorted by Toxicity .........................................................3-7
Distribution of Candidate Substances and Reference Substances by
Source and Toxicity Category ...................................................................3-26
Selected Substances: Distribution of RC Chemicals and RC Outliersby Toxicity Category.................................................................................3-26
Distribution of Chemical Class for the 72 Reference Substances by
Toxicity Category .....................................................................................3-29
Distribution of Product/Use Class for the 72 Reference Substances by
Toxicity Category .....................................................................................3-31
Reference Substances Metabolized to Active Metabolites .........................3-32
Reference Substances Tested in Phases Ib and II .......................................3-33
Internet-Accessible Databases Searched for LD50 Information.....................4-3
Rodent Acute Oral Reference LD50 Values Listed by GHS Category...........4-9
GHS Category Matches for the Rodent Acute Oral LD50 Initial andReference Values ......................................................................................4-12
Maximum:Minimum LD50 Ratios by GHS Toxicity Category ...................4-13
Validation Study Timetable.........................................................................5-4
Reference Substances Affected by the Stopping Rule................................5-10
Positive Control (PC) IC50 Results by Study Phase....................................5-11
3T3 NRU Test Method IC50 and Hill Slope Data by Laboratory ................5-14
NHK NRU Test Method IC50 and Hill Slope Data by Laboratory..............5-19
Key to Validation Study Reference Substances .........................................5-26
Datasets Used for Validation Study Analyses............................................5-32
Comparison of 3T3 and NHK NRU IC50 Geometric Means.......................5-33
Frequency of 3T3:NHK IC50 Ratios for Reference Substances ..................5-36
Solubility Test Results (mg/mL)................................................................5-39
Reference Substances with Precipitate (PPT) and Volatility Issues............5-43
Datasets Used for Accuracy Analyses .........................................................6-4
Linear Regression Analyses of the 3T3 and NHK NRU and Rat Acute
Oral LD50 Test Results ................................................................................6-6
Outlier Substances for the RC and the 3T3 and NHK NRU Methods
When the RC Millimole Regression is Used..............................................6-10
xvii

In Vitro Cytotoxicity Test Methods BRD List of Tables

Table 6-4
Table 6-5
Table 6-6
Table 6-7
Table 6-8
Table 6-9
Table 6-10
Table 6-11
Table 6-12
Table 7-1
Table 7-2
Table 7-3
Table 7-4
Table 7-5
Table 7-6
Table 7-7
Table 7-8
Table 7-9
Table 7-10
Table 7-11
Table 7-12
Table 7-13
Table 7-14
Table 8-1
Table 8-2

November 2006

Substances With Mechanisms of Toxicity Not Expected to Be Activein the 3T3 or NHK Cells in Culture........................................................... 6-15
Linear Regression Analyses to Improve the Prediction of Rodent
Acute Oral LD50 Values from In Vitro NRU IC50 Using the RC
Database ................................................................................................... 6-17
Prediction of GHS Acute Oral Toxicity Category by the RC IC50
Values and the RC Millimole Regression .................................................. 6-21
Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK
NRU Test Methods and the RC Rat-Only Millimole Regression ............... 6-23
Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK
NRU Test Methods and the RC Rat-Only Weight Regression ................... 6-26
Comparison of Regressions and In Vitro NRU Test Methods for Their
Performance in Predicting GHS Acute Oral Toxicity Categories............... 6-28
Reference Substances with Dose-Mortality and NRU Hill Slopes ............. 6-29
Over- and Under- Prediction of Toxicity for Low and High Molecular
Weight Substances Using RC Rat-Only Weight and Millimole
Regressions............................................................................................... 6-33
Linear Regressions for 11 Substances Tested in Phases Ib and II............... 6-36
Reference Substances Excluded from Reproducibility Analyses
Because of Insufficient Cytotoxicity ...........................................................7-4
Number of Reference Substances Tested vs Number of Reference
Substances Yielding IC50 Values from Each Laboratory, by GHS
Acute Oral Toxicity Category .....................................................................7-4
Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU
Test Method................................................................................................7-8
Reference Substances with Significant ANOVA Differences Among
Laboratories for the 3T3 NRU Test Method.............................................. 7-20
Reproducibility of the IC50 Values from the NHK NRU Test Method ....... 7-22
Reference Substances with Significant ANOVA Differences Among
Laboratories for the NHK NRU Test Method............................................ 7-34
Summary of CV Results for the 3T3 and NHK NRU Test Methods .......... 7-34
Intralaboratory CV by Chemical Characteristics for the 3T3 and NHK
NRU Test Methods ................................................................................... 7-36
Interlaboratory 3T3 and NHK NRU Test Method CV Values Sorted by
Chemical Characteristics........................................................................... 7-38
CV Values for 3T3 and NHK NRU Test Method IC50 Values andNormalized IC50 Values ............................................................................ 7-43
ANOVA Results for the SLS IC50 Values in the 3T3 NRU Test
Method ..................................................................................................... 7-47
Linear Regression Analysis of SLS IC50 Values Over Time ...................... 7-48
ANOVA Results for the SLS IC50 Values in the NHK NRU Test
Method ..................................................................................................... 7-49
Solvent Determinations by Laboratory...................................................... 7-51
SMT-Recommended Documentation for FAL.............................................8-4
Phase III Error Rates in the Transfer of Data to the EXCEL® Template..... 8-10

xviii

In Vitro Cytotoxicity Test Methods BRD List of Tables

Table 8-3
Table 8-4
Table 8-5
Table 8-6
Table 9-1
Table 9-2
Table 9-3
Table 9-4
Table 10-1
Table 10-2
Table 10-3
Table 10-4
Table 10-5
Table 10-6
Table 10-7
Table 10-8
Table 10-9
Table 10-10
Table 11-1
Table 11-2
Table 11-3
Table 11-4

November 2006

Definitive Test and Positive Control (PC) Test Failure Rates in Phase
III..............................................................................................................8-10
Combined Definitive and Positive Control (PC) Test Success Rates forthe 3T3 and NHK Methods in Phase III.....................................................8-11
CV Values for Definitive Tests .................................................................8-11
GHS Acute Oral Toxicity Category Predictions by Laboratory..................8-13
Rat Acute Oral LD50 Ranges for Test Substances Used in Previous In
Vitro NRU Cytotoxicity Studies and the NICEATM/ECVAM Study ..........9-4
Chemical Classes Represented by the Substances Used in Published
Studies for Correlation of In Vitro NRU Cytotoxicity with Rodent
Acute Lethality ...........................................................................................9-4
EU Classes of Acute Oral Toxicity..............................................................9-8
Correct Identification of In Vivo Phototoxicants by the NHK NRU
Phototoxicity Assay ..................................................................................9-18
Change in Animal Use with Dose-Mortality Slope for the UDP ................10-8
Mean Animal Use in the UDP Using Starting Doses Based on the 3T3
and NHK NRU Test Methods with the Different Regressions .................10-10
Animal Use for the UDP by GHS Acute Oral Toxicity Category Using
Starting Doses Based on the 3T3 and NHK NRU Test Methods with
the RC Rat-Only Millimole Regression ...................................................10-12
Animal Use for the UDP by GHS Acute Oral Toxicity Category Using
Starting Doses Based on the 3T3 and NHK NRU Test Methods with
the RC Rat-Only Weight Regression .......................................................10-14
Animal Deaths in the UDP Using Starting Doses Based on the 3T3 andNHK NRU Test Methods ........................................................................10-15
Change in Animal Use with Dose-Mortality Slope in the ATC Method ...10-19
Animal Use for the ATC Method Using Starting Doses Based on the
3T3 and NHK NRU Test Methods with the Different Regressions ..........10-21
Animal Savings for the ATC Method by GHS Acute Oral Toxicity
Category Using Starting Doses Based on the 3T3 and NHK NRU Test
Methods with the RC Rat-Only Millimole Regression.............................10-23
Animal Savings for the ATC Method by GHS Acute Oral Toxicity
Category Using Starting Doses Based on the 3T3 and NHK NRU Test
Methods with the RC Rat-Only Weight Regression.................................10-25
Animal Deaths for the ATC Method Using Starting Doses Based on
the 3T3 and NHK NRU Test Methods.....................................................10-27
Costs for Major Laboratory Equipment .....................................................11-9
Costs for Cell Culture Materials and Commercial Laboratory In Vitro
Cytotoxicity Testing................................................................................11-10
Commercial Prices for Conducting In Vivo Acute Rat Toxicity Testing...11-11
Comparison of Time Needed for In Vitro and In Vivo Testing .................11-14

xix

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

November 2006

LIST OF ACRONYMS AND ABBREVIATIONS
A-CUTE-TOX
ADME
ANOVA
ASTDR
ASTM
ATC
ATCC
ATWG
BBB
BPE
BRD
o
C
CAS
CASRN
CCOHS
CDER
CESARS
CFU
CHRIS
CI
CICADSCIS
CNSCOLIPA
CPSC
CSF
CTFA
CV
DART®/ETIC
DEA
DHHS
DIMDI
DNA
DMEM
DMSO
D-PBS
DOD
DOT
EC
EC50

A-Cute-Tox Project (EU Research & Development Integrated Project)
Absorption, distribution, metabolism, and elimination
Analysis of varianceAgency for Toxic Substances and Disease Registry
American Society for Testing and Materials
Acute Toxic Class method
American Type Culture Collection
Acute Toxicity Working Group
Blood:brain barrier
Bovine pituitary extract
Background Review Document
Degrees Celsius
Chemical Abstracts Service
Chemical Abstracts Service Registry Number
Canadian Centre for Occupational Health and Safety (CCOHS)
U.S. FDA Center for Drug Evaluation and Research
Chemical Evaluation Search and Retrieval System
Colony forming units
Chemical Hazard Response
Confidence interval
Concise International Chemical Assessment Documents
ILO Occupational Safety and Health Information Centre
Central nervous system
The European Cosmetic Toiletry and Perfumery Association
U.S. Consumer Product Safety Commission
Colony stimulating factor
Cosmetic, Toiletries and Fragrance Association
Coefficient of variation
Developmental and Reproductive Toxicology/Environmental
Teratology Information Center
U.S. Drug Enforcement Administration
U.S. Department of Health and Human Services
Deutsches Institut fur Medizinische Dokumentation undInformation (The German Institute for Medical Documentation andInformation)
Deoxyribose nucleic acid
Dulbecco’s Modification of Eagle’s Medium
Dimethyl sulfoxide
Dulbecco’s phosphate buffered saline
U.S. Department of Defense
U.S. Department of Transportation
European Commission
Concentration of a substance that produces 50% of the maximum
possible response for that substancexx

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

ECBC
ECETOC
EC/HO
ECVAM
EDIT
EHC
EHS
EPA
ERG
ETOH
EU
EXTONET
FAL
FAO
FB1
FDA
FDP
FIFRA
FR
FRAME
GABA
GCCP
GHS
GLP
gm
HBSS
HPV
hr
HSDB
HSG
HTD
IARC
IC20
IC50
IC80
ICCVAM
ICSC
ID
ID50
IIVS
ILO
i.m.
INVITOXX

November 2006

U.S. Army Edgewood Chemical Biological Center
European Centre for Ecotoxicology and Toxicology of Chemicals
European Commission/British Home Office
European Centre for the Validation of Alternative Methods
Evaluation-guided development of new in vitro tests
Environmental Health Criteria
EPA’s Extremely Hazardous Substance list
U.S. Environmental Protection Agency
Emergency Response Guidebook
Ethanol (Ethyl alcohol)
European Union
The Extension Toxicology Network
FRAME Alternatives Laboratory
UN Food and Agriculture Organization
Fumonisin B1
U.S. Food and Drug Administration
Fixed Dose Procedure
U.S. Federal Insecticide, Fungicide, and Rodenticide Act
Federal Register
Fund for the Replacement of Animals in Medical Experiments
Gamma amino butyric acid
Good cell culture practices
Globally Harmonized System (of Classification and Labeling ofChemicals)
Good Laboratory Practices
Grams
Hanks’ balanced salt solution
High Production Volume
Hour(s)
Hazardous Substances Data Bank
Health and Safety Guides
Highest tolerated doseInternational Agency for Research on Cancer
Concentration producing 20% inhibition of the endpoint measured
Concentration producing 50% inhibition of the endpoint measured
Concentration producing 80% inhibition of the endpoint measured
Interagency Coordinating Committee for the Validation of Alternative
Methods
International Chemical Safety Cards
Insufficient data
Index of cytotoxicity; dose producing a 50% reduction in protein value
Institute for In Vitro Sciences
International Labour Organisation
Intramuscular
In Vitro Techniques in Toxicology (ERGATT FRAME ECVAM Data
bank)

xxi

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

IOM
i.p.
IPCS
IRAG
IRPTC
ISO
IUCLID
i.v.
JECFA
JMPR
KBM®
kg
Kow
L
LC
LD50
LDH
MAS
MEIC
MeSH®
µL
µm
µM
mg
MIT
mL
mM
MMAS
mmol
MPE
MSDS
MTT
N
NA
NADH
NC
NCS
NCTR
n.d.
NHK
NICEATM
NIEHS
NIH
NIOSH
NLM

November 2006

Institute of Medicine
Intraperitoneal
International Programme on Chemical Safety
Interagency Regulatory Alternatives GroupInternational Register of Potentially Toxic Chemicals
International Standards Organization
International Uniform Chemical Information Database
Intravenous
Joint Expert Committee on Food Additives
Joint Meeting on Pesticide Residues
Keratinocyte basal medium
Kilogram
Octanol-water partition coefficient
Liter
Lethal blood concentration
Dose that produces lethality in 50% of test animals
Lactate dehydrogenase
Maximum average Draize score
Multicentre Evaluation of In Vitro Cytotoxicity
Medical Subject Heading
Microliters
Micrometers
Micromoles
Milligram
Metabolic inhibition test
Milliliter
Millimolar
Modified maximum average score
Millimoles
Mean photo effect
Material Safety Data Sheets
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
Number (of substances)
Not applicable
Nicotine adenine dinucleotide (reduced)
Not calculated
Newborn calf serum
U.S. FDA National Center for Toxicological ResearchNot detectable
Normal human epidermal keratinocytes
National Toxicology Program Center for the Evaluation of Alternative
Toxicological Methods
U.S. National Institute of Environmental Health Sciences
U.S. National Institutes of Health
U.S. National Institute for Occupational Safety and HealthNational Library of Medicine

xxii

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

NR
NRU
NTP
OAT
OD
OD540
OECD
OHM/TADS
OPP
OPPTS
ORD
OSHA
OTA
PBS
PC
PDS
pg
PG
PIF
PIMS
pK
PLS
PPIS
PPT
QA
QC
R2
rs
RC
REACH
RTECS®
RTK NET
SD
SIDS
SIS
SLS
SMT
SOP
3T3
TESS
TG
TRI
TSCA
UDP
UN

November 2006

Neutral red
Neutral red uptake
U.S. National Toxicology Program
Organic anionic transporters
Optical density
Optical density (absorbance) at a wavelength of 540 nm
Organisation for Economic Co-operation and Development
EPA Oil and Hazardous Materials/Technical Assistance Data
SystemU.S. EPA Office of Pesticide Programs
EPA Office of Prevention, Pesticides and Toxic Substances
U.S. EPA Office of Research and Development
U.S. Occupational Safety and Health Administration
Ochratoxin A
Phosphate buffered saline
Positive control
Pesticide Data Sheets
Picogram
Packing group
Photoinhibition factor
Poisons Information Monographs
Acid/base dissociation constantPartial Least Squares (analysis)
EPA Pesticide Product Information System
Precipitate
Quality assurance
Quality control
Coefficient of determination
Spearman correlation coefficient
Registry of Cytotoxicity
Registration, evaluation, authorisation and restriction of chemicals
Registry of Toxic Effects of Chemical Substances
The Right-to-Know Network
Standard deviation
OECD Screening Information Data Sets
Scientific Information Service
Sodium lauryl sulfate
Study management teamStandard operating procedure
BALB/c mouse fibroblasts, clone A31 (ATCC # CCL-163)
Toxic Exposure Surveillance System
Test guideline
U.S. EPA Toxics Release Inventory
Toxic Substances Control Act
Up-and-Down Procedure
United Nations

xxiii

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

UNEP
USP
UV
VC
WHO
XTT
ZEBET

November 2006

United Nations Environment Programme
U.S. Pharmacopoeia
Ultraviolet (light)
Vehicle control
World Health Organization
2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carboxanilide
Zentralstelle zur Erfassung und Bewertung von Ersatz- undErgänzungsmethoden zum Tierversuch (German Center forDocumentation and Evaluation of Alternative Methods to Animal
Experiments)xxiv

In Vitro Cytotoxicity Test Methods BRD Acknowledgements

November 2006

ACKNOWLEDGMENTS
The following individuals are acknowledged for their contributions to the in vitro acutetoxicity test method review process.

Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM) Acute Toxicity Working Group (ATWG)

Consumer Product Safety Commission(CPSC)

Food and Drug Administration (FDA)
Leonard Schechtman, Ph.D. (ICCVAM Chair)
Kenneth Hastings, Ph.D.
Abigail Jacobs, Ph.D.
David Morse, Ph.D.
Thomas Umbreit, Ph.D.

Kailash Gupta, D.V.M., Ph.D.
Cassandra Prioleau, Ph.D.
Marilyn Wind, Ph.D. (ATWG Chair,
ICCVAM Vice Chair)
Department of Energy (DOE)

National Institute for Occupational
Safety & Health (NIOSH)

Po-Yung Lu, Ph.D.
Steven Reynolds, Ph.D.
Environmental Protection Agency(EPA)

National Institute of Environmental
Health Sciences (NIEHS)

Karen Hamernik, Ph.D.
Masih Hashim, Ph.D.
Marianne Lewis
Elizabeth Margosches, Ph.D.
Deborah McCallJohn Redden, Ph.D.
Amy Rispin, Ph.D.

Rajendra Chhabra, Ph.D., D.A.B.T.
William Stokes, D.V.M., D.A.C.L.A.M.
(ICCVAM Executive Director; NICEATM
Director)
Raymond Tice, Ph.D. (NICEATM Deputy
Director)
European Centre for the Validation of
Alternative Methods (ECVAM)
Liaisons
Silvia Casati, Ph.D.
Pilar Prieto, Ph.D.

xxv

In Vitro Cytotoxicity Test Methods BRD Acknowledgements

November 2006

National Toxicology Program (NTP) Interagency Center for the
Evaluationof Alternative Toxicological Methods (NICEATM)

Dave Allen, Ph.D.
ILS, Inc.

Linda Litchfield
ILS, Inc.

Bradley Blackard, M.S.P.H.
ILS, Inc.

Deborah McCarley
NIEHS

Sue Brenzel
ILS, Inc.

Michael Paris
ILS, Inc.

Thomas Burns, M.S.
ILS, Inc.

William Stokes, D.V.M., D.A.C.L.A.M.
(Director)
NIEHS

Patricia Ceger, M.S.
ILS, Inc.

Judy Strickland, Ph.D., D.A.B.T.
ILS, Inc.

Jeffery Charles, Ph.D., M.B.A., D.A.B.T.
ILS, Inc.

Raymond Tice, Ph.D. (Deputy Director)
NIEHS

Neepa Choksi, Ph.D.
ILS, Inc.

Jim Truax, M.A.
ILS, Inc.

Frank Deal, M.S.
ILS, Inc.

xxvi

In Vitro Cytotoxicity Test Methods BRD Acknowledgements

November 2006

Participants in the In Vitro Cytotoxicity Validation StudyNICEATM gratefully acknowledges the generous contributions of the individuals who
directly participated in the NICEATM/ECVAM Validation Study. Their time and efforts are
greatly appreciated.
BioReliance Corp.
(Chemical Distribution)
Martin Wenk, Ph.D. – Principal
Investigator

Constella Group (Statistical Analyses)
Patrick Crockett, Ph.D.
Eric Harvey, Ph.D.
Wendell Jones, Ph.D.
Robert Lee, M.S.
Jessica L. Matthews, M.S.
Michael Riggs, Ph.D.
Janine Wilcox
Nicole Williams

Institute for In Vitro Sciences (IIVS)(Lead Laboratory – Protocols)
Hans Raabe, M.S. – Study Director
Greg Mun – Laboratory Manager
Angela Sizemore – Research Technician
Gregory O. Moyer – Research Technician
John Harbell, Ph.D. – Scientific Director

Statistical Consultant
Joseph Haseman, Ph.D.

U.S. Army Edgewood Chemical
Biological Center (ECBC)(Testing Laboratory)
Cheng Cao, Ph.D. – Study Director
Janna Madren-Whalley – ResearchTechnician
Chundakkadu Krishna, Ph.D. – ResearchTechnician
James J. Valdes, Ph.D. – Scientific
AdvisorNIEHS
Grace Kissling, Ph.D. – Contract Project
Officer
Molly Vallant - Contract Project Officer
Study Management Team (SMT)NICEATM
William Stokes, D.V.M., D.A.C.L.A.M.
(NIEHS) – Director, NICEATM
Raymond Tice, Ph.D. (NIEHS) – Deputy
Director, NICEATM – Advisor
Judy Strickland, Ph.D., D.A.B.T. (ILS,
Inc.) – Project Coordinator
Michael Paris (ILS, Inc.) – Assistant
Project Coordinator
Jeffrey Charles, Ph.D., D.A.B.T. (ILS,
Inc.) – Advisor

FRAME Alternatives Laboratory (FAL)
University of Nottingham, UK(Lead Laboratory – Software)
Richard Clothier, Ph.D. – Study Director
Nicola Bourne – Research Technician
Monika Owen – Research Technician
Rachel Budworth – Research Technician

ECVAM
Thomas Hartung, Ph.D., M.D. – Head of
Unit from 2002
Silvia Casati, Ph.D. – Task Leader
Michael Balls, D. Phil.– Head of Unit until
June 2002

xxvii

In Vitro Cytotoxicity Test Methods BRD Acknowledgements[This Page Intentionally Left Blank]xxviiiNovember 2006

In Vitro Cytotoxicity Test Methods BRD Appendix A

November 2006

Appendix A

NICEATM/ECVAM Validation Study Management

A-1

In Vitro Cytotoxicity Test Methods BRD Appendix A

November 2006

[This Page Intentionally Left Blank]

A-2

In Vitro Cytotoxicity Test Methods BRD Appendix A

November 2006

NICEATM/ECVAM Validation Study Management
NICEATM and ECVAM staff managed the study as shown in Figure A-1. The NICEATMECVAM Study Management Team (SMT), in consultation with the Project Design and
Evaluation Team and other advisors shown in Figure A-1, designed the study, selected thereference substances (see Section 3), and selected the laboratories that would purchase anddistribute chemicals and perform solubility and cytotoxicity testing. BioReliance Corporation(Rockville, MD) purchased the reference substances, tested the solubility, and distributed thecoded reference substances to the laboratories that performed the cytotoxicity testing. The
Institute for In Vitro Sciences (IIVS; Gaithersburg, MD), U.S. Army Edgewood Chemical
Biological Center (ECBC; Edgewood, MD), and Fund for the Replacement of Animals in
Medical Experiments (FRAME) Alternatives Laboratory, University of Nottingham, Queen’s
Medical Center (FAL; Nottingham, UK) were the participating laboratories that performed
the solubility and cytotoxicity testing.

A-3

In Vitro Cytotoxicity Test Methods BRD Appendix A

Figure A-1

November 2006

Study Management Chart
FUNDING SPONSORS•••National Institute of Environmental Health Sciences (NIEHS)
U.S. Environmental Protection Agency (U.S. EPA)The European Centre for the Validation of Alternative Methods (ECVAM)

PROJECT ADVISORY TEAMPROJECT DESIGN AND EVALUATION TEAM(PDET)
Dr. Tom Burka – National Toxicology Program (NTP)
Toxicokinetics/Chemistry
Dr. Raj Chhabra – NTP General Toxicology
Dr. Pat Crockett – NTP Biostatistics (Constella Group)
Dr. Eric Harvey – NTP Biostatistics (Constella Group)
Dr. Joe Haseman – NTP Biostatistics (Consultant)
Dr. Suzanne McMaster – U.S. EPA Liaison
Dr. Cynthia Smith – NTP Chemistry
Ms. Molly Vallant – NTP Project Officer (Testing
Contract)
Study Management Team Members

STUDY MANAGEMENT TEAM
National Toxicology Program Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)
Dr. William Stokes – Director
Dr. Raymond Tice – Deputy Director – Advisor
Dr. Judy Strickland (Integrated Laboratory Systems [ILS],
Inc.) – Project Coordinator
Mr. Michael Paris (ILS, Inc.) – Asst. Project Coordinator
ECVAM
Dr. Thomas Hartung – Head of Unit
Dr. Silvia Casati – Task Leader

Fund for the Replacement of Animals in Medical
Experiments (FRAME)
Dr. Michael Balls
German Centre for the Documentation andValidation of Alternative Methods [at BfR] (ZEBET)
Dr. Horst Spielmann
Dr. Elke Genschow
Dr. Manfred Liebsch
Acute Toxicity Working Group (ATWG),
Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM)OTHER REVIEWERSNTP Project Review Committee (PRC)
In Vitro Workshop (2000) Experts

NIEHS/NICEATM

ECVAM

PROJECT MANAGEMENT AND
CHEMICAL DISTRIBUTION TEAM

BioReliance Corp.
(Chemical Distribution)
Dr. Martin Wenk – Principal
Investigator

PROJECT MANAGEMENT

U.S. Army Edgewood Chemical Biological Center(ECBC)(Testing Laboratory)

Institute for In Vitro Sciences (IIVS)(Lead Lab - Protocols)
Mr. Hans Raabe – Study Director
Mr. Greg Mun – Laboratory Manager
Ms. Angela Sizemore – Research Technician
Mr. Gregory O. Moyer – Research Technician
Dr. John Harbell – Scientific Director

Dr. Cheng Cao – Study Director
Ms. Janna Madren-Whalley – Research Technician
Dr. Chundakkadu Krishna – Research Technician
Dr. James J. Valdes – Scientific Advisor

A-4

FRAME Alternatives Laboratory(FAL) University of Nottingham, UK(Lead Lab - Software)
Dr. Richard Clothier – Study Director
Ms. Nicola Bourne – ResearchTechnician
Ms. Monika Owen – ResearchTechnician

In Vitro Cytotoxicity Test Methods BRD Appendix B

November 2006

Appendix B

Validation Study Test Method Protocols(Phase III)

B1

Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake(NRU) Cytotoxicity Test ................................................................... B-3

B2

Test Method Protocol for the Normal Human Epidermal
Keratinocyte (NHK) Neutral Red Uptake (NRU) Cytotoxicity
Test................................................................................................... B-25

B3

Test Method Protocol for Solubility Determination (Phase III)... B-47

B4

Test Method Procedure for Prequalification of Normal Human
Epidermal Keratinocyte Growth Medium (Phase III).................. B-59

B-1

In Vitro Cytotoxicity Test Methods BRD Appendix B

November 2006

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B-2

In Vitro Cytotoxicity Test Methods BRD Appendix B1

November 2006

Appendix B1

Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake (NRU)
Cytotoxicity Test

B-3

In Vitro Cytotoxicity Test Methods BRD Appendix B1

November 2006

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B-4

In Vitro Cytotoxicity Test Methods BRD Appendix B1

November 2006

TEST METHOD PROTOCOL
for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

A Test for Basal Cytotoxicity for an In Vitro Validation Study
Phase III
November 4, 2003
Prepared by
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)

Based on Standard Operating Procedure Recommendations from anInternational Workshop Organized by the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM)

National Institute of Environmental Health Sciences (NIEHS)
National Institutes of Health (NIH)
U.S. Public Health Service
Department of Health and Human Services

B-5

In Vitro Cytotoxicity Test Methods BRD Appendix B1

November 2006

TEST METHOD PROTOCOLThe BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test
A Test for Basal Cytotoxicity
Phase III
I. PURPOSE
The purpose of this study is to evaluate the cytotoxicity of test chemicals using the BALB/c 3T3
Neutral Red Uptake (NRU) cytotoxicity test. The data will be used to evaluate the intra- andinter-laboratory reproducibility of the assay and effectiveness of the cytotoxicity assay to predict
the starting doses for rodent acute oral systemic toxicity assays. This test method protocol
outlines the procedures for performing the cytotoxicity test and supports the in vitro validation
study organized by NICEATM and the European Centre for the Validation of Alternative
Methods (ECVAM) and sponsored by NIEHS, U.S. Environmental Protection Agency, andECVAM. This test method protocol applies to all personnel involved with performing thecytotoxicity assay.
A. BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test
The 3T3 NRU test will be performed to analyze the in vitro toxicity of 60 blinded/coded test
chemicals. This test will be used to determine IC20, IC50, and IC80 values for thepredetermined set of test chemicals of varying toxicities.
II. SPONSOR
A. Name:National Institute of Environmental Health Sciences (NIEHS); The
National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)

B. Address:

P.O. Box 12233
Research Triangle Park, NC 27709

C. Representative:

Named Representative

III. IDENTIFICATION OF TEST AND CONTROL SUBSTANCES
A. Test Chemicals:

Blinded Chemicals (60)

B. Controls:

Positive:
Vehicle (Negative):
NBCS,

Sodium Lauryl Sulfate
Assay medium (DMEM containing 5%
4 mM L-Glutamine, 100 IU/mL Penicillin,
100 µg/mL Streptomycin)
Assay medium, DMSO, or ethanol directedby the Study Management Team, forpreparation of test chemicals

Solvent:

B-6

In Vitro Cytotoxicity Test Methods BRD Appendix B1

November 2006

IV. TESTING FACILITY AND KEY PERSONNEL
A. Facility Information
1)
2)
3)
4)
5)
6)
7)
8)

Name:
Address:
Study Director:
Laboratory Technician(s):Scientific Advisor:
Quality Assurance Director:
Safety Manager:
Facility Management:

B. Test Schedule
1) Proposed Experimental Initiation Date:
2) Proposed Experimental Completion Date:
3) Proposed Report Date:
V. TEST SYSTEMThe NRU cytotoxicity assay procedure is a cell survival/viability chemosensitivity assay based on
the ability of viable cells to incorporate and bind neutral red (NR), a supravital dye. NR is a weakcationic dye that readily penetrates cell membranes by non-ionic diffusion and accumulates
intracellularly in lysosomes. Alterations of the cell surface or the sensitive lysosomal membranelead to lysosomal fragility and other changes that gradually become irreversible. Such changes
brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It isthus possible to distinguish between viable, damaged, or dead cells, which is the basis of thisassay.
Healthy mammalian cells, when maintained in culture, continuously divide and multiply overtime. A toxic chemical, regardless of site or mechanism of action, will interfere with this process
and result in a reduction of the growth rate as reflected by cell number. Cytotoxicity is expressed
as a concentration dependent reduction of the uptake of the NR after chemical exposure thus
providing a sensitive, integrated signal of both cell integrity and growth inhibition.
VI. DEFINITIONS
A. Hill function: a four parameter logistic mathematical model relating the concentration of test
chemical to the response being measured in a sigmoidal shape.

Y = Bottom+

Top− Bottom
1 + 10(logIC50− X)HillSlope

where Y= response, X is the logarithm of dose (or concentration), Bottom is the minimum
response, Top is the maximum response, logIC50 is logarithm of X at the response midway
between Top and Bottom, and HillSlope describes the steepness of the curve.

B-7

In Vitro Cytotoxicity Test Methods BRD Appendix B1

November 2006

B. Documentation: all methods and procedures will be noted in a Study Workbook; logs will bemaintained for general laboratory procedures and equipment (e.g., media preparation, test
chemical preparation, incubator function); all optical density data obtained from thespectrophotometer plate reader will be saved in electronic and paper formats; all calculationsof ICx values and other derived data will be in electronic and paper format; all data will bearchived.
VII.

PROCEDURES

A. Materials
1. Cell Lines
BALB/c 3T3 cells, clone 31
CCL-163, LGC Reference Materials, Customer Service, Queens Road, Teddington,
Middlesex, TW110LY, UK
CCL-163, American Type Culture Collection [ATCC], Manassas, VA, USA)
2. Technical Equipment[Note: Suggested brand names/vendors are listed in parentheses. Equivalents may be
used.]
Incubator: 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/airLaminar flow clean bench/cabinet (standard: "biological hazard")
Water bath: 37ºC ± 1ºC
Inverse phase contrast microscope
Sterile glass tubes with caps (e.g., 5 mL)
Centrifuge (optionally: equipped with microtiter plate rotor)
Laboratory balance
96-well plate spectrophotometer (i.e., plate reader) equipped with 540 nm ± 10 nm
filteri) Shaker for microtiter platesj) Cell counter or hemocytometerk) Pipetting aidl) Pipettes, pipettors (multi-channel and single channel; multichannel repeater pipette),
dilution blockm) Cryotubesn) Tissue culture flasks (e.g., 75 - 80 cm2, 25 cm2)o) 96-well flat bottom tissue culture microtiter plates (e.g., Nunc # 167 008; Falcon
tissue culture-treated)p) pH paper (wide and narrow range)q) Multichannel reagent reservoirr) Waterbath sonicators) Magnetic stirrert) Antistatic bar ionizer/antistatic gun (optional for neutralizing static on 96-well plates)u) Dry heat block (optional)v) Adhesive film plate sealers (e.g., Excel Scientific SealPlate,Cat # STR-SEAL-PLT
or equivalent)w) Vortex mixerx) Filters/filtration devices

a)
b)
c)
d)
e)
f)
g)
h)

B-8

In Vitro Cytotoxicity Test Methods BRD Appendix B1

November 2006

[Note: Tissue culture flasks and microtiter plates should be prescreened to ensure thatthey adequately support the growth of 3T3 cells. Multi-channel repeater pipettes may be
used for plating cells in the 96-well plates, dispensing plate rinse solutions, NR medium,and desorb solution. Do not use the repeater pipette for dispensing test chemicals to thecells.]
3. Chemicals, Media, and Seraa) Dulbecco’s Modification of Eagle’s Medium (DMEM) without L-Glutamine; shouldhave high glucose [4.5gm/l] (e.g., ICN-Flow Cat. No. 12-332-54)b) L-Glutamine 200 mM (e.g., ICN-Flow # 16-801-49)c) New Born Calf Serum (NBCS or NCS) (e.g., Biochrom # SO 125)d) 0.05 % Trypsin/0.02 % EDTA solution (e.g., SIGMA T 3924, ICN-Flow, # 1689149)e) Phosphate buffered saline (PBS) without Ca2+ and Mg2+(for trypsinization)f) Hanks’ Balanced Salt Solution (HBSS) without Ca2+ and Mg2+(CMF-HBSS)g) Dulbecco’s Phosphate Buffered Saline (D-PBS) [formulation containing calcium andmagnesium cations; glucose optional] (for rinsing)h) Penicillin/streptomycin solution (e.g. ICN-Flow # 16-700-49)i) Neutral Red (NR) Dye – tissue culture-grade; liquid form (e.g., SIGMA N 2889);
powder form (e.g., SIGMA N 4638)j) Dimethyl sulfoxide (DMSO), U.S.P. analytical grade (Store under nitrogen @ -20ºC)k) Ethanol (ETOH), U.S.P. analytical grade (100 %, non-denatured for test chemical
preparation; 95 % can be used for the desorb solution)l) Glacial acetic acid, analytical gradem) Distilled H2O or any purified water suitable for cell culture and NR desorb solution(sterile)n) Sterile/non-sterile paper towels (for blotting 96-well plates)[Note: Due to lot variability of NBCS/NCS, first check a lot for growth stimulating
properties with 3T3 cells (approximately 20-24 h doubling time) and then reserve a
sufficient amount of NBCS/NCS. May use pre-tested serum lot from Phases Ia, Ib, and II
of the validation study if the serum has been stored under appropriate conditions andshelf-life has not expired.]
B. Preparations of Media and Solutions[Note: All solutions (except NR stock solution, NR medium and NR desorb), glassware,
pipettes, etc., shall be sterile and all procedures should be carried out under aseptic conditions
and in the sterile environment of a laminar flow cabinet (biological hazard standard). All
methods and procedures will be adequately documented.]
1. Media
DMEM (buffered with sodium bicarbonate) supplemented with (final concentrations in
DMEM are quoted):

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In Vitro Cytotoxicity Test Methods BRD Appendix B1

November 2006a) for freezing (Freeze Medium); contains 2X concentration of NBCS/NCS and DMSO
of final freezing solution
40 %NBCS/NCS
20 %DMSOb) for routine culture (Routine Culture Medium)
10 %NBCS/NCS
4 mM
Glutaminec) for test chemical dilution (Chemical Dilution Medium)
4 mM
Glutamine
200 IU/mL
Penicillin
200 µg/mL
Streptomycind) for dilution of NR stock solution (NR Dilution Medium)5%
4 mM
100 IU/mL
100 µg/mL

NBCS/NCS
Glutamine
Penicillin
Streptomycin[Note: The Chemical Dilution Medium with test chemical will dilute the serum
concentration of the Routine Culture Medium in the test plate to 5 %. Serum proteins
may mask the toxicity of the test substance, but serum cannot be totally excluded because
cell growth is markedly reduced in its absence.]
Completed media formulations should be kept at approximately 2-8° C and stored for nolonger than two weeks.
2. Neutral Red (NR) Stock Solution
The liquid tissue culture-grade stock NR Solution will be the first choice for performing
the assay (e.g., SIGMA #N2889, 3.3 mg/mL). Store liquid tissue culture-grade NR Stock
Solution at the storage conditions and shelf-life period recommended by the
manufacturer.
If the liquid form is not available, the following formulation can be prepared.
EXAMPLE:

0.25 g NR Dye powder in 100 mL H2O

The NR Stock Solution (powder in water) should be stored in the dark at room
temperature for up to two months.
3. Neutral Red (NR) Medium
EXAMPLE:
0.758 mL (3.3 mg NR dye/mL solution)
99.242 mL

NR Stock Solution
NR Dilution Medium (pre-warmed to 37° C)

The final concentration of the NR Medium is 25 µg NR dye/mL and aliquots will be
prepared on the day of application.
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In Vitro Cytotoxicity Test Methods BRD Appendix B1

November 2006

[Note: The NR Medium shall be filtered (e.g., Millipore filtering, 0.2 – 0.45 µm poresize) to reduce NR crystals. Aliquots of the NR Medium should be maintained at 37° C(e.g., in a waterbath) before adding to the cells and used within 30 min of preparation but
also used within 15 min after removing from 37° C storage.]
4. Ethanol/Acetic Acid Solution (NR Desorb)
1%
50 %
49 %

Glacial acetic acid solution
Ethanol
H2O

C. Methods
1. Cell Maintenance and Culture Procedures
BALB/c 3T3 cells are routinely grown as a monolayer in tissue culture grade flasks (e.g.,
75 - 80 cm2) at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air. The cells
should be examined on a daily (i.e., on workdays) basis under a phase contrast
microscope, and any changes in morphology or their adhesive properties noted in a Study
Workbook.
2. Receipt of Cryopreserved BALB/c 3T3 Cells
Upon receipt of cryopreserved BALB/c 3T3 cells, the vial(s) of cells shall be stored in aliquid nitrogen freezer until needed.
3. Thawing Cells
Thaw cells by putting ampules into a water bath at 37°C ± 1ºC. Leave for as brief a time
as possible.
a) Resuspend the cells in pre-warmed Routine Culture Medium and transfer into
pre-warmed Routine Culture Medium in a tissue-culture flask.
b) Incubate at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air.
c) When the cells have attached to the bottom of the flask (within 4 to 24 h),decant the supernatant and replace with fresh pre-warmed (37ºC) medium.
Culture as described above.
d) Passage at least two times before using the cells in a cytotoxicity test.
A fresh batch of frozen cells from the stock lot of cells should be thawed out and cultured
approximately every two months. This period resembles a sequence of about 18
passages.
4. Routine Culture of BALB/C 3T3 Cells
When cells exceed 50 % confluence (but less than 80 % confluent) they should be
removed from the flask by trypsinization:
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In Vitro Cytotoxicity Test Methods BRD Appendix B1

November 2006a) Decant medium, briefly rinse cultures with 5 mL PBS or Hanks’ BSS (without Ca2+,
Mg2+) per 25 cm2 flask (15 mL per 75 cm2 flask). Wash cells by gentle agitation toremove any remaining serum that might inhibit the action of the trypsin.
b) Discard the washing solution. Repeat the rinsing procedure and discard the washingsolution.
c) Add 1-2 mL trypsin-EDTA solution per 25 cm2 to the monolayer for a few seconds(e.g., 15-30 seconds).
d) Remove excess trypsin-EDTA solution and incubate the cells at room temperature.
e) After 2-3 minutes (min), lightly tap the flask to detach the cells into a single cellsuspension.
5. Cell Counting
After detaching the cells, add 0.1-0.2 mL of pre-warmed (37ºC) Routine Culture
Medium/cm2 to the flask (e.g., 2.5 mL for a 25 cm2 flask). Disperse the monolayer by
gentle trituration. It is important to obtain a single cell suspension for exact counting.
Count a sample of the cell suspension obtained using a hemocytometer or cell counter(e.g., Coulter counter).
6. Subculture of Cells
After determination of cell number, the culture can be sub-cultured into other flasks orseeded into 96-well microtiter plates. BALB/c 3T3 cells are routinely passaged atsuggested cell densities as listed in the table (approximate doubling time is 20-24 h). The
individual laboratories will need to determine and adjust the final density to achieve
appropriate growth.
Table 1. Cell Density Guidelines for Subculturing
Days in Culture2
3
4

Seeding Density(cells/cm2)
16800
8400
4200

Total Cells per 25 cm2
flask
4.2 x 105
2.1 x 105
1.05 x 105

Total Cells per 75 cm2
flask
1.26 x 106
6.3 x 105
3.15 x 105[Note: It is important that cells have overcome the lag growth phase when they are used
for the test.]
7. Freezing Cells (procedure required only if current stock of cells is depleted)
Stocks of BALB/c 3T3 cells can be stored in sterile, freezing tubes in a liquid nitrogen
freezer. DMSO is used as a cryoprotective agent.

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In Vitro Cytotoxicity Test Methods BRD Appendix B1

November 2006a) Centrifuge trypsinized cells at approximately 200 x g.
b) Suspend the cells in cold Routine Culture Medium (half the final freezingvolume) so a final concentration of 1-5x106 cells/mL can be attained.
c) Slowly add cold Freeze Medium to the cells so that the solvent will equilibrate
across the cell membranes. Bring the cell suspension to the final freezingvolume. The final cell suspension will be 10 % DMSO. Aliquot the cellsuspension into freezing tubes and fill to 1.8 mL.
d) Place the tubes into an insulated container (e.g., styrofoam trays) and place in afreezer (-70 to -80°C) for 24 h. This gives a freezing rate of approximately
1°C/min. The laboratory needs to ensure that the freezing protocol is applicableto the 3T3 cells and that the cells are viable when removed fromcryopreservation.
e) Place the frozen tubes into liquid nitrogen for storage.
8. Preparation of Cells for Assaysa) Cultured cells that are going to be used in seeding the 96-well plates should be fed
fresh medium the day before subculturing to the plates. On the day of plate seeding,4
prepare a cell suspension of 2.0 – 3.0x10 cells/mL in Routine Culture Medium.
Using a multi-channel pipette, dispense 100 µl Routine Culture Medium only into theperipheral wells (blanks) of a 96-well tissue culture microtiter plate (See Section
VII.F.1). In the remaining wells, dispense 100 µl of a cell suspension of 2.0 –3
3.0x104 cells/mL (= 2.0 – 3.0x10 cells/well). The seeding density should be noted toensure that the cells in the control wells are not overgrown after three days (i.e., 24 hincubation in step b and 48 h exposure to test chemicals). Prepare one plate perchemical to be tested.
b) Incubate cells for 24 ± 2 h (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air)so that cells form a less than half (< 50%) confluent monolayer. This incubation
period assures cell recovery and adherence and progression to exponential growthphase.
c) Examine each plate under a phase contrast microscope to assure that cell growth isrelatively even across the microtiter plate. This check is performed to identify
experimental and systemic cell seeding errors. Record observations in the Study
Workbook.
9. Determination of Doubling Timea) A cell doubling time procedure was performed on the initial lot of cells that was usedin the first cell culture assays of Phase Ia of the Validation Study. The doubling time
only needs to be determined in Phase III if there is a change in the lot of cells used.
Establish cells in culture and trypsinize cells as per Section VII.C.4 for subculture.
Resuspend cells in NR Dilution Medium (5 % NBCS/NCS). Seed cells at 4200
cells/cm2.

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In Vitro Cytotoxicity Test Methods BRD Appendix B1

November 2006b) Seed five sets of cell culture vessels in triplicate for each cell type (e.g., 15 tissue
culture dishes [60mm x 15mm]). Use appropriate volume of culture medium for theculture vessels. Note number of cells placed into each culture dish. Place dishes into
the incubators (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air).
c) After 4 - 6 hours (use the same initial measurement time for each subsequent
doubling time experiment), remove three culture dishes and trypsinize cells. Count
cells using a cell counter or hemocytometer. Cell viability may be determined by dye
exclusion (e.g., Trypan Blue; Nigrosin) if Study Director sees a need. Useappropriate size exclusion limits if using a Coulter counter. Determine the total
number of cells and document. Repeat sampling at 24 h, 48 h, 72 h, and 96 h post
inoculation. Change culture medium at 72 h or sooner in remaining dishes if
indicated by pH drop.
d) Plot cell concentration (per mL of medium) on a log scale against time on a linear
scale. Determine lag time and population doubling time. Additional dishes and time
are needed if the entire growth curve is to be determined (lag phase, log phase,plateau phase).
D. Preparation of Test Chemicals
The Study Management Team will provide direction on the solvent to be used for each test
chemical. [Note: Preparation under red or yellow light is recommended to preserve chemicals
that degrade upon exposure to light.]
1. Test Chemicals in Solutiona) Allow test chemicals to equilibrate to room temperature before dissolving anddiluting.
b) Prepare test chemical immediately prior to use. Test chemical solutions should not
be prepared in bulk for use in subsequent tests. Ideally, the solutions must not be
cloudy nor have noticeable precipitate. Each stock dilution should have at least 1-2
mL total volume to ensure adequate solution for the test wells in a single 96-wellplate. The SMT may direct the Study Director to store an aliquot (e.g., 1 mL) of thehighest 2X stock solution (e.g., low solubility chemicals) in a freezer (e.g., -70°C) for
use in future chemical analyses.
c) For chemicals dissolved in DMSO or ethanol, the final DMSO or ethanol
concentration for application to the cells must be 0.5 % (v/v) in the vehicle controls
and in all of the eight test concentrations.
d) The stock solution for each test chemical should be prepared at the highest
concentration found to be soluble in the solubility test conducted per the Test Method
Protocol for Solubility Determination. Thus, the highest test concentration applied tothe cells in each range finding experiment is:• 0.5 times the highest concentration found to be soluble in the solubility test, if thechemical was soluble in Chemical Dilution Medium, or

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In Vitro Cytotoxicity Test Methods BRD Appendix B1•

November 2006

1/200 the highest concentration found to be soluble in the solubility test if thechemical was soluble in ethanol or DMSO.

e) The seven lower concentrations in the range finding experiment would then be
prepared by successive dilutions that decrease by one log unit each. The following
example illustrates the preparation of test chemical in solvent and the dilution ofdissolved test chemical in Chemical Dilution Medium before application to 3T3 cells.
Example: Preparation of Test Chemical in Solvent Using a Log Dilution Scheme
If DMSO was determined to be the preferred solvent at Tier 2 of the solubility test (i.e.,
200,000 µg/mL), dissolve the chemical in DMSO at 200,000 µg/mL for the chemical
stock solution.
1) Label eight tubes 1 – 8. Add 0.9 mL solvent (e.g., DMSO) to tubes 2 -- 8.
2) Prepare stock solution of 200,000 µg test chemical/mL solvent in tube # 1.
3) Add 0.1 mL of 200,000 µg/mL dilution from tube #1 to tube #2 to make a 1:10
dilution in solvent (i.e., 20,000 µg/mL).
4) Add 0.1 mL of 20,000 µg/mL dilution from tube #2 to tube #3 to make another 1:10
dilution (i.e., 1:100 dilution from stock solution) in solvent (i.e., 2,000 µg/mL)5) Continuing making serial 1:10 dilutions in the prepared solvent tubes.
6) Since each concentration is 200 fold greater than the concentration to be tested, makea 1:100 dilution by diluting 1 part dissolved chemical in each tube with 99 parts ofChemical Dilution Medium (e.g., 0.1 mL test chemical in DMSO + 9.9 mL Chemical
Dilution Medium) to derive the eight 2X concentrations for application to 3T3 cells.
Each 2X test chemical concentration will then contain 1 % v/v solvent. The 3T3
cells will have 0.05 mL Routine Culture Medium in the wells prior to application of
the test chemical. By adding 0.05 mL of the appropriate 2X test chemical
concentration to the appropriate wells, the test chemical will be diluted appropriately(e.g., highest concentration in well will be 1,000 µg/mL) in a total of 0.1 mL and thesolvent concentration in the wells will be 0.5% v/v.
7) A test article prepared in Chemical Dilution Medium, DMSO, or ethanol may
precipitate upon transfer into the Routine Culture Medium. The 2X dosing solutions
should be evaluated for precipitates and the results recorded in the workbook. It will
be permissible to test all of the dosing solutions in the dose range finding assay andmain experiments. However, doses containing test article precipitates should be
avoided and generally will not be used in the ICx determinations for the definitive
tests. Precipitates in 2X dosing solutions are permissible for range finder tests but
not for definitive tests.
Document all test chemical preparations in the Study Workbook.

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2. pH of Test Chemical Solutions
Prior to or immediately after application of the test chemical to the 96-well plate,measure the pH of the highest 2X dosing concentration of the test chemical (i.e., C1 in
the test plate, see Figure 1) in culture medium. Use pH paper (e.g., pH 0 - 14 to estimate
and pH 5 – 10 to determine more precise value; or Study Director’s discretion) for
measurements. The pH paper should be in contact with the solution for approximately
one minute. Document the pH and note the color of the 2X concentration medium (i.e.,in the EXCEL template). Medium color for all dosing dilutions should be noted in the
workbooks. Do not adjust the pH.
3. Concentrations of Test Chemicala) Range Finder Experiment
Test eight concentrations of the test chemical by diluting the stock solution with aconstant factor covering a large range. The initial dilution series shall be log
dilutions (e.g., 1:10, 1:100, 1:1000, etc.).
If a range finder experiment does not generate enough cytotoxicity, then higher doses
should be attempted. If cytotoxicity is limited by solubility, then more stringent
solubility procedures to increase the stock concentration (to the maximum
concentration specified in Section VII.D.3.b.) should be employed. Place the test
chemical concentration into an incubator (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ±
1 % CO2/air) and stir or rock for up to 3 hours, if necessary, to facilitate dissolution.
For stocks prepared in medium, vessel caps should be loose to allow for CO2
exchange. Proceed with dosing solution preparation and dosing.
•If a range finding test produces a biphasic curve, then the doses selected for thesubsequent main experiments should cover the most toxic dose-response range(see Example 1 – the most toxic range is 0.001 – 0.1 µg/mL).
Example 1 – Biphasic Curve
Neutral Red Uptake
125%
100%
75%
50%
25%
0%
0.001

0.010

0.100

1.000

10.000Concentration (µg/mL)

B-16

100.000

1000.000

10000.000

In Vitro Cytotoxicity Test Methods BRD Appendix B1

November 2006b) Main Experiment[Note: After the range finding assay is completed, the definitive concentrationresponse experiment shall be performed three times on three different days for eachchemical (i.e., one plate per day per chemical.]
Depending on the slope of the concentration-response curve estimated from the range
finder, the dilution/progression factor in the concentration series of the main
experiment should be smaller (e.g., dilution factor of 6√10 = 1.47). Cover the
relevant concentration range around the IC50 (> 0 % and < 100 % effect) preferablywith several points of a graded effect, but with a minimum of two points, one on eachside of the estimated IC50 value, avoiding too many non-cytotoxic and/or 100 %cytotoxic concentrations. Experiments revealing less than one cytotoxic
concentration on each side of the IC50 value shall be repeated, where possible, with asmaller dilution factor (see Section VII.E.5.a.4). Each experiment should have atleast one cytotoxicity value > 0 % and ≤ 50.0 % viability and at least one cytotoxicity
value > 50.0 % and < 100 % viability. A progression factor of 1.21 [12√10] isregarded the smallest factor achievable and will be the lowest dosing interval
required.)
Determine which test chemical concentration is closest to the IC50 value (e.g., 50 %cytotoxicity). Use that value as a central concentration and adjust dilutions higher
and lower in equal steps for the definitive assay.
Maximum Doses to be Tested in the Main Experiments
If minimal or no cytotoxicity was measured in the dose range finding assay, amaximum dose for the main experiments will be established as follows:• For test chemicals prepared in Chemical Dilution Medium, the highest test article
concentration that may be applied to the cells in the main experiments will beeither 100 mg/mL, or the maximum soluble dose. Test chemical will be weighed
into a glass tube and the weight will be documented. A volume of Chemical
Dilution Medium will be added to the vessel so that the concentration is200,000 µg/mL (200 mg/mL). The solution is mixed using the mechanicalprocedures that produced solubility when performing the solubility test specified
in Test Method Protocol for Solubility Determination. If complete solubility isachieved in medium, then 7 additional serial stock dosing solutions may be
prepared from the 200 mg/mL 2X stock. If the test chemical is insoluble inmedium at 200 mg/ml, proceed by adding medium, in small incremental
amounts, to attempt to dissolve the chemical by using the sequence of
mechanical procedures specified in Test Method Protocol for Solubility
Determination. More stringent solubility procedures may be employed if needed
based on results from the range finder experiment (Section VII.D.3.a.). The
highest soluble stock solution will be used to prepare the 7 additional serial stock
dosing solutions.
•

For test chemicals prepared in either DMSO or ethanol, the highest test article
concentration that may be applied to the cells in the main experiments will beeither 2.5 mg/mL, or less, depending upon the maximum solubility in solvent.
Weigh the test chemical into a glass tube and document the weight. Add theappropriate solvent (determined from the original solubility test) to the vessel so
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In Vitro Cytotoxicity Test Methods BRD Appendix B1

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that the concentration is 500,000 µg/mL (500 mg/mL). Mix the solution using
the sequence of mechanical procedures specified in Test Method Protocol for
Solubility Determination. If complete solubility is achieved in the solvent, then 7
additional serial stock dosing solutions may be prepared from the 500 mg/mL
200X stock. If the test chemical is insoluble in solvent at 500 mg/ml, proceed byadding solvent, in small incremental amounts, to attempt to dissolve the chemical
by again using the sequence of mixing procedures. The highest soluble stock
solution will be used to prepare the 7 additional serial stock dosing solutions.
•

If precipitates are observed in the 2X dilutions, continue with the experiment,
make the appropriate observations and documentation, and report data to theSMT.

c) Test Chemical Dilutions
The dosing factor of 3.16 (= 2√10) divides a log into two equidistant steps, a factor of
2.15 (= 3√10) divides a decade into three steps. The factor of 1.47 (= 6√10) divides alog into six equidistant steps, the factor of 1.78 (4√10) divides a log into four
equidistant steps, and the factor of 1.21 (= 12√10) divides the log into 12 steps.
EXAMPLE:
10
10
10
10

31.6
12.1

14.7
14.7

17.8

21.5
21.5
21.5

26.1

31.6
31.6

38.3

46.4
46.4
46.4

56.2

68.1
68.1

82.5

100
100
100
100

The technical production of decimal geometric concentration series is simple. An
example is given for factor 1.47:
Dilute 1 volume of the highest concentration by adding 0.47 volumes of diluent. Afterequilibration, dilute 1 volume of this solution by adding 0.47 volumes ofdiluent...(etc.).
E. Test Procedure
1. 96-Well Plate Configuration
The 3T3 NRU assay for test chemicals will use the 96-well plate configuration as shown in
Figure 1.

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November 2006

Figure 1. 96-Well Plate Configuration for Positive Control (PC) and Test Chemical
Assays
1

2

3

4

5

6

7

8

9

10

11

12

A

VCb VCb C1b

C2b

C3b

C4b

C5b

C6b

C7b

C8b

VCb VCbB

VCb VC1

C1

C2

C3

C4

C5

C6

C7

C8

VC2 VCbC

VCb VC1

C1

C2

C3

C4

C5

C6

C7

C8

VC2 VCbD

VCb VC1

C1

C2

C3

C4

C5

C6

C7

C8

VC2 VCbE

VCb VC1

C1

C2

C3

C4

C5

C6

C7

C8

VC2 VCbF

VCb VC1

C1

C2

C3

C4

C5

C6

C7

C8

VC2 VCbG

VCb VC1

C1

C2

C3

C4

C5

C6

C7

C8

VC2 VCbH

VCb VCb C1b

C2b

C3b

C4b

C5b

C6b

C7b

C8b

VCb VCb

VC1 and VC2 = VEHICLE CONTROL
C1 – C8= Test Chemicals or PC (SLS) at eight concentrations(C1 = highest, C8 = lowest)b= BLANKS (Test chemical or PC, but contain no cells)
VCb= VEHICLE CONTROL BLANK (contain no cells)
2. Application of Test Chemicala) Two optional methods for rapidly applying the 2X dosing solutions onto the 96-wellplates may be utilized.
1) The first method is to add each of the 2X dosing solutions into labeled, sterile
reservoirs (e.g., Corning/Costar model 4870 sterile polystyrene 50 mL reagent
reservoirs; or Corning/Transtar model 4878 disposable reservoir liners, 8channel; or other multichannel reservoirs).
2) The second method utilizes a “dummy” plate (i.e., an empty sterile 96-well plate)
prepared to hold the dosing solutions immediately prior to treatment of the test
plate (with cells). The test chemical and control dosing solutions should be
dispensed into the dummy plate in the same pattern/order as will be applied to theplate containing cells. More volume than needed for the test plate (i.e. greaterthan 50 µl/well) should be in the wells of the dummy plate.
At the time of treatment initiation, a multi-channel micropipettor is used to transfer
the 2X dosing solutions, from the reservoirs or dummy plate, to the appropriate wellson the treatment plate (as described in step c. below). These methods will ensure thatthe dosing solutions can be transferred rapidly to the appropriate wells of the test
plate to initiate treatment times and to minimize the range of treatment initiation
times across a large number of treatment plates, and to prevent “out of order” dosing.
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Do not use a multichannel repeater pipette for dispensing test chemical to the plates.
b) After 24 h ± 2 h incubation of the cells, remove Routine Culture Medium from thecells by careful inversion of the plate (i.e., “dump”) over an appropriate receptacle.
Gently blot the plate on a sterile paper towel so that the monolayer is minimallydisrupted. Do not use automatic plate washers for this procedure nor vacuum
aspiration.
c) Immediately add 50 µL of fresh pre-warmed Routine Culture Medium to all of thewells, including the blanks. Fifty microliters (50 µL) of dosing solution will be
rapidly transferred from the 8-channel reservoir (or dummy plate) to the appropriate
wells of the test plate using a single delivery multi-channel pipettor. For example,
the VC may be transferred first (into columns 1, 2, 11, and 12), followed by the test
article dosing solutions from lowest to highest dose, so that the same pipette tips on
the multi-channel pipettor can be used for the whole plate. [The Vehicle Control
blank (VCb) wells (column 1, column 12, wells A2, A11, H2, H11) will receive the
Vehicle Control dosing solutions (which should include any solvents used). Blanks
for wells A3 – A10 and H3 – H10 shall receive the appropriate test chemicalsolutions for each concentration (e.g., wells A3 and H3 receive C1 solution).
d) Incubate cells for 48 h ± 0.5 h (37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 %CO2/air).
e) Positive Control: For each set of test chemical plates used in an assay, a separate
plate of positive control concentrations will be set up following the concentration
range established in the development of the positive control database in Phase I of the
Validation Study. If multiple sets of test chemical plates are set up, then clearly
designate the positive control plates for each set; each set will be an individual entity.
The Study Director will decide how many test chemical plates will be run with apositive control plate. The mean IC50 ± two and a half standard deviations (SD) for
the SLS acceptable tests from Phases Ia, Ib, and II (after the removal of outliers) are
the values that will be used as an acceptance criterion for test sensitivity for the 3T3
NRU assay. This plate will follow the same schedule and procedures as used for thetest chemical plates (including appropriate chemical concentrations in the appropriate
wells and meeting test acceptance criteria – see sections VII.E.1, E.2, and E.5).
3. Microscopic Evaluation
After at least 46 h treatment, examine each plate under a phase contrast microscope toidentify systematic cell seeding errors and growth characteristics of control and treatedcells. Record any changes in morphology of the cells due to the cytotoxic effects of thetest chemical, but do not use these records for any quantitative measure of cytotoxicity.
Undesirable growth characteristics of control cells may indicate experimental error andmay be cause for rejection of the assay. Use the following Visual Observations Codes inthe description of cell culture conditions. Numerical scoring of the cells (see Section
VII.E.3) should be determined and documented in the Study Workbook and in theappropriate section of Addendum II of the EXCEL study template.

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In Vitro Cytotoxicity Test Methods BRD Appendix B1

November 2006

Visual Observations Codes
Note Code
1
2
3
4
1P
2P
3P
4P
5P

Note Text
Normal Cell Morphology
Low Level of Cell Toxicity
Moderate Level of Cell Toxicity
High level of Cell Toxicity
Normal Cell Morphology with Precipitate
Low Level of Cell Toxicity with Precipitate
Moderate Level of Cell Toxicity with PrecipitateHigh level of Cell Toxicity with Precipitate
Unable to View Cells Due to Precipitate

4. Measurement of NRUa) Carefully remove (i.e., “dump”) the medium with test chemical and rinse the cells
very carefully with 250 µL pre-warmed D-PBS. Remove the rinsing solution bydumping and remove excess by gently blotting on paper towels. Add 250 µL NR
medium (to all wells including the blanks) and incubate (37ºC ± 1ºC, 90 % ± 5 %humidity, and 5.0 % ± 1 % CO2/air) for 3±0.1 h. Observe the cells briefly during the
NR incubation (e.g., between 2 and 3 h – Study Director’s discretion) for NR crystal
formation. Record observations in the Study Workbook. Study Director can decideto reject the experiment if excessive NR crystallization has occurred.
b) After incubation, remove the NR medium, and carefully rinse cells with 250 µl prewarmed D-PBS.
c) Decant and blot D-PBS from the plate.
d) Add exactly 100 µl NR Desorb (ETOH/acetic acid) solution to all wells, including
blanks.
e) Shake microtiter plate rapidly on a microtiter plate shaker for 20 – 45 min to extract
NR from the cells and form a homogeneous solution. Plates should be protectedfrom light by using a cover during shaking.
f) Plates should be still for at least five minutes after removal from the plate shaker (ororbital mixer). If any bubbles are observed, assure that they have been ruptured prior
to reading the plate. Measure the absorption (within 60 minutes of adding NR
Desorb solution) of the resulting colored solution at 540 nm ± 10 nm in a microtiterplate reader (spectrophotometer), using the blanks as a reference. [Note: Phases Ia
and Ib data show the mean OD value for the plate blanks to be 0.057 ± 0.043 for 3T3
cells (± 2.5 standard deviations; data from 3 labs; N = 189). Use this range as a
guide for assessment of the blank values.] Save raw data in the Excel format asprovided by the SMT.

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5. Quality Check of 3T3 NRU Assaya) Test Acceptance Criteria
All acceptance criteria (i.e., criteria 1, 2, and 3) must be met for a test to be
acceptable.
1) The PC (SLS) IC50 must be within ± two and a half (2.5) standard deviations of
the historical mean established by the Test Facility (as per VII.E.2.e), and mustmeet criteria 2 and 3, and must have an r2 (coefficient of determination) value
calculated for the Hill model fit (i.e., from PRISM software) ≥ 0.85.
2) The left and right mean of the VCs do not differ by more than 15% from themean of all VCs.
3) At least one calculated cytotoxicity value > 0 % and ≤ 50.0 % viability andat least one calculated cytotoxicity value > 50.0 % and < 100 % viability must bepresent.
Exception: If a test has only one point between 0 and 100 % and the smallest
dilution factor (i.e., 1.21) was used and all other test acceptance criteria weremet, then the test will be considered acceptable.
Stopping Rule for Insoluble Chemicals: If the most rigorous solubility procedures
have been performed and the assay cannot achieve adequate toxicity to meet the test
acceptance criteria after three definitive trials, then the Study Director may end all
testing for that particular chemical.
[Note: A corrected mean OD540 ± 10nm of 0.103 - 0.813 for the VCs is a target range but
will not be a test acceptance criterion. Range determined from Phase Ib VC OD
values from 3 laboratories (mean ± 2.5 standard deviations, N = 98).]b) Checks for Systematic Cell Seeding Errors
To check for systematic cell seeding errors, untreated VCs are placed both at the left
side (row 2) and the right side (row 11 for the test plates) of the 96-well plate.
Aberrations in the cell monolayer for the VCs may reflect a volatile and toxic test
article present in the assay. If volatility is suspected, then proceed to Section
VII.E.6.
Checks for cell seeding errors may also be performed by examining each plate undera phase contrast microscope to assure that cell quantity is consistent.
6. Volatility of Test Chemicals
Highly volatile test chemicals may generate vapors from the treatment medium during thetest chemical treatment incubation period. These vapors may become resorbed into thetreatment medium in adjacent wells, such that culture wells nearest the highest doses may
become contaminated by exposure to resorbed test article vapors. If the test chemical isparticularly toxic at the doses tested, the cross contamination may be evident as a
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significant reduction in viability in the vehicle control cultures (i.e., VC1) adjacent to thehighest test chemical doses.
If potential test article volatility is suspected (e.g., for low density liquids) or if the initial
range finder test (non-sealed plate) results show evidence of toxic effects in the control
cultures (i.e., > 15 % difference in viability between VC1 [column 2] and VC2 [column11]), then seal the subsequent test plates by the following procedure.
a) Plate Sealer Method
1) Plates and chemicals will be prepared as usual according to Sections VII.D and
VII.E.
2) Immediately after the 96-well culture plate has been treated with the suspectedvolatile chemical (Section VII.E.2.b), apply the adhesive plate sealer (e.g., usinga hand, microplate roller, etc.) directly over the culture wells. Assure that thesealer adheres to each culture well (well tops should be dry). Place the 96-wellplate cover over the sealed plate and incubate the plate under specified conditions(Section VII.E.2.b). [Note: Do not jam the plate lid over the film to avoid
deforming the sealer and causing the sealer to detach from culture wells. Loose
fit of the plate lid is acceptable.]3) At the end of the treatment period, the plate sealer should be carefully removed
to avoid spillage. Continue with the NRU assay as per Section VII.E.4.
F. Data Analysis

The Study Director will use good biological/scientific judgment for determining“unusable” wells that will be excluded from the data analysis and provide explanations
for the removal of any data from the analysis.
A calculation of cell viability expressed as NRU is made for each concentration of the test
chemical by using the mean NRU of the six replicate values (minimum of four acceptable
replicate well) per test concentration (blanks will be subtracted). This value is compared withthe mean NRU of all VC values. Relative cell viability is then expressed as percent of
untreated VC. If achievable, the eight concentrations of each chemical tested will span therange of no effect up to total inhibition of cell viability. Data from the microtiter plate reader
shall be transferred to the Excel spreadsheet template provided by the SMT. The template
will automatically determine cell viability, IC50 values by linear interpolation, and performstatistical analyses (including statistical identification of outliers). The template will also
calculate the concentrations associated with 20 %, 50 %, and 80 % viability using the Hill
slope and EC50 (i.e., IC50) from the Hill function analysis.
The Hill function analysis shall be performed using statistical software (e.g., GraphPad
PRISM 3.0) and a template specified by the SMT to calculate IC20, IC50, and IC80 values(and the associated confidence limits) for each test chemical.
The Testing Facility shall report data using at least three (3) significant figures and shallforward the results from each assay to the SMT through the designated contacts in electronic
format and hard copy upon completion of testing. The SMT will be directly responsible forthe statistical analyses of the Validation Study data.

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VIII.

November 2006

REFERENCES
Hackenberg, U. and H. Bartling. 1959. Messen und Rechnen im pharmakologischen
Laboratorium mit einem speziellen Zahlensystem (WL24-System). Arch. Exp. Pathol.
Pharmakol. 235: 437-463.
Spielmann, H., S. Gerner, S. Kalweit, R. Moog, T. Wirnserberger, K. Krauser, R. Kreiling, H.
Kreuzer, N.P. Luepke, H.G. Miltenburger, N. Müller, P. Murmann, W. Pape, B. Siegmund, J.
Spengler, W. Steiling, and F.J. Wiebel. 1991. Interlaboratory assessment of alternatives tothe Draize eye irritation test in Germany. Toxicol. In Vitro 5: 539-542.
Test Method Protocol for Solubility Determination. In Vitro Cytotoxicity Validation Study.
Phase III. August 29, 2003. Prepared by The National Toxicology Program (NTP)
Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM).

IX.

APPROVAL__________________________________SPONSOR REPRESENTATIVE(Print or type name)___________________DATE_____________________________
Test Facility STUDY DIRECTOR(Print or type name)____________________
DATE

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Appendix B2

Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake (NRU) Cytotoxicity Test

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[This Page Intentionally Left Blank]

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TEST METHOD PROTOCOL
for the NHK Neutral Red Uptake Cytotoxicity Test

A Test for Basal Cytotoxicity for an In Vitro Validation Study
Phase III
November 4, 2003

Prepared by
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)

Based on Standard Operating Procedure Recommendations from anInternational Workshop Organized by the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM)

National Institute of Environmental Health Sciences (NIEHS)
National Institutes of Health (NIH)
U.S. Public Health Service
Department of Health and Human Services

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TEST METHOD PROTOCOLThe Normal Human Keratinocyte (NHK) Neutral Red Uptake
Cytotoxicity Test
A Test for Basal Cytotoxicity
Phase IIII.

PURPOSEThe purpose of this study is to evaluate the cytotoxicity of test chemicals using the Normal
Human Keratinocyte (NHK) Neutral Red Uptake (NRU) cytotoxicity test. The data will be used
to evaluate the intra- and inter-laboratory reproducibility of the assay and effectiveness of thecytotoxicity assay to predict the starting doses for rodent acute oral systemic toxicity assays. This
test method protocol outlines the procedures for performing the cytotoxicity test and is in supportof the in vitro validation study organized by NICEATM and the European Centre for the
Validation of Alternative Methods (ECVAM) and sponsored by NIEHS, U.S. Environmental
Protection Agency, and ECVAM. This test method protocol applies to all personnel involved
with performing the cytotoxicity assay.
A. NHK Neutral Red Uptake Cytotoxicity Test
The NHK NRU test will be performed to analyze the in vitro toxicity of 60 blinded/coded test
chemicals. This test will be used to determine IC20, IC50, and IC80 values for thepredetermined set of test chemicals of varying toxicities.

II. SPONSOR
A. Name:National Institute of Environmental Health Sciences (NIEHS); The
National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)

B. Address:

P.O. Box 12233
Research Triangle Park, NC 27709

C. Representative:

Named Representative

III. IDENTIFICATION OF TEST AND CONTROL SUBSTANCES
A. Test Chemicals:

Blinded chemicals (60)

B. Controls:

Positive:
Vehicle (Negative):
Solvent (as directed):

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Sodium Lauryl Sulfate
Assay medium
Assay medium, DMSO, or ethanol asdirected by the Study Management Team,for preparation of test chemicals

In Vitro Cytotoxicity Test Methods BRD Appendix B2

November 2006

IV. TESTING FACILITY AND KEY PERSONNEL
A. Facility Information
1) Name:2) Address:
3) Study Director:
4) Laboratory Technician(s):5) Scientific Advisor:
6) Quality Assurance Director:
7) Safety Manager:
8) Facility Management:
B. Test Schedule
1) Proposed Experimental Initiation Date:
2) Proposed Experimental Completion Date:
3) Proposed Report Date:
V. TEST SYSTEMThe NRU cytotoxicity assay procedure is a cell survival/viability chemosensitivity assay based on
the ability of viable cells to incorporate and bind neutral red (NR), a supravital dye. NR is a weakcationic dye that readily penetrates cell membranes by non-ionic diffusion and accumulates
intracellularly in lysosomes. Alterations of the cell surface or the sensitive lysosomal membranelead to lysosomal fragility and other changes that gradually become irreversible. Such changes
brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It isthus possible to distinguish between viable, damaged, or dead cells, which is the basis of thisassay.
Healthy mammalian cells, when maintained in culture, continuously divide and multiply overtime. A toxic chemical, regardless of site or mechanism of action, will interfere with this process
and result in a reduction of the growth rate as reflected by cell number. Cytotoxicity is expressed
as a concentration dependent reduction of the uptake of the NR after chemical exposure thus
providing a sensitive, integrated signal of both cell integrity and growth inhibition.
VI. DEFINITIONS
A.. Hill function: a four parameter logistic mathematical model relating the concentration of test
chemical to the response being measured in a sigmoidal shape.

Y = Bottom +Top ! Bottom
1 + 10(logIC50! X)HillSlope

where Y= response, X is the logarithm of dose (or concentration), Bottom is the minimum
response, Top is the maximum response, logIC50 is logarithm of X at the response midway
between Top and Bottom, and HillSlope describes the steepness of the curve.
B. Documentation: all methods and procedures will be noted in a Study Workbook; logs will bemaintained for general laboratory procedures and equipment (e.g., media preparation, test
chemical preparation, incubator function); all optical density data obtained from theB-29

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spectrophotometer plate reader will be saved in electronic and paper formats; all calculationsof ICx values and other derived data will be in electronic and paper format; all data will bearchived.
VII.

PROCEDURES

A. Materials[Note: Suggested brand names/vendors are listed in parentheses. Equivalents may be used
unless otherwise noted.]
1. Cell Lines
Normal Human Epidermal Keratinocytes (NHK)Non-transformed cells; from cryopreserved primary or secondary cells (Clonetics #CC2507 or equivalent). Cells will be Clonetics NHK cells.
Cambrex [Cambrex Bio Science, 8830 Biggs Ford Road, Walkersville, MD 21793-0127
Cambrex Europe [Cambrex Bio Science Verviers, S.P.R.L. Parc Industriel de Petit
Rechain, B-4800 Verviers, BELGIUM]
2. Technical Equipment[Note: Suggested brand names/vendors are listed in parentheses. Equivalents may be
used.]
a)
b)
c)
d)
e)
f)
g)
h)

Incubator: 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/airLaminar flow clean bench (standard: "biological hazard")
Water bath: 37ºC ± 1ºC
Inverse phase contrast microscope
Sterile glass tubes with caps (e.g., 5mL)
Centrifuge (optionally: equipped with microtiter plate rotor)
Laboratory balance
96-well plate spectrophotometer (i.e., plate reader) equipped with 540 nm ± 10 nm
filteri) Shaker for microtiter platesj) Cell counter or hemocytometerk) Pipetting aidl) Pipettes, pipettors (multi-channel and single channel; multichannel repeater pipette),
dilution blockm) Cryotubesn) Tissue culture flasks (75 - 80 cm2, 25 cm2)o) 96-well flat bottom tissue culture microtiter plates (e.g., Nunc # 167 008;
Corning/COSTAR tissue culture-treated)p) pH paper (wide and narrow range)q) Multichannel reagent reservoirr) Waterbath sonicators) Magnetic stirrer

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In Vitro Cytotoxicity Test Methods BRD Appendix B2

November 2006t) Antistatic bar ionizer/antistatic gun (optional for neutralizing static on 96-well plates)u) Dry heat block (optional)v) Adhesive film plate sealers (e.g., Excel Scientific SealPlate™,Cat # STR-SEAL-PLT
or equivalent)w) Vortex mixerx) Filters/filtration devices[Note: Tissue culture flasks and microtiter plates should be prescreened to ensure thatthey adequately support the growth of NHK. Multi-channel repeater pipettes may be
used for plating cells in the 96-well plates, dispensing plate rinse solutions, NR medium,and desorb solution. Do not use the repeater pipette for dispensing test chemicals to thecells.]
3. Chemicals, Media, and Seraa) Keratinocyte Basal Medium without Ca++ (KBM®, Clonetics CC-3104) that iscompleted by adding the KBM® SingleQuots® (Clonetics CC-4131) to achieve theproper concentrations of epidermal growth factor, insulin, hydrocortisone,
antimicrobial agents, bovine pituitary extract, and calcium (e.g., Clonetics Calcium
SingleQuots®, 300 mM CaCl2, Clonetics # CC-4202).
b) HEPES Buffered Saline Solution (HEPES-BSS) (e.g., Clonetics # CC-5022)c) 0.025 % Trypsin/EDTA solution (e.g., Clonetics # CC-5012)
d) Trypsin Neutralizing Solution (TNS) (e.g., Clonetics # CC-5002)e) Phosphate Buffered Saline (PBS)f) Dulbecco’s Phosphate Buffered Saline (D-PBS) [formulation containing calcium andmagnesium cations; glucose optional] (for rinsing)g) Neutral Red (NR) Dye – tissue culture-grade; liquid form (e.g., SIGMA N 2889);
powder form (e.g., SIGMA N 4638)h) Dimethyl sulfoxide (DMSO), U.S.P analytical grade (Store under nitrogen @ -20ºC)i) Ethanol (ETOH), U.S.P. analytical grade (100 %, non-denatured for test chemical
preparation; 95 % can be used for the desorb solution)j) Glacial acetic acid, analytical gradek) Hanks' Balanced Salt Solution without Ca2+ or Mg2+ (CMF-HBSS) (e.g., Invitrogen #14170)l) Distilled H2 O or any purified water suitable for cell culture and NR desorb solution(sterile)m) Sterile/non-sterile paper towels (for blotting 96-well plates)
B. Preparations of Media and Solutions[Note: All solutions (except NR stock solution, NR medium and NR desorb), glassware,
pipettes, etc., shall be sterile and all procedures should be carried out under aseptic conditions
and in the sterile environment of a laminar flow cabinet (biological hazard standard). All
methods and procedures will be adequately documented.]
1. Mediaa) Routine Culture Medium/Treatment Medium

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KBM® (Clonetics CC-3104) supplemented with KBM® SingleQuots® (Clonetics
CC-4131) and Clonetics Calcium SingleQuots® (CC-4202) to make 500 mL medium.
Final concentration of supplements in medium are:0.0001 ng/mL
5 µg/mL
0.5 µg/mL
30 µg/mL
15 ng/mL
0.10 mM
30 µg/mL

Human recombinant epidermal growth factor
InsulinHydrocortisone
Gentamicin
Amphotericin B
Calcium
Bovine pituitary extract

Complete media should be kept at 2-8°C and stored for no longer than two weeks.
NOTE:
KBM® SingleQuots® contain the following stock concentrations and volumes:
0.1 ng/mL
5.0 mg/mL
0.5 mg/mL
30 mg/mL
7.5 mg/mL

hEGF
0.5 mL
Insulin
0.5 mL
Hydrocortisone
0.5 mL
Gentamicin, 15 ug/mL Amphotericin-B 0.5 mLBovine Pituitary Extract (BPE)
2.0 mL

Clonetics Calcium SingleQuots® are 2 mL of 300mM calcium.
165 µl of solution per 500 mL calcium-free medium equals 0.10 mM calcium in themedium.
2. Neutral Red (NR) Stock Solution
The liquid tissue culture-grade stock NR Solution will be the first choice for performing
the assay (e.g., SIGMA #N2889, 3.3 mg/mL). Store liquid tissue culture-grade NR Stock
Solution at the storage conditions and shelf-life period recommended by the
manufacturer.
If the liquid form is not available, the following formulation can be prepared.
EXAMPLE:

0.33 g NR Dye powder in 100 mL H2 O

The NR Stock Solution (powder in water) should be stored in the dark at room
temperature for up to two months.
3. Neutral Red (NR) Medium
EXAMPLE:
1.0 mL (3.3 mg NR dye/mL)
99.0 mL

NR Stock Solution
Routine Culture Medium (pre-warmed to 37° C.)

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The final concentration of the NR Medium is 33 µg NR dye/mL and aliquots will be
prepared on the day of application.
[Note: The NR Medium shall be filtered (e.g., Millipore filtering, 0.2 – 0.45 µm poresize) used to reduce NR crystals. Aliquots of the NR Medium should be maintained at
37° C (e.g., in a waterbath) before adding to the cells and used within 30 min of
preparation but also used within 15 min after removing from 37° C storage.]
4. Ethanol/Acetic Acid Solution (NR Desorb)
1%
50 %
49 %

Glacial acetic acid solution
Ethanol
H2 O

C. Methods
1. Cell Maintenance and Culture Procedures
NHK cells are routinely grown as a monolayer in tissue culture grade flasks (e.g., 25 cm2)
at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air. The cells should be
examined on a daily (i.e., on workdays) basis under a phase contrast microscope, and anychanges in morphology or their adhesive properties must be noted in a Study Workbook.
2. Receipt of Cryopreserved Keratinocytes
Upon receipt of cryopreserved keratinocytes, the vial(s) of cells shall be stored in a liquid
nitrogen freezer until needed.
3. Thawing Cells and Establishing Cell Culturesa) Thaw cells by putting ampules into a water bath at 37°C for as brief a time as
possible. Do not thaw cells at room temperature or by hand. Seed the thawed cells
into culture flasks as quickly as possible and with minimal handling.
b) Slowly (taking approximately 1-2 min) add 9 mL of pre-warmed Routine Culture
Medium to the cells suspended in the cryoprotective solution and transfer cells into
flasks containing pre-warmed Routine Culture Medium (See Table 1).
c) Incubate the cultures at 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air until
the cells attach to the flask (within 4 to 24 h), at which time the Routine Culture
Medium should be removed and replaced with fresh Routine Culture Medium.
d) Unless otherwise specified, the cells should be incubated at 37ºC ± 1ºC, 90 % ± 5 %humidity, 5.0 % ± 1 % CO2/air and fed every 2-3 days until they exceed 50 %confluence (but less than 80 % confluent).

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Table 1. Guidelines for Establishing Cell Cultures
Cells/25 cm2 flask(in approximately 5 mL)
1 flask each cell concentration
Approximate Time to Subculture
Cells to 96-Well Plates

6.25 x 104(2500/cm2)

1.25 x 105(5000/cm2)

2.25 x 105(9000/cm2)

96+ hours
6 – 8 plates

72 - 96 hours
6 – 8 plates

48 - 72 hours
6 – 8 plates

Cell growth guidelines – actual growth of individual cell lots may vary.
4. Subculture of NHK Cells to 96-Well Plates[Note: It is important that cells have overcome the lag growth phase when they are used
for the test. Keratinocytes will be passaged only into the 96-well plates and will not be
subcultured into flasks for use in later assays]a) When the keratinocyte culture in a 25 cm2 flask exceeds 50 % confluence (but less than
80 % confluent), remove the medium and rinse the culture twice with 5 mL HEPESBSS. The first rinse may be left on the cells for up to 5 minutes and the second rinseshould remain on the cells for approximately 5 minutes. Discard the washing solutions.
b) Add 2 mL trypsin/EDTA solution to each flask and remove after 15 to 30 seconds.
Incubate the flask at room temperature for 3 to 7 min. When more than 50 % of thecells become dislodged, rap the flask sharply against the palm of the hand.
c) When most of the cells have become detached from the surface, rinse the flask with
5 mL of room temperature TNS. If more than one flask is subcultured, the same 5 mLof TNS may be used to rinse a total of up to two flasks.
d) Then rinse the flask with 5 mL CMF-HBSS and transfer the cell suspension to acentrifuge tube.
e) Pellet the cells by centrifugation for 5 min at approximately 220 x g. Remove thesupernatant by aspiration.
f)Resuspend the keratinocyte pellet by gentle trituration (to have single cells) in Routine
Culture Medium. It is important to obtain a single cell suspension for exact counting.
Count a sample of the cell suspension using a hemocytometer or cell counter.
4g) Prepare a cell suspension –1.6 – 2.0 x10 cells/mL in Routine Culture Medium.
Using a multi-channel pipette, dispense 125 µl Routine Culture Medium only into theperipheral wells (blanks) of a 96-well tissue culture microtiter plate. In the remaining
3
3
wells, dispense 125 µl of the cell suspension (2x10 – 2.5x10 cells/well). Prepare
one plate per chemical to be tested (see Figure 1, Section VII.E.1).
h) Incubate cells (37ºC ± 1ºC, 90 % ± 5.0 % humidity, and 5 % ± 1 % CO2/air) so thatcells form a 20+ % monolayer (~48-72 h). This incubation period assures cellrecovery and adherence and progression to exponential growth phase.

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Examine each plate under a phase contrast microscope to assure that cell growth isrelatively even across the microtiter plate. This check is performed to identify
experimental and systemic cell seeding errors. Record observations in the Study
Workbook.

5. Determination of Doubling Timea) A cell doubling time procedure was performed on the initial lot of cells that was usedin the first cell culture assays of Phase Ia of the Validation Study. The doubling time
only needs to be determined in Phase III if there is a change in the lot of cells used.
Establish cells in culture and trypsinize cells as per Section VII.C.4 for subculture.
Resuspend cells in appropriate culture medium. Use Table 1 to determine seeding
densities.
b) Seed five sets of cell culture vessels in triplicate for each cell type (e.g., 15 tissue
culture dishes [60mm x 15mm]). Use appropriate volume of culture medium for theculture vessels. Note number of cells placed into each culture dish. Place dishes
into the incubators (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air).
c) After 4-6 hours (use the same initial measurement time for each subsequent doublingtime experiment), remove three culture dishes and trypsinize cells. Count cells using
a cell counter or hemocytometer. Cell viability may be determined by dye exclusion(e.g., Trypan Blue; Nigrosin). Determine the total number of cells and document.
Repeat sampling at 24 hr, 48 hr, 72 hr, and 96 hr post inoculation. Change culture
medium at 72 hr or sooner in remaining dishes if indicated by pH drop.
d) Plot cell concentration (per mL of medium) on a log scale against time on a linear
scale. Determine lag time and population doubling time. The doubling time will bein the log (exponential) phase of the growth curve. Additional dishes and time areneeded if the entire growth curve is to be determined (lag phase, log phase, plateau
phase).
D. Preparation of Test Chemicals
The Study Management Team will provide direction on the solvent to be used for each test
chemical. [Note: Preparation under red or yellow light is recommended to preservechemicals that degrade upon exposure to light.]
1. Test Chemical in Solutiona) Allow test chemicals to equilibrate to room temperature before dissolving anddiluting.
b) Prepare test chemical immediately prior to use. Test chemical solutions should not
be prepared in bulk for use in subsequent tests. Ideally, the solutions must not be
cloudy nor have noticeable precipitate. Each stock dilution should have at least 1-2
mL total volume to ensure adequate solution for the test wells in a single 96-wellplate. The SMT may direct the Study Director to store an aliquot (e.g., 1 mL) of the

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highest 2X stock solution (e.g., low solubility chemicals) in a freezer (e.g., -70°C) for
use in future chemical analyses.
c) For chemicals dissolved in DMSO or ethanol, the final DMSO or ethanol
concentration for application to the cells must be 0.5 % (v/v) in the vehicle controls
and in all of the eight test concentrations.
d) The stock solution for each test chemical should be prepared at the highest
concentration found to be soluble in the solubility test (Test Method Protocol for
Solubility Determination). Thus, the highest test concentration applied to the cells ineach range finding experiment is:• 0.5 times the highest concentration found to be soluble in the solubility test, if thechemical was soluble in medium, or• 1/200 the highest concentration found to be soluble in the solubility test if thechemical was soluble in ethanol or DMSO.
e) The seven lower concentrations in the range finding experiment would then be
prepared by successive dilutions that decrease by one log unit each. The following
example illustrates the preparation of test chemical in solvent and the dilution ofdissolved test chemical in medium before application to NHK cells.
Example: Preparation of Test Chemical in Solvent Using a Log Dilution Scheme
If DMSO was determined to be the preferred solvent at Tier 2 of the solubility test
(i.e., 200,000 µg/mL), dissolve the chemical in DMSO at 200,000 µg/mL for thechemical stock solution.
1) Label eight tubes 1 – 8. Add 0.9 mL solvent (e.g., DMSO) to tubes 2 -- 8.
2) Prepare stock solution of 200,000 µg test chemical/mL solvent in tube # 1.
3) Add 0.1 mL of 200,000 µg/mL dilution from tube #1 to tube #2 to make a 1:10
dilution in solvent (i.e., 20,000 µg/mL).
4) Add 0.1 mL of 20,000 µg/mL dilution from tube #2 to tube #3 to make another1:10 dilution (i.e., 1:100 dilution from stock solution) in solvent (i.e., 2,000µg/mL)
5) Continuing making serial 1:10 dilutions in the prepared solvent tubes.
6) Since each concentration is 200 fold greater than the concentration to be tested,make a 1:100 dilution by diluting 1 part dissolved chemical in each tube with 99
parts of culture medium (e.g., 0.1 mL of test chemical in DMSO + 9.9 mL culture
medium) to derive the eight 2X concentrations for application to NHK cells.
Each 2X test chemical concentration will then contain 1 % v/v solvent. The
NHK cells will have 0.125 mL of culture medium in the wells prior toapplication of the test chemical. By adding 0.125 mL of the appropriate 2X test
chemical concentration to the appropriate wells, the test chemical will be diluted
appropriately (e.g., highest concentration in well will be 1,000 µg/mL) in a totalof 0.250 mL and the solvent concentration in the wells will be 0.5% v/v.

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7) A test article prepared in DMSO or ethanol may precipitate upon transfer into the
Routine Culture Medium. The 2X dosing solutions should be evaluated for
precipitates and the results recorded in the workbook. It will be permissible totest all of the dosing solutions in the dose range finding assay and main
experiments. However, doses containing test article precipitates should be
avoided and generally will not be used in the ICx determinations for thedefinitive tests. Precipitates in 2X dosing solutions are permissible for range
finder tests but not for definitive tests.
Document all test chemical preparations in the Study Workbook.
2. pH of Test Chemical Solutions
Prior to or immediately after application of the test chemical to the 96-well plate,measure the pH of the highest 2X dosing concentration of the test chemical (i.e., C1 in
the test plate, see Figure 1) in culture medium. Use pH paper (e.g., pH 0 – 14 to estimate
and pH 5 – 10 to determine more precise value; or Study Director’s discretion). The pH
paper should be in contact with the solution for approximately one minute. Document
the pH and note the color of the 2X concentration medium (i.e., in the EXCEL®
template). Medium color for all dosing dilutions should be noted in the workbooks. Do
not adjust the pH.
3. Concentrations of Test Chemicala) Range Finder Experiment
Test eight concentrations of the test chemical by diluting the stock solution with aconstant factor covering a large range. The initial dilution series shall be log
dilutions (e.g., 1:10, 1:100, 1:1000, etc.).
If a range finder experiment does not generate enough cytotoxicity, then higher doses
should be attempted. If cytotoxicity is limited by solubility, then more stringent
solubility procedures to increase the stock concentration (to the maximum
concentration specified in Section VII.D.3.b.) should be employed. Place the highest
test chemical concentration into an incubator (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0% ± 1 % CO2/air) and stir or rock for up to 3 hours, if necessary, to facilitate
dissolution. For stocks prepared in medium, vessel caps should be loose to allow forCO2 exchange. Proceed with dosing solution preparation and dosing.
•If a range finding test produces a biphasic curve, then the doses selected for thesubsequent main experiments should cover the most toxic dose-response range(see Example 1 – the most toxic range is 0.001 – 0.1 µg/mL).

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Example 1 – Biphasic Curve
Neutral Red Uptake
125%% Viability

100%
75%
50%
25%
0%
0.001

0.010

0.100

1.000

10.000100.000

1000.000

10000.000

Concentration (!g/mL)b) Main Experiment[Note: After the range finding assay is completed, the definitive concentrationresponse experiment shall be performed three times on three different days for eachchemical (i.e., one plate per day per chemical).]Depending on the slope of the concentration-response curve estimated from the range
finder, the dilution/progression factor in the concentration series of the main
experiment should be smaller (e.g., dilution factor of 6√10 = 1.47). Cover the
relevant concentration range around the IC50 (> 0 % and < 100 % effect) preferablywith several points of a graded effect, but with a minimum of two points, one on eachside of the estimated IC50 value, avoiding too many non-cytotoxic and/or 100 %cytotoxic concentrations. Experiments revealing less than one cytotoxic
concentration on each side of the IC50 value shall be repeated, where possible, with asmaller dilution factor (see Section VII.E.5.a.4). Each experiment should have atleast one cytotoxicity value > 0 % and ≤ 50.0 % viability and at least one cytotoxicity
value > 50.0 % and < 100 % viability. A progression factor of 1.21 [12√10] isregarded the smallest factor achievable and will be the lowest dosing interval
required.)
Determine which test chemical concentration is closest to the IC50 value (e.g., 50 %cytotoxicity). Use that value as a central concentration and adjust dilutions higher
and lower in equal steps for the definitive assay.
Maximum Doses to be Tested in the Main Experiments
If minimal or no cytotoxicity was measured in the dose range finding assay, amaximum dose for the main experiments will be established as follows:• For test chemicals prepared in Routine Culture Medium, the highest test article
concentration that may be applied to the cells in the main experiments will beeither 100 mg/mL, or the maximum soluble dose. Test chemical will be weighed
into a glass tube and the weight will be documented. A volume of Routine

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Culture Medium will be added to the vessel so that the concentration is200,000 µg/mL (200 mg/mL). The solution is mixed using the mechanicalprocedures specified in Test Method Protocol for Solubility Determination. If
complete solubility is achieved in medium, then 7 additional serial stock dosing
solutions may be prepared from the 200 mg/mL 2X stock. If the test chemical isinsoluble in medium at 200 mg/ml, proceed by adding medium, in small
incremental amounts, to attempt to dissolve the chemical by using the sequenceof mixing procedures specified in Test Method Protocol for Solubility
Determination. More stringent solubility procedures may be employed if needed
based on results from the range finder experiment (Section VII.D.3.a.). The
highest soluble stock solution will be used to prepare the 7 additional serial stock
dosing solutions.
•

For test chemicals prepared in either DMSO or ethanol, the highest test article
concentration that may be applied to the cells in the main experiments will beeither 2.5 mg/mL, or less, depending upon the maximum solubility in solvent.
Test chemical will be weighed into a glass tube and the weight will be
documented. A volume of the appropriate solvent (determined from the original
solubility test) will be added to the vessel so that the concentration is500,000 µg/mL (500 mg/mL). The solution is mixed as specified in Test Method
Protocol for Solubility Determination. If complete solubility is achieved in thesolvent, then 7 additional serial stock dosing solutions may be prepared from the
500 mg/mL 200X stock. If the test chemical is insoluble in solvent at
500 mg/ml, proceed by adding solvent, in small incremental amounts, to attempt
to dissolve the chemical by using the sequence of mixing procedures. The
highest soluble stock solution will be used to prepare the 7 additional serial stock
dosing solutions.

•

If precipitates are observed in the 2X dilutions, continue with the experiment,
make the appropriate observations and documentation, and report data to theSMT.

c) Test Chemical Dilutions
The dosing factor of 3.16 (= 2√10) divides a log into two equidistant steps, a factor of
2.15 (= 3√10) divides a decade into three steps. The factor of 1.47 (= 6√10) divides alog into six equidistant steps, the factor of 1.78 (4√10) divides a log into four
equidistant steps, and the factor of 1.21 (= 12√10) divides the log into 12 steps.
EXAMPLE:
10
10
10
10

31.6

12.1

14.7
14.7

17.8

21.5
21.5
21.5

26.1

31.6
31.6

38.3

46.4
46.4
46.4

56.2

68.1
68.1

82.5

The technical production of decimal geometric concentration series is simple. An
example is given for factor 1.47:

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Dilute 1 volume of the highest concentration by adding 0.47 volumes of diluent. Afterequilibration, dilute 1 volume of this solution by adding 0.47 volumes ofdiluent...(etc.).
E. Test Procedure
1. 96-Well Plate Configuration
The NHK NRU assay for test chemicals will use the 96-well plate configuration shown in
Figure 1.
Figure 1. 96-Well Plate Configuration for Positive Control (PC) and Test Chemical
Assays
1

2

3

4

5

6

7

8

9

10

11

12

A

VCb VCb C1b

C2b

C3b

C4b

C5b

C6b

C7b

C8b

VCb VCbB

VCb VC1 C1

C2

C3

C4

C5

C6

C7

C8

VC2 VCbC

VCb VC1 C1

C2

C3

C4

C5

C6

C7

C8

VC2 VCbD

VCb VC1 C1

C2

C3

C4

C5

C6

C7

C8

VC2 VCbE

VCb VC1 C1

C2

C3

C4

C5

C6

C7

C8

VC2 VCbF

VCb VC1 C1

C2

C3

C4

C5

C6

C7

C8

VC2 VCbG

VCb VC1 C1

C2

C3

C4

C5

C6

C7

C8

VC2 VCbH

VCb VCb C1b

C2b

C3b

C4b

C5b

C6b

C7b

C8b

VCb VCb

VC1 and VC2 =C1 – C8
=
b
VCb

=
=

VEHICLE CONTROL
Test Chemicals or PC (SLS) at eight concentrations(C1 = highest, C8 = lowest)
BLANKS (Test chemical or PC, but contain no cells)
VEHICLE CONTROL BLANK (contain no cells)

2. Application of Test Chemicala) Two optional methods for rapidly applying the 2X dosing solutions onto the 96-wellplates may be utilized.
1) The first method is to add each of the 2X dosing solutions into labeled, sterile
reservoirs (e.g., Corning/Costar model 4870 sterile polystyrene 50 mL reagent
reservoirs or Corning/Transtar model 4878 disposable reservoir liners, 8-channel; orother multichannel reservoirs).
2) The second method utilizes a “dummy” plate (i.e., an empty sterile 96-well plate)
prepared to hold the dosing solutions immediately prior to treatment of the test plate

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(with cells). The test chemical and control dosing solutions should be dispensed into
the dummy plate in the same pattern/order as will be applied to the plate containing
cells. More volume than needed for the test plate (i.e. greater than 125 µl/well)should be in the wells of the dummy plate.
At the time of treatment initiation, a multi-channel micropipettor is used to transfer the
2X dosing solutions, from the reservoirs or dummy plate, to the appropriate wells on thetreatment plate (as described in step c. below). These methods will ensure that the dosing
solutions can be transferred rapidly to the appropriate wells of the test plate to initiate
treatment times and to minimize the range of treatment initiation times across a large
number of treatment plates, and to prevent “out of order” dosing. Do not use a
multichannel repeater pipette for dispensing test chemical to the plates.
b) After 48 - 72 h (i.e., after cells attain 20+ % confluency [see Section VII.C.4(h)])
incubation of the cells, add 125 µl of the appropriate concentration of test chemical, thePC, or the VC (see Figure 1 for the plate configuration) directly to the test wells. Do notremove Routine Culture Medium for re-feeding the cells. The dosing solutions will be
rapidly transferred from the 8-channel reservoir (or dummy plate) to the test plate using asingle delivery multi-channel pipettor. For example, the VC may be transferred first (intocolumns 1, 2, 11, and 12), followed by the test article dosing solutions from lowest to
highest dose, so that the same pipette tips on the multi-channel pipettor can be used forthe whole plate. [The Vehicle Control blank (VCb) wells (column 1, column 12, wells
A2, A11, H2, H11) will receive the Vehicle Control dosing solutions (which should
include any solvents used). Blanks for wells A3 – A10 and H3 – H10 shall receive theappropriate test chemical solution for each concentration (e.g., wells A3 and H3 receive
C1 solution).] Incubate cells for 48 h ± 0.5 h (37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0% ± 1 % CO2/air).
c) Positive Control: For each set of test chemical plates used in an assay, a separate plate of
positive control concentrations will be set up following the concentration range
established in the development of the positive control database in Phase I of the
Validation Study. If multiple sets of test chemical plates are set up, then clearly
designate the positive control plates for each set; each set will be an individual entity. The
Study Director will decide how many test chemical plates will be run with a positive
control plate. The mean IC50 ± two and a half standard deviations (SD) for the SLS
acceptable tests from Phases Ia, Ib, and II (after the removal of outliers) are the values
that will be used as an acceptance criterion for test sensitivity for the NHK NRU assay.
This plate will follow the same schedule and procedures as used for the test chemical
plates (including appropriate chemical concentrations in the appropriate wells andmeeting test acceptance criteria see Sections VII.E.1, E.2, and E.5).
3. Microscopic Evaluation
After at least 46 h treatment, examine each plate under a phase contrast microscope toidentify systematic cell seeding errors and growth characteristics of control and treatedcells. Record any changes in morphology of the cells due to the cytotoxic effects of thetest chemical, but do not use these records for any quantitative measure of cytotoxicity.
Undesirable growth characteristics of control cells may indicate experimental error andmay be cause for rejection of the assay. Use the following Visual Observations Codes inthe description of cell culture conditions. Numerical scoring of the cells (see Section

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VII.E.3) should be determined and documented in the Study Workbook and in theappropriate section of Addendum II of the EXCEL® study template.
Visual Observations Codes
Note Code
1
2
3
4
1P
2P
3P
4P
5P

Note Text
Normal Cell Morphology
Low Level of Cell Toxicity
Moderate Level of Cell Toxicity
High level of Cell Toxicity
Normal Cell Morphology with Precipitate
Low Level of Cell Toxicity with Precipitate
Moderate Level of Cell Toxicity with PrecipitateHigh level of Cell Toxicity with Precipitate
Unable to View Cells Due to Precipitate

4. Measurement of NRUa) Carefully remove (i.e., “dump”) the Routine Culture Medium (with test chemical)
and rinse the cells very carefully with 250 µL pre-warmed D-PBS. Remove therinsing solution by dumping and remove excess by gently blotting on paper towels.
Add 250 µL NR medium (to all wells including the blanks) and incubate (37ºC ±1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air) for 3±0.1 h. Observe the cells
briefly during the NR incubation (e.g., between 2 and 3 h – Study Director‘s
discretion) for NR crystal formation. Record observations in the Study Workbook.
Study Director can decide to reject the experiment if excessive NR crystallization has
occurred.
b) After incubation, remove the NR medium, and carefully rinse cells with 250 µL prewarmed D-PBS.
c) Decant and blot D-PBS from the plate. (Optionally: centrifuge the reversed plate.)d) Add exactly 100 µL NR Desorb (ETOH/acetic acid) solution to all wells, including
blanks.
e) Shake microtiter plate rapidly on a microtiter plate shaker for 20 – 45 min to extract
NR from the cells and form a homogeneous solution. Plates should be protectedfrom light by using a cover during shaking.
f)

Plates should be still for at least five minutes after removal from the plate shaker (ororbital mixer). If any bubbles are observed, assure that they have been ruptured prior
to reading the plate. Measure the absorption (within 60 minutes of adding NR
Desorb solution) of the resulting colored solution at 540 nm ± 10 nm in a microtiterplate reader (spectrophotometer), using the blanks as a reference. [Phases Ia and Ib
data show the mean OD value for the plate blanks to be 0.055 ± 0.035 for NHK cells(± 2.5 standard deviations; data from 3 labs; N = 156). Use this range as a guide forassessment of the blank values.] Save raw data in the Excel format as provided by
the SMT.

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5. Quality Check of Assaya) Test Acceptance Criteria
All acceptance criteria (i.e., criteria 1, 2, and 3) must be met for a test to be
acceptable.
1) The PC (SLS) IC50 must be within two and a half (2.5) standard deviations of thehistorical mean established by the Test Facility (as per VII.E.2.c), and must meetcriteria 2 and 3, and must have an r2 (coefficient of determination) value
calculated for the Hill model fit (i.e., from PRISM® software) ≥ 0.85.
2) The left and the right mean of the VCs do not differ by more than 15 % from themean of all VCs.
3) At least one calculated cytotoxicity value > 0 % and ≤ 50.0 % viability and at
least one calculated cytotoxicity value > 50.0 % and < 100 % viability must bepresent.
Exception: If a test has only one point between 0 and 100 % and the smallest
dilution factor (i.e., 1.21) was used and all other test acceptance criteria weremet, then the test will be considered acceptable.
Stopping Rule for Insoluble Chemicals: If the most rigorous solubility procedures havebeen performed and the assay cannot achieve adequate toxicity to meet the test
acceptance criteria after three definitive trials, then the Study Director may end all testingfor that particular chemical.
[Note: A corrected mean OD540 ± 10nm of 0.205 - 1.645 for the VCs is a target range but
will not be a test acceptance criterion. Range determined from Phase Ib VC OD values
from 3 laboratories (mean ± 2.5 standard deviations, N = 69).]b) Checks for Systematic Cell Seeding Errors
To check for systematic cell seeding errors, untreated VCs are placed both at the left
side (row 2) and the right side (row 11 for the test plates) of the 96-well plate.
Aberrations in the cell monolayer for the VCs may reflect a volatile and toxic test
article present in the assay. If volatility is suspected, then proceed to Section
VII.E.6.
Checks for cell seeding errors may also be performed by examining each plate undera phase contrast microscope to assure that cell quantity is consistent.
6. Volatility of Test Chemicals
Highly volatile test chemicals may generate vapors from the treatment media during thetest chemical treatment incubation period. These vapors may become resorbed into thetreatment medium in adjacent wells, such that culture wells nearest the highest doses may
become contaminated by exposure to resorbed test article vapors. If the test chemical is

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particularly toxic at the doses tested, the cross contamination may be evident as asignificant reduction in viability in the vehicle control cultures (i.e., VC1) adjacent to thehighest test chemical doses.
If potential test article volatility is suspected (e.g., for low density liquids) or if the initial
range finder test (non-sealed plate) results show evidence of toxic effects in the control
cultures (i.e., > 15 % difference in viability between VC1 [column 2] and VC2 [column11]), then seal the subsequent test plates by the following procedure.
a) Plate Sealer Method
1) Plates and chemicals will be prepared as usual according to Sections VII.D and
VII.E.
2) Immediately after the 96-well culture plate has been treated with the suspectedvolatile chemical (Section VII.E.2.b), apply the adhesive plate sealer (e.g., usinga hand, microplate roller, etc.) directly over the culture wells. Assure that thesealer adheres to each culture well (well tops should be dry). Place the 96-wellplate cover over the sealed plate and incubate the plate under specified conditions(Section VII.E.2.b). [Note: Do not jam the plate lid over the film to avoid
deforming the sealer and causing the sealer to detach from culture wells. Loose
fit of the plate lid is acceptable.]3) At the end of the treatment period, the plate sealer should be carefully removed
to avoid spillage. Continue with the NRU assay as per Section VII.E.4.
F. Data Analysis
The Study Director will use good biological/scientific judgment for determining “unusable”
wells that will be excluded from the data analysis and provide explanations for the removal ofany data from the analysis.
A calculation of cell viability expressed as NRU is made for each concentration of the test
chemical by using the mean NRU of the six replicate values (minimum of four acceptable
replicates wells) per test concentration. This value is compared with the mean NRU of all
VC values. Relative cell viability is then expressed as percent of untreated VC. If
achievable, the eight concentrations of each chemical tested will span the range of no effectup to total inhibition of cell viability. Data from the microtiter plate reader shall betransferred to the Excel® spreadsheet template provided by the SMT. The template willautomatically determine cell viability, IC50 values by linear interpolation, and performstatistical analyses (including statistical identification of outliers). The template will also
calculate the concentrations associated with 20 %, 50 %, and 80 % viability using the Hill
slope and EC50 (i.e., IC50) from the Hill function analysis.
The Hill function analysis shall be performed using statistical software (e.g., GraphPad
PRISM® 3.0) and a template specified by the SMT to calculate IC20, IC50, and IC80 values(and the associated confidence limits) for each test chemical.
The Testing Facility shall report data using at least three (3) significant figures and shallforward the results from each assay to the SMT through the designated contacts in electronic
format and hard copy upon completion of testing. The SMT will be directly responsible forthe statistical analyses of the Validation Study data.

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VIII.

November 2006

REFERENCES

Clonetics Normal Human Keratinocyte Systems Instructions for Use, AA-1000-4-Rev.03/00.
(http://www.clonetics.com).
Hackenberg, U. and H. Bartling. 1959. Messen und Rechnen im pharmakologischen
Laboratorium mit einem speziellen Zahlensystem (WL24-System). Arch. Exp. Pathol.
Pharmakol. 235: 437-463.
Triglia, D., P.T. Wegener, J. Harbell, K. Wallace, D. Matheson, and C. Shopsis. 1989.
Interlaboratory validation study of the keratinocyte neutral red bioassay from Clonetics
Corporation. In Alternative Methods in Toxicology, Volume 7. A.M. Goldberg, ed., pp. 357365. Mary Ann Liebert, Inc., New York.
Test Method Protocol for Solubility Determination. In Vitro Cytotoxicity Validation Study.
Phase III. August 29, 2003. Prepared by The National Toxicology Program (NTP)
Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM).

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IX. APPROVAL__________________________________SPONSOR REPRESENTATIVE___________________DATE(Print or type name)_____________________________
Testing Facility STUDY DIRECTOR(Print or type name)____________________
DATE

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Appendix B3

Test Method Protocol for Solubility Determination (Phase III)

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[This Page Intentionally Left Blank]

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TEST METHOD PROTOCOL
for Solubility Determination

In Vitro Cytotoxicity Validation Study
Phase III
September 24, 2003
Prepared by
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)

Based on Standard Operating Procedure Recommendations from anInternational Workshop Organized by the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM)

National Institute of Environmental Health Sciences (NIEHS)
National Institutes of Health (NIH)
U.S. Public Health Service
Department of Health and Human Services

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TEST METHOD PROTOCOL
Solubility Determination
Phase III
I. PURPOSE
The purpose of this study is to evaluate the cytotoxicity of test chemicals using the BALB/c 3T3
Neutral Red Uptake (NRU) and normal human keratinocyte (NHK) cytotoxicity tests. The data
will be used to evaluate the intra- and inter-laboratory reproducibility of the assay andeffectiveness of the cytotoxicity assay to predict the starting doses for rodent acute oral systemic
toxicity assays. This test method protocol outlines the procedures for performing solubility
determinations for the in vitro validation study organized by NICEATM and the European Centre
for the Validation of Alternative Methods (ECVAM) and sponsored by NIEHS, U.S.
Environmental Protection Agency, and ECVAM. This test method protocol applies to all
personnel involved with performing the solubility testing.
A. Solubility Test
The solubility tests will be performed to determine the best solvent to use for each of the 60
blinded/coded test chemicals to be tested in the 3T3 and NHK NRU cytotoxicity tests
II. SPONSOR
A. Name:National Institute of Environmental Health Sciences (NIEHS); The
National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)

B. Address:

P.O. Box 12233
Research Triangle Park, NC 27709

C. Representative:

Named Representative

III. IDENTIFICATION OF TEST SUBSTANCES AND SOLVENTS
A. Test Chemicals:

60 Coded Chemicals (60)

B. Solvents:

Chemical Dilution Medium for 3T3 assay (See Section VII.B.1)
Treatment Medium for NHK assay (See Section VII.B.2)

IV. TESTING FACILITY AND KEY PERSONNEL
A. Facility Information
1)
2)
3)
4)
5)
6)

Name:
Address:
Study Director:
Laboratory Technician(s):Scientific Advisor:
Quality Assurance Director:
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7) Safety Manager:
8) Facility Management:
B. Test Schedule
1) Proposed Experimental Initiation Date:
2) Proposed Experimental Completion Date:
3) Proposed Report Date:
V. TEST SYSTEM
The solubility test procedure is based on attempting to dissolve chemicals in various solvents witha increasingly rigorous mechanical techniques. The solvents to be used, in the order ofpreference, are cell culture media, DMSO, and ethanol. Solubility shall be determined in a stepwise procedure that involves attempting to dissolve a test chemical in the solvents (in the order ofpreference) at relatively high concentrations using the sequence of mechanical procedures(Section VII.C.2.a). If the chemical does not dissolve, the volume of solvent is increased so as todecrease the concentration by a factor of 10, and then the sequence of mechanical procedures arerepeated in an attempt to solubilize the chemical at the lower concentrations.
Determination of whether a chemical has dissolved is based entirely on visual observation. A
chemical has dissolved if the solution is clear and shows no signs of cloudiness or precipitation.
VI. DEFINITIONS
A. Soluble: Chemical exists in a clear solution without visible cloudiness or precipitate.
B. Documentation: all methods and procedures will be noted in a Study Workbook; logs will bemaintained for general laboratory procedures and equipment (e.g., media preparation,solubility testing, laboratory balance calibration); solubility reports will be in electronic andpaper format; all data will be archived.
VII.

PROCEDURES

A. Materials
1. Technical Equipment[Note: Suggested brand names/vendors are listed in parentheses. Equivalents may be
used.]
a)
b)
c)
d)
e)

Water bath: 37ºC ± 1ºC
Glass tubes with caps (e.g., 5 mL)
Laboratory balance
Pipetting aid
Pipettes, pipettors (multi-channel and single channel; multichannel repeater pipette),
dilution blockf) Waterbath sonicatorg) Dry heat block (optional)

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2. Chemicals, Media, and Seraa) Dulbecco’s Modification of Eagle’s Medium (DMEM) without L-Glutamine; shouldhave high glucose [4.5gm/l] (e.g., ICN-Flow Cat. No. 12-332-54)b) L-Glutamine 200 mM (e.g., ICN-Flow # 16-801-49)c) Penicillin/streptomycin solution (e.g. ICN-Flow # 16-700-49)d) Dimethyl sulfoxide (DMSO), U.S.P. analytical grade (Store under nitrogen @ -20ºC)e) Ethanol (ETOH), U.S.P. analytical grade (100 %, non-denatured for test chemical
preparation; 95 % can be used for the desorb solution)f) Keratinocyte Basal Medium without Ca++ (KBM, Clonetics CC-3104) that iscompleted by adding the KBM SingleQuots (Clonetics CC-4131) to achieve theproper concentrations of epidermal growth factor, insulin, hydrocortisone,
antimicrobial agents, bovine pituitary extract, and calcium (e.g., Clonetics Calcium
SingleQuots, 300 mM CaCl2, Clonetics # CC-4202).
B. Preparations of Media and Solutions[Note: All solutions glassware, pipettes, etc., shall be sterile and all procedures should be
carried out under aseptic conditions and in the sterile environment of a laminar flow cabinet(biological hazard standard). All methods and procedures will be adequately documented.
Completed media formulations should be kept at approximately 2-8° C and stored for nolonger than two weeks.]
1. 3T3 Chemical Dilution Medium
DMEM (buffered with sodium bicarbonate) supplemented with (final concentrations in
DMEM are quoted):
4 mM
200 IU/mL
200 µg/mL

Glutamine
Penicillin
Streptomycin

2. NHK Treatment Medium
KBM (Clonetics CC-3104) supplemented with KBM SingleQuots (Clonetics CC4131) and Clonetics Calcium SingleQuots (CC-4202) to make 500 mL medium. Finalconcentration of supplements in medium are:0.0001 ng/mL
5 µg/mL
0.5 µg/mL
30 µg/mL
15 ng/mL
0.10 mM
30 µg/mL

Human recombinant epidermal growth factor
InsulinHydrocortisone
Gentamicin
Amphotericin B
Calcium
Bovine pituitary extract

NOTE:
KBM SingleQuots contain the following stock concentrations and volumes:

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In Vitro Cytotoxicity Test Methods BRD Appendix B3

0.1 ng/mL
5.0 mg/mL
0.5 mg/mL
30 mg/mL
7.5 mg/mL

November 2006

hEGF
0.5 mL
Insulin
0.5 mL
Hydrocortisone
0.5 mL
Gentamicin, 15 ug/mL Amphotericin-B 0.5 mLBovine Pituitary Extract (BPE)
2.0 mL

Clonetics Calcium SingleQuots are 2 mL of 300 mM calcium.
165 µl of solution per 500 mL calcium-free medium equals 0.10 mM calcium in themedium.
C. Determination of Solubility
The preference of solvent for dissolving test chemicals is medium, DMSO, and then ethanol.
Solubility shall be determined in a step-wise procedure that involves attempting to dissolve atest chemical at a relatively high concentration with the sequence of mechanical procedures
specified in Section VII.C.2.a. If the chemical does not dissolve, the volume of solvent isincreased so as to decrease the concentration by a factor of 10, and then the sequence of
mechanical procedures in Section VII.C.2.a are repeated in an attempt to solubilize thechemical at the lower concentrations. For testing solubility in medium, the starting
concentration is 20,000 µg/ml (i.e., 20 mg/mL) in Tier 1, but for DMSO and ethanol thestarting concentration is 200,000 µg/ml (i.e., 200 mg/mL) in Tier 2. Weighing out chemical
for each solvent (i.e., medium, DMSO, ethanol) can be done all at once, if convenient, butsolubility testing (at each tier that calls for more than one solvent) is designed to be sequential- medium, then DMSO, then ethanol – in accordance with the solvent hierarchy (see Figure1). This allows for testing to stop, rather than continue testing with less preferred solvents, if
the test chemical dissolves in a more preferred solvent. For example, if a chemical is soluble
in medium at a particular tier, testing may stop. Likewise, if a chemical is soluble in DMSO
at any tier, testing need not continue with ethanol. However, since the issue of primary
importance is testing the solvents and concentrations of test chemical required by any one
tier, sequential testing of solvents may be abandoned if the lab can test more efficiently inanother way.
1. Methoda) Tier 1 begins with testing 20 mg/mL each in Chemical Dilution Medium andTreatment Medium (see Table 1). For each medium, weigh approximately 10 mg(10,000 µg) of the test chemical into glass tubes. Document the chemical weight.
Add approximately 0.5 mL of each medium into its respective tube so that theconcentration is 20,000 µg/ml (20 mg/mL). Mix the solution as specified in Section
VII.C.2.a. If complete solubility is achieved in each medium, then additionalsolubility procedures are not needed.
b) If the test chemical is insoluble in either Chemical Dilution Medium or Treatment
Medium, proceed to Tier 2 by adding enough medium, approximately 4.5 mL, toattempt to dissolve the chemical at 2 mg/mL by using the sequence of mixing
procedures specified in Section VII.C.2.a. If the test chemical dissolves in medium
at 2 mg/mL, no further procedures are necessary. If the test chemical does NOT
dissolve in one medium or the other (if both are tested in this tier), weigh out
approximately 100 mg test chemical in a second glass tube and add enough DMSO to
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make the total volume approximately 0.5 mL (for 200 mg/mL) and attempt todissolve the chemical as specified in Section VII.C.2.a. If the test chemical does notdissolve in DMSO, weigh out approximately 100 mg test chemical in another glass
tube and add enough ethanol to make the total volume approximately 0.5 mL (for 200
mg/mL) and attempt to dissolve the chemical as specified in Section VII.C.2.a. If
the chemical is soluble in either solvent, no additional solubility procedures areneeded.
c) If the chemical is NOT soluble in one or both media, DMSO, or ethanol at Tier 2,then continue to Tier 3 in Table 1 by adding enough solvent to increase the volume of
the three (or four) Tier 2 solutions by 10 and attempt to solubilize again using thesequence of mixing procedures in Section VII.C.2.a. If the test chemical dissolves,no additional solubility procedures are necessary. If the test chemical does NOT
dissolve, continue with Tier 4 and, if necessary, Tier 5 using DMSO and ethanol.
Tier 4 begins by diluting the Tier 3 samples with DMSO or ethanol to bring the total
volume to 50 mL. The mixing procedures in Section VII.C.2.a are again followed toattempt to solubilize the chemical. Tier 5 is performed, if necessary, by weighing out
another two samples of test chemical at ~10 mg each and adding ~50 mL DMSO or
ethanol for a 200 µg/mL solution, and following the mixing procedures in Section
VII.C.2.a.
Example: If complete solubility is not achieved at 20,000 µg/mL in either Chemical
Dilution Medium or Treatment Medium at Tier 1 using the mixing procedures
specified in Section VII.C.2.a, then the procedure continues to Tier 2 by diluting thesolution to 5 mL (with either of the appropriate media) and mixing again as specified
in Section VII.C.2.a. If the chemical is not soluble in Chemical Dilution Medium or
Treatment Medium, two samples of ~ 100 mg test chemical are weighed to attempt tosolubilize in DMSO and ethanol at 200,000 µg/mL (i.e., 200 mg/mL). Solutions are
mixed following the sequence of procedures prescribed in Section VII.C.2.a in an
attempt to dissolve. If solubility is not achieved at Tier 2, then the solutions(Chemical Dilution Medium and/or Treatment Medium, DMSO, and ethanol)
prepared in Tier 2 are diluted by 10 so as to test 200 µg/mL in media, and 20,000µg/mL in DMSO and ethanol. This advances the procedure to Tier 3. Solutions areagain mixed as prescribed in Section VII.C.2.a in an attempt to dissolve. If
solubility is not achieved in Tier 3, the procedure continues to Tier 4, and to 5 if
necessary (see Figure 1 and Table 1).

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Table 1. Determination of Solubility in Chemical Dilution Medium, Treatment Medium, DMSO,
or Ethanol
TIER

1

2

3

4

Total Volume

0.5 mL

5 mL

50 mL

20,000 µg/mL

2,000 µg/mL

200 µg/mL(20 mg/mL)(2 mg/mL)(0.20 mg/mL)

0.5 mL

5 mL

50 mL

200,000 µg/mL

20,000 µg/mL

2,000 µg/mL(200 mg/mL)(20 mg/mL)(2 mg/mL)

5

Chemical Dilution
Medium/Treatment
MediumConcentration of Test
Chemical(Add ~10 mg to a tube.
Add enough medium toequal the first volume.
Dilute to subsequent
volumes if necessary.)
Total Volume
DMSO/Ethanol
Concentration of Test
Chemical(Add ~100 mg to a large
tube. Add enough DMSO
or ethanol to equal the first
volume. Dilute withsubsequent volumes ifnecessary.)
Total Volume
DMSO/Ethanol
Concentration of Test
Chemical(Add ~10 mg to a large
tube. Add enough DMSO
or ethanol to equal 50 mL.)
Equivalent Concentration
on Cells

50 mL
200 µg/mL(0.2 mg/mL)
10,000 µg/mL1000 µg/mL

100 µg/mL

10 µg/mL(10 mg/mL)(1 mg/mL)(0.1 mg/mL)(0.01 mg/mL)

1 µg/mL(0.001
mg/mL)[NOTE: The amounts of test chemical weighed and Chemical Dilution Medium andTreatment Medium added may be modified from the amounts given above, provided
that the targeted concentrations specified for each tier are tested.]

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Figure 1. Solubility Flow Chart

STEP 1:

STEP 2:

STEP 3:

STEP 4:

STEP 5:

TIER 120 mg/mL test chemical (TC) in 0.5 mL Chemical Dilution Medium and Treatment
Medium:• if TC soluble in both media, then STOP.
• if TC insoluble in one medium, then go to STEP 2.
TIER 2
2 mg/mL TC in medium (one or both) – increase volume from STEP 1 by 10 (i.e., to 5 mL)• if TC soluble, then STOP.
• if TC insoluble in one medium, then go to STEP 3.
200 mg/mL TC in DMSO
• if TC soluble, then STOP.
• if TC insoluble, test at 200 mg/mL in ETOH.
• if TC soluble, then STOP.
• If TC insoluble, go to STEP 4.
TIER 30.2 mg/mL TC in medium (one or both) – increase volume from STEP 2 by 10 (i.e., to 50
mL)• if TC soluble in both media, then STOP.
• if TC insoluble in one medium, test at 20 mg/mL in DMSO – increase volume from
STEP 3 by 10 (i.e., to 5 mL).
• if TC soluble, then STOP.
• if TC insoluble, test at 20 mg/mL in ETOH – increase volume from STEP 3 by
10 (i.e., to 5 mL).
• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 5.
TIER 42 mg/mL TC in DMSO – increase volume from STEP 4 by 10 (i.e., to 50 mL)• if TC soluble, then STOP.
• if TC insoluble, test at 2 mg/mL in ETOH – increase volume from STEP 4 by 10(i.e., to 50 mL).
• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 6.
TIER 5

STEP 6:

0.2 mg/mL TC in 50 mL DMSO
• if TC soluble, then STOP.
• if TC insoluble, test at 0.2 mg/mL in 50 mL ETOH
• STOP

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2. Mechanical Proceduresa) The following hierarchy of mixing procedures will be followed to dissolve the test
chemical:1) Add test chemical to solvent as in Tier 1 of Table 1. (Test chemical and solvent
should be at room temperature.)
2) Gently mix at room temperature. Vortex the tube (1 –2 minutes).
3) If test chemical hasn’t dissolved, use waterbath sonication for up to 5 minutes.
4) If test chemical is not dissolved after sonication, then warm solution to 37°C for
5 - 60 min. This can be performed by warming tubes in a 37°C water bath or in a
CO2 incubator at 37°C. The solution may be stirred during warming (stirring in a
CO2 incubator will help maintain proper pH).
5) Proceed to Tier 2 (and Tiers 3-5, if necessary of Table 1 and repeat procedures 24).
b) The preference of solvent for dissolving test chemicals is Chemical Dilution Medium
or Treatment Medium, DMSO, and then ethanol. Thus, if all solvents for a particulartier are tested simultaneously and a test chemical dissolves in more than one solvent,then the choice of solvent follows this hierarchy. For example, if, at any tier, achemical is soluble in Chemical Dilution Medium and DMSO, but not in Treatment
Medium or ethanol, the choice of solvent would be medium for the 3T3 assay andDMSO for the NHK assay. If the chemical were insoluble in both media, but soluble
in DMSO and ethanol, the choice of solvent would be DMSO for both assays.
After the lab has determined the preferred solvent for the test chemical and before
proceeding to the cytotoxicity testing, the Study Director will submit the solubility
test results (laboratory worksheets are preferable), and discuss the solvent selection
with the Study Management Team (SMT) of the validation study. The SMT will
provide direction on the solvent to be used in each assay for each chemical prior tocytotoxicity testing. If the laboratory has attempted all solubility testing without
success, then the SMT will provide additional guidance for achieving test chemicalsolubility. The SMT anticipates that all validation study test chemicals will be tested
in the NRU assays.
The Testing Facility shall forward the results from the solubility tests assay to the SMT
through the designated contacts in electronic format and hard copy upon completion of
testing. The SMT will be directly responsible for the statistical analyses of the Validation
Study data.
VIII.

REFERENCES
U. S. Environmental Protection Agency. 1996. Product Properties Test Guidelines. OPPTS
803.7840. Water Solubility: Column Elution Method; Shake Flask Method. EPA712-C-96041, Prevention, Pesticides and Toxic Substances, Washington DC.

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IX.

November 2006

APPROVAL__________________________________SPONSOR REPRESENTATIVE(Print or type name)___________________DATE_____________________________
Test Facility STUDY DIRECTOR(Print or type name)____________________
DATE

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Appendix B4

Test Method Procedure for Prequalification of Normal Human Epidermal
Keratinocyte Growth Medium (Phase III)

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TEST METHOD PROCEDURE
Prequalification of Normal Human Epidermal Keratinocyte Growth Medium

In Vitro Cytotoxicity Validation Study
Phase III
January 28, 2004

Prepared by
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)

Based on Standard Operating Procedure Recommendations from anInternational Workshop Organized by the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM)

National Institute of Environmental Health Sciences (NIEHS)
National Institutes of Health (NIH)
U.S. Public Health Service
Department of Health and Human Services

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I. PROPOSAL
The following document provides the guidelines and testing requirements for qualifying lots of
Keratinocyte Basal Medium without Ca ++ (KBM [CAMBREX/Clonetics # CC-3104]) and the
medium supplements (SingleQuots [CAMBREX/Clonetics # CC-4131]) for use in the normal
human epidermal keratinocyte (NHK) neutral red uptake (NRU) assays for Phase III of the In
Vitro Cytotoxicity Validation Study. The medium and supplements will be tested so as todemonstrate their ability to perform adequately in the NHK NRU assay prior to purchase by thevalidation study laboratories for use in Phase III.
The Testing Facility will request the quality control test data from CAMBREX/Clonetics for eachpotential lot of medium and supplements. Based upon the QC test data, the Testing Facility willpurchase and test the one or two most current lots of medium and supplements in stock with
CAMBREX/Clonetics that appear to have the potential to support NHK cultures according to therequirements of the In Vitro Cytotoxicity Validation Study NHK neutral red uptake assay.
This test method procedure is based on the Phase III NHK NRU protocol (IIVS Protocol No.
SP100066) and outlines the procedures needed for performing the cytotoxicity test specificallyfor prequalifying NHK culture medium. The test method procedure and NHK NRU protocol
support the in vitro validation study organized by NICEATM and the European Centre for the
Validation of Alternative Methods (ECVAM) and sponsored by NIEHS, U.S. Environmental
Protection Agency, and ECVAM. This test method procedure applies to all personnel involved
with performing media/supplement testing.
A. NHK Neutral Red Uptake Cytotoxicity Test
The NHK NRU test will be performed to analyze NHK growth characteristics and the in vitro
toxicity of Sodium Lauryl Sulfate (SLS), as measured by the IC50, with each NHK
medium/supplement being tested.
The Testing Facility will select the lots of medium/supplements and combinations based on
the maximum available quantity and shelf life, as well as growth test results provided byCambrex. Potential medium testing/supplement combinations are:
• One lot of medium/one lot of SingleQuots: Test the lot of medium using the lot ofSingleQuots (one test of three plates).
• Two or more lots of medium/one lot of SingleQuots: Test each lot of medium using the
one lot of SingleQuots (one test of three plates for each lot of medium)• One lot of medium/two or more lots of SingleQuots: Test the lot of medium using each
lot of SingleQuots (one test of three plates for each lot of SingleQuots).
NHK cultures will be established using each medium/supplement combination, and will be
subcultured on 3 different days into 96-well plates for three subsequent SLS cytotoxicity tests
using each appropriate test medium/supplement combination.
B. Testing Conditions
The work will be performed in the IIVS Good Laboratory Practice (GLP)-compliant
laboratories, but will not be performed in full compliance with national and international GLP
guidelines, and neither a protocol nor an audited report will be generated.

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The Study Director will provide recommendations and appropriate test data for
acceptance/rejection of the tested media/supplements to the Study Management Team (SMT).
The Testing Facility will maintain the following documentation: study workbooks noting all
methods and procedures; logs for general laboratory procedures and equipment (e.g., media
preparation, SLS preparation, incubator function); electronic and paper formats of all opticaldensity data obtained from the spectrophotometer plate reader; electronic and paper format of
all calculations of ICx values and other derived data.
II. SPONSOR
A. Name:National Institute of Environmental Health Sciences (NIEHS); The
National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)

B. Address:

P.O. Box 12233
Research Triangle Park, NC 27709

C. Representative:

Molly Vallant, Project Officer, NIEHS

D. Study Management
Team Representatives: William Stokes, Silvia Casati, Raymond Tice, Judy Strickland,
Michael Paris
III. IDENTIFICATION OF TEST AND CONTROL SUBSTANCES
A. Test Substances:

Keratinocyte Basal Medium without Ca++ (KBM, Clonetics CC3104)
KBM SingleQuots (Clonetics CC-4131)

B. Controls:

Positive:
Vehicle (Negative):

Sodium Lauryl Sulfate
Assay medium

IV. TESTING FACILITY AND KEY PERSONNEL
•

Name:

Institute for In Vitro Sciences, Inc.

•

Address:

21 Firstfield Road, Suite 220
Gaithersburg, Maryland 20878•

Study Director:

Hans Raabe, M.S.

•

Laboratory Technician(s):Greg Mun, B.A., Laboratory Manager
Robin Anderson, B.S.
Filomena Diaco, B.S.
Gregory Moyer, B.S.
Massod Rahimi, B.S.
Angela Sizemore, B.S.
Teri Beth Wallace, B.S.

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Nathan Wilt, B.S.
V. PROCEDURES
A. Materials
NHK cells used for this procedure will come from the same lot of NHK cells used in Phases I
and II of the validation study. Equipment, chemicals, and other media will be the same as in
IIVS Protocol No. SP100066.
B. Preparations of Media and Solutions
All media and solutions will be prepared as in IIVS Protocol No. SP100066.
C. Methods
All culture procedures will be performed as in IIVS Protocol No. SP100066..
NHK cultures will be established with cryopreserved cells seeded into individual tissue
culture flasks using the existing medium/supplement combination (the “control” medium)and each test medium/supplement combination. It may be acceptable to suspend freshlythawed cells initially into 9 mL of control medium. The cell suspension will then be added to
culture flasks containing pre-warmed control or test medium. The cells will be subcultured on
three different days into 96-well plates for three subsequent NRU tests using each appropriate
test medium/ supplement combination and control.
D. Preparation of SLS
The preparation of SLS (IIVS code 02AD92) will follow the procedures in Sections
VII.D.1.a, b, and d of IIVS Protocol No. SP100066. SLS will be dissolved only in Routine
Culture Medium. Determination of the pH will follow Section VII.D.2.
Preparation of SLS concentrations/dilutions will follow the main experiment procedures
specifically for testing compounds in Routine Culture Medium as outlined in Section
VII.D.3.b of IIVS Protocol No. SP100066. The concentrations/dilutions should be the sameor similar to those used for SLS as a positive control in Phase II of the validation study.
E. Test Procedure
The 96-well plate configuration will be the same as that outlined in Section VII.E.1 of IIVS
Protocol No. SP100066. The C1 test concentration will be the highest SLS concentration.
Application of the SLS, subsequent toxicity testing, and measurement of NRU will follow
procedures outlined in Sections VII.E.2.a and b and Section VII.4 of IIVS Protocol No.
SP100066.
Cells cultured in control medium and in each test medium/supplement combination will betested in parallel for their sensitivity to SLS.
F. Microscopic Evaluation
Observations of the cell cultures in the culture flasks, as well as in the 96-well plates will be
performed and documented and should include cell morphology (e.g., overall appearance,colony formation and proliferation, presence of mitotic figures, and distribution).
Representative observations of the cultures in the culture flasks will be performed every
working day. Representative observations of the cultures in the 96-well plates will be
performed daily prior to treatment with SLS; at the end of the 48 hour treatment incubation;

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and during the neutral red incubation period (to evaluate relative neutral red uptake in thevehicle control cultures).
Changes in morphology of the cells due to cytotoxic effects of the SLS (prior to measurementof NRU) should be recorded as per procedures outlined in Section VII.E.3 of IIVS Protocol
No. SP100066.
G. Data Analysis and Test Evaluation
Data analysis will be performed as in Section VII.F of IIVS Protocol No. SP100066. The
following parameters will be evaluated to determine whether the NHK media andsupplements are adequate to support the NHK NRU assay:
1) SLS IC502) r2 (coefficient of determination) value calculated for the Hill model fit (i.e., from
PRISM software.
3) Difference between the mean of all vehicle controls (VC) and (a) the left mean VC, and(b) the right mean VC.
4) Number of points between 0 % and 50.0 % viability and between 50.0 % 100 % viability.
5) Mean corrected OD540-550 of the VCs.
6) Cell morphology and confluence of the VCs at the end of the 48 h treatment
The Study Director will utilize all observed growth characteristics and test results todetermine whether the media/supplements perform adequately, and provide the test data and arecommendation for the use or rejection of the media/supplements to the SMT. IIVS will
request CAMBREX/Clonetics reserve a portion of an acceptable lot based on estimates of
media needed by the three laboratories.
V. REFERENCESIIVS Protocol No. SP100066. Test Method Protocol for the NHK Neutral Red Uptake
Cytotoxicity Test. A Test for Basal Cytotoxicity for an In Vitro Validation Study. November 11,
2003. Prepared by the National Toxicology Program (NTP) Interagency Center for the Evaluationof Alternative Toxicological Methods (NICEATM).
VI. APPROVAL__________________________________SPONSOR REPRESENTATIVE___________________DATE(Print or type name)_____________________________
Testing Facility STUDY DIRECTOR(Print or type name)____________________
DATE

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In Vitro Cytotoxicity Test Methods BRD Appendix C

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Appendix C

Validation Study Test Method Protocols(Phases Ia, Ib, and II)

C1

Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase Ia).............................................................. C-3

C2

Test Method Protocol for the Normal Human Epidermal
Keratinocyte (NHK) Neutral Red Uptake Cytotoxicity Test
(Phase Ia) ......................................................................................... C-23

C3

Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase Ib)............................................................ C-41

C4

Test Method Protocol for the Normal Human Epidermal
Keratinocyte (NHK) Neutral Red Uptake Cytotoxicity Test
(Phase Ib)......................................................................................... C-63

C5

Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase II) ............................................................ C-85

C6

Test Method Protocol for the Normal Human Epidermal
Keratinocyte (NHK) Neutral Red Uptake Cytotoxicity Test
(Phase II)........................................................................................ C-109

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Appendix C1

Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase Ia)

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TEST METHOD PROTOCOL
for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

A Test for Basal Cytotoxicity for an In Vitro Validation Study
June 14, 2002

Prepared by
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)

Based on Standard Operating Procedure Recommendations from anInternational Workshop Organized by the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM)

National Institute of Environmental Health Sciences (NIEHS)
National Institutes of Health (NIH)
U.S. Public Health Service
Department of Health and Human Services

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TEST METHOD PROTOCOLThe BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test
A Test for Basal Cytotoxicity

I. PURPOSE
The purpose of this study is to evaluate the cytotoxicity of test chemicals using the BALB/c 3T3
Neutral Red Uptake (NRU) cytotoxicity test. The data will be used to evaluate the intra- andinter-laboratory reproducibility of the assay and effectiveness of the cytotoxicity assay to predict
the starting doses for rodent acute oral systemic toxicity assays. This test method protocol
outlines the procedures for performing the cytotoxicity test and supports the in vitro validation
study organized by NICEATM and the European Centre for the Validation of Alternative
Methods (ECVAM) and sponsored by NIEHS, U.S. Environmental Protection Agency, andECVAM. This test method protocol applies to all personnel involved with performing thecytotoxicity assay.
A. Determination of Positive Control DatabaseAn historical database of IC50 values for the positive control chemical (Sodium Lauryl[dodecyl] Sulfate {SLS}) must be established and maintained by performing 10
concentration-response assays on the 3T3 cells before performing the NRU assay on test
chemicals. Once the mean IC50 and the 95 % confidence interval (CI) of the IC50 of SLS areestablished, the values will be used as an acceptance criterion for test sensitivity for the 3T3
NRU assay.
B. BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test
After acceptable positive control mean IC50 and 95 % CI values have been established, the
3T3 NRU test will be performed to analyze the in vitro toxicity of test chemicals. This test
will be used to determine IC20, IC50, and IC80 values for a predetermined set of test chemicals
of varying toxicities.
II. SPONSOR
A. Name:National Institute of Environmental Health Sciences (NIEHS); The
National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)

B. Address:

P.O. Box 12233
Research Triangle Park, NC 27709

C. Representative:

Named Representative

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III. IDENTIFICATION OF TEST AND CONTROL SUBSTANCES
A. Test Chemicals:

Blinded Chemicals

B. Controls:

Positive:
Sodium Lauryl Sulfate
Vehicle (Negative):
Assay medium
Solvent (as needed):
Assay medium with appropriate solvent
used to prepare the test chemicals (Section VII.E)

IV. TESTING FACILITY AND KEY PERSONNEL
•

Name:

•

Address:•

Study Director:•

Laboratory Technician(s):•Scientific Advisor:•

Quality Assurance Director:•

Safety Manager:•

Facility Management:

A. Test Schedule•

Proposed Experimental Initiation Date:•

Proposed Experimental Completion Date:•

Proposed Report Date:

V. TEST SYSTEMThe NRU cytotoxicity assay procedure is a cell survival/viability chemosensitivity assay based on
the ability of viable cells to incorporate and bind neutral red (NR), a supravital dye. NR is a weakcationic dye that readily penetrates cell membranes by non-ionic diffusion and accumulates
intracellularly in lysosomes. Alterations of the cell surface or the sensitive lysosomal membranelead to lysosomal fragility and other changes that gradually become irreversible. Such changes
brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It isthus possible to distinguish between viable, damaged, or dead cells, which is the basis of thisassay.
Healthy mammalian cells, when maintained in culture, continuously divide and multiply overtime. A toxic chemical, regardless of site or mechanism of action, will interfere with this process
and result in a reduction of the growth rate as reflected by cell number. Cytotoxicity is expressed

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as a concentration dependent reduction of the uptake of the NR after chemical exposure thus
providing a sensitive, integrated signal of both cell integrity and growth inhibition.
VI. DEFINITIONS
A. Hill function: a four parameter logistic mathematical model relating the concentration of test
chemical to the response being measured in a sigmoidal shape.

Y = Bottom+

Top− Bottom
1 + 10(logIC50− X)HillSlope

where Y= response, X is the logarithm of dose (or concentration), Bottom is the minimum
response, Top is the maximum response, logIC50 is logarithm of X at the response midway
between Top and Bottom, and HillSlope describes the steepness of the curve.
B. Documentation: all methods and procedures will be noted in a Study Workbook; logs will bemaintained for general laboratory procedures and equipment (e.g., media preparation, test
chemical preparation, incubator function); all optical density data obtained from thespectrophotometer plate reader will be saved in electronic and paper formats; all calculationsof ICx values and other derived data will be in electronic and paper format; all data will bearchived.
VII.

PROCEDURES

A. Materials
1. Cell Lines
BALB/c 3T3 cells, clone 31
CCL-163, LGC Reference Materials, Customer Service, Queens Road, Teddington,
Middlesex, TW110LY, UK
CCL-163, American Type Culture Collection [ATCC], Manassas, VA, USA)
2. Technical Equipment[Note: Suggested brand names/vendors are listed in parentheses. Equivalents may be
used.]
a)
b)
c)
d)
e)
f)
g)
h)
i)
j)

Incubator: 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/airLaminar flow clean bench/cabinet (standard: "biological hazard")
Water bath: 37ºC ± 1ºC
Inverse phase contrast microscope
Sterile glass tubes with caps (e.g., 5 ml)
Centrifuge (optionally: equipped with microtiter plate rotor)
Laboratory balance
96-well plate spectrophotometer (i.e., plate reader) equipped with 540 nm ± 10 nm
filterShaker for microtiter plates
Cell counter or hemocytometer

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November 2006k) Pipetting aidl) Pipettes, pipettors (multi-channel and single channel), dilution blockm) Cryotubesn) Tissue culture flasks (e.g., 75 - 80 cm2, 25 cm2)o) 96-well flat bottom tissue culture microtiter plates (e.g., Nunc # 167 008; Falcon
tissue culture-treated)p) pH paper (wide and narrow range)[Note: Tissue culture flasks and microtiter plates should be prescreened to ensurethat they adequately support the growth of 3T3 cells.]
3. Chemicals, Media, and Seraa) Dulbecco’s Modification of Eagle’s Medium (DMEM) without L-Glutamine; shouldhave high glucose [4.5gm/l] (e.g., ICN-Flow Cat. No. 12-332-54)b) L-Glutamine 200 mM (e.g., ICN-Flow # 16-801-49)c) New Born Calf Serum (NBCS or NCS) (e.g., Biochrom # SO 125)d) 0.05 % Trypsin/0.02 % EDTA solution (e.g., SIGMA T 3924, ICN-Flow, # 1689149)e) Phosphate buffered saline (PBS) without Ca2+ and Mg2+(for trypsinization)f) Hanks’ Balanced Salt Solution (HBSS) without Ca2+ and Mg2+(CMF-HBSS)g) Dulbecco’s Phosphate Buffered Saline (D-PBS) with glucose) formulation containing
calcium and magnesium cations, and supplemented with 1000mg/L glucose) (forrinsing)h) Penicillin/streptomycin solution (e.g. ICN-Flow # 16-700-49)i) Neutral Red (NR) Dye – tissue culture-grade; liquid form (e.g., SIGMA N 2889);
powder form (e.g., SIGMA N 4638)j) Dimethyl sulfoxide (DMSO), U.S.P. analytical grade (Store under nitrogen @ -20ºC)k) Ethanol (ETOH), U.S.P. analytical grade (100 %, non-denatured for test chemical
preparation; 95 % can be used for the desorb solution)l) Glacial acetic acid, analytical gradem) Distilled H2O or any purified water suitable for cell culture (sterile)n) Sterile paper towels (for blotting 96-well plates)[Note: Due to lot variability of NBCS/NCS, first check a lot for growth stimulating
properties with 3T3 cells (approximately 20-24 h doubling time) and then reserve a
sufficient amount of NBCS/NCS.]
B. Preparations of Media and Solutions[Note: All solutions (except NR stock solution, NR medium and NR desorb), glassware,
pipettes, etc., shall be sterile and all procedures should be carried out under aseptic conditions
and in the sterile environment of a laminar flow cabinet (biological hazard standard). All
methods and procedures will be adequately documented.]
1. Media
DMEM (buffered with sodium bicarbonate) supplemented with (final concentrations in
DMEM are quoted):

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November 2006a) for freezing (Freeze Medium); contains 2X concentration of NBCS/NCS and DMSO
of final freezing solution
40 %NBCS/NCS
20 %DMSOb) for routine culture (Routine Culture Medium)
10 %NBCS/NCS
4 mM
Glutaminec) for treatment with Test Chemicals (Treatment Medium)5%
NBCS/NCS
4 mM
Glutamine
100 IU
Penicillin
100 µg/ml
Streptomycin[Note: The serum concentration of treatment medium is reduced to 5 %, since serum
proteins may mask the toxicity of the test substance. Serum cannot be totally excluded
because cell growth is markedly reduced in its absence.]
Complete media should be kept at approximately 4° C and stored for no longer than two
weeks.
2. Neutral Red (NR) Stock Solution
The liquid tissue culture-grade stock NR Solution will be the first choice for performing
the assay. If the liquid form is not available, the following formulation can be prepared.
0.4 g NR Dye powder in 100 ml of H2O
Make up prior to use and store dark at room temperature. May store for up to twomonths.
3. Neutral Red (NR) Medium
EXAMPLE:
1 ml (4mg NR dye/ml)
79 ml

NR Stock Solution
DMEM

The final concentration of the NR Medium is 50 µg NR dye/ml.
[Note: The NR medium should be incubated overnight at 37ºC ± 1ºC and centrifuged at
approximately 600 x g for 10 min (to remove NR crystals) before adding to the cells.
Alternative procedures (e.g., Millipore filtering) can be used as long as they guaranteethat NR medium is free of crystals.]
4. Ethanol/Acetic Acid Solution (NR Desorb)
1%
50 %
49 %

Glacial acetic acid solution
Ethanol
H2O

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November 2006

C. Methods
1. Cell Maintenance and Culture Procedures
BALB/c 3T3 cells are routinely grown as a monolayer in tissue culture grade flasks (e.g.,
75 - 80 cm2) at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air. The cells
should be examined on a daily basis under a phase contrast microscope, and any changes
in morphology or their adhesive properties noted in a Study Workbook (see Section
VII.F.3).
2. Receipt of Cryopreserved BALB/c 3T3 Cells
Upon receipt of cryopreserved BALB/c 3T3 cells, the vial(s) of cells shall be stored in aliquid nitrogen freezer until needed.
3. Thawing Cells
Thaw cells by putting ampules into a water bath at 37°C ± 1ºC. Leave for as brief a time
as possible.
a) Resuspend the cells and transfer into Routine Culture Medium in a tissueculture flask (see Section 6).
b) Incubate at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air.
c) When the cells have attached to the bottom of the flask (this may take up to 4h), decant the supernatant and replace with fresh medium. Culture as
described above.
d) Passage two to three times before using the cells in a cytotoxicity test.
A fresh batch of frozen cells from the stock lot of cells should be thawed out and cultured
approximately every two months. This period resembles a sequence of about 18
passages.
4. Routine Culture of BALB/C 3T3 Cells
When cells exceed 50 % confluence (but less than 80 % confluent) they should be
removed from the flask by trypsinization:a) Decant medium, rinse cultures with 5 ml PBS or Hanks’ BSS (without Ca2+, Mg2+)
per 25 cm2 flask (15 ml per 75 cm2 flask). Wash cells by gentle agitation to remove
any remaining serum that might inhibit the action of the trypsin.
b) Discard the washing solution.
c) Add 1-2 ml trypsin-EDTA solution per 25 cm2 to the monolayer for a few seconds(e.g., 15-30 seconds).
d) Remove excess trypsin-EDTA solution and incubate the cells at room temperature.
e) After 2-3 minutes (min), lightly tap the flask to detach the cells into a single cellsuspension.
5. Cell Counting
After detaching the cells, add 0.1-0.2 ml of Routine Culture Medium/cm2 to the flask(e.g., 2.5 ml for a 25 cm2 flask). Disperse the monolayer by gentle trituration. It is

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important to obtain a single cell suspension for exact counting. Count a sample of thecell suspension obtained using a hemocytometer or cell counter (e.g., Coulter counter).
6. Subculture of Cells
After determination of cell number, the culture can be sub-cultured into other flasks orseeded into 96-well microtiter plates. BALB/c 3T3 cells are routinely passaged atsuggested cell densities as listed in the table (approximate doubling time is 20-24 h). The
individual laboratories will need to determine and adjust the final density to achieve
growth as outlined in Section VII.C.1.
Table 1. Cell Densities for Subculturing
Days in Culture2
3
4

Seeding Density(cells/cm2)
16800
8400
4200

Total Cells per 25 cm2
flask
4.2 x 105
2.1 x 105
1.05 x 105

Total Cells per 75 cm2
flask
1.26 x 106
6.3 x 105
3.15 x 105[Note: It is important that cells have overcome the lag growth phase when they are used
for the test.]
7. Freezing Cells
Stocks of BALB/c 3T3 cells can be stored in sterile, freezing tubes in a liquid nitrogen
freezer. DMSO is used as a cryoprotective agent.
a) Centrifuge trypsinized cells at approximately 200 x g.
b) Suspend the cells in cold Routine Medium (half the final freezing volume) so a
final concentration of 1-5x106 cells/ml can be attained.
c) Slowly add cold Freeze Medium to the cells so that the solvent will equilibrate
across the cell membranes. Bring the cell suspension to the final freezingvolume. The final cell suspension will be 10 % DMSO. Aliquot the cellsuspension into freezing tubes and fill to 1.8 ml.
d) Place the tubes into an insulated container (e.g., styrofoam trays) and place in afreezer (-70 to -80°C) for 24 h. This gives a freezing rate of approximately
1°C/min. The laboratory needs to ensure that the freezing protocol is applicableto the 3T3 cells and that the cells are viable when removed fromcryopreservation.
e) Place the frozen tubes into liquid nitrogen for storage.
8. Preparation of Cells for Assaysa) Cultured cells that are going to be used in seeding the 96-well plates should be fed
fresh medium the day before subculturing to the plates. On the day of plate seeding,4
prepare a cell suspension of 2.5x10 cells/ml in Routine Culture Medium. Using a
multi-channel pipette, dispense 100 µl Routine Culture Medium only into theperipheral wells (blanks) of a 96-well tissue culture microtiter plate (See SectionIV.F). In the remaining wells, dispense 100 µl of a cell suspension of 2.5x104

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3

cells/ml (= 2.5x10 cells/well). The seeding density should be noted to ensure thatthe cells in the control wells are not overgrown after three days (i.e., 24 h incubationin b and 48 h exposure to test chemicals). Prepare one plate per chemical to betested.
b) Incubate cells for 24 h (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air) sothat cells form a less than half confluent monolayer. This incubation period assures
cell recovery and adherence and progression to exponential growth phase.
c) Examine each plate under a phase contrast microscope to assure that cell growth isrelatively even across the microtiter plate. This check is performed to identify
experimental and systemic cell seeding errors. Record observations in the Study
Workbook.
9. Determination of Doubling Timea) Establish cells in culture and trypsinize cells as per Section C.4 for subculture.
Resuspend cells in about 5ml Treatment Medium (5 % NBCS/NCS). Seed cells at
4200 cells/cm2.
b) Seed five sets of cell culture vessels in triplicate for each cell type (e.g., 15 tissue
culture dishes [60mm x 15mm]). Use appropriate volume of culture medium for theculture vessels. Note number of cells placed into each culture dish. Place dishes
into the incubators (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air).
c) After 4 - 6 hours (use the same initial measurement time for each subsequent
doubling time experiment), remove three culture dishes and trypsinize cells. Count
cells using a cell counter or hemocytometer. Cell viability may be determined by dye
exclusion (e.g., Trypan Blue) if Study Director sees a need. Use appropriate size
exclusion limits if using a Coulter counter. Determine the total number of cells anddocument. Repeat sampling at 24 h, 48 h, 72 h, and 96 h post inoculation. Change
culture medium at 72 h or sooner in remaining dishes if indicated by pH drop.
d) Plot cell concentration (per ml of medium) on a log scale against time on a linear
scale. Determine lag time and population doubling time. Additional dishes and time
are needed if the entire growth curve is to be determined (lag phase, log phase,plateau phase).
D. Establishing the Positive Control DatabaseAn historical database of IC50 values for the positive control chemical (Sodium Lauryl[dodecyl] Sulfate {SLS}) must be established and maintained by performing 10
concentration-response assays on the 3T3 cells.
1. Positive Control Chemical Preparation
The positive control chemical (SLS) is prepared in the same manner as the test chemical(Sections E.1 and E.2) by following the instructions and substituting “test chemical”
with “SLS.”
2. Range Finder Experiment
Before initiating the 10 concentration-response assays, a range finder experiment will be
performed using eight concentrations of SLS by diluting the stock solution with aconstant factor as per Sections E.3.a and E.3.b. The eight chemical concentrations will

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be tested as per the test procedure outlined in Section F and analyzed as per procedures
outlined in Section G.
3. Test Procedure
Once a range has been determined that satisfies the criteria in Section E.3.b, thedefinitive concentration-response assays shall use a 6√10 = 1.47 dilution scheme centeredon the IC50. The Test Facility will perform two tests per day on five different days. The
95 % CI of the IC50 of SLS will be established and defined as an acceptance criterion fortest sensitivity for the 3T3 NRU assay. The confidence intervals shall be calculated using
the average of the individual IC50 values from each positive control assay performed. An
example of an historical mean IC50 of SLS in mammalian cultures is 93 µg/ml and the 95% CI is 70 - 116 µg/ml (Spielmann et. al., 1991). All testing will follow the instructionsin Section F using the 96-well plate configuration in Figure 1. The test meets acceptancecriteria if the conditions in Sections F.5.a.2 and F.5.a.3 are met.
Figure 1. 96-Well Plate Configuration for Positive Control and Test Chemical Assays
1

2

3

4

5

6

7

8

9

10

11

12

A

b

b

b

b

b

b

b

b

b

b

b

b

B

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

C

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

D

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

E

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

F

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

G

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

H

b

b

b

b

b

b

b

b

b

b

b

b

VC
=
C1 – C8 =b=untreated VEHICLE CONTROL (mean viability set to 100 %)Test Chemicals or Positive Control (SLS) at eight concentrations(C1 = highest, C8 = lowest)
BLANKS (contain no cells)

E. Preparation of Test Chemicals[Note: Test chemical must be freshly prepared immediately prior to use. Each stock dilution
should have at least 1-2 ml total volume to ensure adequate solution for the test wells in asingle 96-well plate. The solutions must not be cloudy nor have noticeable precipitate. Test
chemicals must be at room temperature before dissolving and diluting. Preparation under redor yellow light may be necessary, if rapid photodegradation is likely to occur.]

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1. Dissolving Test Chemicala) Approximately 200,000 µg (200 mg) of the test chemical will be weighed into a glass
tube and the weight will be documented. Assay-specific culture medium will be
added to the vessel so that the concentration is 2,000,000 µg/ml (2000 mg/ml) andmixed using the mixing procedures outlined in Section E.1.c. If complete solubility
is achieved, then additional solubility procedures are not needed. The test chemical
can then be prepared and diluted for use in an assay. If only partial solubility isachieved, then add additional medium in the steps outlined in Table 1 until theconcentration is a minimum of 200,000 µg /ml. If complete solubility at 200,000µg/ml in culture medium can’t be attained, then repeat the solubility steps in Table 1
using the other solvent(s) in the solubility hierarchy outlined in Section E.1.c. Test
chemicals that are only soluble in DMSO or ethanol will be prepared at 2,000,000µg/ml as the highest concentration of stock solution.

Table 2 Determination of Solubility
Solubility Data
Total volume of medium added (ml)Total volume of DMSO or ethanol added (ml)Approximate solubility (µg/ml)

Step 10.1
0.1≥ 2,000,000

Step 20.5
0.5
400,000

Step 31.0
1.0
200,000

Example: If complete solubility is not achieved in 0.1 ml medium (Step 1), then 0.4
ml must be added to obtain a total volume of 0.5 ml (Step 2). No additional weighingof chemical is needed. Chemical and medium are again mixed in an attempt todissolve.
b) Each test chemical will be prepared such that the highest test concentration applied tothe cells in each range finding experiment is 100,000 µg/ml in culture medium(10,000 µg/ml if DMSO or ethanol is used). If 100,000 µg/ml in culture medium
cannot be achieved, then the highest concentration attainable will be used. If therange finding experiment shows that 10,000 µg/ml is not high enough for the rangeof chemicals dissolved in DMSO or ethanol to meet the acceptance criteria, thenhigher concentrations will be used for the definitive experiment.
c) The following mixing and solvent hierarchy will be followed in dissolving the test
chemical:
1)
2)
3)
4)

Dissolve test chemical in Treatment Medium.
Gently mix. Vortex the tube (1 – 2 minutes).
If test chemical hasn’t dissolved, use sonication for up to 5 minutes.
If sonication doesn’t work, then warm solution to 37°C.

If the test chemical doesn’t dissolve (i.e., solution is cloudy or has precipitate) in theTreatment Medium, then follow the steps 1) through 4) using DMSO instead ofTreatment Medium.

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If the test chemical doesn’t dissolve in DMSO, then follow steps 1) through 4) using
ethanol instead of DMSO.
d) For the range finding experiments, the highest 2x concentration of test chemicaldissolved only in culture medium will be 200,000 µg/ml (200 mg/ml). The highest
2x concentration of test chemical first dissolved in DMSO or ethanol then transferred
to culture medium will be 20,000 µg/ml (20 mg/ml). Dissolve test chemical inappropriate medium/solvent (at 200-fold the desired final test concentration in thecase of DMSO or ethanol solvents, i.e., 20,000µg/ml). The final solvent (DMSO or
ethanol) concentration for application to the cells should be kept at a constant level of
0.5 % (v/v) in the vehicle controls and in all of the eight test concentrations. The
following example illustrates the preparation of test chemical in solvent and the
dilution of dissolved test chemical in medium before application to 3T3 cells.
Example: Preparation of Test Chemical in Solvent Using a Log Dilution Scheme
1) Label eight tubes 1 – 8. Add 0.9 ml solvent (e.g., DMSO or ethanol) to tubes 2 -8.
2) Prepare stock solution of 2,000,000 µg test chemical/ml solvent in tube # 1.
3) Add 0.1 ml of 2,000,000 µg/ml dilution from tube #1 to tube #2 to make a 1:10
dilution in solvent (i.e., 200,000 µg/ml).
4) Add 0.1 ml of 200,000 µg/ml dilution from tube #2 to tube #3 to make another1:10 dilution (i.e., 1:100 dilution from stock solution) in solvent (i.e., 20,000µg/ml)
5) Continuing making serial 1:10 dilutions in the prepared solvent tubes.
6) Since each concentration is 200 fold greater than the concentration to be tested,
dilute 1 part dissolved chemical in each tube with 99 parts of culture medium(e.g., 0.1 ml of test chemical in DMSO + 9.9 ml culture medium) to derive the 8
2x concentrations for application to 3T3 cells. Each test chemical concentration
will then contain 1 % v/v solvent. The 3T3 cells will have 0.05 ml Treatment
Medium in the wells prior to application of the test chemical. By adding 0.05 mlof the appropriate 2x test chemical concentration to the appropriate wells, the test
chemical will be diluted appropriately (e.g., highest concentration in well will be
10,000 µg/ml) in a total of 0.100 ml and the solvent concentration in the wells
will be 0.5% v/v.
Check carefully to determine whether the chemical is still dissolved after the transferfrom solvent stock solution to medium, and reduce the highest test concentration, if
necessary. Document all test chemical preparations in the Study Workbook.
2. pH of Test Chemical Solutions
Measure the pH of the highest concentration of the test chemical in culture medium using
pH paper. Document the pH and note the color of the medium for all dilutions. Do not
adjust the pH.

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3. Concentrations of Test Chemicala) Range Finder Experiment
Test eight concentrations of the test chemical/PC by diluting the stock solution with aconstant factor covering a large range. The initial dilution series shall be log dilutions(e.g., 1:10, 1:100, 1:1000, etc.).
b) Main Experiment
Depending on the slope of the concentration-response curve estimated from the range
finder, the dilution/progression factor in the concentration series of the main
experiment should be smaller (e.g., 6√10 = 1.47; NOTE: this dilution factor will be
used for the definitive positive control assays [Section VII.D.3]). Cover the relevant
concentration range (≥10 % and ≤ 90 % effect) with at least three points of a graded
effect, avoiding too many non-cytotoxic and/or 100 %-cytotoxic concentrations.
Experiments revealing less than three cytotoxic concentrations in the relevant range
shall be repeated, where possible, with a smaller dilution factor. (Taking into
account pipetting errors, a progression factor of 1.21 is regarded the smallest factor
achievable.)c) Test Chemical Dilutions•

A factor of 2√10 = 3.16 could be used for covering a large range:(e.g., 1 ⇒3.16 ⇒10 ⇒31.6 ⇒100 ⇒316 ⇒1000 ⇒3160 µg/ml).

•

The simplest geometric concentration series (i.e., constant dilution / progression
factor) are dual geometric series (e.g., a factor of 2). These series have thedisadvantage of numerical values that permanently change between logs of the series:(e.g., log0-2, 4, 8; log1- 16, 32, 64; log2- 128, 256, 512; log3- 1024, 2048,).

•

The decimal geometric series, first described by Hackenberg and Bartling (1959) for
use in toxicological and pharmacological studies, has the advantage that independent
experiments with wide or narrow dose factors can be easily compared because theyshare identical concentrations. Furthermore, under certain circumstances,
experiments can even be merged together:

EXAMPLE:
10
10
10
10

31.6
12.1

14.7
14.7

17.8

21.5
21.5
21.5

26.1

31.6
31.6

38.3

46.4
46.4
46.4

56.2

68.1
68.1

82.5

100
100
100
100

The dosing factor of 3.16 (= 2√10) divides a log into two equidistant steps, a factor of
2.15 (= 3√10) divides a decade into three steps. The factor of 1.47 (= 6√10) divides alog into six equidistant steps, and the factor of 1.21 (= 12√10) divides the log into 12
steps.

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For an easier biometrical evaluation of several related concentration response
experiments use decimal geometric concentration series rather than dual geometricseries. The technical production of decimal geometric concentration series is simple.
An example is given for factor 1.47:
Dilute 1 volume of the highest concentration by adding 0.47 volumes of diluent. Afterequilibration, dilute 1 volume of this solution by adding 0.47 volumes ofdiluent...(etc.).
•

Determine which test chemical concentration is closest to the IC50 value (e.g., 50 %cytotoxicity). Use that value as a central concentration and adjust dilutions higher
and lower in equal steps for the definitive assay.

F. Test Procedure
1. 96-Well Plate Configuration
The 3T3 NRU assay for test chemicals will use the 96-well plate configuration shown in
Figure 1.
2. Application of Test Chemicala) Two optional methods for rapidly applying the 2X dosing solutions onto the 96-wellplates may be utilized. The first method is to add each of the 2X dosing solutions intolabeled, sterile reservoirs (e.g., Corning/Costar model 4870 sterile polystyrene 50 mL
reagent reservoirs and/or Corning/Transtar model 4878 disposable reservoir liners, 8channel). The second method utilizes a “dummy” plate (i.e., an empty sterile 96-wellplate) prepared to hold the dosing solutions immediately prior to treatment of the test
plate (with cells). The test chemical and control dosing solutions should be dispensed
into the dummy plate in the same pattern/order as will be applied to the plate containing
cells. More volume than needed for the test plate (i.e. greater than 50 µl/well) should bein the wells of the dummy plate. At the time of treatment initiation, a multi-channel
micropipettor is used to transfer the 2X dosing solutions, from the reservoirs or dummy
plate, to the appropriate wells on the treatment plate (as described in step c. below).
These methods will ensure that the dosing solutions can be transferred rapidly to theappropriate wells of the test plate to initiate treatment times and to minimize the range oftreatment initiation times across a large number of treatment plates, and to prevent “outof order” dosing. A third option, though not a recommended option, is to transfer test
chemical solutions well by well using a single channel pipettor or repeat pipettor. This
option will increase the amount of time needed for test chemical application. The use ofa repeat pipettor increases the risk of dislodging cells from the culture plate.
b) After 24 h ± 1 h incubation of the cells, remove Routine Culture Medium from the cellsby careful inversion of the plate (i.e., “dump”) over an appropriate receptacle. Gently
blot the plate on a sterile paper towel so that the monolayer is minimally disrupted. Do
not use automatic plate washers for this procedure nor vacuum aspiration.
c) Immediately add 50 µl of Treatment Medium to each well. Then add 50 µl Treatment
Medium containing either the appropriate concentration of test chemical, the PC, or theVC (see Figure 1 for the plate configuration). The solutions will be transferred from thedummy plate to the test plate by adding the vehicle control first then lowest to highest
dose so that the same pipette tips on the eight channel pipettor can be used for the wholeplate.
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November 2006d) Incubate cells for 48 h ± 0.5 h (37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 %CO2/air).
e) Positive Control: For each set of test chemical plates used in an assay, a separate plate of
positive control concentrations will be set up following the concentration range
established in developing the positive control database. This plate will follow the sameschedule and procedures as used for the test chemical plates.
3. Microscopic Evaluation
After at least 46h treatment, examine each plate under a phase contrast microscope toidentify systematic cell seeding errors and growth characteristics of control and treatedcells. Record any changes in morphology of the cells due to the cytotoxic effects of thetest chemical, but do not use these records for any quantitative measure of cytotoxicity.
Undesirable growth characteristics of control cells may indicate experimental error andmay be cause for rejection of the assay. Use the following Visual Observations Codes inthe description of cell culture conditions.
Visual Observations Codes
Note Code
1
2
3
4
1P
2P
3P
4P
5P

Note Text
Normal Cell Morphology
Low Level of Cell Toxicity
Moderate Level of Cell Toxicity
High level of Cell Toxicity
Normal Cell Morphology with Precipitate
Low Level of Cell Toxicity with Precipitate
Moderate Level of Cell Toxicity with PrecipitateHigh level of Cell Toxicity with Precipitate
Unable to View Cells Due to Precipitate

4. Measurement of NRUa) Carefully remove (i.e., “dump”) the Treatment Medium and rinse the cells very
carefully with 250 µl pre-warmed D-PBS. Remove the rinsing solution by gentle
tapping. Add 250 µl NR medium (to all wells including the blanks) and incubate(37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air) for 3 h.
b) After incubation, remove the NR medium, and carefully rinse cells with 250 µl DPBS.
c) Decant and blot D-PBS from the plate. (Optionally: centrifuge the reversed plate.)d) Add exactly 100 µl NR Desorb (ETOH/acetic acid) solution to all wells, including
blanks.
e) Shake microtiter plate rapidly on a microtiter plate shaker for 20 – 45 min to extract
NR from the cells and form a homogeneous solution.
f) Measure the absorption (within 60 minutes of adding NR Desorb solution) of theresulting colored solution at 540 nm ± 10 nm in a microtiter plate reader

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(spectrophotometer), using the blanks as a reference. Save raw data in the Excel
format as provided by the Study Management Team.
5. Quality Check of 3T3 NRU Assaya) Test Acceptance Criteria
1) A test meets acceptance criteria, if the IC50 for SLS is within the 95 % CI of thehistorical mean established by the Test Facility (as per Section D).
2) A test meets acceptance criteria if the mean OD540 of VCs is ≥ 0.3 and ≤ 1.1.
3) A test meets acceptance criteria if the left and the right mean of the VCs do notdiffer by more than 15 % from the mean of all VCs.
b) Checks for Systematic Cell Seeding Errors
The absolute value of optical density (OD540 of NRU) obtained in the untreatedvehicle control may indicate whether the 2.5×103 cells seeded per well have grownexponentially with normal doubling time during the two days of the assay. If
doubling time experiments were performed using the NRU assay, then the historical
optical densities observed during the doubling time experiments can be used forcomparison to determine exponential growth.
To check for systematic cell seeding errors, untreated VCs are placed both at the left
side (row 2) and the right side (row 11 for the test plates) of the 96-well plate.
Aberrations in the cell monolayer for the VCs may reflect a volatile and toxic test
article present in the assay.
Checks for cell seeding errors may also be performed by examining each plate undera phase contrast microscope to assure that cell quantity is consistent.
c) Quality Check of Concentration-Response
The IC50 derived from the concentration-response of the test chemicals will bebacked by at least three responses ≥ 10 % and ≤ 90 % inhibition of NRU. If this is
not the case, and the concentration progression factor can be easily reduced, reject theexperiment and repeat it with a smaller progression factor. Numerical scoring of thecells (see Section F.3) should be determined and documented in the Study
Workbook.
G. Data Analysis
A calculation of cell viability expressed as NRU is made for each concentration of the test
chemical by using the mean NRU of the six replicate values (minimum of four acceptable
replicate well) per test concentration (blanks will be subtracted). This value is compared withthe mean NRU of all VC values (provided VC values have met the VC acceptance criteria).
Relative cell viability is then expressed as percent of untreated VC. If achievable, the eight
concentrations of each chemical tested will span the range of no effect up to total inhibitionof cell viability. Data from the microtiter plate reader shall be transferred to the Excel
spreadsheet provided by the Study Management Team for determining cell viability and
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November 2006

performing statistical analyses.
The concentration of a test chemical reflecting a 20 %, 50 %, and 80 % inhibition of cell
viability (i.e., the IC20, IC50, and IC80) is determined from the concentration-response by
applying a Hill function to the concentration-response data. It will not be necessary for the
Testing Facilities to derive the equation since statistical software (e.g., GraphPad PRISM
3.0) specified by the Study Management Team shall be used to calculate IC20, IC50, and IC80
values (and the associated confidence limits) for each test chemical. In addition, the StudyManagement Team shall provide guidelines for calculating ICx values and confidence limits.
The Testing Facility shall report data using at least three (3) significant figures and shallforward the results from each assay to the Study Management Team/biostatistician throughthe designated contacts in electronic format and hard copy upon completion of testing. The
Study Management Team will be directly responsible for the statistical analyses of the
Validation Study data.
VIII.

REFERENCES
Hackenberg, U. and H. Bartling. 1959. Messen und Rechnen im pharmakologischen
Laboratorium mit einem speziellen Zahlensystem (WL24-System). Arch. Exp. Pathol.
Pharmakol. 235: 437-463.
Spielmann, H., S. Gerner, S. Kalweit, R. Moog, T. Wirnserberger, K. Krauser, R. Kreiling, H.
Kreuzer, N.P. Luepke, H.G. Miltenburger, N. Müller, P. Murmann, W. Pape, B. Siegmund, J.
Spengler, W. Steiling, and F.J. Wiebel. 1991. Interlaboratory assessment of alternatives tothe Draize eye irritation test in Germany. Toxicol. In Vitro 5: 539-542.

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IX.

November 2006

APPROVAL__________________________________SPONSOR REPRESENTATIVE(Print or type name)___________________DATE_____________________________
Test Facility STUDY DIRECTOR(Print or type name)____________________
DATE

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Appendix C2

Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test (Phase Ia)

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[This Page Intentionally Left Blank]

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TEST METHOD PROTOCOL
for the NHK Neutral Red Uptake Cytotoxicity Test

A Test for Basal Cytotoxicity for an In Vitro Validation Study
June 14, 2002

Prepared by
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)

Based on Standard Operating Procedure Recommendations from anInternational Workshop Organized by the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM)

National Institute of Environmental Health Sciences (NIEHS)
National Institutes of Health (NIH)
U.S. Public Health Service
Department of Health and Human Services

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TEST METHOD PROTOCOLThe Normal Human Keratinocyte (NHK) Neutral Red Uptake Cytotoxicity
Test
A Test for Basal Cytotoxicity
I.

PURPOSEThe purpose of this study is to evaluate the cytotoxicity of test chemicals using the Normal
Human Keratinocyte (NHK) Neutral Red Uptake (NRU) cytotoxicity test. The data will be used
to evaluate the intra- and inter-laboratory reproducibility of the assay and effectiveness of thecytotoxicity assay to predict the starting doses for rodent acute oral systemic toxicity assays. This
test method protocol outlines the procedures for performing the cytotoxicity test and is in supportof the in vitro validation study organized by NICEATM and the European Centre for the
Validation of Alternative Methods (ECVAM) and sponsored by NIEHS, U.S. Environmental
Protection Agency, and ECVAM. This test method protocol applies to all personnel involved
with performing the cytotoxicity assay.
A. Determination of Positive Control DatabaseAn historical database of IC50 values for the positive control chemical (Sodium Lauryl[dodecyl] Sulfate {SLS}) must be established and maintained by performing 10
concentration-response assays on the NHK cells before performing the NRU assay on test
chemicals. Once the mean IC50 and the 95 % confidence interval (CI) of the IC50 of SLS areestablished, the values will be used as an acceptance criterion for test sensitivity for the NHK
NRU assay.
B. NHK Neutral Red Uptake Cytotoxicity Test
After acceptable positive control mean IC50 and 95 % CI values have been established, the
NHK NRU test will be performed to analyze the in vitro toxicity of test chemicals. This test
will be used to determine IC20, IC50, and IC80 values for a predetermined set of test chemicals
of varying toxicities.

II. SPONSOR
A. Name:National Institute of Environmental Health Sciences (NIEHS); The
National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)

B. Address:

P.O. Box 12233
Research Triangle Park, NC 27709

C. Representative:

Named Representative

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III. IDENTIFICATION OF TEST AND CONTROL SUBSTANCES
A. Test Chemicals:

Blinded chemicals 1

B. Controls:

Positive:
Sodium Lauryl Sulfate
Vehicle (Negative):
Assay medium
Solvent (as needed):
Assay medium with appropriate solvent
used to prepare the test chemicals (Section VII.E)

IV. TESTING FACILITY AND KEY PERSONNEL
•
•
•
•
•
•
•
•

Name:
Address:
Study Director:
Laboratory Technician(s):Scientific Advisor:
Quality Assurance Director:
Safety Manager:
Facility Management:

A. Test Schedule•••

Proposed Experimental Initiation Date:
Proposed Experimental Completion Date:
Proposed Report Date:

V. TEST SYSTEMThe NRU cytotoxicity assay procedure is a cell survival/viability chemosensitivity assay based on
the ability of viable cells to incorporate and bind neutral red (NR), a supravital dye. NR is a weakcationic dye that readily penetrates cell membranes by non-ionic diffusion and accumulates
intracellularly in lysosomes. Alterations of the cell surface or the sensitive lysosomal membranelead to lysosomal fragility and other changes that gradually become irreversible. Such changes
brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It isthus possible to distinguish between viable, damaged, or dead cells, which is the basis of thisassay.
Healthy mammalian cells, when maintained in culture, continuously divide and multiply overtime. A toxic chemical, regardless of site or mechanism of action, will interfere with this process
and result in a reduction of the growth rate as reflected by cell number. Cytotoxicity is expressed
as a concentration dependent reduction of the uptake of the NR after chemical exposure thus
providing a sensitive, integrated signal of both cell integrity and growth inhibition.
VI. DEFINITIONS
A.. Hill function: a four parameter logistic mathematical model relating the concentration of test
chemical to the response being measured in a sigmoidal shape.

Y = Bottom +Top ! Bottom
1 + 10(logIC50! X)HillSlope

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where Y= response, X is the logarithm of dose (or concentration), Bottom is the minimum
response, Top is the maximum response, logIC50 is logarithm of X at the response midway
between Top and Bottom, and HillSlope describes the steepness of the curve.
B. Documentation: all methods and procedures will be noted in a Study Workbook; logs will bemaintained for general laboratory procedures and equipment (e.g., media preparation, test
chemical preparation, incubator function); all optical density data obtained from thespectrophotometer plate reader will be saved in electronic and paper formats; all calculationsof ICx values and other derived data will be in electronic and paper format; all data will bearchived.
VII.

PROCEDURES

A. Materials[Note: Suggested brand names/vendors are listed in parentheses. Equivalents may be used
unless otherwise noted.]
1. Cell Lines
Normal Human Epidermal Keratinocytes (NHK)Non-transformed cells; from cryopreserved primary or secondary cells (Clonetics #CC2507 or equivalent). Cells will be Clonetics NHK cells.
Clonetics/BioWhittaker [BioWhittaker, 8830 Biggs Ford Road, Walkersville, MD 217930127
BioWhittaker Europe [BioWhittaker Europe, S.P.R.L. Parc Industriel de Petit Rechain, B4800 Verviers, BELGIUM]
2. Technical Equipment
a)
b)
c)
d)
e)
f)
g)
h)

Incubator: 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/airLaminar flow clean bench (standard: "biological hazard")
Water bath: 37ºC ± 1ºC
Inverse phase contrast microscope
Sterile glass tubes with caps (e.g., 5ml)
Centrifuge (optionally: equipped with microtiter plate rotor)
Laboratory balance
96-well plate spectrophotometer (i.e., plate reader) equipped with 540 nm ± 10 nm
filteri) Shaker for microtiter platesj) Cell counter or hemocytometerk) Pipetting aidl) Pipettes, pipettors (multi-channel and single channel), dilution blockm) Cryotubesn) Tissue culture flasks (75 - 80 cm2, 25 cm2)o) 96-well flat bottom tissue culture microtiter plates (e.g., Nunc # 167 008;
Corning/COSTAR tissue culture-treated)p) pH paper (wide and narrow range)

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[Note: Tissue culture flasks and microtiter plates should be prescreened to ensurethat they adequately support the growth of NHK.]
3. Chemicals, Media, and Seraa) Keratinocyte Basal Medium without Ca++ (KBM®, Clonetics CC-3104) that iscompleted by adding the KBM® SingleQuots® (Clonetics CC-4131) to achieve theproper concentrations of epidermal growth factor, insulin, hydrocortisone,
antimicrobial agents, bovine pituitary extract, and calcium (e.g., Clonetics Calcium
SingleQuots®, CC-4202).
b) HEPES Buffered Saline Solution (HEPES-BSS) (e.g., Clonetics # CC-5022)c) 0.025 % Trypsin/EDTA solution (e.g., Clonetics # CC-5012)
d) Trypsin Neutralizing Solution (TNS) (e.g., Clonetics # CC-5002)e) Phosphate Buffered Saline (PBS)f) Dulbecco’s Phosphate Buffered Saline (D-PBS) with glucose) formulation containing
calcium and magnesium cations, and supplemented with 1000 mg/L glucose)g) Fetal bovine serum (FBS)h) Neutral Red (NR) Dye – tissue culture-grade; liquid form (e.g., SIGMA N 2889);
powder form (e.g., SIGMA N 4638)
i) Dimethyl sulfoxide (DMSO), U.S.P analytical grade (Store under nitrogen @ -20ºC)j) Ethanol (ETOH), U.S.P. analytical grade (100 %, non-denatured for test chemical
preparation; 95 % can be used for the desorb solution)k) Glacial acetic acid, analytical gradel) Hanks' Balanced Salt Solution without Ca2+ or Mg2+ (CMF-HBSS) (e.g., Invitrogen #14170)m) Distilled H2 O or any purified water suitable for cell culture (sterile)n) Sterile paper towels (for blotting 96-well plates)
B. Preparations of Media and Solutions[Note: All solutions (except NR stock solution, NR medium and NR desorb), glassware,
pipettes, etc., shall be sterile and all procedures should be carried out under aseptic conditions
and in the sterile environment of a laminar flow cabinet (biological hazard standard).]
1. Mediaa) Routine Culture Medium/Treatment Medium
KBM® (Clonetics CC-3104) supplemented with KBM® SingleQuots® (Clonetics
CC-4131) and Clonetics Calcium SingleQuots® (CC-4202) to make 500ml of
medium. Final concentration of supplements in medium are:0.0001 ng/ml
5 µg/ml
0.5 µg/ml
30 µg/ml
15 ng/ml
0.10 mM
30 µg/ml

Human recombinant epidermal growth factor
InsulinHydrocortisone
Gentamicin
Amphotericin B
Calcium
Bovine pituitary extract

Complete media should be kept at 4°C and stored for no longer than two weeks.

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NOTE:
KBM® SingleQuots® contain the following stock concentrations and volumes:
0.1 ng/ml
5.0 mg/ml
0.5 mg/ml
30 mg/ml
7.5 mg/ml

hEGF
InsulinHydrocortisone
Gentamicin, 15 ug/ml Amphotericin-B
Bovine Pituitary Extract (BPE)

0.5 ml
0.5 ml
0.5 ml
0.5 ml2.0 mlClonetics Calcium SingleQuots® are 2 ml of 300mM concentration of calcium.
165 ul of solution per 500 ml calcium-free medium equals 0.10 mM calcium in themedium.
2. Neutral Red (NR) Stock Solution
The liquid tissue culture-grade stock NR Solution will be the first choice for performing
the assay. If the liquid form is not available, the following formulation can be prepared.
0.4 g NR Dye powder in 100 ml of H2O
Make up prior to use and store dark at room temperature. May store for up to twomonths.
3. Neutral Red (NR) Medium
EXAMPLE:
1 ml (4mg NR dye/ml)
79 ml

NR Stock Solution
KGM

The final concentration of the NR Medium is 50 µg NR dye/ml.
[Note: The NR medium should be incubated overnight at 37ºC ± 1ºC and centrifuged at
approximately 600 x g for 10 min (to remove NR crystals) before adding to the cells.
Alternative procedures (e.g., Millipore filtering) can be used as long as they guaranteethat NR medium is free of crystals.]
4. Ethanol/Acetic Acid Solution (NR Desorb)
1%
50 %
49 %

Glacial acetic acid solution
Ethanol
H2 O

C. Methods
1. Cell Maintenance and Culture Procedures
NHK cells are routinely grown as a monolayer in tissue culture grade flasks (e.g., 25 cm2)
at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air. The cells should be

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examined on a daily basis under a phase contrast microscope, and any changes inmorphology or their adhesive properties must be noted in a Study Workbook (See
Section VII.F.3)
2. Receipt of Cryopreserved Keratinocytes
Upon receipt of cryopreserved keratinocytes, the vial(s) of cells shall be stored in a liquid
nitrogen freezer until needed.
3. Thawing Cells and Establishing Cell Culturesa) Thaw cells by putting ampules into a water bath at 37°C for as brief a time as
possible. Do not thaw cells at room temperature or by hand. Seed the thawed cells
into culture flasks as quickly as possible and with minimal handling.
b) Slowly (taking approximately 1-2 min) add 9 ml of Routine Culture Medium to thecells suspended in the cryoprotective solution and transfer cells into flasks containing
pre-warmed Routine Culture Medium (See Table 1).
c) Incubate the cultures at 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air until
the cells attach to the flask, at which time the Routine Culture Medium should be
removed and replaced with fresh Routine Culture Medium.
d) Unless otherwise specified, the cells should be incubated at 37ºC ± 1ºC, 90 % ± 5 %humidity, 5.0 % ± 1 % CO2/air and fed every 2-3 days until they exceed 50 %confluence (but less than 80 % confluent).
Table 1. Establishing Cell Cultures
Cells/25 cm2 flask(in approximately 5 ml)
1 flask each cell concentration
Approximate Time to Subculture
Cells to 96-Well Plates

6.25 x 104(2500 cm2)

1.25 x 105(5000 cm2)2.25 x 105(9000 cm2)

96+ hours
6 – 8 plates

72 - 96 hours
6 – 8 plates

48 - 72 hours
6 – 8 plates

Cell growth guidelines – actual growth of individual cell lots may vary.
4. Subculture of NHK Cells to 96-Well Plates[Note: It is important that cells have overcome the lag growth phase when they are used
for the test. Keratinocytes will be passaged only into the 96-well plates and will not be
subcultured into flasks for use in later assays](a) When the keratinocyte culture in a 25 cm2 flask exceeds 50 % confluence (but less than
80 % confluent), remove the medium and rinse the culture twice with 5 ml HEPESBSS. The second rinse should be left on the cells for approximately 5 minutes. Discard
the washing solution.
(b) Add 2 ml trypsin/EDTA solution to each flask and remove after 15 to 30 seconds.
Incubate the flask at room temperature for 3 to 7 min. When more than 50 % of thecells become dislodged, rap the flask sharply against the palm of the hand.
(c) When most of the cells have become detached from the surface, rinse the flask with 5 mlof room temperature TNS.
(d) Then rinse the flask with 5 ml CMF-HBSS and transfer the cell suspension to acentrifuge tube.
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In Vitro Cytotoxicity Test Methods BRD Appendix C2

November 2006(e) Pellet the cells by centrifugation for 5 min at approximately 220 x g. Remove thesupernatant by aspiration.
(f) Resuspend the keratinocyte pellet by gentle trituration (to have single cells) in Routine
Culture Medium. It is important to obtain a single cell suspension for exact counting.
Count a sample of the cell suspension using a hemocytometer or cell counter.
4(g) Prepare a cell suspension of 0.8 - 1x10 cells/ml in Routine Culture Medium. Using a
multi-channel pipette, dispense 250 µl PBS only into the peripheral wells (blanks) of
a 96-well tissue culture microtiter plate. In the remaining wells, dispense 250 µl of33
the cell suspension (2x10 – 2.5x10 cells/well). Prepare one plate per chemical to betested.
(h) Incubate cells (37ºC ± 1ºC, 90 % ± 5.0 % humidity, and 5 % ± 1 % CO2/air) so thatcells form a 30+ % monolayer (~48-72 h). This incubation period assures cellrecovery and adherence and progression to exponential growth phase.
(i) Examine each plate under a phase contrast microscope to assure that cell growth isrelatively even across the microtiter plate. This check is performed to identify
experimental and systemic cell seeding errors. Record observations in the Study
Workbook.
5. Determination of Doubling Timea) Establish cells in culture and trypsinize cells as per Section C.4 for subculture.
Resuspend cells in appropriate culture medium. Use Table 1 to determine seeding
densities.
b) Seed five sets of cell culture vessels in triplicate for each cell type (e.g., 15 tissue
culture dishes [60mm x 15mm]). Use appropriate volume of culture medium for theculture vessels. Note number of cells placed into each culture dish. Place dishes
into the incubators (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air).
c) After 4-6 hours (use the same initial measurement time for each subsequent doublingtime experiment), remove three culture dishes and trypsinize cells. Count cells using
a cell counter or hemocytometer. Cell viability may be determined by dye exclusion(e.g., Trypan Blue). Determine the total number of cells and document. Repeat
sampling at 24 hr, 48 hr, 72 hr, and 96 hr post inoculation. Change culture medium
at 72 hr or sooner in remaining dishes if indicated by pH drop.
d) Plot cell concentration (per ml of medium) on a log scale against time on a linear
scale. Determine lag time and population doubling time. The doubling time will bein the log (exponential) phase of the growth curve. Additional dishes and time areneeded if the entire growth curve is to be determined (lag phase, log phase, plateau
phase).
D. Establishing the Positive Control DatabaseAn historical database of IC50 values for the positive control chemical (Sodium Lauryl[dodecyl] Sulfate {SLS}) must be established and maintained by performing 10
concentration-response assays on the NHK cells.
1. Positive Control Chemical Preparation
The positive control chemical (SLS) is prepared in the same manner as the test chemical(Sections E.1 and E.2) by following the instructions and substituting “test chemical”
with “SLS.”

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2. Range Finder Experiment
Before initiating the 10 concentration-response assays, a range finder experiment will be
performed using eight concentrations of SLS by diluting the stock solution with aconstant factor as per Section E.3.a and E.3.b. The eight chemical concentrations will betested as per the test procedure outlined in Section F and analyzed as per procedures
outlined in Section G.
3. Test Procedure
Once a range has been determined that satisfies the criteria in Section E.3.b, thedefinitive concentration-response assays shall use a 6√10 = 1.47 dilution scheme centeredon the IC50. The Test Facility will perform two tests per day on five different days. The
95 % CI of the IC50 of SLS will be established and defined as an acceptance criterion fortest sensitivity for the NHK NRU assay. The confidence intervals shall be calculatedusing the average of the individual IC50 values from each positive control assay
performed. An example of an historical mean IC50 of SLS in NHK cultures is 4.4 µg/ml± 0.97 µg/ml [two standard deviations] (Triglia, 1989). All testing will follow the
instructions in Section F using the 96-well plate configuration in Figure 1. The test
meets acceptance criteria if the conditions in Sections F.5.a.2 and F.5.a.3 are met.

Figure 1. 96-Well Plate Configuration for Positive Control and Test Chemical
Assays
1

2

3

4

5

6

7

8

9

10

11

12

A

b

b

b

b

b

b

b

b

b

b

b

b

B

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

C

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

D

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

E

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

F

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

G

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

H

b

b

b

b

b

b

b

b

b

b

b

b

VC
=
C1 – C8 =b=untreated VEHICLE CONTROL (mean viability set to 100 %)Test Chemicals or Positive Control (SLS) at eight concentrations(C1 = highest, C8 = lowest)
BLANKS (contain no cells)

E. Preparation of Test Chemicals[Note: Test chemical must be freshly prepared immediately prior to use. Each stock dilution
should have at least 1-2 ml total volume to ensure adequate solution for the test wells in a

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single 96-well plate. The solutions must not be cloudy nor have noticeable precipitate. Test
chemicals must be at room temperature before dissolving and diluting. Preparation under redor yellow light may be necessary, if rapid photodegradation is likely to occur.]
1. Dissolving Test Chemicala) Approximately 200,000 µg (200 mg) of the test chemical will be weighed into a glass
tube and the weight will be documented. Assay-specific culture medium will be
added to the vessel so that the concentration is 2,000,000 µg/ml (2000 mg/ml) andmixed using the mixing procedures outlined in Section E.1.c. If complete solubility
is achieved, then additional solubility procedures are not needed. The test chemical
can then be prepared and diluted for use in an assay. If only partial solubility isachieved, then add additional medium in the steps outlined in Table 1 until theconcentration is a minimum of 200,000 µg /ml. If complete solubility at 200,000µg/ml in culture medium can’t be attained, then repeat the solubility steps in Table 1
and Section E.1.c using the other solvent(s) in the solubility hierarchy. Test
chemicals that are only soluble in DMSO or ethanol will be prepared at 2,000,000µg/ml as the highest concentration of stock solution.
Table 2 Determination of Solubility
Solubility Data
Total volume of medium added (ml)Total volume of DMSO or ethanol added (ml)Approximate solubility (µg /ml)

Step 10.1
0.1≥ 2,000,000

Step 20.5
0.5
400,000

Step 31.0
1.0
200,000

Example: If complete solubility is not achieved in 0.1 ml medium (Step 1), then 0.4
ml is added to obtain a total volume of 0.5 ml (Step 2). No additional weighing ofchemical is needed. Chemical and medium are again mixed in an attempt to dissolve.
b) Each test chemical will be prepared such that the highest test concentration applied tothe cells in each range finding experiment is 100,000 µg/ml in culture medium(10,000 µg/ml if DMSO or ethanol is used). If 100,000 µg/ml in culture medium
cannot be achieved, then the highest concentration attainable will be used. If therange finding experiment shows that 10,000 µg/ml is not high enough for the rangeof chemicals dissolved in DMSO or ethanol to meet the acceptance criteria, thenhigher concentrations will be used for the definitive experiment.
c) The following mixing and solvent hierarchy will be followed in dissolving the test
chemical:
1)
2)
3)
4)

Dissolve test chemical in Treatment Medium.
Gently mix. Vortex the tube (1 –2 minutes).
If test chemical hasn’t dissolved, use sonication for up to 5 minutes.
If sonication doesn’t work, then warm solution to 37°C.

If the test chemical doesn’t dissolve (i.e., solution is cloudy or has precipitate) in theTreatment Medium, then follow the steps 1) through 4) using DMSO instead ofTreatment Medium.

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If the test chemical doesn’t dissolve in DMSO, then follow steps 1) through 4) using
ethanol instead of DMSO.
d) For the range finding experiments, the highest 2x concentration of test chemicaldissolved only in culture medium will be 200,000 µg/ml (200 mg/ml). The highest
2x concentration of test chemical first dissolved in DMSO or ethanol then transferred
to culture medium will be 20,000 µg/ml (20 mg/ml). Dissolve test chemical inappropriate medium/solvent (at 200-fold the desired final test concentration in thecase of DMSO or ethanol solvents, i.e., 20,000 µg/ml). The final solvent (DMSO or
ethanol) concentration for application to the cells should be kept at a constant level of
0.5 % (v/v) in the vehicle controls and in all of the eight test concentrations. The
following example illustrates the preparation of test chemical in solvent and the
dilution of dissolved test chemical in medium before application to NHK cells.
Example: Preparation of Test Chemical in Solvent Using a Log Dilution Scheme
1) Label eight tubes 1 – 8. Add 0.9 ml solvent (e.g., DMSO or ethanol) to tubes 2 -8.
2) Prepare stock solution of 2,000,000 µg test chemical/ml solvent in tube # 1.
3) Add 0.1 ml of 2,000,000µg/ml dilution from tube #1 to tube #2 to make a 1:10
dilution in solvent (i.e., 200,000 µg/ml).
4) Add 0.1 ml of 200,000 µg/ml dilution from tube #2 to tube #3 to make another1:10 dilution (i.e., 1:100 dilution from stock solution) in solvent (i.e., 20,000µg/ml)
5) Continuing making serial 1:10 dilutions in the prepared solvent tubes.
6) Since each concentration is 200 fold greater than the concentration to be tested,
dilute 1 part dissolved chemical in each tube with 99 parts of culture medium(e.g., 0.1 ml of test chemical in DMSO + 9.9 ml culture medium) to derive the 8
2x concentrations for application to NHK cells. Each test chemical concentration
will then contain 1 % v/v solvent. The NHK cells will have 0.125 ml of culture
medium in the wells prior to application of the test chemical. By adding 0.125
ml of the appropriate 2x test chemical concentration to the appropriate wells, thetest chemical will be diluted appropriately (e.g., highest concentration in well
will be 10,000 µg/ml) in a total of 0.250 ml and the solvent concentration in thewells will be 0.5% v/v.
Check carefully to determine whether the chemical is still dissolved after the transferfrom solvent stock solution to medium, and reduce the highest test concentration, if
necessary. Document all test chemical preparations in the Study Workbook.
2. pH of Test Chemical Solutions
Measure the pH of the highest concentration of the test chemical in culture medium using
pH paper. Document the pH and note the color of the medium for all dilutions. Do not
adjust the pH.
3. Concentrations of Test Chemicala) Range Finder Experiment

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Test eight concentrations of the test chemical/PC by diluting the stock solution with aconstant factor covering a large range. The initial dilution series shall be log dilutions(e.g., 1:10, 1:100, 1:1000, etc.).
b) Main Experiment
Depending on the slope of the concentration-response curve estimated from the range
finder, the dilution/progression factor in the concentration series of the main experimentshould be smaller (e.g., 6√10 = 1.47; NOTE: this dilution factor will be used for thedefinitive positive control assays [Section VII.D.3]). Cover the relevant concentration
range (≥10 % and ≤ 90 % effect) with at least three points of a graded effect, avoiding too
many non-cytotoxic and/or 100 %-cytotoxic concentrations. Experiments revealing lessthan three cytotoxic concentrations in the relevant range shall be repeated, where
possible, with a smaller dilution factor. (Taking into account pipetting errors, a
progression factor of 1.21 is regarded the smallest factor achievable.)c) Test Chemical Dilutions•

A factor of 2√10 = 3.16 could be used for covering a large range:(e.g., 1 ⇒3.16 ⇒10 ⇒31.6 ⇒100 ⇒316 ⇒1000 ⇒3160 µg/ml).

•

The simplest geometric concentration series (i.e., constant dilution / progression
factor) are dual geometric series (e.g., a factor of 2). These series have thedisadvantage of numerical values that permanently change between logs of the series:(e.g., log0-2, 4, 8; log1- 16, 32, 64; log2- 128, 256, 512; log3- 1024, 2048,).

•

The decimal geometric series, first described by Hackenberg and Bartling (1959) for
use in toxicological and pharmacological studies, has the advantage that independent
experiments with wide or narrow dose factors can be easily compared because theyshare identical concentrations. Furthermore, under certain circumstances,
experiments can even be merged together:

EXAMPLE:
10
10
10
10

31.6

12.1

14.7
14.7

17.8

21.5
21.5
21.5

26.1

31.6
31.6

38.3

46.4
46.4
46.4

56.2

68.1
68.1

82.5

100
100
100
100

The dosing factor of 3.16 (= 2√10) divides a log into two equidistant steps, a factor of
2.15 (= 3√10) divides a decade into three steps. The factor of 1.47 (= 6√10) divides alog into six equidistant steps, and the factor of 1.21 (= 12√10) divides the log into 12
steps.
For an easier biometrical evaluation of several related concentration response
experiments use decimal geometric concentration series rather than dual geometricseries. The technical production of decimal geometric concentration series is simple.
An example is given for factor 1.47:

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Dilute 1 volume of the highest concentration by adding 0.47 volumes of diluent. Afterequilibration, dilute 1 volume of this solution by adding 0.47 volumes ofdiluent...(etc.).
•

Determine which test chemical concentration is closest to the IC50 value (e.g., 50 %cytotoxicity). Use that value as a central concentration and adjust dilutions higher
and lower in equal steps for the definitive assay.

F. Test Procedure
1. The NHK NRU assay for test chemicals will use the 96-well plate configuration shown in
Figure 1.
2. Application of Test Chemicala) Two optional methods for rapidly applying the 2X dosing solutions onto the 96-wellplates may be utilized. The first method is to add each of the 2X dosing solutions
into labeled, sterile reservoirs (e.g., Corning/Costar model 4870 sterile polystyrene
50 mL reagent reservoirs and/or Corning/Transtar model 4878 disposable reservoir
liners, 8-channel). The second method utilizes a “dummy” plate (i.e., an empty
sterile 96-well plate) prepared to hold the dosing solutions immediately prior totreatment of the test plate (with cells). The test chemical and control dosing solutions
should be dispensed into the dummy plate in the same pattern/order as will be applied
to the plate containing cells. More volume than needed for the test plate (i.e. greaterthan 125 µl/well) should be in the wells of the dummy plate. At the time of treatment
initiation, a multi-channel micropipettor is used to transfer the 2X dosing solutions,from the reservoirs or dummy plate, to the appropriate wells on the treatment plate(as described in step c. below). These methods will ensure that the dosing solutions
can be transferred rapidly to the appropriate wells of the test plate to initiate
treatment times and to minimize the range of treatment initiation times across a large
number of treatment plates, and to prevent “out of order” dosing. A third option,
though not a recommended option, is to transfer test chemical solutions well by wellusing a single channel pipettor or repeat pipettor. This option will increase theamount of time needed for test chemical application. The use of a repeat pipettor
increases the risk of dislodging cells from the culture plate.
b) After 24 - 72 h incubation of the cells, remove Routine Culture Medium from thecells by careful inversion of the plate (i.e., “dump”) over an appropriate receptacle.
Gently blot the plate on a sterile paper towel so that the monolayer is minimallydisrupted. Do not use automatic plate washers for this procedure nor vacuum
aspiration.
c) Immediately add 125 µl of fresh Routine Culture Medium to each well. Add 125 µlof the appropriate concentration of test chemical, the PC, or the VC (see Figure 1 forthe plate configuration).
d) Incubate cells for 48 h ± 0.5 h (37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 %CO2/air).
e) Positive Control: For each set of test chemical plates used in an assay, a separate
plate of positive control concentrations will be set up following the concentration
range established in developing the positive control database. This plate will follow
the same schedule and procedures as used for the test chemical plates.

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3. Microscopic Evaluation
After at least 46 h treatment, examine each plate under a phase contrast microscope toidentify systematic cell seeding errors and growth characteristics of control and treatedcells. Record any changes in morphology of the cells due to the cytotoxic effects of thetest chemical, but do not use these records for any quantitative measure of cytotoxicity.
Undesirable growth characteristics of control cells may indicate experimental error andmay be cause for rejection of the assay. Use the following Visual Observations Codes inthe description of cell culture conditions.
Visual Observations Codes
Note Code
1
2
3
4
1P
2P
3P
4P
5P

Note Text
Normal Cell Morphology
Low Level of Cell Toxicity
Moderate Level of Cell Toxicity
High level of Cell Toxicity
Normal Cell Morphology with Precipitate
Low Level of Cell Toxicity with Precipitate
Moderate Level of Cell Toxicity with PrecipitateHigh level of Cell Toxicity with Precipitate
Unable to View Cells Due to Precipitate

4. Measurement of NRUa) Carefully remove (i.e., “dump”) the Routine Culture Medium (with test chemical)
and rinse the cells very carefully with 250 µl pre-warmed D-PBS. Remove therinsing solution by gentle tapping and blot the plate. Add 250 µl NR medium (to allwells including the blanks) and incubate (37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0% ± 1 % CO2/air) for 3 h.
b) After incubation, remove the NR medium, and carefully rinse cells with 250 µl DPBS.
c) Decant and blot D-PBS from the plate. (Optionally: centrifuge the reversed plate.)d) Add exactly 100 µl NR Desorb (ETOH/acetic acid) solution to all wells, including
blanks.
e) Shake microtiter plate rapidly on a microtiter plate shaker for 20 – 45 min to extract
NR from the cells and form a homogeneous solution.
f) Measure the absorption (within 60 minutes of adding NR Desorb solution) of theresulting colored solution at 540 nm ± 10 nm in a microtiter plate reader(spectrophotometer), using the blanks as a reference. Save raw data in the Excel
format as provided by the Study Management Team.
5. Quality Check of Assaya) Test Acceptance Criteria
1) A test meets acceptance criteria, if the IC50 for SLS is within the 95 % CI of thehistorical mean established by the Test Facility (as per Section D).
2) A test meets acceptance criteria if the mean OD540 of VCs is ≥ 0.3 and ≤ 1.1.
3) A test meets acceptance criteria if the left and the right mean of the VCs do notdiffer by more than 15 % from the mean of all VCs.
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November 2006b) Checks for Systematic Cell Seeding Errors
The absolute value of optical density (OD540 of NRU) obtained in the untreated3
3
vehicle control may indicate whether the 2x10 – 2.5x10 cells seeded per well havegrown exponentially with normal doubling time during the assay. Historical opticaldensities observed during doubling time experiments can be used for comparison todetermine exponential growth.
To check for systematic cell seeding errors, untreated VCs are placed both at the left
side (row 2) and the right side (row 11 for the test plates) of the 96-well plate.
Aberrations in the cell monolayer for the VCs may reflect a volatile and toxic test
article present in the assay.
Checks for cell seeding errors may also be performed by examining each plate undera phase contrast microscope to assure that cell quantity is consistent.
c) Quality Check of Concentration-Response
The IC50 derived from the concentration-response of the test chemicals should bebacked by at least three responses between 10 and 90 % inhibition of NRU. If this is
not the case, and the concentration progression factor can be easily reduced, reject theexperiment and repeat it with a smaller progression factor. Numerical scoring of thecells (see Section F.3) should be determined and documented in the Study
Workbook.
G. Data Analysis
A calculation of cell viability expressed as NRU is made for each concentration of the test
chemical by using the mean NRU of the six replicate values (minimum of four acceptable
replicates wells) per test concentration. This value is compared with the mean NRU of all
VC values (provided VC values have met the VC acceptance criteria). Relative cell viability
is then expressed as percent of untreated VC. If achievable, the eight concentrations of eachchemical tested will span the range of no effect up to total inhibition of cell viability. Data
from the microtiter plate reader shall be transferred to the Excel® spreadsheet provided by
the Study Management Team for determining cell viability and performing statisticalanalyses.
The concentration of a test chemical reflecting a 20 %, 50 %, and 80 % inhibition of cell
viability (i.e., the IC20, IC50, and IC80) is determined from the concentration-response by
applying a Hill function to the concentration-response data. It will not be necessary for the
Testing Facilities to derive the equation since statistical software (e.g., GraphPad PRISM®
3.0) specified by the Study Management Team shall be used to calculate IC20, IC50, and IC80
values (and the associated confidence limits) for each test chemical. In addition, the StudyManagement Team shall provide guidelines for calculating ICx values and confidence limits.
The Testing Facility shall report data using at least three (3) significant figures and shallforward the results from each assay to the Study Management Team/biostatistician throughthe designated contacts in electronic format and hard copy upon completion of testing. The
Study Management Team will be directly responsible for the statistical analyses of the
Validation Study data.

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VIII.

November 2006

REFERENCES
Clonetics Normal Human Keratinocyte Systems Instructions for Use, AA-1000-4-Rev.03/00.
(http://www.clonetics.com).
Hackenberg, U. and H. Bartling. 1959. Messen und Rechnen im pharmakologischen
Laboratorium mit einem speziellen Zahlensystem (WL24-System). Arch. Exp. Pathol.
Pharmakol. 235: 437-463.
Triglia, D., P.T. Wegener, J. Harbell, K. Wallace, D. Matheson, and C. Shopsis. 1989.
Interlaboratory validation study of the keratinocyte neutral red bioassay from Clonetics
Corporation. In Alternative Methods in Toxicology, Volume 7. A.M. Goldberg, ed., pp. 357365. Mary Ann Liebert, Inc., New York.

IX. APPROVAL__________________________________SPONSOR REPRESENTATIVE___________________DATE(Print or type name)_____________________________
Testing Facility STUDY DIRECTOR(Print or type name)____________________
DATE

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Appendix C3

Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase Ib)

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TEST METHOD PROTOCOL
for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

A Test for Basal Cytotoxicity for an In Vitro Validation Study
Phase Ib
November 15, 2002
Revised November 22, 2002
Revised by IIVS Nov. 26, 2002
Prepared by
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)

Based on Standard Operating Procedure Recommendations from anInternational Workshop Organized by the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM)

National Institute of Environmental Health Sciences (NIEHS)
National Institutes of Health (NIH)
U.S. Public Health Service
Department of Health and Human Services

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TEST METHOD PROTOCOLThe BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test
A Test for Basal Cytotoxicity
Phase Ib
I. PURPOSE
The purpose of this study is to evaluate the cytotoxicity of test chemicals using the BALB/c 3T3
Neutral Red Uptake (NRU) cytotoxicity test. The data will be used to evaluate the intra- andinter-laboratory reproducibility of the assay and effectiveness of the cytotoxicity assay to predict
the starting doses for rodent acute oral systemic toxicity assays. This test method protocol
outlines the procedures for performing the cytotoxicity test and supports the in vitro validation
study organized by NICEATM and the European Centre for the Validation of Alternative
Methods (ECVAM) and sponsored by NIEHS, U.S. Environmental Protection Agency, andECVAM. This test method protocol applies to all personnel involved with performing thecytotoxicity assay.
A. BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test
The 3T3 NRU test will be performed to analyze the in vitro toxicity of three (3)blinded/coded test chemicals. This test will be used to determine IC20, IC50, and IC80 values
for the predetermined set of test chemicals of varying toxicities.
II. SPONSOR
A. Name:National Institute of Environmental Health Sciences (NIEHS); The
National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)

B. Address:

P.O. Box 12233
Research Triangle Park, NC 27709

C. Representative:

Named Representative

III. IDENTIFICATION OF TEST AND CONTROL SUBSTANCES
A. Test Chemicals:

Blinded Chemicals (3)B. Controls:

Positive:
Vehicle (Negative):
NBCS,

Sodium Lauryl Sulfate
Assay medium (DMEM containing 5%

4 mM L-Glutamine, 100 IU/mL Penicillin,
100 µg/mL Streptomycin)
Solvent (as needed):
Assay medium with appropriate solvent
used to prepare the test chemicals (Section VII.E)

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IV. TESTING FACILITY AND KEY PERSONNEL
1)
2)
3)
4)
5)
6)
7)
8)

Name:
Address:
Study Director:
Laboratory Technician(s):Scientific Advisor:
Quality Assurance Director:
Safety Manager:
Facility Management:
A. Test Schedule
1) Proposed Experimental Initiation Date:
2) Proposed Experimental Completion Date:
3) Proposed Report Date:

V. TEST SYSTEMThe NRU cytotoxicity assay procedure is a cell survival/viability chemosensitivity assay based on
the ability of viable cells to incorporate and bind neutral red (NR), a supravital dye. NR is a weakcationic dye that readily penetrates cell membranes by non-ionic diffusion and accumulates
intracellularly in lysosomes. Alterations of the cell surface or the sensitive lysosomal membranelead to lysosomal fragility and other changes that gradually become irreversible. Such changes
brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It isthus possible to distinguish between viable, damaged, or dead cells, which is the basis of thisassay.
Healthy mammalian cells, when maintained in culture, continuously divide and multiply overtime. A toxic chemical, regardless of site or mechanism of action, will interfere with this process
and result in a reduction of the growth rate as reflected by cell number. Cytotoxicity is expressed
as a concentration dependent reduction of the uptake of the NR after chemical exposure thus
providing a sensitive, integrated signal of both cell integrity and growth inhibition.
VI. DEFINITIONS
A. Hill function: a four parameter logistic mathematical model relating the concentration of test
chemical to the response being measured in a sigmoidal shape.

Y = Bottom+

Top− Bottom
1 + 10(logIC50− X)HillSlope

where Y= response, X is the logarithm of dose (or concentration), Bottom is the minimum
response, Top is the maximum response, logIC50 is logarithm of X at the response midway
between Top and Bottom, and HillSlope describes the steepness of the curve.
B. Documentation: all methods and procedures will be noted in a Study Workbook; logs will bemaintained for general laboratory procedures and equipment (e.g., media preparation, test
chemical preparation, incubator function); all optical density data obtained from theC-45

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spectrophotometer plate reader will be saved in electronic and paper formats; all calculationsof ICx values and other derived data will be in electronic and paper format; all data will bearchived.
VII.

PROCEDURES

A. Materials
1. Cell Lines
BALB/c 3T3 cells, clone 31
CCL-163, LGC Reference Materials, Customer Service, Queens Road, Teddington,
Middlesex, TW110LY, UK
CCL-163, American Type Culture Collection [ATCC], Manassas, VA, USA)
2. Technical Equipment[Note: Suggested brand names/vendors are listed in parentheses. Equivalents may be
used.]
1)
2)
3)
4)
5)
6)
7)
8)

Incubator: 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/airLaminar flow clean bench/cabinet (standard: "biological hazard")
Water bath: 37ºC ± 1ºC
Inverse phase contrast microscope
Sterile glass tubes with caps (e.g., 5 mL)
Centrifuge (optionally: equipped with microtiter plate rotor)
Laboratory balance
96-well plate spectrophotometer (i.e., plate reader) equipped with 540 nm ± 10 nm
filter9) Shaker for microtiter plates10) Cell counter or hemocytometer
11) Pipetting aid
12) Pipettes, pipettors (multi-channel and single channel), dilution block13) Cryotubes
14) Tissue culture flasks (e.g., 75 - 80 cm2, 25 cm2)15) 96-well flat bottom tissue culture microtiter plates (e.g., Nunc # 167 008; Falcon
tissue culture-treated)
16) pH paper (wide and narrow range)[Note: Tissue culture flasks and microtiter plates should be prescreened to ensurethat they adequately support the growth of 3T3 cells.]
3. Chemicals, Media, and Sera
•
•
•
•
•

Dulbecco’s Modification of Eagle’s Medium (DMEM) without L-Glutamine; shouldhave high glucose [4.5gm/l] (e.g., ICN-Flow Cat. No. 12-332-54)
L-Glutamine 200 mM (e.g., ICN-Flow # 16-801-49)
New Born Calf Serum (NBCS or NCS) (e.g., Biochrom # SO 125)0.05 % Trypsin/0.02 % EDTA solution (e.g., SIGMA T 3924, ICN-Flow, # 1689149)
Phosphate buffered saline (PBS) without Ca2+ and Mg2+(for trypsinization)
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•
•
•
•
•
•
•
•
•

November 2006

Hanks’ Balanced Salt Solution (HBSS) without Ca2+ and Mg2+(CMF-HBSS)
Dulbecco’s Phosphate Buffered Saline (D-PBS) [formulation containing calcium andmagnesium cations; glucose optional] (for rinsing)
Penicillin/streptomycin solution (e.g. ICN-Flow # 16-700-49)
Neutral Red (NR) Dye – tissue culture-grade; liquid form (e.g., SIGMA N 2889);
powder form (e.g., SIGMA N 4638)
Dimethyl sulfoxide (DMSO), U.S.P. analytical grade (Store under nitrogen @ -20ºC)
Ethanol (ETOH), U.S.P. analytical grade (100 %, non-denatured for test chemical
preparation; 95 % can be used for the desorb solution)
Glacial acetic acid, analytical grade
Distilled H2O or any purified water suitable for cell culture (sterile)
Sterile paper towels (for blotting 96-well plates)[Note: Due to lot variability of NBCS/NCS, first check a lot for growth stimulating
properties with 3T3 cells (approximately 20-24 h doubling time) and then reserve a
sufficient amount of NBCS/NCS. May use pre-tested serum lot from Phase Ia of thevalidation study if the serum has been stored under appropriate conditions and shelflife has not expired.]

B. Preparations of Media and Solutions[Note: All solutions (except NR stock solution, NR medium and NR desorb), glassware,
pipettes, etc., shall be sterile and all procedures should be carried out under aseptic conditions
and in the sterile environment of a laminar flow cabinet (biological hazard standard). All
methods and procedures will be adequately documented.]
1. Media
DMEM (buffered with sodium bicarbonate) supplemented with (final concentrations in
DMEM are quoted):a) for freezing (Freeze Medium); contains 2X concentration of NBCS/NCS and DMSO
of final freezing solution
40 %NBCS/NCS
20 %DMSOb) for routine culture (Routine Culture Medium)
10 %NBCS/NCS
4 mM
Glutaminec) for solubility testing and test chemical dilution (Chemical Dilution Medium)
4 mM
Glutamine
200 IU/mL
Penicillin
200 µg/mL
Streptomycind) for dilution of NR stock solution (NR Dilution Medium)5%
4 mM
100 IU/mL
100 µg/mL

NBCS/NCS
Glutamine
Penicillin
Streptomycin
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[Note: The Chemical Dilution Medium with test chemical will dilute the serum
concentration of the Routine Culture Medium in the test plate to 5 %. Serum proteins
may mask the toxicity of the test substance, but serum cannot be totally excluded because
cell growth is markedly reduced in its absence.]
Completed media formulations should be kept at approximately 2-8° C and stored for nolonger than two weeks.
2. Neutral Red (NR) Stock Solution
The liquid tissue culture-grade stock NR Solution will be the first choice for performing
the assay (e.g., SIGMA #N2889, 3.3 mg/mL). Store liquid tissue culture-grade NR Stock
Solution at the storage conditions and shelf-life period recommended by the
manufacturer.
If the liquid form is not available, the following formulation can be prepared.
EXAMPLE:

0.33 g NR Dye powder in 100 mL H2O

The NR Stock Solution (powder in water) should be stored in the dark at room
temperature for up to two months.
3. Neutral Red (NR) Medium
EXAMPLE:
1 mL (3.3 mg NR dye/mL)
99 mL

NR Stock Solution
NR Dilution Medium (pre-warmed to 37° C)

The final concentration of the NR Medium is 33 µg NR dye/mL.
[Note: The NR medium may be centrifuged at approximately 600 x g for 10 min (toremove NR crystals). The NR Medium shall be filtered (e.g., Millipore filtering, 0.2 –0.45 µm pore size) to reduce NR crystals. The temperature of the NR Medium should bemaintained at 37° C (e.g., in a waterbath) before adding tothe cells and will be usedwithin 15 minutes after removing from 37° C storage. Aliquots of NR Medium can bemade on the day of testing and maintained at 37° C for later use.]
4. Ethanol/Acetic Acid Solution (NR Desorb)
1%
50 %
49 %

Glacial acetic acid solution
Ethanol
H2O

C. Methods
1. Cell Maintenance and Culture Procedures
BALB/c 3T3 cells are routinely grown as a monolayer in tissue culture grade flasks (e.g.,
75 - 80 cm2) at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air. The cells
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should be examined on a daily (i.e., on workdays) basis under a phase contrast
microscope, and any changes in morphology or their adhesive properties noted in a Study
Workbook.
2. Receipt of Cryopreserved BALB/c 3T3 Cells
Upon receipt of cryopreserved BALB/c 3T3 cells, the vial(s) of cells shall be stored in aliquid nitrogen freezer until needed.
3. Thawing Cells
Thaw cells by putting ampules into a water bath at 37°C ± 1ºC. Leave for as brief a time
as possible.
a) Resuspend the cells in pre-warmed Routine Culture Medium and transfer into
pre-warmed Routine Culture Medium in a tissue-culture flask.
b) Incubate at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air.
c) When the cells have attached to the bottom of the flask (within 4 to 24 h),decant the supernatant and replace with fresh pre-warmed (37ºC) medium.
Culture as described above.
d) Passage at least two times before using the cells in a cytotoxicity test.
A fresh batch of frozen cells from the stock lot of cells should be thawed out and cultured
approximately every two months. This period resembles a sequence of about 18
passages.
4. Routine Culture of BALB/C 3T3 Cells
When cells exceed 50 % confluence (but less than 80 % confluent) they should be
removed from the flask by trypsinization:a) Decant medium, rinse cultures with 5 mL PBS or Hanks’ BSS (without Ca2+, Mg2+)
per 25 cm2 flask (15 mL per 75 cm2 flask). Wash cells by gentle agitation to remove
any remaining serum that might inhibit the action of the trypsin.
b) Discard the washing solution.
c) Add 1-2 mL trypsin-EDTA solution per 25 cm2 to the monolayer for a few seconds(e.g., 15-30 seconds).
d) Remove excess trypsin-EDTA solution and incubate the cells at room temperature.
e) After 2-3 minutes (min), lightly tap the flask to detach the cells into a single cellsuspension.
5. Cell Counting
After detaching the cells, add 0.1-0.2 mL of pre-warmed (37ºC) Routine Culture
Medium/cm2 to the flask (e.g., 2.5 mL for a 25 cm2 flask). Disperse the monolayer by
gentle trituration. It is important to obtain a single cell suspension for exact counting.
Count a sample of the cell suspension obtained using a hemocytometer or cell counter(e.g., Coulter counter).

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6. Subculture of Cells
After determination of cell number, the culture can be sub-cultured into other flasks orseeded into 96-well microtiter plates. BALB/c 3T3 cells are routinely passaged atsuggested cell densities as listed in the table (approximate doubling time is 20-24 h). The
individual laboratories will need to determine and adjust the final density to achieve
appropriate growth.
Table 1. Cell Densities for Subculturing
Days in Culture2
3
4

Seeding Density(cells/cm2)
16800
8400
4200

Total Cells per 25 cm2
flask
4.2 x 105
2.1 x 105
1.05 x 105

Total Cells per 75 cm2
flask
1.26 x 106
6.3 x 105
3.15 x 105[Note: It is important that cells have overcome the lag growth phase when they are used
for the test.]
7. Freezing Cells (procedure required only if current stock of cells is depleted)
Stocks of BALB/c 3T3 cells can be stored in sterile, freezing tubes in a liquid nitrogen
freezer. DMSO is used as a cryoprotective agent.
a) Centrifuge trypsinized cells at approximately 200 x g.
b) Suspend the cells in cold Routine Culture Medium (half the final freezingvolume) so a final concentration of 1-5x106 cells/mL can be attained.
c) Slowly add cold Freeze Medium to the cells so that the solvent will equilibrate
across the cell membranes. Bring the cell suspension to the final freezingvolume. The final cell suspension will be 10 % DMSO. Aliquot the cellsuspension into freezing tubes and fill to 1.8 mL.
d) Place the tubes into an insulated container (e.g., styrofoam trays) and place in afreezer (-70 to -80°C) for 24 h. This gives a freezing rate of approximately
1°C/min. The laboratory needs to ensure that the freezing protocol is applicableto the 3T3 cells and that the cells are viable when removed fromcryopreservation.
e) Place the frozen tubes into liquid nitrogen for storage.
8. Preparation of Cells for Assayse) Cultured cells that are going to be used in seeding the 96-well plates should be fed
fresh medium the day before subculturing to the plates. On the day of plate seeding,4
prepare a cell suspension of 2.5x10 cells/mL in Routine Culture Medium. Using a
multi-channel pipette, dispense 100 µl Routine Culture Medium only into theperipheral wells (blanks) of a 96-well tissue culture microtiter plate (See Section
VII.F.1). In the remaining wells, dispense 100 µl of a cell suspension of 2.5x104
3cells/mL (= 2.5x10 cells/well). The seeding density should be noted to ensure thatthe cells in the control wells are not overgrown after three days (i.e., 24 h incubation

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in step b and 48 h exposure to test chemicals). Prepare one plate per chemical to betested.
e) Incubate cells for 24 + 1 h (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air)so that cells form a less than half confluent monolayer. This incubation period
assures cell recovery and adherence and progression to exponential growth phase.
e) Examine each plate under a phase contrast microscope to assure that cell growth isrelatively even across the microtiter plate. This check is performed to identify
experimental and systemic cell seeding errors. Record observations in the Study
Workbook.
9. Determination of Doubling Timea) A cell doubling time procedure was performed on the initial lot of cells that was usedin the first cell culture assays of Phase Ia of the Validation Study. The doubling time
only needs to be determined in Phase Ib if there is a change in the lot of cells used.
Establish cells in culture and trypsinize cells as per Section VII.C.4 for subculture.
Resuspend cells in NR Dilution Medium (5 % NBCS/NCS). Seed cells at 4200
cells/cm2.
b) Seed five sets of cell culture vessels in triplicate for each cell type (e.g., 15 tissue
culture dishes [60mm x 15mm]). Use appropriate volume of culture medium for theculture vessels. Note number of cells placed into each culture dish. Place dishes into
the incubators (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air).
c) After 4 - 6 hours (use the same initial measurement time for each subsequent
doubling time experiment), remove three culture dishes and trypsinize cells. Count
cells using a cell counter or hemocytometer. Cell viability may be determined by dye
exclusion (e.g., Trypan Blue) if Study Director sees a need. Use appropriate size
exclusion limits if using a Coulter counter. Determine the total number of cells anddocument. Repeat sampling at 24 h, 48 h, 72 h, and 96 h post inoculation. Change
culture medium at 72 h or sooner in remaining dishes if indicated by pH drop.
d) Plot cell concentration (per mL of medium) on a log scale against time on a linear
scale. Determine lag time and population doubling time. Additional dishes and time
are needed if the entire growth curve is to be determined (lag phase, log phase,plateau phase).
D. Solubility Test
The preference of solvent for dissolving test chemicals is medium, DMSO, and then ethanol.
Solubility shall be determined in a step-wise procedure that involves attempting to dissolve atest chemical at a relatively high concentration with the sequence of mechanical procedures
specified in Section VII.D.2.a. If the chemical does not dissolve, the volume of solvent isincreased so as to decrease the concentration by a factor of 10, and then the sequence of
mechanical procedures in Section VII.D.2.a are repeated in an attempt to solubilize thechemical at the lower concentrations. For testing solubility in medium, the starting
concentration is 20,000 µg/ml (i.e., 20 mg/mL) in Tier 1, but for DMSO and ethanol thestarting concentration is 200,000 µg/ml (i.e., 200 mg/mL) in Tier 2. Weighing out chemical
for each solvent (i.e., medium, DMSO, ethanol) can be done all at once, if convenient, butsolubility testing (at each tier that calls for more than one solvent) is designed to be sequential- medium, then DMSO, then ethanol – in accordance with the solvent hierarchy (see Figure1). This allows for testing to stop, rather than continue testing with less preferred solvents, if
the test chemical dissolves in a more preferred solvent. For example, if a chemical is soluble
in medium at a particular tier, testing may stop. Likewise, if a chemical is soluble in DMSO
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at any tier, testing need not continue with ethanol. However, since the issue of primary
importance is testing the solvents and concentrations of test chemical required by any one
tier, sequential testing of solvents may be abandoned if the lab can test more efficiently inanother way.
1. Determination of Solubilitya) Tier 1 begins with testing 20 mg/mL in Chemical Dilution Medium (see Table 2).
Approximately 10 mg (10,000 µg) of the test chemical will be weighed into a glass
tube and the weight will be documented. A volume of Chemical Dilution Medium,approximately 0.5 mL, will be added to the vessel so that the concentration is 20,000µg/ml (20 mg/mL). The solution is mixed as specified in Section VII.D.2.a. If
complete solubility is achieved in medium, then additional solubility procedures are
not needed.
b) If the test chemical is insoluble in Chemical Dilution Medium, proceed to Tier 2 byadding enough medium, approximately 4.5 mL, to attempt to dissolve the chemical at
2 mg/mL by using the sequence of mixing procedures specified in Section VII.D.2.a.
If the test chemical dissolves in Chemical Dilution Medium at 2 mg/mL, no further
procedures are necessary. If the test chemical does NOT dissolve, weigh out
approximately 100 mg test chemical in a second glass tube and add enough DMSO to
make the total volume approximately 0.5 mL (for 200 mg/mL). In another glass
tube, also add approximately 100 mg test chemical to enough ethanol to make thetotal volume approximately 0.5 mL (for 200 mg/mL). Mix both solutions as
specified in Section VII.D.2.a in an attempt to solubilize the test chemical. If thechemical is soluble in either solvent, no additional solubility procedures are needed.
c) If the chemical is NOT soluble in Chemical Dilution Medium, DMSO, or ethanol atTier 2, then continue to Tier 3 in Table 2 by adding enough solvent to increase thevolume of the three Tier 2 solutions by 10 and attempt to solubilize again using thesequence of mixing procedures in Section VII.D.2.a. If the test chemical dissolves,no additional solubility procedures are necessary. If the test chemical does NOT
dissolve, continue with Tier 4 and, if necessary, Tier 5 using DMSO and ethanol.
Tier 4 begins by diluting the Tier 3 samples with DMSO or ethanol to bring the total
volume to 50 mL. The mixing procedures in Section VII.D.2.a are again followed toattempt to solubilize the chemical. Tier 5 is performed, if necessary, by weighing out
another two more samples of test chemical at ~10 mg each and adding ~50 mL
DMSO or ethanol for a 200 µg/mL solution, and following the mixing procedures in
Section VII.D.2.a.
Example: If complete solubility is not achieved at 20,000 µg/mL in Chemical
Dilution Medium at Tier 1 using the mixing procedures specified in Section
VII.D.2.a, then the procedure continues to Tier 2 by diluting the solution to 5 mL andmixing again as specified in Section VII.D.2.a. If the chemical is not soluble in
Chemical Dilution Medium, two samples of ~ 100 mg test chemical are weighed toattempt to solubilize in DMSO and ethanol at 200,000 µg/mL (i.e., 200 mg/mL).
Solutions are mixed following the sequence of procedures prescribed in Section
VII.D.2.a in an attempt to dissolve. If solubility is not achieved at Tier 2, then thesolutions (Chemical Dilution Medium, DMSO, and ethanol) prepared in Tier 2 are
diluted by 10 so as to test 200 µg/mL in media, and 20,000 µg/mL in DMSO andC-52

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ethanol. This advances the procedure to Tier 3. Solutions are again mixed asprescribed in Section VII.D.2.a in an attempt to dissolve. If solubility is notachieved in Tier 3, the procedure continues to Tier 4, and to 5 if necessary (seeFigure 1 and Table 2).
Table 2 Determination of Solubility in Chemical Dilution Medium, DMSO, or Ethanol
TIER

1

2

3

4

Total Volume

0.5 mL

5 mL

50 mL

20,000 µg/mL

2,000 µg/mL

200 µg/mL(20 mg/mL)(2 mg/mL)(0.20 mg/mL)

0.5 mL

5 mL

50 mL

200,000 µg/mL

20,000 µg/mL

2,000 µg/mL(200 mg/mL)(20 mg/mL)(2 mg/mL)

5

Chemical Dilution
MediumConcentration of Test
Chemical(Add ~10 mg to a tube.
Add enough medium toequal the first volume.
Dilute to subsequent
volumes if necessary.)
Total Volume
DMSO/Ethanol
Concentration of Test
Chemical(Add ~100 mg to alarge tube. Add enoughDMSO or ethanol toequal the first volume.
Dilute to subsequent
volumes if necessary.)
Total Volume
DMSO/Ethanol
Concentration of Test
Chemical(Add ~10 mg to a large
tube. Add enoughDMSO or ethanol toequal 50 mL.)
Equivalent
Concentration on Cells

50 mL
200 µg/mL(0.2 mg/mL)
10,000 µg/mL1000 µg/mL

100 µg/mL

10 µg/mL

1 µg/mL(10 mg/mL)(1 mg/mL)(0.1 mg/mL)(0.01 mg/mL)(0.001 mg/mL)

NOTE: The amounts of test chemical weighed and Chemical Dilution Medium added
may be modified from the amounts given above, provided that the targeted
concentrations specified for each tier are tested.

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Figure 1. Solubility Flow Chart

STEP 1:TIER 120 mg/mL test chemical (TC) in 0.5 mL Chemical Dilution Medium:• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 2.

STEP 2:TIER 2
2 mg/mL TC in medium – increase volume from STEP 1 by 10 (i.e., to 5 mL)• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 3.

STEP 3:

STEP 4:

STEP 5:

STEP 6:200 mg/mL TC in DMSO
• if TC soluble, then STOP.
• if TC insoluble, test at 200 mg/mL in ETOH.
• if TC soluble, then STOP.
• If TC insoluble, go to STEP 4.
TIER 30.2 mg/mL TC in medium – increase volume from STEP 2 by 10 (i.e., to 50 mL)• if TC soluble, then STOP.
• if TC insoluble, test at 20 mg/mL in DMSO – increase volume from STEP 3 by 10(i.e., to 5 mL).
• if TC soluble, then STOP.
• if TC insoluble, test at 20 mg/mL in ETOH – increase volume from STEP 3 by
10 (i.e., to 5 mL).
• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 5.
TIER 42 mg/mL TC in DMSO – increase volume from STEP 4 by 10 (i.e., to 50 mL)• if TC soluble, then STOP.
• if TC insoluble, test at 2 mg/mL in ETOH – increase volume from STEP 4 by 10(i.e., to 50 mL).
• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 6.
TIER 50.2 mg/mL TC in 50 mL DMSO
• if TC soluble, then STOP.
• if TC insoluble, test at 0.2 mg/mL in 50 mL ETOH
• STOP

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November 2006

Mechanical Proceduresa) The following hierarchy of mixing procedures will be followed to dissolve the test
chemical:
1)
2)
3)
4)

Add test chemical to solvent as in Tier 1 of Table 2.
Gently mix. Vortex the tube (1 –2 minutes).
If test chemical hasn’t dissolved, use sonication for up to 5 minutes.
If sonication doesn’t work, then warm solution to 37°C. This can be performed
by warming 5 mL tubes in a 37°C water bath for at least 5-10 minutes before
evaluating solubility. Warm larger vessels for at least 15-20 minutes in a 37°C
water bath before evaluating solubility.
5) Proceed to Tier 2 (and Tiers 3-5, if necessary of Table 2 and repeat procedures 24).
b) The preference of solvent for dissolving test chemicals is Chemical Dilution
Medium, DMSO, and then ethanol. Thus, if (all solvents for a particular tier are
tested simultaneously and) a test chemical dissolves in more than one solvent, thenthe choice of solvent follows this hierarchy. For example, if, at any tier, a chemical
is soluble in Chemical Dilution Medium and DMSO, but not ethanol, the choice ofsolvent would be medium. If the chemical were insoluble in medium, but soluble in
DMSO and ethanol, the choice of solvent would be DMSO.
After the lab has determined the preferred solvent for the test chemical andbefore proceeding to the cytotoxicity testing, the Study Director will discuss thesolvent selection with the Study Management Team (SMT) of the validation
study. The SMT will relate what solvent should be used in the assay for eachchemical.
E. Preparation of Test Chemicals[Note: Preparation under red or yellow light is recommended to preserve chemicals that
degrade upon exposure to light.]
1. Test Chemicals in Solutiona) Allow test chemicals to equilibrate to room temperature before dissolving anddiluting.
b) Prepare test chemical immediately prior to use. The solutions must not be cloudy nor
have noticeable precipitate. Each stock dilution should have at least 1-2 mL total
volume to ensure adequate solution for the test wells in a single 96-well plate.
c) For chemicals dissolved in DMSO or ethanol, the final DMSO or ethanol
concentration for application to the cells must be 0.5 % (v/v) in the vehicle controls
and in all of the eight test concentrations.
d) The stock solution for each test chemical should be prepared at the highest
concentration found to be soluble in the solubility test. Thus, the highest test
concentration applied to the cells in each range finding experiment is:C-55

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0.5 times the highest concentration found to be soluble in the solubility test, if thechemical was soluble in Chemical Dilution Medium, or
1/200 the highest concentration found to be soluble in the solubility test if thechemical was soluble in ethanol or DMSO.

e) The seven lower concentrations in the range finding experiment would then be
prepared by successive dilutions that decrease by one log unit each. The following
example illustrates the preparation of test chemical in solvent and the dilution ofdissolved test chemical in Chemical Dilution Medium before application to 3T3 cells.
Example: Preparation of Test Chemical in Solvent Using a Log Dilution Scheme
If DMSO was determined to be the preferred solvent at Tier 2 of the solubility test (i.e.,
200,000 µg/mL), dissolve the chemical in DMSO at 200,000 µg/mL for the chemical
stock solution.
1) Label eight tubes 1 – 8. Add 0.9 mL solvent (e.g., DMSO) to tubes 2 -- 8.
2) Prepare stock solution of 200,000 µg test chemical/mL solvent in tube # 1.
3) Add 0.1 mL of 200,000 µg/mL dilution from tube #1 to tube #2 to make a 1:10
dilution in solvent (i.e., 20,000 µg/mL).
4) Add 0.1 mL of 20,000 µg/mL dilution from tube #2 to tube #3 to make another 1:10
dilution (i.e., 1:100 dilution from stock solution) in solvent (i.e., 2,000 µg/mL)5) Continuing making serial 1:10 dilutions in the prepared solvent tubes.
6) Since each concentration is 200 fold greater than the concentration to be tested, makea 1:100 dilution by diluting 1 part dissolved chemical in each tube with 99 parts ofChemical Dilution Medium (e.g., 0.1 mL test chemical in DMSO + 9.9 mL Chemical
Dilution Medium) to derive the eight 2X concentrations for application to 3T3 cells.
Each 2X test chemical concentration will then contain 1 % v/v solvent. The 3T3
cells will have 0.05 mL Routine Culture Medium in the wells prior to application of
the test chemical. By adding 0.05 mL of the appropriate 2X test chemical
concentration to the appropriate wells, the test chemical will be diluted appropriately(e.g., highest concentration in well will be 1,000 µg/mL) in a total of 0.1 mL and thesolvent concentration in the wells will be 0.5% v/v.
7) A test article prepared in Chemical Dilution Medium, DMSO, or ethanol may
precipitate upon transfer into the Routine Culture Medium. The 2X dosing solutions
should be evaluated for precipitates and the results will be recorded in the workbook.
It will be permissible to test all of the dosing solutions in the dose range finding assay
only. Doses containing test article precipitates should be avoided, and will not be
used in the ICx determinations for either the range finding experiments or thedefinitive tests.
Document all test chemical preparations in the Study Workbook.
2. pH of Test Chemical Solutions
Measure the pH of the highest concentration of the test chemical in culture medium using
pH paper. Document the pH and note the color of the medium for all dilutions. Do not
adjust the pH.

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3. Concentrations of Test Chemicala) Range Finder Experiment
Test eight concentrations of the test chemical by diluting the stock solution with aconstant factor covering a large range. The initial dilution series shall be log
dilutions (e.g., 1:10, 1:100, 1:1000, etc.).
The data from any well that has precipitate will be excluded from any calculations.
b) Main Experiment[Note: After the range finding assay is completed, the concentration-response
experiment shall be performed three times on three different days for each chemical(i.e., one plate per day per chemical.]
Depending on the slope of the concentration-response curve estimated from the range
finder, the dilution/progression factor in the concentration series of the main
experiment should be smaller (6√10 = 1.47). Cover the relevant concentration range(≥10 % and ≤ 90 % effect) preferably with three points of a graded effect, but with aminimum of two points, one on each side of the IC50 value, avoiding too many noncytotoxic and/or 100 %-cytotoxic concentrations. Experiments revealing less than
one cytotoxic concentration on each side of the IC50 value shall be repeated, where
possible, with a smaller dilution factor. In addition, the dilution scheme shall be
adjusted in subsequent replicate assays (i.e., definitive assays), if necessary, toincrease the number of points on both sides of the IC50 in the 10-90% response range.
(Taking into account pipetting errors, a progression factor of 1.21 is regarded thesmallest factor achievable.)
Determine which test chemical concentration is closest to the IC50 value (e.g., 50 %cytotoxicity). Use that value as a central concentration and adjust dilutions higher
and lower in equal steps for the definitive assay.
Maximum Doses to be Tested in the Main Experiments
If minimal or no cytotoxicity was measured in the dose range finding assay, amaximum dose for the main experiments will be established as follows:• For test chemicals prepared in Chemical Dilution Medium, the highest test article
concentration that may be applied to the cells in the main experiments will beeither 100 mg/mL, or the maximum soluble dose. Test chemical will be weighed
into a glass tube and the weight will be documented. A volume of Chemical
Dilution Medium will be added to the vessel so that the concentration is200,000 µg/mL (200 mg/mL). The solution is mixed as specified in Section
VII.D.2.a. If complete solubility is achieved in medium, then 7 additional serial
stock dosing solutions may be prepared from the 200 mg/mL 2X stock. If thetest chemical is insoluble in medium at 200 mg/ml, proceed by adding medium,in small incremental amounts, to attempt to dissolve the chemical by using thesequence of mixing procedures specified in Section VII.D.2.a. The highest
soluble stock solution will be used to prepare the 7 additional serial stock dosing
solutions.

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For test chemicals prepared in either DMSO or ethanol, the highest test article
concentration that may be applied to the cells in the main experiments will beeither 2.5 mg/mL, or less, depending upon the maximum solubility in solvent.
Test chemical will be weighed into a glass tube and the weight will be
documented. A volume of the appropriate solvent (determined from the original
solubility test) will be added to the vessel so that the concentration is500,000 µg/mL (500 mg/mL). The solution is mixed as specified in Section
VII.D.2.a. If complete solubility is achieved in the solvent, then 7 additional
serial stock dosing solutions may be prepared from the 500 mg/mL 200X stock.
If the test chemical is insoluble in solvent at 500 mg/ml, proceed by adding
solvent, in small incremental amounts, to attempt to dissolve the chemical byusing the sequence of mixing procedures specified in Section VII.D.2.a. The
highest soluble stock solution will be used to prepare the 7 additional serial stock
dosing solutions.

c) Test Chemical Dilutions
The dosing factor of 3.16 (= 2√10) divides a log into two equidistant steps, a factor of
2.15 (= 3√10) divides a decade into three steps. The factor of 1.47 (= 6√10) divides alog into six equidistant steps, and the factor of 1.21 (= 12√10) divides the log into 12
steps.
EXAMPLE:
10
10
10
10

31.6
12.1

14.7
14.7

17.8

21.5
21.5
21.5

26.1

31.6
31.6

38.3

46.4
46.4
46.4

56.2

68.1
68.1

82.5

100
100
100
100

The technical production of decimal geometric concentration series is simple. An
example is given for factor 1.47:
Dilute 1 volume of the highest concentration by adding 0.47 volumes of diluent. Afterequilibration, dilute 1 volume of this solution by adding 0.47 volumes ofdiluent...(etc.).
F. Test Procedure
1. 96-Well Plate Configuration
The 3T3 NRU assay for test chemicals will use the 96-well plate configuration shown in
Figure 2.

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Figure 2. 96-Well Plate Configuration for Positive Control and Test Chemical Assays
1

2

3

4

5

6

7

8

9

10

11

12

A

b

b

b

b

b

b

b

b

b

b

b

b

B

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

C

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

D

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

E

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

F

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

G

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

H

b

b

b

b

b

b

b

b

b

b

b

b

VC
=
C1 – C8 =b=untreated VEHICLE CONTROL (mean viability set to 100 %)Test Chemicals or Positive Control (SLS) at eight concentrations(C1 = highest, C8 = lowest)
BLANKS (contain no cells)

2. Application of Test Chemicala) Two optional methods for rapidly applying the 2X dosing solutions onto the 96-wellplates may be utilized. The first method is to add each of the 2X dosing solutions
into labeled, sterile reservoirs (e.g., Corning/Costar model 4870 sterile polystyrene
50 mL reagent reservoirs and/or Corning/Transtar model 4878 disposable reservoir
liners, 8-channel). The second method utilizes a “dummy” plate (i.e., an empty
sterile 96-well plate) prepared to hold the dosing solutions immediately prior totreatment of the test plate (with cells). The test chemical and control dosing solutions
should be dispensed into the dummy plate in the same pattern/order as will be applied
to the plate containing cells. More volume than needed for the test plate (i.e. greaterthan 50 µl/well) should be in the wells of the dummy plate. At the time of treatment
initiation, a multi-channel micropipettor is used to transfer the 2X dosing solutions,from the reservoirs or dummy plate, to the appropriate wells on the treatment plate(as described in step c. below). These methods will ensure that the dosing solutions
can be transferred rapidly to the appropriate wells of the test plate to initiate
treatment times and to minimize the range of treatment initiation times across a large
number of treatment plates, and to prevent “out of order” dosing.
b) After 24 h ± 1 h incubation of the cells, remove Routine Culture Medium from thecells by careful inversion of the plate (i.e., “dump”) over an appropriate receptacle.
Gently blot the plate on a sterile paper towel so that the monolayer is minimallydisrupted. Do not use automatic plate washers for this procedure nor vacuum
aspiration.
c) Immediately add 50 µL of fresh pre-warmed Routine Culture Medium to all of thewells, including the blanks. Add 50 µL of Chemical Dilution Medium to the blankwells. Then add 50 µL Chemical Dilution Medium containing either the appropriate
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concentration of test chemical, the PC, or the VC (see Figure 2 for the plate
configuration). The solutions will be transferred from the dummy plate to the test
plate by adding the vehicle control first then lowest to highest dose so that the same
pipette tips on the eight channel pipettor can be used for the whole plate.
d) Incubate cells for 48 h ± 0.5 h (37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 %CO2/air).
e) Positive Control: For each set of test chemical plates used in an assay, a separate
plate of positive control concentrations will be set up following the concentration
range established in the development of the positive control database in Phase Ia ofthe Validation Study. The mean IC50 and ± two standard deviations (SD) of the IC50
of SLS (mutually agreed upon by the Testing Facility and the SMT) are the values
that will be used as an acceptance criterion for test sensitivity for the 3T3 NRU assay.
This plate will follow the same schedule and procedures as used for the test chemical
plates.
3. Microscopic Evaluation
After at least 46 h treatment, examine each plate under a phase contrast microscope toidentify systematic cell seeding errors and growth characteristics of control and treatedcells. Record any changes in morphology of the cells due to the cytotoxic effects of thetest chemical, but do not use these records for any quantitative measure of cytotoxicity.
Undesirable growth characteristics of control cells may indicate experimental error andmay be cause for rejection of the assay. Use the following Visual Observations Codes inthe description of cell culture conditions.
Visual Observations Codes
Note Code
1
2
3
4
1P
2P
3P
4P
5P

Note Text
Normal Cell Morphology
Low Level of Cell Toxicity
Moderate Level of Cell Toxicity
High level of Cell Toxicity
Normal Cell Morphology with Precipitate
Low Level of Cell Toxicity with Precipitate
Moderate Level of Cell Toxicity with PrecipitateHigh level of Cell Toxicity with Precipitate
Unable to View Cells Due to Precipitate

4. Measurement of NRUa) Carefully remove (i.e., “dump”) the medium with test chemical and rinse the cells
very carefully with 250 µL pre-warmed D-PBS. Remove the rinsing solution bydumping and remove excess by gently blotting on sterile paper towels. Add 250 µL
NR medium (to all wells including the blanks) and incubate (37ºC ± 1ºC, 90 % ± 5 %humidity, and 5.0 % ± 1 % CO2/air) for 3±0.1 h. Observe the cells briefly during the
NR incubation (e.g., at 1, 2, and 3 h – Study Director’s discretion) for NR crystal
formation. Record observations in the Study Workbook. Study Director can decideto reject the experiment if excessive NR crystallization has occurred.
b) After incubation, remove the NR medium, and carefully rinse cells with 250 µl prewarmed D-PBS.
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d) Add exactly 100 µl NR Desorb (ETOH/acetic acid) solution to all wells, including
blanks.
e) Shake microtiter plate rapidly on a microtiter plate shaker for 20 – 45 min to extract
NR from the cells and form a homogeneous solution.
f) Measure the absorption (within 60 minutes of adding NR Desorb solution) of theresulting colored solution at 540 nm ± 10 nm in a microtiter plate reader(spectrophotometer), using the blanks as a reference. [Phase Ia data show the mean
OD value for the plate blanks to be 0.051 ± 0.022 for 3T3 cells (± two standard
deviations; data from 3 labs; N = 59). Use this value as a guide for assessment of theblank values.] Save raw data in the Excel format as provided by the SMT.
5. Quality Check of 3T3 NRU Assaya) Test Acceptance Criteria
1) A test meets acceptance criteria, if the IC50 for SLS (PC) is within ± two (2)standard deviations of the historical mean established by the Test Facility (as per
VII.F.2.e).
2) A test meets acceptance criteria if the corrected mean OD540 of VCs is ≥ 0.30 and≤ 0.80.
3) A test meets acceptance criteria if the left and the right mean of the VCs do notdiffer by more than 15 % from the mean of all VCs.
4) A test meets acceptance criteria if a minimum of two points, one on each side of
the IC50 value, are determined and fall within the range ≥10 % and ≤ 90 % effect.
[Note: All acceptance criteria must be met for an assay to be considered
acceptable.]b) Checks for Systematic Cell Seeding Errors
To check for systematic cell seeding errors, untreated VCs are placed both at the left
side (row 2) and the right side (row 11 for the test plates) of the 96-well plate.
Aberrations in the cell monolayer for the VCs may reflect a volatile and toxic test
article present in the assay.
Checks for cell seeding errors may also be performed by examining each plate undera phase contrast microscope to assure that cell quantity is consistent.
c) Quality Check of Concentration-Response
The IC50 derived from the concentration-response of the test chemicals will bebacked by preferably three responses ≥ 10 % and ≤ 90 % inhibition of NRU and at
least two responses, one on either side of the IC50 value (see VII.E.3.b). If this is not
the case, and the concentration progression factor can be easily reduced, reject theexperiment and repeat it with a smaller progression factor. In addition, the dilution
scheme shall be adjusted in subsequent replicate assays, if necessary, to increase thenumber of points on both sides of the IC50 in the 10-90% response range. Numericalscoring of the cells (see VII.F.3) should be determined and documented in the Study
Workbook.
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G. Data Analysis
A calculation of cell viability expressed as NRU is made for each concentration of the test
chemical by using the mean NRU of the six replicate values (minimum of four acceptable
replicate well) per test concentration (blanks will be subtracted). This value is compared withthe mean NRU of all VC values (provided VC values have met the VC acceptance criteria).
Relative cell viability is then expressed as percent of untreated VC. If achievable, the eight
concentrations of each chemical tested will span the range of no effect up to total inhibitionof cell viability. Data from the microtiter plate reader shall be transferred to the Excel
spreadsheet provided by the SMT for determining cell viability and performing statisticalanalyses.
The concentration of a test chemical reflecting a 20 %, 50 %, and 80 % inhibition of cell
viability (i.e., the IC20, IC50, and IC80) is determined from the concentration-response by
applying a Hill function to the concentration-response data. Statistical software (e.g.,
GraphPad PRISM 3.0) specified by the SMT shall be used to calculate IC20, IC50, and IC80
values (and the associated confidence limits) for each test chemical. In addition, the SMT
shall provide guidelines for calculating ICx values and confidence limits. The Testing
Facility shall report data using at least three (3) significant figures and shall forward theresults from each assay to the SMT/biostatistician through the designated contacts inelectronic format and hard copy upon completion of testing. The SMT will be directly
responsible for the statistical analyses of the Validation Study data.
VIII.

REFERENCES
Hackenberg, U. and H. Bartling. 1959. Messen und Rechnen im pharmakologischen
Laboratorium mit einem speziellen Zahlensystem (WL24-System). Arch. Exp. Pathol.
Pharmakol. 235: 437-463.
Spielmann, H., S. Gerner, S. Kalweit, R. Moog, T. Wirnserberger, K. Krauser, R. Kreiling, H.
Kreuzer, N.P. Luepke, H.G. Miltenburger, N. Müller, P. Murmann, W. Pape, B. Siegmund, J.
Spengler, W. Steiling, and F.J. Wiebel. 1991. Interlaboratory assessment of alternatives tothe Draize eye irritation test in Germany. Toxicol. In Vitro 5: 539-542.

IX.

APPROVAL__________________________________SPONSOR REPRESENTATIVE(Print or type name)___________________DATE_____________________________
Test Facility STUDY DIRECTOR(Print or type name)____________________
DATE

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Appendix C4

Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test (Phase Ib)

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[This Page Intentionally Left Blank]

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TEST METHOD PROTOCOL
for the NHK Neutral Red Uptake Cytotoxicity Test

A Test for Basal Cytotoxicity for an In Vitro Validation Study
Phase Ib
November 15, 2002
Revised November 22, 2002
Revised by IIVS Nov. 26, 2002

Prepared by
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)

Based on Standard Operating Procedure Recommendations from anInternational Workshop Organized by the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM)

National Institute of Environmental Health Sciences (NIEHS)
National Institutes of Health (NIH)
U.S. Public Health Service
Department of Health and Human Services

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TEST METHOD PROTOCOLThe Normal Human Keratinocyte (NHK) Neutral Red Uptake Cytotoxicity
Test
A Test for Basal Cytotoxicity
Phase IbI.

PURPOSEThe purpose of this study is to evaluate the cytotoxicity of test chemicals using the Normal
Human Keratinocyte (NHK) Neutral Red Uptake (NRU) cytotoxicity test. The data will be used
to evaluate the intra- and inter-laboratory reproducibility of the assay and effectiveness of thecytotoxicity assay to predict the starting doses for rodent acute oral systemic toxicity assays. This
test method protocol outlines the procedures for performing the cytotoxicity test and is in supportof the in vitro validation study organized by NICEATM and the European Centre for the
Validation of Alternative Methods (ECVAM) and sponsored by NIEHS, U.S. Environmental
Protection Agency, and ECVAM. This test method protocol applies to all personnel involved
with performing the cytotoxicity assay.
A. NHK Neutral Red Uptake Cytotoxicity Test
The NHK NRU test will be performed to analyze the in vitro toxicity of three (3)blinded/coded test chemicals. This test will be used to determine IC20, IC50, and IC80 values
for the predetermined set of test chemicals of varying toxicities.

II. SPONSOR
A. Name:National Institute of Environmental Health Sciences (NIEHS); The
National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)

B. Address:

P.O. Box 12233
Research Triangle Park, NC 27709

C. Representative:

Named Representative

III. IDENTIFICATION OF TEST AND CONTROL SUBSTANCES
A. Test Chemicals:
B. Controls:

Blinded chemicals (3)Positive:
Sodium Lauryl Sulfate
Vehicle (Negative):
Assay medium
Solvent (as needed):
Assay medium with appropriate solvent
used to prepare the test chemicals (Section VII.E)

IV. TESTING FACILITY AND KEY PERSONNEL
•
•

Name:
Address:C-66

In Vitro Cytotoxicity Test Methods BRD Appendix C4

•
•
•
•
•
•

November 2006

Study Director:
Laboratory Technician(s):Scientific Advisor:
Quality Assurance Director:
Safety Manager:
Facility Management:

A. Test Schedule
1. Proposed Experimental Initiation Date:
2. Proposed Experimental Completion Date:
3. Proposed Report Date:
V. TEST SYSTEMThe NRU cytotoxicity assay procedure is a cell survival/viability chemosensitivity assay based on
the ability of viable cells to incorporate and bind neutral red (NR), a supravital dye. NR is a weakcationic dye that readily penetrates cell membranes by non-ionic diffusion and accumulates
intracellularly in lysosomes. Alterations of the cell surface or the sensitive lysosomal membranelead to lysosomal fragility and other changes that gradually become irreversible. Such changes
brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It isthus possible to distinguish between viable, damaged, or dead cells, which is the basis of thisassay.
Healthy mammalian cells, when maintained in culture, continuously divide and multiply overtime. A toxic chemical, regardless of site or mechanism of action, will interfere with this process
and result in a reduction of the growth rate as reflected by cell number. Cytotoxicity is expressed
as a concentration dependent reduction of the uptake of the NR after chemical exposure thus
providing a sensitive, integrated signal of both cell integrity and growth inhibition.
VI. DEFINITIONS
A.. Hill function: a four parameter logistic mathematical model relating the concentration of test
chemical to the response being measured in a sigmoidal shape.

Y = Bottom +Top ! Bottom
1 + 10(logIC50! X)HillSlope

where Y= response, X is the logarithm of dose (or concentration), Bottom is the minimum
response, Top is the maximum response, logIC50 is logarithm of X at the response midway
between Top and Bottom, and HillSlope describes the steepness of the curve.
B. Documentation: all methods and procedures will be noted in a Study Workbook; logs will bemaintained for general laboratory procedures and equipment (e.g., media preparation, test
chemical preparation, incubator function); all optical density data obtained from thespectrophotometer plate reader will be saved in electronic and paper formats; all calculationsof ICx values and other derived data will be in electronic and paper format; all data will bearchived.

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VII.

November 2006

PROCEDURES

A. Materials[Note: Suggested brand names/vendors are listed in parentheses. Equivalents may be used
unless otherwise noted.]
1. Cell Lines
Normal Human Epidermal Keratinocytes (NHK)Non-transformed cells; from cryopreserved primary or secondary cells (Clonetics #CC2507 or equivalent). Cells will be Clonetics NHK cells.
Cambrex [Cambrex Bio Science, 8830 Biggs Ford Road, Walkersville, MD 21793-0127
Cambrex Europe [Cambrex Bio Science Verviers, S.P.R.L. Parc Industriel de Petit
Rechain, B-4800 Verviers, BELGIUM]
2. Technical Equipment[Note: Suggested brand names/vendors are listed in parentheses. Equivalents may be
used.]
a)
b)
c)
d)
e)
f)
g)
h)

Incubator: 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/airLaminar flow clean bench (standard: "biological hazard")
Water bath: 37ºC ± 1ºC
Inverse phase contrast microscope
Sterile glass tubes with caps (e.g., 5mL)
Centrifuge (optionally: equipped with microtiter plate rotor)
Laboratory balance
96-well plate spectrophotometer (i.e., plate reader) equipped with 540 nm ± 10 nm
filteri) Shaker for microtiter platesj) Cell counter or hemocytometerk) Pipetting aidl) Pipettes, pipettors (multi-channel and single channel), dilution blockm) Cryotubesn) Tissue culture flasks (75 - 80 cm2, 25 cm2)o) 96-well flat bottom tissue culture microtiter plates (e.g., Nunc # 167 008;
Corning/COSTAR tissue culture-treated)p) pH paper (wide and narrow range)[Note: Tissue culture flasks and microtiter plates should be prescreened to ensurethat they adequately support the growth of NHK.]
3. Chemicals, Media, and Seraa) Keratinocyte Basal Medium without Ca++ (KBM®, Clonetics CC-3104) that iscompleted by adding the KBM® SingleQuots® (Clonetics CC-4131) to achieve theproper concentrations of epidermal growth factor, insulin, hydrocortisone,

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antimicrobial agents, bovine pituitary extract, and calcium (e.g., Clonetics Calcium
SingleQuots®, 300 mM CaCl2, Clonetics # CC-4202).
b) HEPES Buffered Saline Solution (HEPES-BSS) (e.g., Clonetics # CC-5022)c) 0.025 % Trypsin/EDTA solution (e.g., Clonetics # CC-5012)
d) Trypsin Neutralizing Solution (TNS) (e.g., Clonetics # CC-5002)e) Phosphate Buffered Saline (PBS)f) Dulbecco’s Phosphate Buffered Saline (D-PBS) [formulation containing calcium andmagnesium cations; glucose optional] (for rinsing)g) Neutral Red (NR) Dye – tissue culture-grade; liquid form (e.g., SIGMA N 2889);
powder form (e.g., SIGMA N 4638)h) Dimethyl sulfoxide (DMSO), U.S.P analytical grade (Store under nitrogen @ -20ºC)i) Ethanol (ETOH), U.S.P. analytical grade (100 %, non-denatured for test chemical
preparation; 95 % can be used for the desorb solution)j) Glacial acetic acid, analytical gradek) Hanks' Balanced Salt Solution without Ca2+ or Mg2+ (CMF-HBSS) (e.g., Invitrogen #14170)l) Distilled H2 O or any purified water suitable for cell culture (sterile)m) Sterile paper towels (for blotting 96-well plates)
B. Preparations of Media and Solutions[Note: All solutions (except NR stock solution, NR medium and NR desorb), glassware,
pipettes, etc., shall be sterile and all procedures should be carried out under aseptic conditions
and in the sterile environment of a laminar flow cabinet (biological hazard standard). ). All
methods and procedures will be adequately documented.]
1. Mediaa) Routine Culture Medium/Treatment Medium
KBM® (Clonetics CC-3104) supplemented with KBM® SingleQuots® (Clonetics
CC-4131) and Clonetics Calcium SingleQuots® (CC-4202) to make 500 mL medium.
Final concentration of supplements in medium are:0.0001 ng/mL
5 µg/mL
0.5 µg/mL
30 µg/mL
15 ng/mL
0.10 mM
30 µg/mL

Human recombinant epidermal growth factor
InsulinHydrocortisone
Gentamicin
Amphotericin B
Calcium
Bovine pituitary extract

Complete media should be kept at 2-8°C and stored for no longer than two weeks.
NOTE:
KBM® SingleQuots® contain the following stock concentrations and volumes:
0.1 ng/mL
5.0 mg/mL
0.5 mg/mL

hEGF
InsulinHydrocortisone

0.5 mL
0.5 mL
0.5 mLC-69

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7.5 mg/mL

November 2006

Gentamicin, 15 ug/mL Amphotericin-B 0.5 mLBovine Pituitary Extract (BPE)
2.0 mL

Clonetics Calcium SingleQuots® are 2 mL of 300mM calcium.
165 ul of solution per 500 mL calcium-free medium equals 0.10 mM calcium in themedium.
2. Neutral Red (NR) Stock Solution
The liquid tissue culture-grade stock NR Solution will be the first choice for performing
the assay (e.g., SIGMA #N2889, 3.3 mg/mL). Store liquid tissue culture-grade NR Stock
Solution at the storage conditions and shelf-life period recommended by the
manufacturer.
If the liquid form is not available, the following formulation can be prepared.
EXAMPLE:

0.33 g NR Dye powder in 100 mL H2 O

The NR Stock Solution (powder in water) should be stored in the dark at room
temperature for up to two months.
3. Neutral Red (NR) Medium
EXAMPLE:
1 mL (3.3 mg NR dye/mL)
99 mL

NR Stock Solution
Routine Culture Medium (pre-warmed to 37° C.)

The final concentration of the NR Medium is 33 µg NR dye/mL.
[Note: The NR medium may be centrifuged at approximately 600 x g for 10 min (toremove NR crystals). The NR Medium shall be filtered (e.g., Millipore filtering, 0.2 –0.45 µm pore size) used to reduce NR crystals. The temperature of the NR Medium
should be maintained at 37° C (e.g., in a waterbath) before adding to the cells and will be
used within 15 minutes after removing from 37° C storage. Aliquots of NR Medium canbe made on the day of testing and maintained at 37° C. for later use.]
4. Ethanol/Acetic Acid Solution (NR Desorb)
1%
50 %
49 %

Glacial acetic acid solution
Ethanol
H2 O

C. Methods
1. Cell Maintenance and Culture Procedures
NHK cells are routinely grown as a monolayer in tissue culture grade flasks (e.g., 25 cm2)
at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air. The cells should be

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examined on a daily (i.e., on workdays) basis under a phase contrast microscope, and anychanges in morphology or their adhesive properties must be noted in a Study Workbook.
2. Receipt of Cryopreserved Keratinocytes
Upon receipt of cryopreserved keratinocytes, the vial(s) of cells shall be stored in a liquid
nitrogen freezer until needed.
3. Thawing Cells and Establishing Cell Culturesa) Thaw cells by putting ampules into a water bath at 37°C for as brief a time as
possible. Do not thaw cells at room temperature or by hand. Seed the thawed cells
into culture flasks as quickly as possible and with minimal handling.
b) Slowly (taking approximately 1-2 min) add 9 mL of pre-warmed Routine Culture
Medium to the cells suspended in the cryoprotective solution and transfer cells into
flasks containing pre-warmed Routine Culture Medium (See Table 1).
c) Incubate the cultures at 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air until
the cells attach to the flask (within 4 to 24 h), at which time the Routine Culture
Medium should be removed and replaced with fresh Routine Culture Medium.
d) Unless otherwise specified, the cells should be incubated at 37ºC ± 1ºC, 90 % ± 5 %humidity, 5.0 % ± 1 % CO2/air and fed every 2-3 days until they exceed 50 %confluence (but less than 80 % confluent).
Table 1. Establishing Cell Cultures
Cells/25 cm2 flask(in approximately 5 mL)
1 flask each cell concentration
Approximate Time to Subculture
Cells to 96-Well Plates

6.25 x 104(2500/cm2)

1.25 x 105(5000/cm2)

2.25 x 105(9000/cm2)

96+ hours
6 – 8 plates

72 - 96 hours
6 – 8 plates

48 - 72 hours
6 – 8 plates

Cell growth guidelines – actual growth of individual cell lots may vary.
4. Subculture of NHK Cells to 96-Well Plates[Note: It is important that cells have overcome the lag growth phase when they are used
for the test. Keratinocytes will be passaged only into the 96-well plates and will not be
subcultured into flasks for use in later assays]a) When the keratinocyte culture in a 25 cm2 flask exceeds 50 % confluence (but less than
80 % confluent), remove the medium and rinse the culture twice with 5 mL HEPESBSS. The second rinse should be left on the cells for approximately 5 minutes. Discard
the washing solution.
b) Add 2 mL trypsin/EDTA solution to each flask and remove after 15 to 30 seconds.
Incubate the flask at room temperature for 3 to 7 min. When more than 50 % of thecells become dislodged, rap the flask sharply against the palm of the hand.
c) When most of the cells have become detached from the surface, rinse the flask with
5 mL of room temperature TNS. If more than one flask is subcultured, the same 5 mLof TNS may be used to rinse a total of up to 2 flasks.

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November 2006d) Then rinse the flask with 5 mL CMF-HBSS and transfer the cell suspension to acentrifuge tube.
e) Pellet the cells by centrifugation for 5 min at approximately 220 x g. Remove thesupernatant by aspiration.
f) Resuspend the keratinocyte pellet by gentle trituration (to have single cells) in Routine
Culture Medium. It is important to obtain a single cell suspension for exact counting.
Count a sample of the cell suspension using a hemocytometer or cell counter.
4g) Prepare a cell suspension –1.6 – 2.0 x10 cells/mL in Routine Culture Medium.
Using a multi-channel pipette, dispense 250 µl Routine Culture Medium only into theperipheral wells (blanks) of a 96-well tissue culture microtiter plate. In the remaining
3
3
wells, dispense 125 µl of the cell suspension (2x10 – 2.5x10 cells/well). Prepare
one plate per chemical to be tested (see Figure 2, Section VII.F.1).
h) Incubate cells (37ºC ± 1ºC, 90 % ± 5.0 % humidity, and 5 % ± 1 % CO2/air) so thatcells form a 20+ % monolayer (~48-72 h). This incubation period assures cellrecovery and adherence and progression to exponential growth phase.
i) Examine each plate under a phase contrast microscope to assure that cell growth isrelatively even across the microtiter plate. This check is performed to identify
experimental and systemic cell seeding errors. Record observations in the Study
Workbook.
5. Determination of Doubling Timea) A cell doubling time procedure was performed on the initial lot of cells that was usedin the first cell culture assays of Phase Ia of the Validation Study. The doubling time
only needs to be determined in Phase Ib if there is a change in the lot of cells used.
Establish cells in culture and trypsinize cells as per Section VII.C.4 for subculture.
Resuspend cells in appropriate culture medium. Use Table 1 to determine seeding
densities.
b) Seed five sets of cell culture vessels in triplicate for each cell type (e.g., 15 tissue
culture dishes [60mm x 15mm]). Use appropriate volume of culture medium for theculture vessels. Note number of cells placed into each culture dish. Place dishes
into the incubators (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air).
c) After 4-6 hours (use the same initial measurement time for each subsequent doublingtime experiment), remove three culture dishes and trypsinize cells. Count cells using
a cell counter or hemocytometer. Cell viability may be determined by dye exclusion(e.g., Trypan Blue). Determine the total number of cells and document. Repeat
sampling at 24 hr, 48 hr, 72 hr, and 96 hr post inoculation. Change culture medium
at 72 hr or sooner in remaining dishes if indicated by pH drop.
d) Plot cell concentration (per mL of medium) on a log scale against time on a linear
scale. Determine lag time and population doubling time. The doubling time will bein the log (exponential) phase of the growth curve. Additional dishes and time areneeded if the entire growth curve is to be determined (lag phase, log phase, plateau
phase).
D. Solubility Test
The preference of solvent for dissolving test chemicals is medium, DMSO, and then ethanol.
Solubility shall be determined in a step-wise procedure that involves attempting to dissolve atest chemical at a relatively high concentration with the sequence of mechanical procedures
specified in Section VII.D.2.a. If the chemical does not dissolve, the volume of solvent is

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increased so as to decrease the concentration by a factor of 10, and then the sequence of
mechanical procedures in Section VII.D.2.a are repeated in an attempt to solubilize thechemical at the lower concentrations. For testing solubility in media, the starting
concentration is 20,000 µg/ml (i.e., 20 mg/mL) in Tier 1, but for DMSO and ethanol thestarting concentration is 200,000 µg/ml (i.e., 200 mg/mL) in Tier 2. Weighing out chemical
for each solvent (i.e., media, DMSO, ethanol) can be done all at once, if convenient, butsolubility testing (at each tier that calls for more than one solvent) is designed to be sequential- media, then DMSO, then ethanol – in accordance with the solvent hierarchy (see Figure 1).
This allows for testing to stop, rather than continue testing with less preferred solvents, if thetest chemical dissolves in a more preferred solvent. For example, if a chemical is soluble inmedium at a particular tier, testing may stop. Likewise, if a chemical is soluble in DMSO at
any tier, testing need not continue with ethanol. However, since the issue of primary
importance is testing the solvents and concentrations of test chemical required by any one
tier, sequential testing of solvents may be abandoned if the lab can test more efficiently inanother way.
1. Determination of Solubilitya) Tier 1 begins with testing 20 mg/mL in Routine Culture Medium (see Table 2).
Approximately 10 mg (10,000 µg) of the test chemical will be weighed into a glass
tube and the weight will be documented. A volume of Routine Culture Medium,approximately 0.5 mL, will be added to the vessel so that the concentration is 20,000µg/ml (20 mg/mL). The solution is mixed as specified in Section VII.D.2.a. If
complete solubility is achieved in media, then additional solubility procedures are notneeded.
b) If the test chemical is insoluble in medium, proceed to Tier 2 by adding enoughmedium, approximately 4.5 mL, to attempt to dissolve the chemical at 2 mg/mL byusing the sequence of mixing procedures specified in Section VII.D.2.a. If the test
chemical dissolves in medium at 2 mg/mL, no further procedures are necessary. If
the test chemical does NOT dissolve, weigh out approximately 100 mg test chemical
in a second glass tube and add enough DMSO to make the total volume
approximately 0.5 mL (for 200 mg/mL). In another glass tube, also add
approximately 100 mg test chemical to enough ethanol to make the total volume
approximately 0.5 mL (for 200 mg/mL). Mix both solutions as specified in Section
VII.D.2.a in an attempt to solubilize the test chemical. If the chemical is soluble ineither solvent, no additional solubility procedures are needed.

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Table 2 Determination of Solubility in Routine Culture Medium, DMSO, or Ethanol
TIER

1

2

3

Total Volume
MediumConcentration of Test
Chemical(Add ~10 mg to a tube.
Add enough medium toequal the first volume.
Dilute to subsequent
volumes if necessary.)
Total Volume
DMSO/Ethanol
Concentration of Test
Chemical(Add ~100 mg to alarge tube. Add enoughDMSO or ethanol toequal the first volume.
Dilute to subsequent
volumes if necessary.)
Total Volume
DMSO/Ethanol
Concentration of Test
Chemical(Add ~10 mg to a large
tube. Add enoughDMSO or ethanol toequal 50 mL.)

0.5 mL

5 mL

50 mL

20,000 µg/mL

2,000 µg/mL

200 µg/mL(20 mg/mL)(2 mg/mL)(0.20 mg/mL)

0.5 mL

5 mL

50 mL

200,000 µg/mL

20,000 µg/mL

2,000 µg/mL(200 mg/mL)(20 mg/mL)(2 mg/mL)

Equivalent
Concentration on Cells

4

5

50 mL
200 µg/mL(0.2 mg/mL)
10,000 µg/mL1000 µg/mL

100 µg/mL

10 µg/mL

1 µg/mL(10 mg/mL)(1 mg/mL)(0.1 mg/mL)(0.01 mg/mL)(0.001 mg/mL)c) If the chemical is NOT soluble in media, DMSO, or ethanol at Tier 2, then continue
to Tier 3 in Table 2 by adding enough solvent to increase the volume of the three
Tier 2 solutions by 10 and attempt to solubilize again using the sequence of mixing
procedures in Section VII.D.2.a. If the test chemical dissolves, no additionalsolubility procedures are necessary. If the test chemical does NOT dissolve, continue
with Tier 4 and, if necessary, Tier 5 using DMSO and ethanol. Tier 4 begins by
diluting the Tier 3 samples with DMSO or ethanol to bring the total volume to 50
mL. The mixing procedures in Section VII.D.2.a are again followed to attempt tosolubilize the chemical. Tier 5 is performed, if necessary, by weighing out anothertwo more samples of test chemical at ~10 mg each and adding ~50 mL DMSO or
ethanol for a 200 µg/mL solution, and following the mixing procedures in Section
VII.D.2.a.
Example: If complete solubility is not achieved at 20,000 µg/mL in Routine Culture
Medium at Tier 1 using the mixing procedures specified in Section VII.D.2.a, thenthe procedure continues to Tier 2 by diluting the solution to 5 mL and mixing again

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as specified in Section VII.D.2.a. If the chemical is not soluble in medium, twosamples of ~ 100 mg test chemical are weighed to attempt to solubilize in DMSO andethanol at 200,000 µg/mL (i.e., 200 mg/mL). Solutions are mixed following thesequence of procedures prescribed in Section VII.D.2.a in an attempt to dissolve. If
solubility is not achieved at Tier 2, then the solutions (media, DMSO, and ethanol)
prepared in Tier 2 are diluted by 10 so as to test 200 µg/mL in media, and 20,000µg/mL in DMSO and ethanol. This advances the procedure to Tier 3. Solutions areagain mixed as prescribed in Section VII.D.2.a in an attempt to dissolve. If
solubility is not achieved in Tier 3, the procedure continues to Tier 4, and to 5 if
necessary (see Figure 1 and Table 2).
NOTE: The amounts of test chemical weighed and Routine Culture Medium added
may be modified from the amounts given above, provided that the targeted
concentrations specified for each tier are tested.

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Figure 1. Solubility Flow Chart

STEP 1:TIER 120 mg/mL test chemical (TC) in 0.5 mL medium:• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 2.

STEP 2:TIER 2
2 mg/mL TC in medium – increase volume from STEP 1 by 10 (i.e., to 5 mL)• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 3.

STEP 3:

STEP 4:

STEP 5:

STEP 6:200 mg/mL TC in DMSOj) if TC soluble, then STOP.
k) if TC insoluble, test at 200 mg/mL in ETOH.
l) if TC soluble, then STOP.
m) If TC insoluble, go to STEP 4.
TIER 30.2 mg/mL TC in medium – increase volume from STEP 2 by 10 (i.e., to 50 mL)• if TC soluble, then STOP.
• if TC insoluble, test at 20 mg/mL in DMSO – increase volume from STEP 3 by 10(i.e., to 5 mL).
• if TC soluble, then STOP.
• if TC insoluble, test at 20 mg/mL in ETOH – increase volume from STEP 3 by
10 (i.e., to 5 mL).
• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 5.
TIER 42 mg/mL TC in DMSO – increase volume from STEP 4 by 10 (i.e., to 50 mL)• if TC soluble, then STOP.
• if TC insoluble, test at 2 mg/mL in ETOH – increase volume from STEP 4 by 10(i.e., to 50 mL).
• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 6.
TIER 50.2 mg/mL TC in 50 mL DMSO
• if TC soluble, then STOP.
•
if TC insoluble, test at 0.2 mg/mL in 50 mL ETOH
• STOP

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2.

November 2006

Mechanical Proceduresa) The following hierarchy of mixing procedures will be followed to dissolve the test
chemical:
1)
2)
3)
4)

Add test chemical to solvent as in Tier 1 of Table 2.
Gently mix. Vortex the tube (1 –2 minutes).
If test chemical hasn’t dissolved, use sonication for up to 5 minutes.
If sonication doesn’t work, then warm solution to 37°C. This can be performed
by warming 5 mL tubes in a 37°C water bath for at least 5-10 minutes before
evaluating solubility. Warm larger vessels for at least 15-20 minutes in a 37°C
water bath before evaluating solubility.
5) Proceed to Tier 2 (and Tiers 3-5, if necessary of Table 2 and repeat procedures 24).
b) The preference of solvent for dissolving test chemicals is medium, DMSO, and thenethanol. Thus, if a test chemical dissolves in more than one solvent at any one
solubility-testing tier, then the choice of solvent follows this hierarchy. For example,
if, at any tier, a chemical is soluble in medium and DMSO, but not ethanol, thechoice of solvent would be medium. If the chemical were insoluble in medium, butsoluble in DMSO and ethanol, the choice of solvent would be DMSO.
After the lab has determined the preferred solvent for the test chemical andbefore proceeding to the cytotoxicity testing, the Study Director will discuss thesolvent selection with the Study Management Team (SMT) of the validation
study. The SMT will relate what solvent should be used in the assay for eachchemical.
E. Preparation of Test Chemicals[Note: Preparation under red or yellow light is recommended to preserve chemicals that
degrade upon exposure to light.]
1. Test Chemical in Solutiona) Allow test chemicals to equilibrate to room temperature before dissolving anddiluting.
b) Prepare test chemical immediately prior to use. The solutions must not be cloudy nor
have noticeable precipitate. Each stock dilution should have at least 1-2 mL total
volume to ensure adequate solution for the test wells in a single 96-well plate.
c) For chemicals dissolved in DMSO or ethanol, the final DMSO or ethanol
concentration for application to the cells must be 0.5 % (v/v) in the vehicle controls
and in all of the eight test concentrations.
d) The stock solution for each test chemical should be prepared at the highest
concentration found to be soluble in the solubility test. Thus, the highest test
concentration applied to the cells in each range finding experiment is:• 0.5 times the highest concentration found to be soluble in the solubility test, if thechemical was soluble in medium, or

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1/200 the highest concentration found to be soluble in the solubility test if thechemical was soluble in ethanol or DMSO.

e) The seven lower concentrations in the range finding experiment would then be
prepared by successive dilutions that decrease by one log unit each. The following
example illustrates the preparation of test chemical in solvent and the dilution ofdissolved test chemical in medium before application to NHK cells.
Example: Preparation of Test Chemical in Solvent Using a Log Dilution Scheme
If DMSO was determined to be the preferred solvent at Tier 2 of the solubility test
(i.e., 200,000 µg/mL), dissolve the chemical in DMSO at 200,000 µg/mL for thechemical stock solution.
1) Label eight tubes 1 – 8. Add 0.9 mL solvent (e.g., DMSO) to tubes 2 -- 8.
2) Prepare stock solution of 200,000 µg test chemical/mL solvent in tube # 1.
3) Add 0.1 mL of 200,000 µg/mL dilution from tube #1 to tube #2 to make a 1:10
dilution in solvent (i.e., 20,000 µg/mL).
4) Add 0.1 mL of 20,000 µg/mL dilution from tube #2 to tube #3 to make another1:10 dilution (i.e., 1:100 dilution from stock solution) in solvent (i.e., 2,000µg/mL)
5) Continuing making serial 1:10 dilutions in the prepared solvent tubes.
6) Since each concentration is 200 fold greater than the concentration to be tested,make a 1:100 dilution by diluting 1 part dissolved chemical in each tube with 99
parts of culture medium (e.g., 0.1 mL of test chemical in DMSO + 9.9 mL culture
medium) to derive the eight 2X concentrations for application to NHK cells.
Each 2X test chemical concentration will then contain 1 % v/v solvent. The
NHK cells will have 0.125 mL of culture medium in the wells prior toapplication of the test chemical. By adding 0.125 mL of the appropriate 2X test
chemical concentration to the appropriate wells, the test chemical will be diluted
appropriately (e.g., highest concentration in well will be 1,000 µg/mL) in a totalof 0.250 mL and the solvent concentration in the wells will be 0.5% v/v.
7) A test article prepared in DMSO or ethanol may precipitate upon transfer into the
Routine Culture Medium. The 2X dosing solutions should be evaluated for
precipitates and the results will be recorded in the workbook. It will bepermissible to test all of the dosing solutions in the dose range finding assay
only. Doses containing test article precipitates should be avoided, and will not be
used in the ICx determinations for either the range finding experiments or thedefinitive tests.
Document all test chemical preparations in the Study Workbook.
2. pH of Test Chemical Solutions
Measure the pH of the highest concentration of the test chemical in culture medium using
pH paper. Document the pH and note the color of the medium for all dilutions. Do not
adjust the pH.

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3. Concentrations of Test Chemicala) Range Finder Experiment
Test eight concentrations of the test chemical by diluting the stock solution with aconstant factor covering a large range. The initial dilution series shall be log
dilutions (e.g., 1:10, 1:100, 1:1000, etc.).
The data from any well that has precipitate will be excluded from any calculations.
b) Main Experiment[Note: After the range finding assay is completed, the concentration-response
experiment shall be performed three times on three different days for each chemical(i.e., one plate per day per chemical).]Depending on the slope of the concentration-response curve estimated from the range
finder, the dilution/progression factor in the concentration series of the main
experiment should be smaller (6√10 = 1.47). Cover the relevant concentration range(≥10 % and ≤ 90 % effect) preferably with three points of a graded effect, but with aminimum of two points, one on each side of the IC50 value, avoiding too many noncytotoxic and/or 100 %-cytotoxic concentrations. Experiments revealing less than
one cytotoxic concentration on each side of the IC50 value shall be repeated, where
possible, with a smaller dilution factor. In addition, the dilution scheme shall be
adjusted in subsequent replicate assays (i.e., definitive assays), if necessary, toincrease the number of points on both sides of the IC50 in the 10-90% response range.
(Taking into account pipetting errors, a progression factor of 1.21 is regarded thesmallest factor achievable.)
Determine which test chemical concentration is closest to the IC50 value (e.g., 50 %cytotoxicity). Use that value as a central concentration and adjust dilutions higher
and lower in equal steps for the definitive assay.
Maximum Doses to be Tested in the Main Experiments
If minimal or no cytotoxicity was measured in the dose range finding assay, amaximum dose for the main experiments will be established as follows:• For test chemicals prepared in Routine Culture Medium, the highest test article
concentration that may be applied to the cells in the main experiments will beeither 100 mg/mL, or the maximum soluble dose. Test chemical will be weighed
into a glass tube and the weight will be documented. A volume of Routine
Culture Medium will be added to the vessel so that the concentration is200,000 µg/mL (200 mg/mL). The solution is mixed as specified in Section
VII.D.2.a. If complete solubility is achieved in medium, then 7 additional serial
stock dosing solutions may be prepared from the 200 mg/mL 2X stock. If thetest chemical is insoluble in medium at 200 mg/ml, proceed by adding medium,in small incremental amounts, to attempt to dissolve the chemical by using thesequence of mixing procedures specified in Section VII.D.2.a. The highest
soluble stock solution will be used to prepare the 7 additional serial stock dosing
solutions.

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For test chemicals prepared in either DMSO or ethanol, the highest test article
concentration that may be applied to the cells in the main experiments will beeither 2.5 mg/mL, or less, depending upon the maximum solubility in solvent.
Test chemical will be weighed into a glass tube and the weight will be
documented. A volume of the appropriate solvent (determined from the original
solubility test) will be added to the vessel so that the concentration is500,000 µg/mL (500 mg/mL). The solution is mixed as specified in Section
VII.D.2.a. If complete solubility is achieved in the solvent, then 7 additional
serial stock dosing solutions may be prepared from the 500 mg/mL 200X stock.
If the test chemical is insoluble in solvent at 500 mg/ml, proceed by adding
solvent, in small incremental amounts, to attempt to dissolve the chemical byusing the sequence of mixing procedures specified in Section VII.D.2.a. The
highest soluble stock solution will be used to prepare the 7 additional serial stock
dosing solutions.

c) Test Chemical Dilutions
The dosing factor of 3.16 (= 2√10) divides a log into two equidistant steps, a factor of
2.15 (= 3√10) divides a decade into three steps. The factor of 1.47 (= 6√10) divides alog into six equidistant steps, and the factor of 1.21 (= 12√10) divides the log into 12
steps.
EXAMPLE:
10
10
10
10

31.6

12.1

14.7
14.7

17.8

21.5
21.5
21.5

26.1

31.6
31.6

38.3

46.4
46.4
46.4

56.2

68.1
68.1

82.5

100
100
100
100

The technical production of decimal geometric concentration series is simple. An
example is given for factor 1.47:
Dilute 1 volume of the highest concentration by adding 0.47 volumes of diluent. Afterequilibration, dilute 1 volume of this solution by adding 0.47 volumes ofdiluent...(etc.).
F. Test Procedure
1. 96-Well Plate Configuration
The NHK NRU assay for test chemicals will use the 96-well plate configuration shown in
Figure 2.

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Figure 2. 96-Well Plate Configuration for Positive Control and Test Chemical Assays
1

2

3

4

5

6

7

8

9

10

11

12

A

b

b

b

b

b

b

b

b

b

b

b

b

B

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

C

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

D

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

E

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

F

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

G

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

H

b

b

b

b

b

b

b

b

b

b

b

b

VC
=
C1 – C8 =b=untreated VEHICLE CONTROL (mean viability set to 100 %)Test Chemicals or Positive Control (SLS) at eight concentrations(C1 = highest, C8 = lowest)
BLANKS (contain no cells)

2. Application of Test Chemicala) Two optional methods for rapidly applying the 2X dosing solutions onto the 96-wellplates may be utilized. The first method is to add each of the 2X dosing solutions intolabeled, sterile reservoirs (e.g., Corning/Costar model 4870 sterile polystyrene 50 mL
reagent reservoirs and/or Corning/Transtar model 4878 disposable reservoir liners, 8channel). The second method utilizes a “dummy” plate (i.e., an empty sterile 96-wellplate) prepared to hold the dosing solutions immediately prior to treatment of the test
plate (with cells). The test chemical and control dosing solutions should be dispensed
into the dummy plate in the same pattern/order as will be applied to the plate containing
cells. More volume than needed for the test plate (i.e. greater than 125 µl/well) should bein the wells of the dummy plate. At the time of treatment initiation, a multi-channel
micropipettor is used to transfer the 2X dosing solutions, from the reservoirs or dummy
plate, to the appropriate wells on the treatment plate (as described in step c. below).
These methods will ensure that the dosing solutions can be transferred rapidly to theappropriate wells of the test plate to initiate treatment times and to minimize the range oftreatment initiation times across a large number of treatment plates, and to prevent “outof order” dosing.
b) After 48 - 72 h (i.e., after cells attain 20-30+ % confluency [see Section VII.C.4(h)[)
incubation of the cells, add 125 µl of the appropriate concentration of test chemical, thePC, or the VC (see Figure 2 for the plate configuration) directly to the test wells. Do notremove Routine Culture Medium for re-feeding the cells.Incubate cells for 48 h ± 0.5 h(37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air).

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November 2006c) Positive Control: For each set of test chemical plates used in an assay, a separate plate of
positive control concentrations will be set up following the concentration range
established in the development of the positive control database in Phase Ia of the
Validation Study. The mean IC50 and two standard deviations (SD) of the IC50 of SLS
are the values that will be used as an acceptance criterion for test sensitivity for the NHK
NRU assay. This plate will follow the same schedule and procedures as used for the test
chemical plates.
3. Microscopic Evaluation
After at least 46 h treatment, examine each plate under a phase contrast microscope toidentify systematic cell seeding errors and growth characteristics of control and treatedcells. Record any changes in morphology of the cells due to the cytotoxic effects of thetest chemical, but do not use these records for any quantitative measure of cytotoxicity.
Undesirable growth characteristics of control cells may indicate experimental error andmay be cause for rejection of the assay. Use the following Visual Observations Codes inthe description of cell culture conditions.
Visual Observations Codes
Note Code
1
2
3
4
1P
2P
3P
4P
5P

Note Text
Normal Cell Morphology
Low Level of Cell Toxicity
Moderate Level of Cell Toxicity
High level of Cell Toxicity
Normal Cell Morphology with Precipitate
Low Level of Cell Toxicity with Precipitate
Moderate Level of Cell Toxicity with PrecipitateHigh level of Cell Toxicity with Precipitate
Unable to View Cells Due to Precipitate

4. Measurement of NRUb) Carefully remove (i.e., “dump”) the Routine Culture Medium (with test chemical)
and rinse the cells very carefully with 250 µL pre-warmed D-PBS. Remove therinsing solution by dumping and remove excess by gently blotting on sterile paper
towels. Add 250 µL NR medium (to all wells including the blanks) and incubate(37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air) for 3±0.1 h. Observe
the cells briefly during the NR incubation (e.g., at 1, 2, and 3 h – Study Director ‘sdiscretion) for NR crystal formation. Record observations in the Study Workbook.
Study Director can decide to reject the experiment if excessive NR crystallization has
occurred.
c) After incubation, remove the NR medium, and carefully rinse cells with 250 µL prewarmed D-PBS.
d) Decant and blot D-PBS from the plate. (Optionally: centrifuge the reversed plate.)e) Add exactly 100 µL NR Desorb (ETOH/acetic acid) solution to all wells, including
blanks.
f) Shake microtiter plate rapidly on a microtiter plate shaker for 20 – 45 min to extract
NR from the cells and form a homogeneous solution.

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November 2006g) Measure the absorption (within 60 minutes of adding NR Desorb solution) of theresulting colored solution at 540 nm ± 10 nm in a microtiter plate reader(spectrophotometer), using the blanks as a reference. [Phase Ia data show the mean
OD value for the plate blanks to be 0.058 ± 0.032 for NHK cells (mutually agreed
upon by Testing Facility and SMT; data from 3 labs; N = 75). Use this value as a
guide for assessment of the blank values.] Save raw data in the Excel format asprovided by the Study Management Team.
5. Quality Check of Assaya) Test Acceptance Criteria
1) A test meets acceptance criteria, if the IC50 for SLS is within two standard
deviations of the historical mean established by the Test Facility (as per
VII.F.2.c).
2) A test meets acceptance criteria if the corrected mean OD540 of VCs is ≥ 0.60 and≤ 1.703) A test meets acceptance criteria if the left and the right mean of the VCs do notdiffer by more than 15 % from the mean of all VCs.
4) A test meets acceptance criteria if a minimum of two points, one on each side of
the IC50 value, are determined and fall within the range ≥10 % and ≤ 90 % effect.
[Note: All acceptance criteria must be met for an assay to be considered
acceptable.]b) Checks for Systematic Cell Seeding Errors
To check for systematic cell seeding errors, untreated VCs are placed both at the left
side (row 2) and the right side (row 11 for the test plates) of the 96-well plate.
Aberrations in the cell monolayer for the VCs may reflect a volatile and toxic test
article present in the assay.
Checks for cell seeding errors may also be performed by examining each plate undera phase contrast microscope to assure that cell quantity is consistent.
c) Quality Check of Concentration-Response
The IC50 derived from the concentration-response of the test chemicals should bebacked by preferably three responses ≥ 10 and ≤ 90 % inhibition of NRU and at least
two responses, one on either side of the IC50 value (see VII.E.3.b). If this is not thecase, and the concentration progression factor can be easily reduced, reject theexperiment and repeat it with a smaller progression factor. In addition, the dilution
scheme shall be adjusted in subsequent replicate assays, if necessary, to increase thenumber of points on both sides of the IC50 in the 10-90% response range. Numericalscoring of the cells (see VII.F.3) should be determined and documented in the Study
Workbook.
G. Data Analysis

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A calculation of cell viability expressed as NRU is made for each concentration of the test
chemical by using the mean NRU of the six replicate values (minimum of four acceptable
replicates wells) per test concentration. This value is compared with the mean NRU of all
VC values (provided VC values have met the VC acceptance criteria). Relative cell viability
is then expressed as percent of untreated VC. If achievable, the eight concentrations of eachchemical tested will span the range of no effect up to total inhibition of cell viability. Data
from the microtiter plate reader shall be transferred to the Excel® spreadsheet provided by
the Study Management Team for determining cell viability and performing statisticalanalyses.
The concentration of a test chemical reflecting a 20 %, 50 %, and 80 % inhibition of cell
viability (i.e., the IC20, IC50, and IC80) is determined from the concentration-response by
applying a Hill function to the concentration-response data. Statistical software (e.g.,
GraphPad PRISM® 3.0) specified by the Study Management Team shall be used to calculate
IC20, IC50, and IC80 values (and the associated confidence limits) for each test chemical. In
addition, the Study Management Team shall provide guidelines for calculating ICx values
and confidence limits. The Testing Facility shall report data using at least three (3)significant figures and shall forward the results from each assay to the Study Management
Team/biostatistician through the designated contacts in electronic format and hard copy uponcompletion of testing. The Study Management Team will be directly responsible for thestatistical analyses of the Validation Study data.
VIII.

REFERENCES
Clonetics Normal Human Keratinocyte Systems Instructions for Use, AA-1000-4-Rev.03/00.
(http://www.clonetics.com).
Hackenberg, U. and H. Bartling. 1959. Messen und Rechnen im pharmakologischen
Laboratorium mit einem speziellen Zahlensystem (WL24-System). Arch. Exp. Pathol.
Pharmakol. 235: 437-463.
Triglia, D., P.T. Wegener, J. Harbell, K. Wallace, D. Matheson, and C. Shopsis. 1989.
Interlaboratory validation study of the keratinocyte neutral red bioassay from Clonetics
Corporation. In Alternative Methods in Toxicology, Volume 7. A.M. Goldberg, ed., pp. 357365. Mary Ann Liebert, Inc., New York.

IX. APPROVAL__________________________________SPONSOR REPRESENTATIVE___________________DATE(Print or type name)_____________________________
Testing Facility STUDY DIRECTOR(Print or type name)____________________
DATE

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Appendix C5

Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase II)

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[This Page Intentionally Left Blank]

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TEST METHOD PROTOCOL
for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

A Test for Basal Cytotoxicity for an In Vitro Validation Study
Phase IIMay 15, 2003
Prepared by
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)

Based on Standard Operating Procedure Recommendations from anInternational Workshop Organized by the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM)

National Institute of Environmental Health Sciences (NIEHS)
National Institutes of Health (NIH)
U.S. Public Health Service
Department of Health and Human Services

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TEST METHOD PROTOCOLThe BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test
A Test for Basal Cytotoxicity
Phase II
I. PURPOSE
The purpose of this study is to evaluate the cytotoxicity of test chemicals using the BALB/c 3T3
Neutral Red Uptake (NRU) cytotoxicity test. The data will be used to evaluate the intra- andinter-laboratory reproducibility of the assay and effectiveness of the cytotoxicity assay to predict
the starting doses for rodent acute oral systemic toxicity assays. This test method protocol
outlines the procedures for performing the cytotoxicity test and supports the in vitro validation
study organized by NICEATM and the European Centre for the Validation of Alternative
Methods (ECVAM) and sponsored by NIEHS, U.S. Environmental Protection Agency, andECVAM. This test method protocol applies to all personnel involved with performing thecytotoxicity assay.
A. BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test
The 3T3 NRU test will be performed to analyze the in vitro toxicity of nine (9) blinded/coded
test chemicals. This test will be used to determine IC20, IC50, and IC80 values for thepredetermined set of test chemicals of varying toxicities.
II. SPONSOR
A. Name:National Institute of Environmental Health Sciences (NIEHS); The
National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)

B. Address:

P.O. Box 12233
Research Triangle Park, NC 27709

C. Representative:

Named Representative

III. IDENTIFICATION OF TEST AND CONTROL SUBSTANCES
A. Test Chemicals:

Blinded Chemicals (9)B. Controls:

Positive:
Vehicle (Negative):
NBCS,
Solvent (as needed):

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Sodium Lauryl Sulfate
Assay medium (DMEM containing 5%
4 mM L-Glutamine, 100 IU/mL Penicillin,
100 µg/mL Streptomycin)
Assay medium with appropriate solvent
used to prepare the test chemicals (Section
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IV. TESTING FACILITY AND KEY PERSONNEL
A. Facility Information
1)
2)
3)
4)
5)
6)
7)
8)

Name:
Address:
Study Director:
Laboratory Technician(s):Scientific Advisor:
Quality Assurance Director:
Safety Manager:
Facility Management:

B. Test Schedule
1) Proposed Experimental Initiation Date:
2) Proposed Experimental Completion Date:
3) Proposed Report Date:
V. TEST SYSTEMThe NRU cytotoxicity assay procedure is a cell survival/viability chemosensitivity assay based on
the ability of viable cells to incorporate and bind neutral red (NR), a supravital dye. NR is a weakcationic dye that readily penetrates cell membranes by non-ionic diffusion and accumulates
intracellularly in lysosomes. Alterations of the cell surface or the sensitive lysosomal membranelead to lysosomal fragility and other changes that gradually become irreversible. Such changes
brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It isthus possible to distinguish between viable, damaged, or dead cells, which is the basis of thisassay.
Healthy mammalian cells, when maintained in culture, continuously divide and multiply overtime. A toxic chemical, regardless of site or mechanism of action, will interfere with this process
and result in a reduction of the growth rate as reflected by cell number. Cytotoxicity is expressed
as a concentration dependent reduction of the uptake of the NR after chemical exposure thus
providing a sensitive, integrated signal of both cell integrity and growth inhibition.
VI. DEFINITIONS
A. Hill function: a four parameter logistic mathematical model relating the concentration of test
chemical to the response being measured in a sigmoidal shape.

Y = Bottom+

Top− Bottom
1 + 10(logIC50− X)HillSlope

where Y= response, X is the logarithm of dose (or concentration), Bottom is the minimum
response, Top is the maximum response, logIC50 is logarithm of X at the response midway
between Top and Bottom, and HillSlope describes the steepness of the curve.
B. Documentation: all methods and procedures will be noted in a Study Workbook; logs will bemaintained for general laboratory procedures and equipment (e.g., media preparation, test
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chemical preparation, incubator function); all optical density data obtained from thespectrophotometer plate reader will be saved in electronic and paper formats; all calculationsof ICx values and other derived data will be in electronic and paper format; all data will bearchived.
VII.

PROCEDURES

A. Materials
1. Cell Lines
BALB/c 3T3 cells, clone 31
CCL-163, LGC Reference Materials, Customer Service, Queens Road, Teddington,
Middlesex, TW110LY, UK
CCL-163, American Type Culture Collection [ATCC], Manassas, VA, USA)
2. Technical Equipment[Note: Suggested brand names/vendors are listed in parentheses. Equivalents may be
used.]
a)
b)
c)
d)
e)
f)
g)
h)

Incubator: 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/airLaminar flow clean bench/cabinet (standard: "biological hazard")
Water bath: 37ºC ± 1ºC
Inverse phase contrast microscope
Sterile glass tubes with caps (e.g., 5 mL)
Centrifuge (optionally: equipped with microtiter plate rotor)
Laboratory balance
96-well plate spectrophotometer (i.e., plate reader) equipped with 540 nm ± 10 nm
filteri) Shaker for microtiter platesj) Cell counter or hemocytometerk) Pipetting aidl) Pipettes, pipettors (multi-channel and single channel; multichannel repeater pipette),
dilution blockm) Cryotubesn) Tissue culture flasks (e.g., 75 - 80 cm2, 25 cm2)o) 96-well flat bottom tissue culture microtiter plates (e.g., Nunc # 167 008; Falcon
tissue culture-treated)p) pH paper (wide and narrow range)q) Multichannel reagent reservoirr) Waterbath sonicators) Magnetic stirrert) Antistatic bar ionizer/antistatic gun (optional for neutralizing static on 96-well plates)u) Dry heat block (optional)[Note: Tissue culture flasks and microtiter plates should be prescreened to ensure thatthey adequately support the growth of 3T3 cells. Multi-channel repeater pipettes may be
used for plating cells in the 96-well plates, dispensing plate rinse solutions, NR medium,and desorb solution. Do not use the repeater pipette for dispensing test chemicals to thecells.]C-90

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3. Chemicals, Media, and Seraa) Dulbecco’s Modification of Eagle’s Medium (DMEM) without L-Glutamine; shouldhave high glucose [4.5gm/l] (e.g., ICN-Flow Cat. No. 12-332-54)b) L-Glutamine 200 mM (e.g., ICN-Flow # 16-801-49)c) New Born Calf Serum (NBCS or NCS) (e.g., Biochrom # SO 125)d) 0.05 % Trypsin/0.02 % EDTA solution (e.g., SIGMA T 3924, ICN-Flow, # 1689149)e) Phosphate buffered saline (PBS) without Ca2+ and Mg2+(for trypsinization)f) Hanks’ Balanced Salt Solution (HBSS) without Ca2+ and Mg2+(CMF-HBSS)g) Dulbecco’s Phosphate Buffered Saline (D-PBS) [formulation containing calcium andmagnesium cations; glucose optional] (for rinsing)h) Penicillin/streptomycin solution (e.g. ICN-Flow # 16-700-49)i) Neutral Red (NR) Dye – tissue culture-grade; liquid form (e.g., SIGMA N 2889);
powder form (e.g., SIGMA N 4638)j) Dimethyl sulfoxide (DMSO), U.S.P. analytical grade (Store under nitrogen @ -20ºC)k) Ethanol (ETOH), U.S.P. analytical grade (100 %, non-denatured for test chemical
preparation; 95 % can be used for the desorb solution)l) Glacial acetic acid, analytical gradem) Distilled H2O or any purified water suitable for cell culture (sterile)n) Sterile paper towels (for blotting 96-well plates)[Note: Due to lot variability of NBCS/NCS, first check a lot for growth stimulating
properties with 3T3 cells (approximately 20-24 h doubling time) and then reserve a
sufficient amount of NBCS/NCS. May use pre-tested serum lot from Phases Ia and Ib of
the validation study if the serum has been stored under appropriate conditions and shelflife has not expired.]
B. Preparations of Media and Solutions[Note: All solutions (except NR stock solution, NR medium and NR desorb), glassware,
pipettes, etc., shall be sterile and all procedures should be carried out under aseptic conditions
and in the sterile environment of a laminar flow cabinet (biological hazard standard). All
methods and procedures will be adequately documented.]
1. Media
DMEM (buffered with sodium bicarbonate) supplemented with (final concentrations in
DMEM are quoted):a) for freezing (Freeze Medium); contains 2X concentration of NBCS/NCS and DMSO
of final freezing solution
40 %NBCS/NCS
20 %DMSOb) for routine culture (Routine Culture Medium)
10 %NBCS/NCS
4 mM
Glutamine

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November 2006c) for solubility testing and test chemical dilution (Chemical Dilution Medium)
4 mM
Glutamine
200 IU/mL
Penicillin
200 µg/mL
Streptomycind) for dilution of NR stock solution (NR Dilution Medium)5%
4 mM
100 IU/mL
100 µg/mL

NBCS/NCS
Glutamine
Penicillin
Streptomycin[Note: The Chemical Dilution Medium with test chemical will dilute the serum
concentration of the Routine Culture Medium in the test plate to 5 %. Serum proteins
may mask the toxicity of the test substance, but serum cannot be totally excluded because
cell growth is markedly reduced in its absence.]
Completed media formulations should be kept at approximately 2-8° C and stored for nolonger than two weeks.
2. Neutral Red (NR) Stock Solution
The liquid tissue culture-grade stock NR Solution will be the first choice for performing
the assay (e.g., SIGMA #N2889, 3.3 mg/mL). Store liquid tissue culture-grade NR Stock
Solution at the storage conditions and shelf-life period recommended by the
manufacturer.
If the liquid form is not available, the following formulation can be prepared.
EXAMPLE:

0.25 g NR Dye powder in 100 mL H2O

The NR Stock Solution (powder in water) should be stored in the dark at room
temperature for up to two months.
3. Neutral Red (NR) Medium
EXAMPLE:
0.758 mL (3.3 mg NR dye/mL solution)
99.242 mL

NR Stock Solution
NR Dilution Medium (pre-warmed to 37° C)

The final concentration of the NR Medium is 25 µg NR dye/mL and aliquots will be
prepared on the day of application.
[Note: The NR Medium shall be filtered (e.g., Millipore filtering, 0.2 – 0.45 µm poresize) to reduce NR crystals. Aliquots of the NR Medium should be maintained at 37° C(e.g., in a waterbath) before adding to the cells and used within 30 min of preparation but
also used within 15 min after removing from 37° C storage.]

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4. Ethanol/Acetic Acid Solution (NR Desorb)
1%
50 %
49 %

Glacial acetic acid solution
Ethanol
H2O

C. Methods
1. Cell Maintenance and Culture Procedures
BALB/c 3T3 cells are routinely grown as a monolayer in tissue culture grade flasks (e.g.,
75 - 80 cm2) at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air. The cells
should be examined on a daily (i.e., on workdays) basis under a phase contrast
microscope, and any changes in morphology or their adhesive properties noted in a Study
Workbook.
2. Receipt of Cryopreserved BALB/c 3T3 Cells
Upon receipt of cryopreserved BALB/c 3T3 cells, the vial(s) of cells shall be stored in aliquid nitrogen freezer until needed.
3. Thawing Cells
Thaw cells by putting ampules into a water bath at 37°C ± 1ºC. Leave for as brief a time
as possible.
a) Resuspend the cells in pre-warmed Routine Culture Medium and transfer into
pre-warmed Routine Culture Medium in a tissue-culture flask.
b) Incubate at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air.
c) When the cells have attached to the bottom of the flask (within 4 to 24 h),decant the supernatant and replace with fresh pre-warmed (37ºC) medium.
Culture as described above.
d) Passage at least two times before using the cells in a cytotoxicity test.
A fresh batch of frozen cells from the stock lot of cells should be thawed out and cultured
approximately every two months. This period resembles a sequence of about 18
passages.
4. Routine Culture of BALB/C 3T3 Cells
When cells exceed 50 % confluence (but less than 80 % confluent) they should be
removed from the flask by trypsinization:a) Decant medium, briefly rinse cultures with 5 mL PBS or Hanks’ BSS (without Ca2+,
Mg2+) per 25 cm2 flask (15 mL per 75 cm2 flask). Wash cells by gentle agitation toremove any remaining serum that might inhibit the action of the trypsin.

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November 2006b) Discard the washing solution. Repeat the rinsing procedure and discard the washingsolution.
c) Add 1-2 mL trypsin-EDTA solution per 25 cm2 to the monolayer for a few seconds(e.g., 15-30 seconds).
d) Remove excess trypsin-EDTA solution and incubate the cells at room temperature.
e) After 2-3 minutes (min), lightly tap the flask to detach the cells into a single cellsuspension.
5. Cell Counting
After detaching the cells, add 0.1-0.2 mL of pre-warmed (37ºC) Routine Culture
Medium/cm2 to the flask (e.g., 2.5 mL for a 25 cm2 flask). Disperse the monolayer by
gentle trituration. It is important to obtain a single cell suspension for exact counting.
Count a sample of the cell suspension obtained using a hemocytometer or cell counter(e.g., Coulter counter).
6. Subculture of Cells
After determination of cell number, the culture can be sub-cultured into other flasks orseeded into 96-well microtiter plates. BALB/c 3T3 cells are routinely passaged atsuggested cell densities as listed in the table (approximate doubling time is 20-24 h). The
individual laboratories will need to determine and adjust the final density to achieve
appropriate growth.
Table 1. Cell Densities for Subculturing
Days in Culture2
3
4

Seeding Density(cells/cm2)
16800
8400
4200

Total Cells per 25 cm2
flask
4.2 x 105
2.1 x 105
1.05 x 105

Total Cells per 75 cm2
flask
1.26 x 106
6.3 x 105
3.15 x 105[Note: It is important that cells have overcome the lag growth phase when they are used
for the test.]
7. Freezing Cells (procedure required only if current stock of cells is depleted)
Stocks of BALB/c 3T3 cells can be stored in sterile, freezing tubes in a liquid nitrogen
freezer. DMSO is used as a cryoprotective agent.
a) Centrifuge trypsinized cells at approximately 200 x g.
b) Suspend the cells in cold Routine Culture Medium (half the final freezingvolume) so a final concentration of 1-5x106 cells/mL can be attained.
c) Slowly add cold Freeze Medium to the cells so that the solvent will equilibrate
across the cell membranes. Bring the cell suspension to the final freezing
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volume. The final cell suspension will be 10 % DMSO. Aliquot the cellsuspension into freezing tubes and fill to 1.8 mL.
d) Place the tubes into an insulated container (e.g., styrofoam trays) and place in afreezer (-70 to -80°C) for 24 h. This gives a freezing rate of approximately
1°C/min. The laboratory needs to ensure that the freezing protocol is applicableto the 3T3 cells and that the cells are viable when removed fromcryopreservation.
e) Place the frozen tubes into liquid nitrogen for storage.
8. Preparation of Cells for Assaysa) Cultured cells that are going to be used in seeding the 96-well plates should be fed
fresh medium the day before subculturing to the plates. On the day of plate seeding,4
prepare a cell suspension of 2.0 – 3.0x10 cells/mL in Routine Culture Medium.
Using a multi-channel pipette, dispense 100 µl Routine Culture Medium only into theperipheral wells (blanks) of a 96-well tissue culture microtiter plate (See Section
VII.F.1). In the remaining wells, dispense 100 µl of a cell suspension of 2.0 –3
3.0x104 cells/mL (= 2.0 – 3.0x10 cells/well). The seeding density should be noted toensure that the cells in the control wells are not overgrown after three days (i.e., 24 hincubation in step b and 48 h exposure to test chemicals). Prepare one plate perchemical to be tested.
b) Incubate cells for 24 ± 2 h (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air)so that cells form a less than half (< 50%) confluent monolayer. This incubation
period assures cell recovery and adherence and progression to exponential growthphase.
c) Examine each plate under a phase contrast microscope to assure that cell growth isrelatively even across the microtiter plate. This check is performed to identify
experimental and systemic cell seeding errors. Record observations in the Study
Workbook.
9. Determination of Doubling Timea) A cell doubling time procedure was performed on the initial lot of cells that was usedin the first cell culture assays of Phase Ia of the Validation Study. The doubling time
only needs to be determined in Phase II if there is a change in the lot of cells used.
Establish cells in culture and trypsinize cells as per Section VII.C.4 for subculture.
Resuspend cells in NR Dilution Medium (5 % NBCS/NCS). Seed cells at 4200
cells/cm2.
b) Seed five sets of cell culture vessels in triplicate for each cell type (e.g., 15 tissue
culture dishes [60mm x 15mm]). Use appropriate volume of culture medium for theculture vessels. Note number of cells placed into each culture dish. Place dishes into
the incubators (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air).
c) After 4 - 6 hours (use the same initial measurement time for each subsequent
doubling time experiment), remove three culture dishes and trypsinize cells. CountC-95

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cells using a cell counter or hemocytometer. Cell viability may be determined by dye
exclusion (e.g., Trypan Blue; Nigrosin) if Study Director sees a need. Useappropriate size exclusion limits if using a Coulter counter. Determine the total
number of cells and document. Repeat sampling at 24 h, 48 h, 72 h, and 96 h post
inoculation. Change culture medium at 72 h or sooner in remaining dishes if
indicated by pH drop.
d) Plot cell concentration (per mL of medium) on a log scale against time on a linear
scale. Determine lag time and population doubling time. Additional dishes and time
are needed if the entire growth curve is to be determined (lag phase, log phase,plateau phase).
D. Solubility Test
The preference of solvent for dissolving test chemicals is medium, DMSO, and then ethanol.
Solubility shall be determined in a step-wise procedure that involves attempting to dissolve atest chemical at a relatively high concentration with the sequence of mechanical procedures
specified in Section VII.D.2.a. If the chemical does not dissolve, the volume of solvent isincreased so as to decrease the concentration by a factor of 10, and then the sequence of
mechanical procedures in Section VII.D.2.a are repeated in an attempt to solubilize thechemical at the lower concentrations. For testing solubility in medium, the starting
concentration is 20,000 µg/ml (i.e., 20 mg/mL) in Tier 1, but for DMSO and ethanol thestarting concentration is 200,000 µg/ml (i.e., 200 mg/mL) in Tier 2. Weighing out chemical
for each solvent (i.e., medium, DMSO, ethanol) can be done all at once, if convenient, butsolubility testing (at each tier that calls for more than one solvent) is designed to be sequential- medium, then DMSO, then ethanol – in accordance with the solvent hierarchy (see Figure1). This allows for testing to stop, rather than continue testing with less preferred solvents, if
the test chemical dissolves in a more preferred solvent. For example, if a chemical is soluble
in medium at a particular tier, testing may stop. Likewise, if a chemical is soluble in DMSO
at any tier, testing need not continue with ethanol. However, since the issue of primary
importance is testing the solvents and concentrations of test chemical required by any one
tier, sequential testing of solvents may be abandoned if the lab can test more efficiently inanother way.
1. Determination of Solubilitya) Tier 1 begins with testing 20 mg/mL in Chemical Dilution Medium (see Table 2).
Approximately 10 mg (10,000 µg) of the test chemical will be weighed into a glass
tube and the weight will be documented. A volume of Chemical Dilution Medium,approximately 0.5 mL, will be added to the vessel so that the concentration is 20,000µg/ml (20 mg/mL). The solution is mixed as specified in Section VII.D.2.a. If
complete solubility is achieved in medium, then additional solubility procedures are
not needed.
b) If the test chemical is insoluble in Chemical Dilution Medium, proceed to Tier 2 byadding enough medium, approximately 4.5 mL, to attempt to dissolve the chemical at
2 mg/mL by using the sequence of mixing procedures specified in Section VII.D.2.a.
If the test chemical dissolves in Chemical Dilution Medium at 2 mg/mL, no further
procedures are necessary. If the test chemical does NOT dissolve, weigh out
approximately 100 mg test chemical in a second glass tube and add enough DMSO to
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make the total volume approximately 0.5 mL (for 200 mg/mL) and attempt todissolve the chemical as specified in Section VII.D.2.a. If the test chemical does notdissolve in DMSO, weigh out approximately 100 mg test chemical in another glass
tube and add enough ethanol to make the total volume approximately 0.5 mL (for 200
mg/mL) and attempt to dissolve the chemical as specified in Section VII.D.2.a. If
the chemical is soluble in either solvent, no additional solubility procedures areneeded.
c) If the chemical is NOT soluble in Chemical Dilution Medium, DMSO, or ethanol atTier 2, then continue to Tier 3 in Table 2 by adding enough solvent to increase thevolume of the three Tier 2 solutions by 10 and attempt to solubilize again using thesequence of mixing procedures in Section VII.D.2.a. If the test chemical dissolves,no additional solubility procedures are necessary. If the test chemical does NOT
dissolve, continue with Tier 4 and, if necessary, Tier 5 using DMSO and ethanol.
Tier 4 begins by diluting the Tier 3 samples with DMSO or ethanol to bring the total
volume to 50 mL. The mixing procedures in Section VII.D.2.a are again followed toattempt to solubilize the chemical. Tier 5 is performed, if necessary, by weighing out
another two more samples of test chemical at ~10 mg each and adding ~50 mL
DMSO or ethanol for a 200 µg/mL solution, and following the mixing procedures in
Section VII.D.2.a.
Example: If complete solubility is not achieved at 20,000 µg/mL in Chemical
Dilution Medium at Tier 1 using the mixing procedures specified in Section
VII.D.2.a, then the procedure continues to Tier 2 by diluting the solution to 5 mL andmixing again as specified in Section VII.D.2.a. If the chemical is not soluble in
Chemical Dilution Medium, two samples of ~ 100 mg test chemical are weighed toattempt to solubilize in DMSO and ethanol at 200,000 µg/mL (i.e., 200 mg/mL).
Solutions are mixed following the sequence of procedures prescribed in Section
VII.D.2.a in an attempt to dissolve. If solubility is not achieved at Tier 2, then thesolutions (Chemical Dilution Medium, DMSO, and ethanol) prepared in Tier 2 are
diluted by 10 so as to test 200 µg/mL in media, and 20,000 µg/mL in DMSO andethanol. This advances the procedure to Tier 3. Solutions are again mixed asprescribed in Section VII.D.2.a in an attempt to dissolve. If solubility is notachieved in Tier 3, the procedure continues to Tier 4, and to 5 if necessary (seeFigure 1 and Table 2).

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Table 2 Determination of Solubility in Chemical Dilution Medium, DMSO, or Ethanol
TIER

1

2

3

4

Total Volume

0.5 mL

5 mL

50 mL

20,000 µg/mL

2,000 µg/mL

200 µg/mL(20 mg/mL)(2 mg/mL)(0.20 mg/mL)

0.5 mL

5 mL

50 mL

200,000 µg/mL

20,000 µg/mL

2,000 µg/mL(200 mg/mL)(20 mg/mL)(2 mg/mL)

5

ChemicalDilution
MediumConcentration of
Test Chemical(Add ~10 mg toa tube. Add
enough medium
to equal the first
volume. Diluteto subsequent
volumes ifnecessary.)
Total Volume
DMSO/Ethanol
Concentration of
Test Chemical(Add ~100 mg
to a large tube.
Add enoughDMSO orethanol to equal
the first volume.
Dilute tosubsequent
volumes ifnecessary.)
Total Volume
DMSO/Ethanol
Concentration of
Test Chemical(Add ~10 mg toa large tube. Add
enough DMSO
or ethanol toequal 50 mL.)
Equivalent
Concentration
on Cells

50 mL

200 µg/mL(0.2 mg/mL)

10,000 µg/mL1000 µg/mL

100 µg/mL

10 µg/mL(10 mg/mL)(1 mg/mL)(0.1 mg/mL)(0.01 mg/mL)

1 µg/mL(0.001
mg/mL)[NOTE: The amounts of test chemical weighed and Chemical Dilution Medium
added may be modified from the amounts given above, provided that the targeted
concentrations specified for each tier are tested.]

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Figure 1. Solubility Flow Chart

STEP 1:TIER 120 mg/mL test chemical (TC) in 0.5 mL Chemical Dilution Medium:• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 2.

STEP 2:TIER 2
2 mg/mL TC in medium – increase volume from STEP 1 by 10 (i.e., to 5 mL)• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 3.

STEP 3:

STEP 4:

STEP 5:

STEP 6:200 mg/mL TC in DMSO
• if TC soluble, then STOP.
• if TC insoluble, test at 200 mg/mL in ETOH.
• if TC soluble, then STOP.
• If TC insoluble, go to STEP 4.
TIER 30.2 mg/mL TC in medium – increase volume from STEP 2 by 10 (i.e., to 50 mL)• if TC soluble, then STOP.
• if TC insoluble, test at 20 mg/mL in DMSO – increase volume from STEP 3 by 10(i.e., to 5 mL).
• if TC soluble, then STOP.
• if TC insoluble, test at 20 mg/mL in ETOH – increase volume from STEP 3 by
10 (i.e., to 5 mL).
• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 5.
TIER 42 mg/mL TC in DMSO – increase volume from STEP 4 by 10 (i.e., to 50 mL)• if TC soluble, then STOP.
• if TC insoluble, test at 2 mg/mL in ETOH – increase volume from STEP 4 by 10(i.e., to 50 mL).
• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 6.
TIER 50.2 mg/mL TC in 50 mL DMSO
• if TC soluble, then STOP.
• if TC insoluble, test at 0.2 mg/mL in 50 mL ETOH
• STOP

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November 2006

Mechanical Proceduresa) The following hierarchy of mixing procedures will be followed to dissolve the test
chemical:1) Add test chemical to solvent as in Tier 1 of Table 2.
2) Gently mix. Vortex the tube (1 –2 minutes).
3) If test chemical hasn’t dissolved, use sonication for up to 5 minutes.
4) If sonication doesn’t work, then warm solution to 37°C for 5 - 60 min. This canbe performed by warming tubes in a 37°C water bath or in a CO2 incubator at
37°C. The solution may be stirred during warming (stirring in a CO2 incubator
will help maintain proper pH).
5) Proceed to Tier 2 (and Tiers 3-5, if necessary of Table 2 and repeat procedures 24).
b) The preference of solvent for dissolving test chemicals is Chemical Dilution
Medium, DMSO, and then ethanol. Thus, if (all solvents for a particular tier are
tested simultaneously and) a test chemical dissolves in more than one solvent, thenthe choice of solvent follows this hierarchy. For example, if, at any tier, a chemical
is soluble in Chemical Dilution Medium and DMSO, but not ethanol, the choice ofsolvent would be medium. If the chemical were insoluble in medium, but soluble in
DMSO and ethanol, the choice of solvent would be DMSO.
After the lab has determined the preferred solvent for the test chemical and before
proceeding to the cytotoxicity testing, the Study Director will discuss the solvent
selection with the Study Management Team (SMT) of the validation study. The
SMT will relate what solvent should be used in the assay for each chemical. If thelaboratory has attempted all solubility testing without success, then the SMT will
provide additional guidance for achieving test chemical solubility. The SMT
anticipates that all validation study test chemicals will be tested in the NRU assays.

E. Preparation of Test Chemicals[Note: Preparation under red or yellow light is recommended to preserve chemicals that
degrade upon exposure to light.]
1. Test Chemicals in Solutiona) Allow test chemicals to equilibrate to room temperature before dissolving anddiluting.
b) Prepare test chemical immediately prior to use. Test chemical solutions should not
be prepared in bulk for use in subsequent tests. The solutions must not be cloudy nor
have noticeable precipitate. Each stock dilution should have at least 1-2 mL total
volume to ensure adequate solution for the test wells in a single 96-well plate. The
SMT may direct the Study Director to store an aliquot (e.g., 1 mL) of the highest 2X

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stock solution (e.g., low solubility chemicals) in a freezer (e.g., -70°C) for use in
future chemical analyses.
c) For chemicals dissolved in DMSO or ethanol, the final DMSO or ethanol
concentration for application to the cells must be 0.5 % (v/v) in the vehicle controls
and in all of the eight test concentrations.
d) The stock solution for each test chemical should be prepared at the highest
concentration found to be soluble in the solubility test. Thus, the highest test
concentration applied to the cells in each range finding experiment is:• 0.5 times the highest concentration found to be soluble in the solubility test, if thechemical was soluble in Chemical Dilution Medium, or• 1/200 the highest concentration found to be soluble in the solubility test if thechemical was soluble in ethanol or DMSO.
e) The seven lower concentrations in the range finding experiment would then be
prepared by successive dilutions that decrease by one log unit each. The following
example illustrates the preparation of test chemical in solvent and the dilution ofdissolved test chemical in Chemical Dilution Medium before application to 3T3 cells.
Example: Preparation of Test Chemical in Solvent Using a Log Dilution Scheme
If DMSO was determined to be the preferred solvent at Tier 2 of the solubility test (i.e.,
200,000 µg/mL), dissolve the chemical in DMSO at 200,000 µg/mL for the chemical
stock solution.
1) Label eight tubes 1 – 8. Add 0.9 mL solvent (e.g., DMSO) to tubes 2 -- 8.
2) Prepare stock solution of 200,000 µg test chemical/mL solvent in tube # 1.
3) Add 0.1 mL of 200,000 µg/mL dilution from tube #1 to tube #2 to make a 1:10
dilution in solvent (i.e., 20,000 µg/mL).
4) Add 0.1 mL of 20,000 µg/mL dilution from tube #2 to tube #3 to make another 1:10
dilution (i.e., 1:100 dilution from stock solution) in solvent (i.e., 2,000 µg/mL)5) Continuing making serial 1:10 dilutions in the prepared solvent tubes.
6) Since each concentration is 200 fold greater than the concentration to be tested, makea 1:100 dilution by diluting 1 part dissolved chemical in each tube with 99 parts ofChemical Dilution Medium (e.g., 0.1 mL test chemical in DMSO + 9.9 mL Chemical
Dilution Medium) to derive the eight 2X concentrations for application to 3T3 cells.
Each 2X test chemical concentration will then contain 1 % v/v solvent. The 3T3
cells will have 0.05 mL Routine Culture Medium in the wells prior to application of
the test chemical. By adding 0.05 mL of the appropriate 2X test chemical
concentration to the appropriate wells, the test chemical will be diluted appropriately(e.g., highest concentration in well will be 1,000 µg/mL) in a total of 0.1 mL and thesolvent concentration in the wells will be 0.5% v/v.
7) A test article prepared in Chemical Dilution Medium, DMSO, or ethanol may
precipitate upon transfer into the Routine Culture Medium. The 2X dosing solutions
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should be evaluated for precipitates and the results will be recorded in the workbook.
It will be permissible to test all of the dosing solutions in the dose range finding assay
only. Doses containing test article precipitates should be avoided, and will not be
used in the ICx determinations for either the range finding experiments or thedefinitive tests.
Document all test chemical preparations in the Study Workbook.
2. pH of Test Chemical Solutions
Measure the pH of the highest concentration of the test chemical in culture medium using
pH paper (e.g., pH 0 - 14 to estimate and pH 5 – 10 to determine more precise value).
The pH paper should be in contact with the solution for approximately one minute.
Document the final pH (i.e., in the EXCEL template) and note the color of the medium
for all dilutions. Do not adjust the pH.
3. Concentrations of Test Chemicala) Range Finder Experiment
Test eight concentrations of the test chemical by diluting the stock solution with aconstant factor covering a large range. The initial dilution series shall be log
dilutions (e.g., 1:10, 1:100, 1:1000, etc.).
The data from any well that has precipitate will be excluded from any calculations.
b) Main Experiment[Note: After the range finding assay is completed, the concentration-response
experiment shall be performed three times on three different days for each chemical(i.e., one plate per day per chemical.]
Depending on the slope of the concentration-response curve estimated from the range
finder, the dilution/progression factor in the concentration series of the main
experiment should be smaller (6√10 = 1.47). Cover the relevant concentration range(≥10 % and ≤ 90 % effect) preferably with three points of a graded effect, but with aminimum of two points, one on each side of the estimated IC50 value, avoiding too
many non-cytotoxic and/or 100 %-cytotoxic concentrations. Experiments revealing
less than one cytotoxic concentration on each side of the IC50 value shall be repeated,
where possible, with a smaller dilution factor. Each experiment should have at least
one cytotoxicity value ≥ 10.0 % and ≤ 50.0 % viability and at least one cytotoxicity
value > 50.0 % and ≤ 90.0 % viability. In addition, the dilution scheme shall be
adjusted in subsequent replicate assays (i.e., definitive assays), if necessary, toincrease the number of points on both sides of the IC50 in the 10-90% response range.
(Taking into account pipetting errors, a progression factor of 1.21 is regarded thesmallest factor achievable.)
Determine which test chemical concentration is closest to the IC50 value (e.g., 50 %cytotoxicity). Use that value as a central concentration and adjust dilutions higher
and lower in equal steps for the definitive assay.

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Maximum Doses to be Tested in the Main Experiments
If minimal or no cytotoxicity was measured in the dose range finding assay, amaximum dose for the main experiments will be established as follows:• For test chemicals prepared in Chemical Dilution Medium, the highest test article
concentration that may be applied to the cells in the main experiments will beeither 100 mg/mL, or the maximum soluble dose. Test chemical will be weighed
into a glass tube and the weight will be documented. A volume of Chemical
Dilution Medium will be added to the vessel so that the concentration is200,000 µg/mL (200 mg/mL). The solution is mixed as specified in Section
VII.D.2.a. If complete solubility is achieved in medium, then 7 additional serial
stock dosing solutions may be prepared from the 200 mg/mL 2X stock. If thetest chemical is insoluble in medium at 200 mg/ml, proceed by adding medium,in small incremental amounts, to attempt to dissolve the chemical by using thesequence of mixing procedures specified in Section VII.D.2.a. The highest
soluble stock solution will be used to prepare the 7 additional serial stock dosing
solutions.
•

For test chemicals prepared in either DMSO or ethanol, the highest test article
concentration that may be applied to the cells in the main experiments will beeither 2.5 mg/mL, or less, depending upon the maximum solubility in solvent.
Test chemical will be weighed into a glass tube and the weight will be
documented. A volume of the appropriate solvent (determined from the original
solubility test) will be added to the vessel so that the concentration is500,000 µg/mL (500 mg/mL). The solution is mixed as specified in Section
VII.D.2.a. If complete solubility is achieved in the solvent, then 7 additional
serial stock dosing solutions may be prepared from the 500 mg/mL 200X stock.
If the test chemical is insoluble in solvent at 500 mg/ml, proceed by adding
solvent, in small incremental amounts, to attempt to dissolve the chemical byusing the sequence of mixing procedures specified in Section VII.D.2.a. The
highest soluble stock solution will be used to prepare the 7 additional serial stock
dosing solutions.

c) Test Chemical Dilutions
The dosing factor of 3.16 (= 2√10) divides a log into two equidistant steps, a factor of
2.15 (= 3√10) divides a decade into three steps. The factor of 1.47 (= 6√10) divides alog into six equidistant steps, and the factor of 1.21 (= 12√10) divides the log into 12
steps.
EXAMPLE:
10
10
10
10

31.6
12.1

14.7
14.7

17.8

21.5
21.5
21.5

26.1

31.6
31.6

38.3

46.4
46.4
46.4

56.2

68.1
68.1

82.5

The technical production of decimal geometric concentration series is simple. An
example is given for factor 1.47:

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Dilute 1 volume of the highest concentration by adding 0.47 volumes of diluent. Afterequilibration, dilute 1 volume of this solution by adding 0.47 volumes ofdiluent...(etc.).
F. Test Procedure
1. 96-Well Plate Configuration
The 3T3 NRU assay for test chemicals will use the 96-well plate configuration as shown in
Figure 2.
Figure 2. 96-Well Plate Configuration for Positive Control and Test Chemical Assays
1

2

3

4

5

6

7

8

9

10

11

12

A

VCb VCb C1b

C2b

C3b

C4b

C5b

C6b

C7b

C8b

VCb VCbB

VCb VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

VCb

C

VCb VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

VCb

D

VCb VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

VCb

E

VCb VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

VCb

F

VCb VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

VCb

G

VCb VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

VCb

H

VCb VCb C1b

C2b

C3b

C4b

C5b

C6b

C7b

C8b

VCb VCbVC=
C1 – C8 =
b
VCb

=
=

untreated VEHICLE CONTROL (mean viability set to 100 %)Test Chemicals or Positive Control (SLS) at eight concentrations(C1 = highest, C8 = lowest)
BLANKS (contain no cells)
VEHICLE CONTROL BLANK

2. Application of Test Chemicala) Two optional methods for rapidly applying the 2X dosing solutions onto the 96-wellplates may be utilized.
1) The first method is to add each of the 2X dosing solutions into labeled, sterile
reservoirs (e.g., Corning/Costar model 4870 sterile polystyrene 50 mL reagent
reservoirs; or Corning/Transtar model 4878 disposable reservoir liners, 8channel; or other multichannel reservoirs).
2) The second method utilizes a “dummy” plate (i.e., an empty sterile 96-well plate)
prepared to hold the dosing solutions immediately prior to treatment of the test
plate (with cells). The test chemical and control dosing solutions should be
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plate containing cells. More volume than needed for the test plate (i.e. greaterthan 50 µl/well) should be in the wells of the dummy plate.
At the time of treatment initiation, a multi-channel micropipettor is used to transfer
the 2X dosing solutions, from the reservoirs or dummy plate, to the appropriate wellson the treatment plate (as described in step c. below). These methods will ensure thatthe dosing solutions can be transferred rapidly to the appropriate wells of the test
plate to initiate treatment times and to minimize the range of treatment initiation
times across a large number of treatment plates, and to prevent “out of order” dosing.
Do not use a multichannel repeater pipette for dispensing test chemical to the plates.
b) After 24 h ± 2 h incubation of the cells, remove Routine Culture Medium from thecells by careful inversion of the plate (i.e., “dump”) over an appropriate receptacle.
Gently blot the plate on a sterile paper towel so that the monolayer is minimallydisrupted. Do not use automatic plate washers for this procedure nor vacuum
aspiration.
c) Immediately add 50 µL of fresh pre-warmed Routine Culture Medium to all of thewells, including the blanks. Fifty microliters (50 µL) of dosing solution will be
rapidly transferred from the 8-channel reservoir (or dummy plate) to the appropriate
wells of the test plate using a single delivery multi-channel pipettor. For example, theVC may be transferred first (into columns 1, 2, 11, and 12), followed by the test
article dosing solutions from lowest to highest dose, so that the same pipette tips on
the multi-channel pipettor can be used for the whole plate. [The Vehicle Control
blank (VCb) wells (column 1, column 12, wells A2, A11, H2, H11) will receive the
Vehicle Control dosing solutions (which should include any solvents used). Blanks
for wells A3 – A10 and H3 – H10 shall receive the appropriate test chemicalsolutions for each concentration (e.g., wells A3 and H3 receive C1 solution). [The test
chemical blanks in rows A and H will be used for their respective test chemical
concentrations.]d) Incubate cells for 48 h ± 0.5 h (37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 %CO2/air).
e) Positive Control: For each set of test chemical plates used in an assay, a separate
plate of positive control concentrations will be set up following the concentration
range established in the development of the positive control database in Phase I of the
Validation Study. If multiple sets of test chemical plates are set up, then clearly
designate the positive control plates for each set; each set will be an individual entity.
The mean IC50 ± two and a half standard deviations (SD) for the SLS acceptable tests
from Phases Ia and Ib (after the removal of outliers) are the values that will be used
as an acceptance criterion for test sensitivity for the 3T3 NRU assay. This plate will
follow the same schedule and procedures as used for the test chemical plates(including appropriate chemical concentrations in the appropriate wells – see sections
VII.F.1 and F.2).
3. Microscopic Evaluation
After at least 46 h treatment, examine each plate under a phase contrast microscope toidentify systematic cell seeding errors and growth characteristics of control and treatedcells. Record any changes in morphology of the cells due to the cytotoxic effects of the
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test chemical, but do not use these records for any quantitative measure of cytotoxicity.
Undesirable growth characteristics of control cells may indicate experimental error andmay be cause for rejection of the assay. Use the following Visual Observations Codes inthe description of cell culture conditions.
Visual Observations Codes
Note Code
1
2
3
4
1P
2P
3P
4P
5P

Note Text
Normal Cell Morphology
Low Level of Cell Toxicity
Moderate Level of Cell Toxicity
High level of Cell Toxicity
Normal Cell Morphology with Precipitate
Low Level of Cell Toxicity with Precipitate
Moderate Level of Cell Toxicity with PrecipitateHigh level of Cell Toxicity with Precipitate
Unable to View Cells Due to Precipitate

4. Measurement of NRUa) Carefully remove (i.e., “dump”) the medium with test chemical and rinse the cells
very carefully with 250 µL pre-warmed D-PBS. Remove the rinsing solution bydumping and remove excess by gently blotting on sterile paper towels. Add 250 µL
NR medium (to all wells including the blanks) and incubate (37ºC ± 1ºC, 90 % ± 5 %humidity, and 5.0 % ± 1 % CO2/air) for 3±0.1 h. Observe the cells briefly during the
NR incubation (e.g., between 2 and 3 h – Study Director’s discretion) for NR crystal
formation. Record observations in the Study Workbook. Study Director can decideto reject the experiment if excessive NR crystallization has occurred.
b) After incubation, remove the NR medium, and carefully rinse cells with 250 µl prewarmed D-PBS.
c) Decant and blot D-PBS from the plate.
d) Add exactly 100 µl NR Desorb (ETOH/acetic acid) solution to all wells, including
blanks.
e) Shake microtiter plate rapidly on a microtiter plate shaker for 20 – 45 min to extract
NR from the cells and form a homogeneous solution. Plates should be protectedfrom light by using a cover during shaking.
f) Plates should be still for at least five minutes after removal from the plate shaker (ororbital mixer). Observe the wells for bubbles. Measure the absorption (within 60
minutes of adding NR Desorb solution) of the resulting colored solution at 540 nm ±10 nm in a microtiter plate reader (spectrophotometer), using the blanks as areference. [Note: Phases Ia and Ib data show the mean OD value for the plate blanks
to be 0.057 ± 0.043 for 3T3 cells (± 2.5 standard deviations; data from 3 labs; N =189). Use this range as a guide for assessment of the blank values.] Save raw data in
the Excel format as provided by the SMT.

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5. Quality Check of 3T3 NRU Assaya) Test Acceptance Criteria
1) A test meets acceptance criteria, if the IC50 for SLS (PC) is within ± two and ahalf (2.5) standard deviations of the historical mean established by the Test
Facility (as per VII.F.2.e).
2) A test meets acceptance criteria if the left and the right mean of the VCs do notdiffer by more than 15.0 % from the mean of all VCs.
3) A test meets acceptance criteria if:• at least one calculated cytotoxicity value ≥ 10.0 % and ≤ 50.0 % viability and• at least one calculated cytotoxicity value > 50.0 % and ≤ 90.0 % viability.
4) A test meets acceptance criteria if the r2 (coefficient of determination) value
calculated for the Hill model fit (i.e., from PRISM software) is ≥ 0.90. A test
does not meet acceptance criteria if the r2 value is < 0.80. If the r2 value is ≥ 0.80
and < 0.90 (“gray zone”), then the SMT will evaluate the model fit and make thedetermination of whether or not the test meets the acceptance criteria and relate
the information to the Study Director.
[Note: All acceptance criteria must be met for an assay to be considered
acceptable.][A corrected mean OD540 ± 10nm of 0.103 - 0.813 for the VCs is a target range but
will not be a test acceptance criterion. Range determined from Phase Ib VC OD
values from 3 laboratories (mean ± 2.5 standard deviations, N = 98).]b) Checks for Systematic Cell Seeding Errors
To check for systematic cell seeding errors, untreated VCs are placed both at the left
side (row 2) and the right side (row 11 for the test plates) of the 96-well plate.
Aberrations in the cell monolayer for the VCs may reflect a volatile and toxic test
article present in the assay.
Checks for cell seeding errors may also be performed by examining each plate undera phase contrast microscope to assure that cell quantity is consistent.
c) Quality Check of Concentration-Response
The IC50 derived from the concentration-response of the test chemicals will bebacked by preferably three responses ≥ 10 % and ≤ 90 % inhibition of NRU and at
least two responses, one on either side of the IC50 value (see sections VII.E.3.b and
VII.F.5.a.3). If this is not the case, and the concentration progression factor can beeasily reduced, reject the experiment and repeat it with a smaller progression factor.
In addition, the dilution scheme shall be adjusted in subsequent replicate assays, if
necessary, to increase the number of points on both sides of the IC50 in the 10-90%
response range. Numerical scoring of the cells (see VII.F.3) should be determinedand documented in the Study Workbook.
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G. Data Analysis
A calculation of cell viability expressed as NRU is made for each concentration of the test
chemical by using the mean NRU of the six replicate values (minimum of four acceptable
replicate well) per test concentration (blanks will be subtracted). The Study Director will use
good biological/scientific judgment for determining “unusable” wells that will be excluded
from the statistical analysis. This value is compared with the mean NRU of all VC values(provided VC values have met the VC acceptance criteria). Relative cell viability is thenexpressed as percent of untreated VC. If achievable, the eight concentrations of eachchemical tested will span the range of no effect up to total inhibition of cell viability. Data
from the microtiter plate reader shall be transferred to the Excel spreadsheet (template withmacros provided by the SMT) that will automatically determine cell viability and performstatistical analyses (including determination of outliers).
The concentration of a test chemical reflecting a 20 %, 50 %, and 80 % inhibition of cell
viability (i.e., the IC20, IC50, and IC80) is determined from the concentration-response by
applying a Hill function to the concentration-response data. Statistical software (e.g.,
GraphPad PRISM 3.0) specified by the SMT shall be used to calculate IC20, IC50, and IC80
values (and the associated confidence limits) for each test chemical. In addition, the SMT
shall provide guidelines for calculating ICx values and confidence limits. The Testing
Facility shall report data using at least three (3) significant figures and shall forward theresults from each assay to the SMT/biostatistician through the designated contacts inelectronic format and hard copy upon completion of testing. The SMT will be directly
responsible for the statistical analyses of the Validation Study data.
VIII.

REFERENCES
Hackenberg, U. and H. Bartling. 1959. Messen und Rechnen im pharmakologischen
Laboratorium mit einem speziellen Zahlensystem (WL24-System). Arch. Exp. Pathol.
Pharmakol. 235: 437-463.
Spielmann, H., S. Gerner, S. Kalweit, R. Moog, T. Wirnserberger, K. Krauser, R. Kreiling, H.
Kreuzer, N.P. Luepke, H.G. Miltenburger, N. Müller, P. Murmann, W. Pape, B. Siegmund, J.
Spengler, W. Steiling, and F.J. Wiebel. 1991. Interlaboratory assessment of alternatives tothe Draize eye irritation test in Germany. Toxicol. In Vitro 5: 539-542.

IX.

APPROVAL__________________________________SPONSOR REPRESENTATIVE(Print or type name)___________________DATE_____________________________
Test Facility STUDY DIRECTOR(Print or type name)____________________
DATE

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Appendix C6

Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test (Phase II)

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TEST METHOD PROTOCOL
for the NHK Neutral Red Uptake Cytotoxicity Test

A Test for Basal Cytotoxicity for an In Vitro Validation Study
Phase IIMay 15, 2003

Prepared by
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)

Based on Standard Operating Procedure Recommendations from anInternational Workshop Organized by the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM)

National Institute of Environmental Health Sciences (NIEHS)
National Institutes of Health (NIH)
U.S. Public Health Service
Department of Health and Human Services

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TEST METHOD PROTOCOLThe Normal Human Keratinocyte (NHK) Neutral Red Uptake
Cytotoxicity Test
A Test for Basal Cytotoxicity
Phase III.

PURPOSEThe purpose of this study is to evaluate the cytotoxicity of test chemicals using the Normal
Human Keratinocyte (NHK) Neutral Red Uptake (NRU) cytotoxicity test. The data will be used
to evaluate the intra- and inter-laboratory reproducibility of the assay and effectiveness of thecytotoxicity assay to predict the starting doses for rodent acute oral systemic toxicity assays. This
test method protocol outlines the procedures for performing the cytotoxicity test and is in supportof the in vitro validation study organized by NICEATM and the European Centre for the
Validation of Alternative Methods (ECVAM) and sponsored by NIEHS, U.S. Environmental
Protection Agency, and ECVAM. This test method protocol applies to all personnel involved
with performing the cytotoxicity assay.
A. NHK Neutral Red Uptake Cytotoxicity Test
The NHK NRU test will be performed to analyze the in vitro toxicity of nine (9)blinded/coded test chemicals. This test will be used to determine IC20, IC50, and IC80 values
for the predetermined set of test chemicals of varying toxicities.

II. SPONSOR
A. Name:National Institute of Environmental Health Sciences (NIEHS); The
National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)

B. Address:

P.O. Box 12233
Research Triangle Park, NC 27709

C. Representative:

Named Representative

III. IDENTIFICATION OF TEST AND CONTROL SUBSTANCES
A. Test Chemicals:

Blinded chemicals (9)B. Controls:

Positive:
Vehicle (Negative):
Solvent (as needed):C-112

Sodium Lauryl Sulfate
Assay medium
Assay medium with appropriate solvent
used to prepare the test chemicals (Section
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IV. TESTING FACILITY AND KEY PERSONNEL
A. Facility Information
1) Name:2) Address:
3) Study Director:
4) Laboratory Technician(s):5) Scientific Advisor:
6) Quality Assurance Director:
7) Safety Manager:
8) Facility Management:
B. Test Schedule
1) Proposed Experimental Initiation Date:
2) Proposed Experimental Completion Date:
3) Proposed Report Date:
V. TEST SYSTEMThe NRU cytotoxicity assay procedure is a cell survival/viability chemosensitivity assay based on
the ability of viable cells to incorporate and bind neutral red (NR), a supravital dye. NR is a weakcationic dye that readily penetrates cell membranes by non-ionic diffusion and accumulates
intracellularly in lysosomes. Alterations of the cell surface or the sensitive lysosomal membranelead to lysosomal fragility and other changes that gradually become irreversible. Such changes
brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It isthus possible to distinguish between viable, damaged, or dead cells, which is the basis of thisassay.
Healthy mammalian cells, when maintained in culture, continuously divide and multiply overtime. A toxic chemical, regardless of site or mechanism of action, will interfere with this process
and result in a reduction of the growth rate as reflected by cell number. Cytotoxicity is expressed
as a concentration dependent reduction of the uptake of the NR after chemical exposure thus
providing a sensitive, integrated signal of both cell integrity and growth inhibition.
VI. DEFINITIONS
A.. Hill function: a four parameter logistic mathematical model relating the concentration of test
chemical to the response being measured in a sigmoidal shape.

Y = Bottom +Top ! Bottom
1 + 10(logIC50! X)HillSlope

where Y= response, X is the logarithm of dose (or concentration), Bottom is the minimum
response, Top is the maximum response, logIC50 is logarithm of X at the response midway
between Top and Bottom, and HillSlope describes the steepness of the curve.
B. Documentation: all methods and procedures will be noted in a Study Workbook; logs will bemaintained for general laboratory procedures and equipment (e.g., media preparation, test
chemical preparation, incubator function); all optical density data obtained from theC-113

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spectrophotometer plate reader will be saved in electronic and paper formats; all calculationsof ICx values and other derived data will be in electronic and paper format; all data will bearchived.
VII.

PROCEDURES

A. Materials[Note: Suggested brand names/vendors are listed in parentheses. Equivalents may be used
unless otherwise noted.]
1. Cell Lines
Normal Human Epidermal Keratinocytes (NHK)Non-transformed cells; from cryopreserved primary or secondary cells (Clonetics #CC2507 or equivalent). Cells will be Clonetics NHK cells.
Cambrex [Cambrex Bio Science, 8830 Biggs Ford Road, Walkersville, MD 21793-0127
Cambrex Europe [Cambrex Bio Science Verviers, S.P.R.L. Parc Industriel de Petit
Rechain, B-4800 Verviers, BELGIUM]
2. Technical Equipment[Note: Suggested brand names/vendors are listed in parentheses. Equivalents may be
used.]
a)
b)
c)
d)
e)
f)
g)
h)

Incubator: 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/airLaminar flow clean bench (standard: "biological hazard")
Water bath: 37ºC ± 1ºC
Inverse phase contrast microscope
Sterile glass tubes with caps (e.g., 5mL)
Centrifuge (optionally: equipped with microtiter plate rotor)
Laboratory balance
96-well plate spectrophotometer (i.e., plate reader) equipped with 540 nm ± 10 nm
filteri) Shaker for microtiter platesj) Cell counter or hemocytometerk) Pipetting aidl) Pipettes, pipettors (multi-channel and single channel; multichannel repeater pipette),
dilution blockm) Cryotubesn) Tissue culture flasks (75 - 80 cm2, 25 cm2)o) 96-well flat bottom tissue culture microtiter plates (e.g., Nunc # 167 008;
Corning/COSTAR tissue culture-treated)p) pH paper (wide and narrow range)q) Multichannel reagent reservoirr) Waterbath sonicators) Magnetic stirrer

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November 2006t) Antistatic bar ionizer/antistatic gun (optional for neutralizing static on 96-well plates)u) Dry heat block (optional)[Note: Tissue culture flasks and microtiter plates should be prescreened to ensure thatthey adequately support the growth of NHK. Multi-channel repeater pipettes may be
used for plating cells in the 96-well plates, dispensing plate rinse solutions, NR medium,and desorb solution. Do not use the repeater pipette for dispensing test chemicals to thecells.]
3. Chemicals, Media, and Seraa) Keratinocyte Basal Medium without Ca++ (KBM®, Clonetics CC-3104) that iscompleted by adding the KBM® SingleQuots® (Clonetics CC-4131) to achieve theproper concentrations of epidermal growth factor, insulin, hydrocortisone,
antimicrobial agents, bovine pituitary extract, and calcium (e.g., Clonetics Calcium
SingleQuots®, 300 mM CaCl2, Clonetics # CC-4202).
b) HEPES Buffered Saline Solution (HEPES-BSS) (e.g., Clonetics # CC-5022)c) 0.025 % Trypsin/EDTA solution (e.g., Clonetics # CC-5012)
d) Trypsin Neutralizing Solution (TNS) (e.g., Clonetics # CC-5002)e) Phosphate Buffered Saline (PBS)f) Dulbecco’s Phosphate Buffered Saline (D-PBS) [formulation containing calcium andmagnesium cations; glucose optional] (for rinsing)g) Neutral Red (NR) Dye – tissue culture-grade; liquid form (e.g., SIGMA N 2889);
powder form (e.g., SIGMA N 4638)h) Dimethyl sulfoxide (DMSO), U.S.P analytical grade (Store under nitrogen @ -20ºC)i) Ethanol (ETOH), U.S.P. analytical grade (100 %, non-denatured for test chemical
preparation; 95 % can be used for the desorb solution)j) Glacial acetic acid, analytical gradek) Hanks' Balanced Salt Solution without Ca2+ or Mg2+ (CMF-HBSS) (e.g., Invitrogen #14170)l) Distilled H2 O or any purified water suitable for cell culture (sterile)m) Sterile paper towels (for blotting 96-well plates)
B. Preparations of Media and Solutions[Note: All solutions (except NR stock solution, NR medium and NR desorb), glassware,
pipettes, etc., shall be sterile and all procedures should be carried out under aseptic conditions
and in the sterile environment of a laminar flow cabinet (biological hazard standard). All
methods and procedures will be adequately documented.]
1. Mediaa) Routine Culture Medium/Treatment Medium
KBM® (Clonetics CC-3104) supplemented with KBM® SingleQuots® (Clonetics
CC-4131) and Clonetics Calcium SingleQuots® (CC-4202) to make 500 mL medium.
Final concentration of supplements in medium are:0.0001 ng/mL Human recombinant epidermal growth factor
5 µg/mL
Insulin
0.5 µg/mL
Hydrocortisone

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15 ng/mL
0.10 mM
30 µg/mL

November 2006

Gentamicin
Amphotericin B
Calcium
Bovine pituitary extract

Complete media should be kept at 2-8°C and stored for no longer than two weeks.
NOTE:
KBM® SingleQuots® contain the following stock concentrations and volumes:
0.1 ng/mL
5.0 mg/mL
0.5 mg/mL
30 mg/mL
7.5 mg/mL

hEGF
0.5 mL
Insulin
0.5 mL
Hydrocortisone
0.5 mL
Gentamicin, 15 ug/mL Amphotericin-B 0.5 mLBovine Pituitary Extract (BPE)
2.0 mL

Clonetics Calcium SingleQuots® are 2 mL of 300mM calcium.
165 µl of solution per 500 mL calcium-free medium equals 0.10 mM calcium in themedium.
2. Neutral Red (NR) Stock Solution
The liquid tissue culture-grade stock NR Solution will be the first choice for performing
the assay (e.g., SIGMA #N2889, 3.3 mg/mL). Store liquid tissue culture-grade NR Stock
Solution at the storage conditions and shelf-life period recommended by the
manufacturer.
If the liquid form is not available, the following formulation can be prepared.
EXAMPLE:

0.33 g NR Dye powder in 100 mL H2 O

The NR Stock Solution (powder in water) should be stored in the dark at room
temperature for up to two months.
3. Neutral Red (NR) Medium
EXAMPLE:
1.0 mL (3.3 mg NR dye/mL)
99.0 mL

NR Stock Solution
Routine Culture Medium (pre-warmed to 37° C.)

The final concentration of the NR Medium is 33 µg NR dye/mL and aliquots will be
prepared on the day of application.
[Note: The NR Medium shall be filtered (e.g., Millipore filtering, 0.2 – 0.45 µm poresize) used to reduce NR crystals. Aliquots of the NR Medium should be maintained at
37° C (e.g., in a waterbath) before adding to the cells and used within 30 min of
preparation but also used within 15 min after removing from 37° C storage.]

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4. Ethanol/Acetic Acid Solution (NR Desorb)
1%
50 %
49 %

Glacial acetic acid solution
Ethanol
H2 O

C. Methods
1. Cell Maintenance and Culture Procedures
NHK cells are routinely grown as a monolayer in tissue culture grade flasks (e.g., 25 cm2)
at 37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air. The cells should be
examined on a daily (i.e., on workdays) basis under a phase contrast microscope, and anychanges in morphology or their adhesive properties must be noted in a Study Workbook.
2. Receipt of Cryopreserved Keratinocytes
Upon receipt of cryopreserved keratinocytes, the vial(s) of cells shall be stored in a liquid
nitrogen freezer until needed.
3. Thawing Cells and Establishing Cell Culturesa) Thaw cells by putting ampules into a water bath at 37°C for as brief a time as
possible. Do not thaw cells at room temperature or by hand. Seed the thawed cells
into culture flasks as quickly as possible and with minimal handling.
b) Slowly (taking approximately 1-2 min) add 9 mL of pre-warmed Routine Culture
Medium to the cells suspended in the cryoprotective solution and transfer cells into
flasks containing pre-warmed Routine Culture Medium (See Table 1).
c) Incubate the cultures at 37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air until
the cells attach to the flask (within 4 to 24 h), at which time the Routine Culture
Medium should be removed and replaced with fresh Routine Culture Medium.
d) Unless otherwise specified, the cells should be incubated at 37ºC ± 1ºC, 90 % ± 5 %humidity, 5.0 % ± 1 % CO2/air and fed every 2-3 days until they exceed 50 %confluence (but less than 80 % confluent).
Table 1. Establishing Cell Cultures
Cells/25 cm2 flask(in approximately 5 mL)
1 flask each cell concentration
Approximate Time to Subculture
Cells to 96-Well Plates

6.25 x 104(2500/cm2)

1.25 x 105(5000/cm2)

2.25 x 105(9000/cm2)

96+ hours
6 – 8 plates

72 - 96 hours
6 – 8 plates

48 - 72 hours
6 – 8 plates

Cell growth guidelines – actual growth of individual cell lots may vary.
4. Subculture of NHK Cells to 96-Well Plates

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[Note: It is important that cells have overcome the lag growth phase when they are used
for the test. Keratinocytes will be passaged only into the 96-well plates and will not be
subcultured into flasks for use in later assays]a) When the keratinocyte culture in a 25 cm2 flask exceeds 50 % confluence (but less than
80 % confluent), remove the medium and rinse the culture twice with 5 mL HEPESBSS. The first rinse may be left on the cells for up to 5 minutes and the second rinseshould remain on the cells for approximately 5 minutes. Discard the washing solutions.
b) Add 2 mL trypsin/EDTA solution to each flask and remove after 15 to 30 seconds.
Incubate the flask at room temperature for 3 to 7 min. When more than 50 % of thecells become dislodged, rap the flask sharply against the palm of the hand.
c) When most of the cells have become detached from the surface, rinse the flask with
5 mL of room temperature TNS. If more than one flask is subcultured, the same 5 mLof TNS may be used to rinse a total of up to two flasks.
d) Then rinse the flask with 5 mL CMF-HBSS and transfer the cell suspension to acentrifuge tube.
e) Pellet the cells by centrifugation for 5 min at approximately 220 x g. Remove thesupernatant by aspiration.
f)Resuspend the keratinocyte pellet by gentle trituration (to have single cells) in Routine
Culture Medium. It is important to obtain a single cell suspension for exact counting.
Count a sample of the cell suspension using a hemocytometer or cell counter.
4g) Prepare a cell suspension –1.6 – 2.0 x10 cells/mL in Routine Culture Medium.
Using a multi-channel pipette, dispense 125 µl Routine Culture Medium only into theperipheral wells (blanks) of a 96-well tissue culture microtiter plate. In the remaining
3
3
wells, dispense 125 µl of the cell suspension (2x10 – 2.5x10 cells/well). Prepare
one plate per chemical to be tested (see Figure 2, Section VII.F.1).
h) Incubate cells (37ºC ± 1ºC, 90 % ± 5.0 % humidity, and 5 % ± 1 % CO2/air) so thatcells form a 20+ % monolayer (~48-72 h). This incubation period assures cellrecovery and adherence and progression to exponential growth phase.
i)

Examine each plate under a phase contrast microscope to assure that cell growth isrelatively even across the microtiter plate. This check is performed to identify
experimental and systemic cell seeding errors. Record observations in the Study
Workbook.

5. Determination of Doubling Timea) A cell doubling time procedure was performed on the initial lot of cells that was usedin the first cell culture assays of Phase Ia of the Validation Study. The doubling time
only needs to be determined in Phase II if there is a change in the lot of cells used.
Establish cells in culture and trypsinize cells as per Section VII.C.4 for subculture.
Resuspend cells in appropriate culture medium. Use Table 1 to determine seeding
densities.

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November 2006b) Seed five sets of cell culture vessels in triplicate for each cell type (e.g., 15 tissue
culture dishes [60mm x 15mm]). Use appropriate volume of culture medium for theculture vessels. Note number of cells placed into each culture dish. Place dishes
into the incubators (37ºC ± 1ºC, 90 % ± 5 % humidity, 5.0 % ± 1 % CO2/air).
c) After 4-6 hours (use the same initial measurement time for each subsequent doublingtime experiment), remove three culture dishes and trypsinize cells. Count cells using
a cell counter or hemocytometer. Cell viability may be determined by dye exclusion(e.g., Trypan Blue; Nigrosin). Determine the total number of cells and document.
Repeat sampling at 24 hr, 48 hr, 72 hr, and 96 hr post inoculation. Change culture
medium at 72 hr or sooner in remaining dishes if indicated by pH drop.
d) Plot cell concentration (per mL of medium) on a log scale against time on a linear
scale. Determine lag time and population doubling time. The doubling time will bein the log (exponential) phase of the growth curve. Additional dishes and time areneeded if the entire growth curve is to be determined (lag phase, log phase, plateau
phase).
D. Solubility Test
The preference of solvent for dissolving test chemicals is medium, DMSO, and then ethanol.
Solubility shall be determined in a step-wise procedure that involves attempting to dissolve atest chemical at a relatively high concentration with the sequence of mechanical procedures
specified in Section VII.D.2.a. If the chemical does not dissolve, the volume of solvent isincreased so as to decrease the concentration by a factor of 10, and then the sequence of
mechanical procedures in Section VII.D.2.a are repeated in an attempt to solubilize thechemical at the lower concentrations. For testing solubility in media, the starting
concentration is 20,000 µg/ml (i.e., 20 mg/mL) in Tier 1, but for DMSO and ethanol thestarting concentration is 200,000 µg/ml (i.e., 200 mg/mL) in Tier 2. Weighing out chemical
for each solvent (i.e., media, DMSO, ethanol) can be done all at once, if convenient, butsolubility testing (at each tier that calls for more than one solvent) is designed to be sequential- media, then DMSO, then ethanol – in accordance with the solvent hierarchy (see Figure 1).
This allows for testing to stop, rather than continue testing with less preferred solvents, if thetest chemical dissolves in a more preferred solvent. For example, if a chemical is soluble inmedium at a particular tier, testing may stop. Likewise, if a chemical is soluble in DMSO at
any tier, testing need not continue with ethanol. However, since the issue of primary
importance is testing the solvents and concentrations of test chemical required by any one
tier, sequential testing of solvents may be abandoned if the lab can test more efficiently inanother way.
1. Determination of Solubilitya) Tier 1 begins with testing 20 mg/mL in Routine Culture Medium (see Table 2).
Approximately 10 mg (10,000 µg) of the test chemical will be weighed into a glass
tube and the weight will be documented. A volume of Routine Culture Medium,approximately 0.5 mL, will be added to the vessel so that the concentration is 20,000µg/ml (20 mg/mL). The solution is mixed as specified in Section VII.D.2.a. If
complete solubility is achieved in media, then additional solubility procedures are notneeded.

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chemical dissolves in medium at 2 mg/mL, no further procedures are necessary. If
the test chemical does NOT dissolve, weigh out approximately 100 mg test chemical
in a second glass tube and add enough DMSO to make the total volume
approximately 0.5 mL (for 200 mg/mL), and attempt to dissolve the chemical as
specified in Section VII.D.2.a. If the chemical does not dissolve in DMSO, weighout approximately 100 mg test chemical in another glass tube and add enough ethanol
to make the total volume approximately 0.5 mL (for 200 mg/mL) and attempt todissolve the chemical as specified in Section VII.D.2.a. If the chemical is soluble ineither solvent, no additional solubility procedures are needed.
c) If the chemical is NOT soluble in media, DMSO, or ethanol at Tier 2, then continue
to Tier 3 in Table 2 by adding enough solvent to increase the volume of the three
Tier 2 solutions by 10 and attempt to solubilize again using the sequence of mixing
procedures in Section VII.D.2.a. If the test chemical dissolves, no additionalsolubility procedures are necessary. If the test chemical does NOT dissolve, continue
with Tier 4 and, if necessary, Tier 5 using DMSO and ethanol. Tier 4 begins by
diluting the Tier 3 samples with DMSO or ethanol to bring the total volume to 50
mL. The mixing procedures in Section VII.D.2.a are again followed to attempt tosolubilize the chemical. Tier 5 is performed, if necessary, by weighing out anothertwo more samples of test chemical at ~10 mg each and adding ~50 mL DMSO or
ethanol for a 200 µg/mL solution, and following the mixing procedures in Section
VII.D.2.a.
Example: If complete solubility is not achieved at 20,000 µg/mL in Routine Culture
Medium at Tier 1 using the mixing procedures specified in Section VII.D.2.a, thenthe procedure continues to Tier 2 by diluting the solution to 5 mL and mixing again
as specified in Section VII.D.2.a. If the chemical is not soluble in medium, twosamples of ~ 100 mg test chemical are weighed to attempt to solubilize in DMSO andethanol at 200,000 µg/mL (i.e., 200 mg/mL). Solutions are mixed following thesequence of procedures prescribed in Section VII.D.2.a in an attempt to dissolve. If
solubility is not achieved at Tier 2, then the solutions (media, DMSO, and ethanol)
prepared in Tier 2 are diluted by 10 to test 200 µg/mL in media, and 20,000 µg/mL in
DMSO and ethanol. This advances the procedure to Tier 3. Solutions are again
mixed as prescribed in Section VII.D.2.a in an attempt to dissolve. If solubility is
not achieved in Tier 3, the procedure continues to Tier 4, and to 5 if necessary (seeFigure 1 and Table 2).

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Table 2 Determination of Solubility in Routine Culture Medium, DMSO, or Ethanol
TIER

1

2

3

Total Volume
MediumConcentration of Test
Chemical(Add ~10 mg to a tube.
Add enough medium toequal the first volume.
Dilute to subsequent
volumes if necessary.)
Total Volume
DMSO/Ethanol
Concentration of Test
Chemical(Add ~100 mg to alarge tube. Add enoughDMSO or ethanol toequal the first volume.
Dilute to subsequent
volumes if necessary.)
Total Volume
DMSO/Ethanol
Concentration of Test
Chemical(Add ~10 mg to a large
tube. Add enoughDMSO or ethanol toequal 50 mL.)

0.5 mL

5 mL

50 mL

20,000 µg/mL

2,000 µg/mL

200 µg/mL(20 mg/mL)(2 mg/mL)(0.20 mg/mL)

0.5 mL

5 mL

50 mL

200,000 µg/mL

20,000 µg/mL

2,000 µg/mL(200 mg/mL)(20 mg/mL)(2 mg/mL)

Equivalent
Concentration on Cells

4

5

50 mL
200 µg/mL(0.2 mg/mL)
10,000 µg/mL1000 µg/mL

100 µg/mL

10 µg/mL

1 µg/mL(10 mg/mL)(1 mg/mL)(0.1 mg/mL)(0.01 mg/mL)(0.001 mg/mL)

NOTE: The amounts of test chemical weighed and Routine Culture Medium added
may be modified from the amounts given above, provided that the targeted
concentrations specified for each tier are tested.

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Figure 1. Solubility Flow Chart

STEP 1:TIER 120 mg/mL test chemical (TC) in 0.5 mL medium:• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 2.

STEP 2:TIER 2
2 mg/mL TC in medium – increase volume from STEP 1 by 10 (i.e., to 5 mL)• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 3.

STEP 3:

STEP 4:

STEP 5:

STEP 6:200 mg/mL TC in DMSO
• if TC soluble, then STOP.
• if TC insoluble, test at 200 mg/mL in ETOH.
• if TC soluble, then STOP.
• If TC insoluble, go to STEP 4.
TIER 30.2 mg/mL TC in medium – increase volume from STEP 2 by 10 (i.e., to 50 mL)• if TC soluble, then STOP.
• if TC insoluble, test at 20 mg/mL in DMSO – increase volume from STEP 3 by 10(i.e., to 5 mL).
• if TC soluble, then STOP.
• if TC insoluble, test at 20 mg/mL in ETOH – increase volume from STEP 3 by
10 (i.e., to 5 mL).
• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 5.
TIER 42 mg/mL TC in DMSO – increase volume from STEP 4 by 10 (i.e., to 50 mL)• if TC soluble, then STOP.
• if TC insoluble, test at 2 mg/mL in ETOH – increase volume from STEP 4 by 10(i.e., to 50 mL).
• if TC soluble, then STOP.
• if TC insoluble, then go to STEP 6.
TIER 50.2 mg/mL TC in 50 mL DMSO
• if TC soluble, then STOP.
•
if TC insoluble, test at 0.2 mg/mL in 50 mL ETOH
• STOP

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2. Mechanical Proceduresa) The following hierarchy of mixing procedures will be followed to dissolve the test
chemical:1) Add test chemical to solvent as in Tier 1 of Table 2.
2) Gently mix. Vortex the tube (1 –2 minutes).
3) If test chemical hasn’t dissolved, use sonication for up to 5 minutes.
4) If sonication doesn’t work, then warm solution to 37°C for 5 – 60 minutes. Thiscan be performed by warming tubes in a 37°C water bath or in a CO2 incubator at
37°C. The solution may be stirred during warming (stirring in a CO2 incubator
will help maintain proper pH).
5) Proceed to Tier 2 (and Tiers 3-5, if necessary of Table 2 and repeat procedures 24).
b) The preference of solvent for dissolving test chemicals is medium, DMSO, and thenethanol. Thus, if a test chemical dissolves in more than one solvent at any one
solubility-testing tier, then the choice of solvent follows this hierarchy. For example,
if, at any tier, a chemical is soluble in medium and DMSO, but not ethanol, thechoice of solvent would be medium. If the chemical were insoluble in medium, butsoluble in DMSO and ethanol, the choice of solvent would be DMSO.
After the lab has determined the preferred solvent for the test chemical and before
proceeding to the cytotoxicity testing, the Study Director will discuss the solvent
selection with the Study Management Team (SMT) of the validation study. The
SMT will relate what solvent should be used in the assay for each chemical. If thelaboratory has attempted all solubility testing without success, then the SMT will
provide additional guidance for achieving test chemical solubility. The SMT
anticipates that all validation study test chemicals will be tested in the NRU assays.
E. Preparation of Test Chemicals[Note: Preparation under red or yellow light is recommended to preserve chemicals that
degrade upon exposure to light.]
1. Test Chemical in Solutiona) Allow test chemicals to equilibrate to room temperature before dissolving anddiluting.
b) Prepare test chemical immediately prior to use. Test chemical solutions should not
be prepared in bulk for use in subsequent tests. The solutions must not be cloudy
nor have noticeable precipitate. Each stock dilution should have at least 1-2 mL total
volume to ensure adequate solution for the test wells in a single 96-well plate. The
SMT may direct the Study Director to store an aliquot (e.g., 1 mL) of the highest 2X

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stock solution (e.g., low solubility chemicals) in a freezer (e.g., -70°C) for use in
future chemical analyses.
c) For chemicals dissolved in DMSO or ethanol, the final DMSO or ethanol
concentration for application to the cells must be 0.5 % (v/v) in the vehicle controls
and in all of the eight test concentrations.
d) The stock solution for each test chemical should be prepared at the highest
concentration found to be soluble in the solubility test. Thus, the highest test
concentration applied to the cells in each range finding experiment is:• 0.5 times the highest concentration found to be soluble in the solubility test, if thechemical was soluble in medium, or• 1/200 the highest concentration found to be soluble in the solubility test if thechemical was soluble in ethanol or DMSO.
e) The seven lower concentrations in the range finding experiment would then be
prepared by successive dilutions that decrease by one log unit each. The following
example illustrates the preparation of test chemical in solvent and the dilution ofdissolved test chemical in medium before application to NHK cells.
Example: Preparation of Test Chemical in Solvent Using a Log Dilution Scheme
If DMSO was determined to be the preferred solvent at Tier 2 of the solubility test
(i.e., 200,000 µg/mL), dissolve the chemical in DMSO at 200,000 µg/mL for thechemical stock solution.
1) Label eight tubes 1 – 8. Add 0.9 mL solvent (e.g., DMSO) to tubes 2 -- 8.
2) Prepare stock solution of 200,000 µg test chemical/mL solvent in tube # 1.
3) Add 0.1 mL of 200,000 µg/mL dilution from tube #1 to tube #2 to make a 1:10
dilution in solvent (i.e., 20,000 µg/mL).
4) Add 0.1 mL of 20,000 µg/mL dilution from tube #2 to tube #3 to make another1:10 dilution (i.e., 1:100 dilution from stock solution) in solvent (i.e., 2,000µg/mL)
5) Continuing making serial 1:10 dilutions in the prepared solvent tubes.
6) Since each concentration is 200 fold greater than the concentration to be tested,make a 1:100 dilution by diluting 1 part dissolved chemical in each tube with 99
parts of culture medium (e.g., 0.1 mL of test chemical in DMSO + 9.9 mL culture
medium) to derive the eight 2X concentrations for application to NHK cells.
Each 2X test chemical concentration will then contain 1 % v/v solvent. The
NHK cells will have 0.125 mL of culture medium in the wells prior toapplication of the test chemical. By adding 0.125 mL of the appropriate 2X test
chemical concentration to the appropriate wells, the test chemical will be diluted
appropriately (e.g., highest concentration in well will be 1,000 µg/mL) in a totalof 0.250 mL and the solvent concentration in the wells will be 0.5% v/v.

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7) A test article prepared in DMSO or ethanol may precipitate upon transfer into the
Routine Culture Medium. The 2X dosing solutions should be evaluated for
precipitates and the results will be recorded in the workbook. It will bepermissible to test all of the dosing solutions in the dose range finding assay
only. Doses containing test article precipitates should be avoided, and will not be
used in the ICx determinations for either the range finding experiments or thedefinitive tests.
Document all test chemical preparations in the Study Workbook.
2. pH of Test Chemical Solutions
Measure the pH of the highest concentration of the test chemical in culture medium using
pH paper (e.g., pH 0 – 14 to estimate and pH 5 – 10 to determine more precise value).
The pH paper should be in contact with the solution for approximately one minute.
Document the pH and note the color of the medium for all dilutions. Do not adjust thepH.
3. Concentrations of Test Chemicala) Range Finder Experiment
Test eight concentrations of the test chemical by diluting the stock solution with aconstant factor covering a large range. The initial dilution series shall be log
dilutions (e.g., 1:10, 1:100, 1:1000, etc.).
The data from any well that has precipitate will be excluded from any calculations.
b) Main Experiment[Note: After the range finding assay is completed, the concentration-response
experiment shall be performed three times on three different days for each chemical(i.e., one plate per day per chemical)]
Depending on the slope of the concentration-response curve estimated from the range
finder, the dilution/progression factor in the concentration series of the main
experiment should be smaller (6√10 = 1.47). Cover the relevant concentration range(≥10 % and ≤ 90 % effect) preferably with three points of a graded effect, but with aminimum of two points, one on each side of the estimated IC50 value, avoiding too
many non-cytotoxic and/or 100 %-cytotoxic concentrations. Experiments revealing
less than one cytotoxic concentration on each side of the IC50 value shall be repeated,
where possible, with a smaller dilution factor. Each experiment should have at least
one cytotoxicity value ≥ 10.0 % and ≤ 50.0 % viability and at least one cytotoxicity
value > 50.0 % and ≤ 90.0 % viability. In addition, the dilution scheme shall be
adjusted in subsequent replicate assays (i.e., definitive assays), if necessary, toincrease the number of points on both sides of the IC50 in the 10-90% response range.
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Determine which test chemical concentration is closest to the IC50 value (e.g., 50 %cytotoxicity). Use that value as a central concentration and adjust dilutions higher
and lower in equal steps for the definitive assay.
Maximum Doses to be Tested in the Main Experiments
If minimal or no cytotoxicity was measured in the dose range finding assay, amaximum dose for the main experiments will be established as follows:• For test chemicals prepared in Routine Culture Medium, the highest test article
concentration that may be applied to the cells in the main experiments will beeither 100 mg/mL, or the maximum soluble dose. Test chemical will be weighed
into a glass tube and the weight will be documented. A volume of Routine
Culture Medium will be added to the vessel so that the concentration is200,000 µg/mL (200 mg/mL). The solution is mixed as specified in Section
VII.D.2.a. If complete solubility is achieved in medium, then 7 additional serial
stock dosing solutions may be prepared from the 200 mg/mL 2X stock. If thetest chemical is insoluble in medium at 200 mg/ml, proceed by adding medium,in small incremental amounts, to attempt to dissolve the chemical by using thesequence of mixing procedures specified in Section VII.D.2.a. The highest
soluble stock solution will be used to prepare the 7 additional serial stock dosing
solutions.
•

For test chemicals prepared in either DMSO or ethanol, the highest test article
concentration that may be applied to the cells in the main experiments will beeither 2.5 mg/mL, or less, depending upon the maximum solubility in solvent.
Test chemical will be weighed into a glass tube and the weight will be
documented. A volume of the appropriate solvent (determined from the original
solubility test) will be added to the vessel so that the concentration is500,000 µg/mL (500 mg/mL). The solution is mixed as specified in Section
VII.D.2.a. If complete solubility is achieved in the solvent, then 7 additional
serial stock dosing solutions may be prepared from the 500 mg/mL 200X stock.
If the test chemical is insoluble in solvent at 500 mg/ml, proceed by adding
solvent, in small incremental amounts, to attempt to dissolve the chemical byusing the sequence of mixing procedures specified in Section VII.D.2.a. The
highest soluble stock solution will be used to prepare the 7 additional serial stock
dosing solutions.

c) Test Chemical Dilutions
The dosing factor of 3.16 (= 2√10) divides a log into two equidistant steps, a factor of
2.15 (= 3√10) divides a decade into three steps. The factor of 1.47 (= 6√10) divides alog into six equidistant steps, and the factor of 1.21 (= 12√10) divides the log into 12
steps.
EXAMPLE:
10
10
10
10

31.6

12.1

14.7
14.7

17.8

21.5
21.5
21.5

26.1

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31.6
31.6

38.3

46.4
46.4
46.4

56.2

68.1
68.1

82.5

100
100
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The technical production of decimal geometric concentration series is simple. An
example is given for factor 1.47:
Dilute 1 volume of the highest concentration by adding 0.47 volumes of diluent. Afterequilibration, dilute 1 volume of this solution by adding 0.47 volumes ofdiluent...(etc.).
F. Test Procedure
1. 96-Well Plate Configuration
The NHK NRU assay for test chemicals will use the 96-well plate configuration shown in
Figure 2.
Figure 2. 96-Well Plate Configuration for Positive Control and Test Chemical Assays
1

2

3

4

5

6

7

8

9

10

11

12

A

VCb VCb C1b

C2b

C3b

C4b

C5b

C6b

C7b

C8b

VCb VCbB

VCb VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

VCb

C

VCb VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

VCb

D

VCb VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

VCb

E

VCb VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

VCb

F

VCb VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

VCb

G

VCb VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

VCb

H

VCb VCb C1b

C2b

C3b

C4b

C5b

C6b

C7b

C8b

VCb VCbVC=
C1 – C8 =
b
VCb

=
=

untreated VEHICLE CONTROL (mean viability set to 100 %)Test Chemicals or Positive Control (SLS) at eight concentrations(C1 = highest, C8 = lowest)
BLANKS (contain no cells)
VEHICLE CONTROL BLANK

2. Application of Test Chemicala) Two optional methods for rapidly applying the 2X dosing solutions onto the 96-wellplates may be utilized.
1) The first method is to add each of the 2X dosing solutions into labeled, sterile
reservoirs (e.g., Corning/Costar model 4870 sterile polystyrene 50 mL reagent
reservoirs or Corning/Transtar model 4878 disposable reservoir liners, 8-channel; orother multichannel reservoirs).

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2) The second method utilizes a “dummy” plate (i.e., an empty sterile 96-well plate)
prepared to hold the dosing solutions immediately prior to treatment of the test plate(with cells). The test chemical and control dosing solutions should be dispensed into
the dummy plate in the same pattern/order as will be applied to the plate containing
cells. More volume than needed for the test plate (i.e. greater than 125 µl/well)should be in the wells of the dummy plate.
At the time of treatment initiation, a multi-channel micropipettor is used to transfer the
2X dosing solutions, from the reservoirs or dummy plate, to the appropriate wells on thetreatment plate (as described in step c. below). These methods will ensure that the dosing
solutions can be transferred rapidly to the appropriate wells of the test plate to initiate
treatment times and to minimize the range of treatment initiation times across a large
number of treatment plates, and to prevent “out of order” dosing. Do not use a
multichannel repeater pipette for dispensing test chemical to the plates.
b) After 48 - 72 h (i.e., after cells attain 20+ % confluency [see Section VII.C.4(h)])
incubation of the cells, add 125 µl of the appropriate concentration of test chemical, thePC, or the VC (see Figure 2 for the plate configuration) directly to the test wells. Do notremove Routine Culture Medium for re-feeding the cells. The dosing solutions will be
rapidly transferred from the 8-channel reservoir (or dummy plate) to the test plate using asingle delivery multi-channel pipettor. For example, the VC may be transferred first (intocolumns 1, 2, 11, and 12), followed by the test article dosing solutions from lowest to
highest dose, so that the same pipette tips on the multi-channel pipettor can be used forthe whole plate. [The Vehicle Control blank (VCb) wells (column 1, column 12, wells
A2, A11, H2, H11) will receive the Vehicle Control dosing solutions (which should
include any solvents used). Blanks for wells A3 – A10 and H3 – H10 shall receive theappropriate test chemical solution for each concentration (e.g., wells A3 and H3 receive
C1 solution). The test chemical blanks in rows A and H will be used for their respective
test chemical concentrations.] Incubate cells for 48 h ± 0.5 h (37ºC ± 1ºC, 90 % ± 5 %humidity, and 5.0 % ± 1 % CO2/air).
c) Positive Control: For each set of test chemical plates used in an assay, a separate plate of
positive control concentrations will be set up following the concentration range
established in the development of the positive control database in Phase I of the
Validation Study. If multiple sets of test chemical plates are set up, then clearly
designate the positive control plates for each set; each set will be an individual entity.
The mean IC50 ± two and a half standard deviations (SD) for the SLS acceptable tests
from Phases Ia and Ib (after the removal of outliers) are the values that will be used as an
acceptance criterion for test sensitivity for the NHK NRU assay. This plate will follow
the same schedule and procedures as used for the test chemical plates (including
appropriate chemical concentrations in the appropriate wells – see sections VII.F.1 andF.2)..
3. Microscopic Evaluation
After at least 46 h treatment, examine each plate under a phase contrast microscope toidentify systematic cell seeding errors and growth characteristics of control and treatedcells. Record any changes in morphology of the cells due to the cytotoxic effects of thetest chemical, but do not use these records for any quantitative measure of cytotoxicity.
Undesirable growth characteristics of control cells may indicate experimental error andC-128

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may be cause for rejection of the assay. Use the following Visual Observations Codes inthe description of cell culture conditions.
Visual Observations Codes
Note Code
1
2
3
4
1P
2P
3P
4P
5P

Note Text
Normal Cell Morphology
Low Level of Cell Toxicity
Moderate Level of Cell Toxicity
High level of Cell Toxicity
Normal Cell Morphology with Precipitate
Low Level of Cell Toxicity with Precipitate
Moderate Level of Cell Toxicity with PrecipitateHigh level of Cell Toxicity with Precipitate
Unable to View Cells Due to Precipitate

4. Measurement of NRUa) Carefully remove (i.e., “dump”) the Routine Culture Medium (with test chemical)
and rinse the cells very carefully with 250 µL pre-warmed D-PBS. Remove therinsing solution by dumping and remove excess by gently blotting on sterile paper
towels. Add 250 µL NR medium (to all wells including the blanks) and incubate(37ºC ± 1ºC, 90 % ± 5 % humidity, and 5.0 % ± 1 % CO2/air) for 3±0.1 h. Observe
the cells briefly during the NR incubation (e.g., between 2 and 3 h – Study Director‘s
discretion) for NR crystal formation. Record observations in the Study Workbook.
Study Director can decide to reject the experiment if excessive NR crystallization has
occurred.
b) After incubation, remove the NR medium, and carefully rinse cells with 250 µL prewarmed D-PBS.
c) Decant and blot D-PBS from the plate. (Optionally: centrifuge the reversed plate.)d) Add exactly 100 µL NR Desorb (ETOH/acetic acid) solution to all wells, including
blanks.
e) Shake microtiter plate rapidly on a microtiter plate shaker for 20 – 45 min to extract
NR from the cells and form a homogeneous solution. Plates should be protectedfrom light by using a cover during shaking.
f)

Plates should be still for at least five minutes after removal from the plate shaker (ororbital mixer). Observe the wells for bubbles. Measure the absorption (within 60
minutes of adding NR Desorb solution) of the resulting colored solution at 540 nm ±10 nm in a microtiter plate reader (spectrophotometer), using the blanks as areference. [Phases Ia and Ib data show the mean OD value for the plate blanks to be
0.055 ± 0.035 for NHK cells (± 2.5 standard deviations; data from 3 labs; N = 156).
Use this range as a guide for assessment of the blank values.] Save raw data in the
Excel format as provided by the SMT.

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5. Quality Check of Assaya) Test Acceptance Criteria
1) A test meets acceptance criteria, if the IC50 for SLS is within two and a half (2.5)
standard deviations of the historical mean established by the Test Facility (as per
VII.F.2.c).
2) A test meets acceptance criteria if the left and the right mean of the VCs do notdiffer by more than 15.0 % from the mean of all VCs.
3) A test meets acceptance criteria if:• at least one calculated cytotoxicity value ≥ 10.0 % and ≤ 50.0 % viability and• at least one calculated cytotoxicity value > 50.0 % and ≤ 90.0 % viability.
4) A test meets acceptance criteria if the r2 (coefficient of determination) value
calculated for the Hill model fit (i.e., from PRISM® software) is ≥ 0.90. A test
does not meet acceptance criteria if the r2 value is < 0.80. If the r2 value is ≥ 0.80
and < 0.90 (“gray zone”), then the SMT will evaluate the model fit and make thedetermination of whether or not the test meets the acceptance criteria and relate
the information to the Study Director.
[Note: All acceptance criteria must be met for an assay to be considered acceptable.][A corrected mean OD540 ± 10nm of 0.205 - 1.645 for the VCs is a target range but will
not be a test acceptance criterion. Range determined from Phase Ib VC OD values
from 3 laboratories (mean ± 2.5 standard deviations, N = 69).]b) Checks for Systematic Cell Seeding Errors
To check for systematic cell seeding errors, untreated VCs are placed both at the left
side (row 2) and the right side (row 11 for the test plates) of the 96-well plate.
Aberrations in the cell monolayer for the VCs may reflect a volatile and toxic test
article present in the assay.
Checks for cell seeding errors may also be performed by examining each plate undera phase contrast microscope to assure that cell quantity is consistent.
c) Quality Check of Concentration-Response
The IC50 derived from the concentration-response of the test chemicals should bebacked by preferably three responses ≥ 10 and ≤ 90 % inhibition of NRU and at least
two responses, one on either side of the IC50 value (see sections VII.E.3.b and
VII.F.5.a.3). If this is not the case, and the concentration progression factor can beeasily reduced, reject the experiment and repeat it with a smaller progression factor.
In addition, the dilution scheme shall be adjusted in subsequent replicate assays, if
necessary, to increase the number of points on both sides of the IC50 in the 10-90%
response range. Numerical scoring of the cells (see VII.F.3) should be determinedand documented in the Study Workbook.

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G. Data Analysis
A calculation of cell viability expressed as NRU is made for each concentration of the test
chemical by using the mean NRU of the six replicate values (minimum of four acceptable
replicates wells) per test concentration. The Study Director will use good biological/scientificjudgment for determining “unusable” wells that will be excluded from the statistical analysis.
This value is compared with the mean NRU of all VC values (provided VC values have met
the VC acceptance criteria). Relative cell viability is then expressed as percent of untreated
VC. If achievable, the eight concentrations of each chemical tested will span the range of noeffect up to total inhibition of cell viability. Data from the microtiter plate reader shall betransferred to the Excel® spreadsheet (template with macros provided by the SMT) that willautomatically determine cell viability and perform statistical analyses (including
determination of outliers).
The concentration of a test chemical reflecting a 20 %, 50 %, and 80 % inhibition of cell
viability (i.e., the IC20, IC50, and IC80) is determined from the concentration-response by
applying a Hill function to the concentration-response data. Statistical software (e.g.,
GraphPad PRISM® 3.0) specified by the SMT shall be used to calculate IC20, IC50, and IC80
values (and the associated confidence limits) for each test chemical. In addition, the SMT
shall provide guidelines for calculating ICx values and confidence limits. The Testing
Facility shall report data using at least three (3) significant figures and shall forward theresults from each assay to the SMT/biostatistician through the designated contacts inelectronic format and hard copy upon completion of testing. The SMT will be directly
responsible for the statistical analyses of the Validation Study data.
VIII.

REFERENCES
Clonetics Normal Human Keratinocyte Systems Instructions for Use, AA-1000-4-Rev.03/00.
(http://www.clonetics.com).
Hackenberg, U. and H. Bartling. 1959. Messen und Rechnen im pharmakologischen
Laboratorium mit einem speziellen Zahlensystem (WL24-System). Arch. Exp. Pathol.
Pharmakol. 235: 437-463.
Triglia, D., P.T. Wegener, J. Harbell, K. Wallace, D. Matheson, and C. Shopsis. 1989.
Interlaboratory validation study of the keratinocyte neutral red bioassay from Clonetics
Corporation. In Alternative Methods in Toxicology, Volume 7. A.M. Goldberg, ed., pp. 357365. Mary Ann Liebert, Inc., New York.

C-131

In Vitro Cytotoxicity Test Methods BRD Appendix C6

November 2006

IX. APPROVAL__________________________________SPONSOR REPRESENTATIVE___________________DATE(Print or type name)_____________________________
Testing Facility STUDY DIRECTOR(Print or type name)____________________
DATE

C-132

In Vitro Cytotoxicity Test Methods BRD Appendix D

November 2006

Appendix D

D1

SAS Code for ANOVA and Contrasts............................................... D-3

D2

SAS Code for Regression Comparisons ............................................ D-7

D-1

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D-2

In Vitro Cytotoxicity Test Methods BRD Appendix D1

November 2006

Appendix D1

SAS Code for ANOVA and Contrasts

D-3

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D-4

In Vitro Cytotoxicity Test Methods BRD Appendix D1

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options nodate nonumber;
libname lib "S:\NIEHS\EXP Studies\BasicResearch\Haseman\Cytotoxicity
Validation\Post Phase III Analysis and Data\data sets";proc sort data=lib.anovadata; by chemical cell lab;
ods trace on;
ods listing close;
ods output OverallANOVA=temp;
ods output Contrasts=temp1;
proc glm data=lib.anovadata;class lab;by chemical cell;
model log_ic50=lab;contrast 'Comparing IIVS to FRAME and ECBC'
lab -.5 -.5 1;
contrast 'Comparing ECBC to FRAME and IIVS'
lab 1 -.5 -.5;
contrast 'Comparing FRAME to ECBC and IIVS'
lab -.5 1 -.5;
run;ods listing;*proc print data=temp1;run;data lib.contrast_results; set temp1;
keep chemical cell Source ProbF;
run;*proc print data=lib.contrast_results;run;data lib.anova_results; set temp;
if Source="Error" then delete;
if Source="Corrected Total" then delete;keep chemical cell ProbF;
run;
proc sort data=lib.anova_results; by chemical cell;/*proc print data=lib.anova_results;
var chemical cell ProbF;
run;*/
data temp;
set lib.anova_results;
keep chemical cell ProbF;
run;
proc export data=temp
outfile='S:\NIEHS\EXP Studies\BasicResearch\Haseman\Cytotoxicity
Validation\Post Phase III Analysis and Data\data sets\Anova Results.txt'
dbms=TAB;
run;

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D-6

In Vitro Cytotoxicity Test Methods BRD Appendix D2

November 2006

Appendix D2

SAS Code for Regression Comparisons

D-7

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D-8

In Vitro Cytotoxicity Test Methods BRD Appendix D2

November 2006

dm 'output; clear';dm 'log; clear';*****************************************************************************filename: task3.sas*creation date: 08/02/06*study:niceatm*investigator:*purpose: perform the individual lab regressions (tasks 1-4)
* note: 3 models are fit:
* (a) full model* (b) reduced model with separate intercepts + common slope* (c) separate intercepts + separate slopes model*authors:mike riggs*input data medium: sas data sets************************************************************************;***compare rc to niceatm regressions, by cell type*note: the input data set anal3 was created by taking the 47 3t3*chemicals and the 51 nhk chemicals and computing their*means by cell line**************************************************************************;
proc mixed data=anal3 maxiter=200;
by celline;
class est_type;model log_ld50=est_type logic50_lab est_type*logic50_lab/outpredm=predat;
title1 'ancova model (estimation type = trt) log-scale lab regressions, bycell line';
title2 '(test for slope differences)';
run;
quit;
****compute the full-model rsquare from the model residuals and predictions***;****note: proc mixed does not compute rsq, so you need to do it
yourself***;data pred3t3 prednhk;
set predat;
if celline='3t3' then output pred3t3;
else output prednhk;
run;proc summary data=pred3t3 nway;
var log_ld50;
output out=sumdat
mean=_mean_;run;data pred3t3;
if _n_=1 then set sumdat;
set pred3t3;
D-9

In Vitro Cytotoxicity Test Methods BRD Appendix D2
run;data comp;set pred3t3 end=eof;
sst+((log_ld50-_mean_)**2);
sse+(resid**2);
n=_n_ ;
if eof then output;run;data comp;set comp;
rsq=(sst-sse)/sst;label _mean_='response*mean'
sst='total sum*of squares'
sse='error sum*of squares'
rsq='r-squared';run;proc print data=comp split='*';
var n _mean_ sst sse rsq;
format rsq 5.3;
title1 'full ancova model r-square for 3t3 cell line (task 3)';run;proc summary data=prednhk nway;
var log_ld50;
output out=sumdat
mean=_mean_;run;data prednhk;
if _n_=1 then set sumdat;
set prednhk;
run;data comp;set prednhk end=eof;
sst+((log_ld50-_mean_)**2);
sse+(resid**2);
n=_n_ ;
if eof then output;run;data comp;set comp;
rsq=(sst-sse)/sst;label _mean_='response*mean'
sst='total sum*of squares'
sse='error sum*of squares'
rsq='r-squared';run;proc print data=comp split='*';
var n _mean_ sst sse rsq;
format rsq 5.3;
D-10

November 2006

In Vitro Cytotoxicity Test Methods BRD Appendix D2

November 2006

title1 'full ancova model r-square for nhk cell line (task 3)';run;proc mixed data=anal3 maxiter=200;
by celline;
class est_type;model log_ld50=est_type est_type*logic50_lab/noint solution cl alpha=0.05;* the following contrast is the simultaneous test of equal intercepts andslopes ***;
contrast 'lab vs. rc' est_type -1 1,
est_type*logic50_lab -1 1;
title1 'ancova model (trt=estimation type) log-scale lab regressions, bycell line';
title2 '(separate slope estimates)';
run;
quit;
proc mixed data=anal3 maxiter=200;
by celline;
model log_ld50=logic50_lab/solution cl alpha=0.05;
title1 'ancova model (estimation type = trt) log-scale lab regressions, bycell line';
title2 '(estimate homogeneous slope with single intercept)';
run;
quit;

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D-12

November 2006

In Vitro Cytotoxicity Test Methods BRD Appendix E

November 2006

Appendix E

Neutral Red Dye Experiments
E1

Institute for In Vitro Sciences (IIVS) Assessment of Protocol
Variables in the NICEATM/ECVAM Evaluation of Cytotoxicity
Assays................................................................................................. E-5

E2

Neutral Red (NR) Dye Experiments – 3T3 Cells – IIVS ............... E-13

E3

Neutral Red (NR) Dye Experiments – NHK Cells – IIVS............. E-19

E4

Neutral Red (NR) Dye Experiments – 3T3 Cells – ECBC ............ E-25

E-1

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E-2

In Vitro Cytotoxicity Test Methods BRD Appendix E

November 2006

APPENDIX E
Neutral Red Dye Experiments
Appendix E1: Institute for In Vitro Sciences (IIVS) Assessment of Protocol Variables in
the NICEATM/ECVAM Evaluation of Cytotoxicity Assays
IIVS performed experiments using the 3T3 cells and the NRU test methods before the
NICEATM/ECVAM validation study was initiated. The laboratory examined: optimalsolvent concentrations (DMSO and ETOH), cell seeding densities, doubling times, andexposure duration of a test chemical (24, 48, and 72-hour exposures). Data are presented in
the appendix.
Appendix E2: Neutral Red (NR) Dye Experiments – 3T3 Cells
IIVS performed three sets of experiments to compare the optical density (OD) readings
obtained in an NRU assay using various concentrations of NR dye and different incubation
periods.
• Experiment 1: NR Stain Time Course in 3T3 Cells; NRU incubation times:
0.25, 0.50, 1.0, 2.0, and 3.0 hour.
• Experiment 2: Neutral Red Stain Prepared in DMEM/5%NCS; Test of NR
Preparation 1 Day Prior to Use; Tested in 90-100% Confluent 3T3 Cultures• Experiment 3: Neutral Red Stain Prepared in DMEM/5%NCS; Filtered
Immediately before Use; Tested in 90-100% Confluent 3T3 Cultures
Appendix E3: Neutral Red (NR) Dye Experiments – NHK Cells
IIVS performed three sets of experiments to compare the optical density (OD) readings
obtained in an NRU assay using various concentrations of NR dye and different incubation
periods.
• Experiment 1: NR Stain Time Course in NHK Cells; NRU incubation times:
0.25, 0.50, 1.0, 2.0, and 3.0 hour.
• Experiment 2: Neutral Red Stain Prepared in KGM; Test of NR Preparation 1
Day Prior to Use; Tested in 90-100% Confluent NHK Cultures• Experiment 3: Neutral Red Stain Prepared in KGM; Filtered Immediately
before Use; Tested in 90-100% Confluent NHK Cultures

E-3

In Vitro Cytotoxicity Test Methods BRD Appendix E

November 2006

Appendix E4: Neutral Red (NR) Dye Experiments – Concentration vs Time – 3T3 Cells
ECBC performed experiments using the 3T3 cells and the NRU test methods.
• in vitro cytotoxicity NRU tests (3T3 cells) using SLS (range = 100 µg/mL to
6.7 µg/mL)• NR dye mixed with DMEM culture medium with 10% NCS; final
concentrations = 25 µg/mL and 50 µg/mL• Tests performed with two NRU incubation times: 1 hour and 3 hoursµg NR dye/mL

NRU Incubation
Time (hours)

25
25
50
50

1
3
1
3

E-4

Mean Vehicle
Control OD540
Value
0.255
0.508
0.330
0.457

In Vitro Cytotoxicity Test Methods BRD Appendix E1

November 2006

Appendix E1

Institute for In Vitro Sciences (IIVS) Assessment of Protocol Variables in
the NICEATM/ECVAM Evaluation of Cytotoxicity Assays

E-5

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E-6

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INSTITUTE FOR IN VITRO SCIENCES (IIVS)ASSESSMENT OF PROTOCOL VARIABLES IN THE NTP EVALUATIONOF CYTOTOXICITY ASSAYSAPRIL 2002
BALB/c 3T3 Cells
I.

What is the acceptable solvent concentration?
Two solvents, DMSO and ETOH, were assayed in the 3T3 assay to determine
acceptable concentrations. Multiple exposure times were assessed since the final
assay exposure time was not yet established. Various cell seeding concentrations
were tested since these experiments were run concurrently with others which used todetermine optimal seeding density.

Table 1.
ETOH
Date
2%
2/26/02 58%
2/26/02 49%

1%
72%
73%

0.50%
100%
102%

Seeding Density
9X103 cells/ml
4.5X103 cells/ml

2/26/02 67%
2/26/02 68%

75%
82%

105%
108%

9X103 cells/ml
4.5X103 cells/ml

Date
3/19/021%
76%

0.5% 0.4%
91% 92%

0.3% 0.2%
0.1% Seeding Density
99% 100% 101.6% 2X104 cells/ml

48hour

2/26/02 25% 54%
2/26/02 27% 56%
3/19/02
116%

83%
78%
123% 122%

9X103 cells/ml
4.5X103 cells/ml
120% 117% 108.8% 1X104 cells/ml

72hour

2/26/02 20%
2/26/02 19%
3/19/0286%
93%
89% 102%

9X103 cells/ml
4.5X103 cells/ml
102% 112% 110.1% 5X103 cells/ml

48hour72hourDMSO
24hour

2%

52%
56%
58%

We concluded from these experiments that 0.5% ETOH was the optimal ETOH
concentration (little to no toxicity), and that 0.5% was probably acceptable for DMSO
as a trade-off between slight toxicity and ability to test chemicals to higher does
levels.

E-7

In Vitro Cytotoxicity Test Methods BRD Appendix E1

November 2006

From about the middle of March 2002 on, we used 0.5% in all of our experiments
where DMSO was called for as a solvent. This gave us a number of opportunities tofurther determine the toxicity of DMSO by comparing the solvent control wells withthe media control wells in the same experiment.
Table 2.
DMSO
Date & ExposureTime
24hour 3/19/02

OD Assay Medium
Wells
0.502
0.441

OD Solvent
Wells
0.474
0.394

% Survival in
Solvent
94.5%
89.4%

48hour 3/19/02

0.587
0.582

0.536
0.545

91.4%
93.6%

72hour 3/19/02

0.687
0.601
0.666
0.588
The average survival in 0.5% DMSO from Table 2 was 90.8%.

II.

87.6%
88.3%

Doubling Time Experiments

We ran a series of experiments designed primarily to determine the appropriate original
seeding density for 24, 48, and 72 hour exposure times. We judged our results on visualobservations of the cells at the conclusion of the experiment (control cells should be justconfluent at 24, 48, or 72 hours), and on the shape of the growth curve.
Figure 1.

E-8

In Vitro Cytotoxicity Test Methods BRD Appendix E1

November 2006

Figure 2.
3T3 Density Growth Curves, 2/26/02 seeding

OD550

1.000

0.100

0.010
0

24

48

72

96

120

Growth (Hrs)
9.0x10^3

4.5x10^3

We have concluded from these growth curves that our 3T3 cells have a doubling time ofabout 19 hours and that cell concentration of: 1x104 cells/ml (24hour); 5x103 cells/ml(48hour); and 2.5x103 (72hour) are acceptable.
III.

Exposure Duration

The exposure question was first raised by Richard Clothier who indicated that a paper by
Riddell, et al. (1986) showed a number of chemicals whose toxicity changed greatly between
a 24 hour and a 72 hour exposure (for 25/50 materials there was little change and for 25/50
materials there was a change). We examined the paper and chose to investigate six
chemicals that showed some of the largest differences between 24 hour and 72 hour.
Our initial studies gave similar results to those of Riddell et al. (1986). However we felt thatthe cell number for the longer exposures was not optimal, and we conducted additional
studies to determine a standard seeding density for each exposure period. Using thismethodology we looked at the 6 materials in a standardized fashion at 24, 48 and 72 hours.
Our results are shown in Fig. 3.

E-9

In Vitro Cytotoxicity Test Methods BRD Appendix E1

November 2006

Figure 3.

In this figure the historic Halle et al. (1992) data are shown as small blue dots and theregression line as a dark black line. To add perspective we have included the Riddell, et al.
(1986) data as a light blue diamond (24hour) or a dark blue diamond (72hour). Arrows
emerging from certain points indicate that the value is less than or greater than that point.
Our values are graphed in increasing shades of green from light (24hour) to dark (72hour).
All green values are averages of at least two separate experiments. It appears that our data
are somewhat different than Riddell, et al. (1986), i.e., most differences are not as great asoriginally seen. Nonetheless the values, as expected, do become more toxic with increased
exposure time. We feel that 48 hours is probably the optimal time for these data if the Halle
regression is considered some type of a standard.
Next we asked whether a 48 hour exposure time would affect our earlier results with the 11
chemicals presented in the Guidance Document (ICCVAM 2001b). If these numbers werechanged significantly, this might cause us to make significant modification to our guidance.
To assess the effect of increasing exposure time on the 11 chemicals, we tested them with
exposure times of 24, 48 and 72 hours as shown in Fig. 4.

E-10

In Vitro Cytotoxicity Test Methods BRD Appendix E1

November 2006

Figure 4.

The data shown on the graph are averages of duplicate experiments. It can be seen that
although each of the chemicals becomes more toxic with increased exposure, all points arestill within the 0.5 log range of the regression line. It again appears that 48 hour exposure fits
the regression more closely, however we regraphed the data in Fig. 5 to show the regression
line and statistics for each of the new sets of data.

E-11

In Vitro Cytotoxicity Test Methods BRD Appendix E1

November 2006

LD50 (mmol/kg)

Figure 5.

24hr r=0.984
y=0.493x + 0.529

48hr r=0.989
y=0.47x + 0.69

72hr r=0.985
y=0.464x + 0.8

24hr (GD)r=0.992
y=0.506x + 0.47

In this figure it can be seen that all the regression lines for the 3 new time points plus the
Guidance Document data (red triangles) fall with in the regression boundaries. It again
appears that the 48 hour values best fit the original regression line.
We now feel that for the 3T3 cells an extended exposure period (>24hour) should be used,
and that 48 hours seems to help identify the more toxic compounds while not over estimating
the less toxic ones.
REFERENCES
Halle W, Spielmann H. 1992. Two procedures for the prediction of acute toxicity (LD50)
from cytotoxicity data. Altern Lab Anim 20:40-49.
ICCVAM. 2001b. Guidance Document On Using In Vitro Data To Estimate In Vivo Starting
Doses For Acute Toxicity. NIH Publication No. 01-4500. Research Triangle Park,
NC:National Institute for Environmental Health Sciences. Available:
http://iccvam.niehs.nih.gov/ [accessed 01 November 2006].
Riddell RJ, Panacer DS, Wilde SM, Clothier RH, Balls M. 1986. The importance of exposure
period and cell type in in vitro cytotoxicity tests. Altern Lab Anim 14:86-92.

E-12

In Vitro Cytotoxicity Test Methods BRD Appendix E2

November 2006

Appendix E2

Neutral Red (NR) Dye Experiments – 3T3 Cells – IIVS

E-13

In Vitro Cytotoxicity Test Methods BRD Appendix E2

November 2006

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E-14

In Vitro Cytotoxicity Test Methods BRD Appendix E2

November 2006

Test Facility : IIVS
Chemical Code : N/A
2nd Chem. Code*: NRU

Study Number.: R&D - NR Stain Time Course in 3T3
96-Well Plate ID : 1
Experiment ID : RD96023T

96-WELL PLATE MAP
1
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

2
Blank

3
Blank

4
Blank

5
Blank

6
Blank

7
Blank

8
Blank

9
Blank

10
Blank

11
Blank

3 hr

3 hr

2 hr

2 hr

1 hr

1 hr

30 min30 min15 min15 min

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

1

2

3

4

5

6

7

8

9

10

11

12

A
B
C
D
E
F
G
H

0.048
0.048
0.047
0.046
0.046
0.044
0.043
0.044

0.046
0.753
0.866
0.844
0.717
0.776
0.717
0.044

0.045
0.794
0.766
0.794
0.805
0.769
0.807
0.045

0.047
0.595
0.668
0.607
0.627
0.618
0.639
0.044

0.047
0.607
0.668
0.622
0.610
0.665
0.616
0.045

0.046
0.415
0.406
0.393
0.384
0.378
0.385
0.045

0.046
0.396
0.391
0.387
0.375
0.398
0.349
0.043

0.044
0.267
0.257
0.228
0.239
0.277
0.265
0.043

0.044
0.282
0.256
0.262
0.266
0.301
0.269
0.045

0.043
0.219
0.227
0.213
0.210
0.186
0.211
0.045

0.044
0.213
0.220
0.217
0.206
0.202
0.195
0.041

0.038
0.039
0.038
0.038
0.038
0.038
0.036
0.036

A
B
C
D
E
F
G
H

1
0.005
0.005
0.004
0.003
0.003
0.001
0.000
0.001

A
B
C
D
E
F
G
H

12
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

RAW ABSORBANCE DATA (OD550)

CORRECTED ABSORBANCE (Sample OD550 - Mean Blank OD550)
2
0.003
0.710
0.823
0.801
0.674
0.733
0.674
0.000

3
0.002
0.751
0.723
0.751
0.762
0.726
0.764
0.002

Mean Blank =

0.043

4
0.004
0.552
0.625
0.564
0.584
0.575
0.596
0.001

5
0.004
0.564
0.625
0.579
0.567
0.622
0.573
0.002

6
0.003
0.372
0.363
0.350
0.341
0.335
0.342
0.002

7
0.003
0.353
0.348
0.344
0.332
0.355
0.306
0.000

8
0.001
0.224
0.214
0.185
0.196
0.234
0.222
0.000

9
0.001
0.239
0.213
0.219
0.223
0.258
0.226
0.002

10
0.000
0.176
0.184
0.170
0.167
0.143
0.168
0.002

11
0.001
0.170
0.177
0.174
0.163
0.159
0.152
-0.00212-0.005-0.004-0.005-0.005-0.005-0.005-0.007-0.007

12

RELATIVE VIABILITY (% OF VEHICLE CONTROL)
1
A
B
C
D
E
F
G
H

2

3

4

5

6

7

8

9

10

11

95.8%
111.1%
108.1%
91.0%
98.9%
91.0%

101.4%
97.6%
101.4%
102.8%
98.0%
103.1%

74.5%
84.3%
76.1%
78.8%
77.6%
80.4%

76.1%
84.3%
78.1%
76.5%
83.9%
77.3%

50.2%
49.0%
47.2%
46.0%
45.2%
46.1%

47.6%
46.9%
46.4%
44.8%
47.9%
41.3%

30.2%
28.9%
24.9%
26.4%
31.6%
29.9%

32.2%
28.7%
29.5%
30.1%
34.8%
30.5%

23.7%
24.8%
22.9%
22.5%
19.3%
22.6%

22.9%
23.9%
23.5%
22.0%
21.4%
20.5%

Study Number.: R&D - NR Stain Time Course in 3T3

3 hr

3 hr

2 hr

2 hr

1 hr

1 hr

30 min30 min15 min15 minMean Corr. OD :
SD :

0.736
0.064

0.746
0.018

0.582
0.026

0.588
0.028

0.350
0.014

0.339
0.018

0.212
0.019

0.229
0.016

0.168
0.014

0.166
0.010

Mean 3 hour :Mean Blank :

0.741
0.043

% of 3 hour:SD :% CV :
hours% of 3 hour:

99.3%
8.6%
8.63%

100.7%
2.4%
2.37%

78.6%
3.5%
4.42%
3
100.0%

79.4%
3.7%
4.72%
2
79.0%

47.3%
1.9%
4.08%
1
46.5%

45.8%
2.5%
5.42%
0.50
29.8%

28.6%
2.5%
8.73%
0.25
22.5%

31.0%
2.2%
7.14%

22.6%
1.9%
8.22%

22.3%
1.3%
5.76%

Conc. (µg/mL) :E-15

In Vitro Cytotoxicity Test Methods BRD Appendix E2

Neutral Red Uptake

125%% of 3 hour group

November 2006

100%
75%
50%
25%
0%
0

1

2

3

Time (Hours)
R&D - NR Stain Time Course in 3T3

E-16

4

In Vitro Cytotoxicity Test Methods BRD Appendix E2

November 2006

Neutral Red Stain Prepared in DMEM5%NCS - TEST OF NR PREP 1 DAY PRIOR TO USE
Tested in 90-100% Confluent 3T3 Cultures

96-WELL PLATE MAP
A
B
C
D
E
F
G
H

1
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

2
Blank

3
Blank

4
Blank

5
Blank

Blank

Blank

1

2

3

4

5

6

7

8

9

10

11

12

0.045
0.043
0.045
0.043
0.042
0.044
0.042
0.044

0.045
0.383
0.389
0.383
0.361
0.368
0.415
0.042

0.045
0.459
0.397
0.429
0.345
0.412
0.451
0.043

0.044
0.417
0.379
0.350
0.334
0.374
0.422
0.043

0.056
0.541
0.557
0.539
0.579
0.582
0.600
0.057

0.056
0.631
0.536
0.575
0.585
0.588
0.620
0.059

0.056
0.639
0.621
0.545
0.577
0.578
0.616
0.055

0.057
0.635
0.559
0.629
0.573
0.572
0.632
0.057

0.053
0.637
0.590
0.613
0.626
0.687
0.572
0.050

0.051
0.686
0.618
0.658
0.635
0.647
0.744
0.057

0.051
0.656
0.612
0.652
0.599
0.641
0.637
0.050

0.052
0.052
0.051
0.053
0.051
0.050
0.050
0.054

11
0.008
0.613
0.569
0.609
0.556
0.598
0.594
0.007

12
0.009
0.009
0.008
0.010
0.008
0.007
0.007
0.011

50 ug/ml
Prepared and filteredin evening before use
Filtered before use

6
Blank

7
Blank

8
Blank

9
Blank

50 ug/ml
Filtered before use

Blank

Blank

Blank

10
Blank

11
Blank

33 ug/ml
Filtered before use

Blank

Blank

Blank

Blank

Blank

12
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

RAW ABSORBANCE DATA (OD550)
A
B
C
D
E
F
G
H

CORRECTED ABSORBANCE (Sample OD550 - Mean Blank OD550)
A
B
C
D
E
F
G
H

1
0.002
0.000
0.002
0.000
-0.0010.001-0.001
0.001

2
0.002
0.340
0.346
0.340
0.318
0.325
0.372
0.000

3
0.002
0.416
0.354
0.386
0.302
0.369
0.408
0.000

Mean Blank =

0.052

4
0.001
0.374
0.336
0.307
0.291
0.331
0.379
0.000

5
0.013
0.498
0.514
0.496
0.536
0.539
0.557
0.014

6
0.013
0.588
0.493
0.532
0.542
0.545
0.577
0.016

7
0.013
0.596
0.578
0.502
0.534
0.535
0.573
0.012

8
0.014
0.592
0.516
0.586
0.530
0.529
0.589
0.014

9
0.010
0.594
0.547
0.570
0.583
0.644
0.529
0.007

10
0.008
0.643
0.575
0.615
0.592
0.604
0.701
0.014

(Only the 14 wells from the 33 ug/ml group)

Neutral Red Stain Concentration
Conc. (µg/mL) :Mean Corr. OD :SD :Group meancorr OD:

50.0
0.340
0.019

0.372
0.042

0.349

50.0
0.336
0.035

0.523
0.025

0.546
0.034

33.0
0.553
0.035

0.557
0.035

0.545

Note: Significant crystal formation was observed in the DMEM5%NCS/NR prepared 1 day prior,and the color was essentailly medium-colored. Much NR stain stripped out of solution.
No ppt or crystalization observed in the wells during the NR loading of cells.

E-17

0.578
0.040

0.621
0.045

0.596

0.590
0.023

In Vitro Cytotoxicity Test Methods BRD Appendix E2

November 2006

Neutral Red Stain Prepared in DMEM5%NCS/Filtered immediately before use
Tested in 90-100% Confluent 3T3 Cultures

96-WELL PLATE MAP
1
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

2
Blank
50 ug/ml

Blank

Blank

1

2

3

4

5

6

7

8

9

10

11

12

A
B
C
D
E
F
G
H

0.076
0.058
0.053
0.048
0.047
0.073
0.072
0.056

0.051
0.553
0.561
0.493
0.491
0.606
0.63
0.089

0.05
0.535
0.503
0.527
0.497
0.697
0.497
0.055

0.045
0.58
0.517
0.489
0.528
0.53
0.563
0.043

0.044
0.587
0.549
0.495
0.571
0.6
0.592
0.045

0.041
0.421
0.338
0.351
0.312
0.36
0.399
0.041

0.041
0.353
0.345
0.331
0.321
0.373
0.39
0.04

0.041
0.225
0.213
0.196
0.188
0.239
0.235
0.039

0.039
0.221
0.203
0.196
0.195
0.218
0.21
0.039

0.038
0.149
0.144
0.143
0.132
0.143
0.145
0.042

0.037
0.145
0.155
0.161
0.172
0.163
0.157
0.04

0.037
0.037
0.035
0.038
0.038
0.036
0.037
0.036

A
B
C
D
E
F
G
H

1
0.033
0.015
0.010
0.005
0.004
0.030
0.029
0.013

A
B
C
D
E
F
G
H

3
Blank
50 ug/ml

4
Blank
28 ug/ml

5Blank
28 ug/ml

6Blank
16 ug/ml

7Blank
16 ug/ml

8Blank
9 ug/ml

9Blank
9 ug/ml10Blank
5 ug/ml

11Blank
5 ug/ml

12

Empty

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

RAW ABSORBANCE DATA (OD550)

CORRECTED ABSORBANCE (Sample OD550 - Mean Blank OD550)
2
0.008
0.510
0.518
0.450
0.448
0.563
0.587
0.000

3
0.007
0.492
0.460
0.484
0.454
0.654
0.454
0.012

Mean Blank =

0.039

4
0.002
0.537
0.474
0.446
0.485
0.487
0.520
0.000

5
0.001
0.544
0.506
0.452
0.528
0.557
0.549
0.002

6
-0.002
0.378
0.295
0.308
0.269
0.317
0.356
-0.0027-0.002
0.310
0.302
0.288
0.278
0.330
0.347
-0.0038-0.002
0.182
0.170
0.153
0.145
0.196
0.192
-0.0049-0.004
0.178
0.160
0.153
0.152
0.175
0.167
-0.00410-0.005
0.106
0.101
0.100
0.089
0.100
0.102
-0.00111-0.006
0.102
0.112
0.118
0.129
0.120
0.114
-0.00312-0.006-0.006-0.008-0.005-0.005-0.007-0.006-0.007(Only the 4 wells from the 5.0 ug/ml group)

Neutral Red Stain Concentration
Conc. (µg/mL) :

50.0

50.0

28.0

28.0

15.8

15.8

8.9

8.9

5.0

5.0

Mean Corr. OD :
SD :

0.512
0.057

0.499
0.077

0.491
0.033

0.522
0.039

0.320
0.040

0.309
0.026

0.173
0.021

0.164
0.011

0.099
0.006

0.116
0.009

Group meancorr OD:

0.506

graph

0.507

x
y

50.0
0.506

0.315

28.0
0.507

0.168

15.8
0.315

8.9
0.168

0.107

5.0
0.107

Neutral Red Uptake

1.250

Corrected OD

1.000
0.750
0.500
0.250
0.000
1

10
Concentration Neutral Red(µg/mL)NR Stain

E-18

100

In Vitro Cytotoxicity Test Methods BRD Appendix E3

November 2006

Appendix E3

Neutral Red (NR) Dye Experiments – NHK Cells – IIVS

E-19

In Vitro Cytotoxicity Test Methods BRD Appendix E3

November 2006

[This Page Intentionally Left Blank]

E-20

In Vitro Cytotoxicity Methods BRD Appendix E3

November 2006

Test Facility : IIVS
Chemical Code : N/A
2nd Chem. Code*: NRU

Study Number.: R&D - NR Stain Time Course in NHK
96-Well Plate ID : 1
Experiment ID : RD9602NK

96-WELL PLATE MAP
1
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

2
Blank

3
Blank

4
Blank

5
Blank

6
Blank

7
Blank

8
Blank

9
Blank

10
Blank

11
Blank

3 hr

3 hr

2 hr

2 hr

1 hr

1 hr

30 min30 min15 min15 min

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

1

2

3

4

5

6

7

8

9

10

11

12

A
B
C
D
E
F
G
H

0.057
0.068
0.057
0.058
0.057
0.057
0.054
0.057

0.053
1.501
1.549
1.540
1.553
1.632
1.462
0.054

0.059
1.564
1.482
1.503
1.532
1.600
1.514
0.053

0.058
1.311
1.376
1.415
1.388
1.396
1.357
0.052

0.054
1.327
1.372
1.422
1.453
1.380
1.439
0.051

0.055
0.998
1.082
1.026
1.060
1.066
1.069
0.055

0.053
1.052
1.076
0.995
1.010
1.074
1.010
0.051

0.052
0.671
0.714
0.724
0.675
0.656
0.708
0.049

0.051
0.649
0.697
0.698
0.634
0.628
0.606
0.047

0.048
0.438
0.494
0.482
0.459
0.470
0.474
0.050

0.049
0.474
0.474
0.474
0.462
0.429
0.437
0.046

0.035
0.037
0.034
0.036
0.034
0.033
0.035
0.034

A
B
C
D
E
F
G
H

1
0.007
0.018
0.007
0.008
0.007
0.007
0.004
0.007

A
B
C
D
E
F
G
H

12
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

RAW ABSORBANCE DATA (OD550)

CORRECTED ABSORBANCE (Sample OD550 - Mean Blank OD550)
2
0.003
1.451
1.499
1.490
1.503
1.582
1.412
0.000

3
0.009
1.514
1.432
1.453
1.482
1.550
1.464
0.003

Mean Blank =

0.050

4
0.008
1.261
1.326
1.365
1.338
1.346
1.307
0.002

5
0.004
1.277
1.322
1.372
1.403
1.330
1.389
0.001

6
0.005
0.948
1.032
0.976
1.010
1.016
1.019
0.005

7
0.003
1.002
1.026
0.945
0.960
1.024
0.960
0.001

8
0.002
0.621
0.664
0.674
0.625
0.606
0.658
-0.001

9
0.001
0.599
0.647
0.648
0.584
0.578
0.556
-0.00310-0.002
0.388
0.444
0.432
0.409
0.420
0.424
0.000

11
-0.001
0.424
0.424
0.424
0.412
0.379
0.387
-0.00412-0.015-0.013-0.016-0.014-0.016-0.017-0.015-0.016

12

RELATIVE VIABILITY (% OF VEHICLE CONTROL)
1

2

3

4

5

6

7

8

9

10

11

97.6%
100.9%
100.3%
101.1%
106.5%
95.0%

101.9%
96.4%
97.8%
99.7%
104.3%
98.5%

84.9%
89.2%
91.9%
90.0%
90.6%
88.0%

85.9%
89.0%
92.3%
94.4%
89.5%
93.5%

63.8%
69.5%
65.7%
68.0%
68.4%
68.6%

67.4%
69.1%
63.6%
64.6%
68.9%
64.6%

41.8%
44.7%
45.4%
42.1%
40.8%
44.3%

40.3%
43.6%
43.6%
39.3%
38.9%
37.4%

26.1%
29.9%
29.1%
27.5%
28.3%
28.6%

28.6%
28.6%
28.6%
27.7%
25.5%
26.1%

3 hr

3 hr

2 hr

2 hr

1 hr

1 hr

30 min30 min15 min15 minMean Corr. OD :
SD :

1.490
0.057

1.483
0.043

1.324
0.036

1.349
0.048

1.001
0.032

0.987
0.036

0.642
0.028

0.602
0.038

0.420
0.019

0.409
0.020

Mean 3 hour :Mean Blank :

1.486
0.050

% of 3 hour:SD :% CV :
hours% of 3 hour:

100.2%
3.8%
3.83%

99.8%
2.9%
2.91%

89.1%
2.4%
2.75%
3
100.0%

90.8%
3.2%
3.53%
2
89.9%

67.3%
2.1%
3.17%
1
66.8%

66.4%
2.4%
3.61%
0.50
41.9%

43.2%
1.9%
4.29%
0.25
27.9%

40.5%
2.5%
6.28%

28.3%
1.3%
4.62%

27.5%
1.4%
4.97%

A
B
C
D
E
F
G
H

Study Number.: R&D - NR Stain Time Course in NHK

Conc. (µg/mL) :E-21

In Vitro Cytotoxicity Methods BRD Appendix E3

November 2006

Neutral Red Uptake% of 3 hour group

125%
100%
75%
50%
25%
0%
0

1

2
Time (Hours)
R&D - NR Stain Time Course in NHK

E-22

3

4

In Vitro Cytotoxicity Methods BRD Appendix E3

November 2006

Neutral Red Stain Prepared in KGM - TEST OF NR PREP 1 DAY PRIOR TO USE
Tested in 90-100% Confluent NHK Cultures

96-WELL PLATE MAP
1
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

2
Blank

Blank

Blank

1

2

3

4

5

6

7

8

9

10

11

A
B
C
D
E
F
G
H

0.062
0.055
0.060
0.062
0.067
0.057
0.065
0.064

0.061
1.306
1.530
1.454
1.423
1.423
1.430
0.059

0.063
1.545
1.520
1.527
1.433
1.591
1.468
0.060

0.064
1.530
1.554
1.513
1.505
1.577
1.393
0.064

0.063
1.514
1.471
1.511
1.577
1.577
1.319
0.064

0.062
1.403
1.536
1.472
1.469
1.403
1.432
0.065

0.060
1.421
1.416
1.491
1.448
1.431
1.304
0.061

0.060
1.297
1.415
1.438
1.474
1.347
1.416
0.064

0.052
1.249
1.308
1.217
1.199
1.250
1.243
0.060

0.053
1.136
1.160
1.192
1.249
1.235
1.117
0.055

0.051
1.134
1.189
1.173
1.158
1.102
1.110
0.060

A
B
C
D
E
F
G
H

1
0.012
0.005
0.010
0.012
0.017
0.007
0.015
0.014

A
B
C
D
E
F
G
H

3
Blank

4
Blank

5
Blank

50 ug/ml
Prepared and filteredin evening before use
Filtered before use

6
Blank

7
Blank

8
Blank

9
Blank

50 ug/ml
Filtered before use

Blank

Blank

Blank

10
Blank

11
Blank

33 ug/ml
Filtered before use

Blank

Blank

Blank

Blank

Blank

RAW ABSORBANCE DATA (OD550)

CORRECTED ABSORBANCE (Sample OD550 - Mean Blank OD550)
2
0.011
1.256
1.480
1.404
1.373
1.373
1.380
0.000

3
0.013
1.495
1.470
1.477
1.383
1.541
1.418
0.010

Mean Blank =

0.055

4
0.014
1.480
1.504
1.463
1.455
1.527
1.343
0.014

5
0.013
1.464
1.421
1.461
1.527
1.527
1.269
0.014

6
0.012
1.353
1.486
1.422
1.419
1.353
1.382
0.015

7
0.010
1.371
1.366
1.441
1.398
1.381
1.254
0.011

8
0.010
1.247
1.365
1.388
1.424
1.297
1.366
0.014

9
0.002
1.199
1.258
1.167
1.149
1.200
1.193
0.010

10
0.003
1.086
1.110
1.142
1.199
1.185
1.067
0.005

11
0.001
1.084
1.139
1.123
1.108
1.052
1.060
0.010

(Only the 14 wells from the 33 ug/ml group)

Neutral Red Stain Concentration
Conc. (µg/mL) :Mean Corr. OD :SD :Group meancorr OD:

50.0
1.378
0.072

1.464
0.056
1.435

50.0
1.462
0.064

1.445
0.096

1.403
0.051

33.0
1.369
0.062

1.391

Note: No crystal formation was observed in the KGM/NR prepared 1 day prior.
No ppt or crystalization observed in the wells during the NR loading of cells.

E-23

1.348
0.064

1.195
0.037

1.132
0.053
1.141

1.095
0.035

In Vitro Cytotoxicity Methods BRD Appendix E3

November 2006

Neutral Red Stain Prepared in KGM/Filtered immediately before use
Tested in 90-100% Confluent NHK Cultures

96-WELL PLATE MAP
A
B
C
D
E
F
G
H

1
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

2
Blank
50 ug/ml

3
Blank
50 ug/ml

4
Blank
28 ug/ml

5Blank
28 ug/ml

6Blank
16 ug/ml

7Blank
16 ug/ml

8Blank
9 ug/ml

9Blank
9 ug/ml10Blank
5 ug/ml

11Blank
5 ug/ml

12

Blank

Blank

1

2

3

4

5

6

7

8

9

10

11

12

0.067
0.048
0.050
0.047
0.047
0.046
0.047
0.063

0.064
1.255
1.035
1.131
1.117
1.245
1.136
0.056

0.066
1.119
1.004
1.352
1.227
1.129
1.282
0.060

0.049
1.103
1.020
1.094
0.923
0.976
1.061
0.061

0.049
1.054
0.956
1.078
0.893
0.988
0.995
0.048

0.040
0.623
0.624
0.643
0.595
0.607
0.624
0.042

0.040
0.605
0.601
0.635
0.618
0.617
0.582
0.042

0.038
0.325
0.345
0.331
0.323
0.308
0.283
0.038

0.038
0.334
0.312
0.314
0.302
0.313
0.282
0.039

0.036
0.156
0.151
0.157
0.155
0.156
0.131
0.040

0.037
0.150
0.154
0.147
0.150
0.156
0.127
0.038

0.035
0.034
0.034
0.035
0.035
0.035
0.037
0.036

empty

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

RAW ABSORBANCE DATA (OD550)
A
B
C
D
E
F
G
H

CORRECTED ABSORBANCE (Sample OD550 - Mean Blank OD550)
A
B
C
D
E
F
G
H

1
0.017
-0.0020.000-0.003-0.003-0.004-0.003
0.013

2
0.014
1.205
0.985
1.081
1.067
1.195
1.086
0.000

3
0.016
1.069
0.954
1.302
1.177
1.079
1.232
0.010

Mean Blank =

0.038

4
-0.001
1.053
0.970
1.044
0.873
0.926
1.011
0.011

5
-0.001
1.004
0.906
1.028
0.843
0.938
0.945
-0.0026-0.010
0.573
0.574
0.593
0.545
0.557
0.574
-0.0087-0.010
0.555
0.551
0.585
0.568
0.567
0.532
-0.0088-0.012
0.275
0.295
0.281
0.273
0.258
0.233
-0.0129-0.012
0.284
0.262
0.264
0.252
0.263
0.232
-0.01110-0.014
0.106
0.101
0.107
0.105
0.106
0.081
-0.01011-0.013
0.100
0.104
0.097
0.100
0.106
0.077
-0.01212-0.015-0.016-0.016-0.015-0.015-0.015-0.013-0.014(Only the 4 wells from the 5.0 ug/ml group)

Neutral Red Stain Concentration
Conc. (µg/mL) :

50.0

50.0

28.0

28.0

15.8

15.8

8.9

8.9

5.0

5.0

Mean Corr. OD :
SD :

1.104
0.083

1.136
0.126

0.980
0.070

0.944
0.067

0.570
0.017

0.560
0.018

0.270
0.021

0.260
0.017

0.101
0.010

0.098
0.010

Group meancorr OD:

1.120

graph

0.962

x
y

50.0
1.120

0.565

28.0
0.962

0.265

15.8
0.565

8.9
0.265

0.100

5.0
0.100

Neutral Red Uptake

Corrected OD

1.250
1.000
0.750
0.500
0.250
0.000
1

10
Concentration Neutral Red(µg/mL)NR Stain

E-24

100

In Vitro Cytotoxicity Test Methods BRD Appendix E4

November 2006

Appendix E4

Neutral Red (NR) Dye Experiments – 3T3 Cells – ECBC

E-25

In Vitro Cytotoxicity Test Methods BRD Appendix E4

November 2006

[This Page Intentionally Left Blank]

E-26

In Vitro Cytotoxicity Test Methods BRD Appendix E4

November 2006

Test Facility : ECBC
Chemical Code : SLS
2nd Chem. Code*: none

Study Number.: ECBC-3T3 Ia 0#
96-Well Plate ID : 090602-1
Experiment ID : SLS-B(25ug NR/ml 1hr)

96-WELL PLATE MAP
A
B
C
D
E
F
G
H

1
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

2
Blank
VC1
VC1
VC1
VC1
VC1
VC1
Blank

3
Blank
C1
C1
C1
C1
C1
C1
Blank

A
B
C
D
E
F
G
H

1
0.049
0.050
0.052
0.050
0.000
0.000
0.000
0.000

2
0.051
0.262
0.283
0.307
0.000
0.000
0.000
0.000

3
0.048
0.050
0.053
0.055
0.000
0.100
0.000
0.000

A
B
C
D
E
F
G
H

1
0.000
0.001
0.003
0.001
-0.049-0.049-0.049-0.049

4
Blank
C2
C2
C2
C2
C2
C2
Blank

5
Blank
C3
C3
C3
C3
C3
C3
Blank

6
Blank
C4
C4
C4
C4
C4
C4
Blank

7
Blank
C5
C5
C5
C5
C5
C5
Blank

8
Blank
C6
C6
C6
C6
C6
C6
Blank

9
Blank
C7
C7
C7
C7
C7
C7
Blank

10
Blank
C8
C8
C8
C8
C8
C8
Blank

11
Blank
VC2
VC2
VC2
VC2
VC2
VC2
Blank

12
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

9
0.044
0.315
0.283
0.280
0.000
0.000
0.000
0.000

10
0.045
0.329
0.293
0.302
0.000
0.000
0.000
0.000

11
0.046
0.333
0.321
0.314
0.000
0.000
0.000
0.000

12
0.047
0.046
0.050
0.049
0.000
0.000
0.000
0.000

10
-0.004
0.281
0.245
0.254
-0.049-0.049-0.049-0.04911-0.003
0.285
0.273
0.266
-0.049-0.049-0.049-0.04912-0.002-0.0030.001
0.000-0.049-0.049-0.049-0.04912

RAW ABSORBANCE DATA (OD540)
4
0.052
0.046
0.051
0.053
0.000
0.000
0.000
0.000

5
0.048
0.130
0.145
0.135
0.000
0.000
0.000
0.000

6
0.050
0.274
0.231
0.242
0.000
0.000
0.000
0.000

7
0.050
0.254
0.252
0.252
0.000
0.000
0.000
0.000

8
0.046
0.322
0.276
0.291
0.000
0.000
0.000
0.000

CORRECTED ABSORBANCE (Sample OD540 - Mean Blank OD540)
2
0.002
0.214
0.235
0.259
-0.049-0.049-0.0490.000

3-0.001
0.001
0.004
0.006
-0.0490.052-0.049-0.049Mean Blank =

0.049

4
0.003
-0.0030.002
0.004-0.049-0.049-0.049-0.0495-0.001
0.082
0.097
0.087
-0.049-0.049-0.049-0.049

6
0.001
0.226
0.183
0.194
-0.049-0.049-0.049-0.049

7
0.001
0.206
0.204
0.204
-0.049-0.049-0.049-0.0498-0.003
0.274
0.228
0.243
-0.049-0.049-0.049-0.0499-0.005
0.267
0.235
0.232
-0.049-0.049-0.049-0.049

RELATIVE VIABILITY (% OF VEHICLE CONTROL)
1
A
B
C
D
E
F
G
H

2

3

4

5

6

7

8

9

10

11

83.8%
92.0%
101.4%-19.0%-19.0%-19.0%

0.6%
1.8%
2.6%
-19.0%
20.2%-19.0%

-1.0%
1.0%
1.8%
-19.0%-19.0%-19.0%

32.0%
37.9%
33.9%
-19.0%-19.0%-19.0%

88.5%
71.6%
75.9%
-19.0%-19.0%-19.0%

80.6%
79.9%
79.9%
-19.0%-19.0%-19.0%

107.3%
89.3%
95.2%-19.0%-19.0%-19.0%

104.6%
92.0%
90.8%-19.0%-19.0%-19.0%

110.1%
95.9%
99.5%-19.0%-19.0%-19.0%

111.6%
106.9%
104.2%-19.0%-19.0%-19.0%E-27

In Vitro Cytotoxicity Test Methods BRD Appendix E4
Test Facility : ECBC
Chemical Code : SLS
2nd Chem. Code*: none

November 2006
Study Number.: ECBC-3T3 Ia 0#
96-Well Plate ID : 090602-1
Experiment ID : SLS-B(25ug NR/ml 1hr)

VC1

C1

C2

C3

C4

C5

C6

C7

C8

VC2

Conc. (µg/mL) :

0.0

100.0

68.0

46.3

31.5

21.4

14.6

9.9

6.7

0.0

Mean Corr. OD :
SD :

0.236
0.023

0.004
0.003

0.001
0.004

0.088
0.008

0.201
0.022

0.204
0.001

0.248
0.023

0.244
0.019

0.260
0.019

0.274
0.010

Mean Vehicle Control :Mean Blank :

0.255
0.049

% of Vehicle Control :SD :% CV :

92.4%
8.8%
9.56%

1.6%
1.0%
60.40%

0.6%
1.4%
240.37%

34.6%
3.0%
8.66%

78.7%
8.8%
11.14%

80.1%
0.5%
0.57%

97.3%
9.2%
9.47%

95.8%
7.6%
7.95%

101.8%
7.4%
7.22%

107.6%
3.8%
3.50%

Mean VC - VC1 (%) :Mean VC - VC2 (%) :7.59%-7.59%

Mean Absolute OD :0.303

Neutral Red Uptake% Viability

125%
100%
75%
50%
25%
0%
1

10

100
Concentration (µg/mL)

SLS

E-28

1000

In Vitro Cytotoxicity Test Methods BRD Appendix E4

November 2006

Test Facility : ECBC
Chemical Code : SLS
2nd Chem. Code*: none

Study Number.: ECBC-3T3 Ia 0#
96-Well Plate ID : 090602-2
Experiment ID : SLS-B(50ug NR/ml 1hr)

96-WELL PLATE MAP
A
B
C
D
E
F
G
H

1
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

2
Blank
VC1
VC1
VC1
VC1
VC1
VC1
Blank

3
Blank
C1
C1
C1
C1
C1
C1
Blank

A
B
C
D
E
F
G
H

1
0.056
0.088
0.058
0.061
0.000
0.000
0.000
0.000

2
0.061
0.377
0.378
0.373
0.000
0.000
0.000
0.000

3
0.063
0.057
0.062
0.054
0.000
0.100
0.000
0.000

A
B
C
D
E
F
G
H

1
0.007
0.040
0.009
0.013
-0.049-0.049-0.049-0.049

4
Blank
C2
C2
C2
C2
C2
C2
Blank

5
Blank
C3
C3
C3
C3
C3
C3
Blank

6
Blank
C4
C4
C4
C4
C4
C4
Blank

7
Blank
C5
C5
C5
C5
C5
C5
Blank

8
Blank
C6
C6
C6
C6
C6
C6
Blank

9
Blank
C7
C7
C7
C7
C7
C7
Blank

10
Blank
C8
C8
C8
C8
C8
C8
Blank

11
Blank
VC2
VC2
VC2
VC2
VC2
VC2
Blank

12
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

9
0.050
0.402
0.400
0.425
0.000
0.000
0.000
0.000

10
0.052
0.403
0.391
0.420
0.000
0.000
0.000
0.000

11
0.050
0.396
0.386
0.409
0.000
0.000
0.000
0.000

12
0.051
0.053
0.051
0.050
0.000
0.000
0.000
0.000

10
0.003
0.355
0.343
0.372
-0.049-0.049-0.049-0.049

11
0.001
0.348
0.338
0.361
-0.049-0.049-0.049-0.049

12
0.002
0.004
0.002
0.001
-0.049-0.049-0.049-0.04912

RAW ABSORBANCE DATA (OD540)
4
0.055
0.053
0.058
0.051
0.000
0.000
0.000
0.000

5
0.052
0.192
0.158
0.182
0.000
0.000
0.000
0.000

6
0.051
0.315
0.277
0.308
0.000
0.000
0.000
0.000

7
0.058
0.325
0.337
0.343
0.000
0.000
0.000
0.000

8
0.050
0.364
0.379
0.367
0.000
0.000
0.000
0.000

CORRECTED ABSORBANCE (Sample OD540 - Mean Blank OD540)
2
0.013
0.329
0.330
0.325
-0.049-0.049-0.049
0.000

3
0.015
0.008
0.014
0.005
-0.0490.052-0.049-0.049Mean Blank =

0.056

4
0.006
0.004
0.009
0.002
-0.049-0.049-0.049-0.049

5
0.003
0.144
0.110
0.134
-0.049-0.049-0.049-0.049

6
0.002
0.267
0.229
0.260
-0.049-0.049-0.049-0.049

7
0.009
0.277
0.289
0.295
-0.049-0.049-0.049-0.049

8
0.001
0.316
0.331
0.319
-0.049-0.049-0.049-0.049

9
0.001
0.354
0.352
0.377
-0.049-0.049-0.049-0.049

RELATIVE VIABILITY (% OF VEHICLE CONTROL)
1
A
B
C
D
E
F
G
H

2

3

4

5

6

7

8

9

10

11

128.9%
129.3%
127.3%-19.0%-19.0%-19.0%

3.3%
5.3%
2.2%
-19.0%
20.2%-19.0%

1.8%
3.7%
1.0%
-19.0%-19.0%-19.0%

56.3%
43.0%
52.4%
-19.0%-19.0%-19.0%

104.6%
89.7%
101.8%-19.0%-19.0%-19.0%

108.5%
113.2%
115.6%-19.0%-19.0%-19.0%

123.8%
129.7%
125.0%-19.0%-19.0%-19.0%

138.7%
137.9%
147.7%-19.0%-19.0%-19.0%

139.1%
134.4%
145.8%-19.0%-19.0%-19.0%

136.4%
132.4%
141.5%-19.0%-19.0%-19.0%E-29

In Vitro Cytotoxicity Test Methods BRD Appendix E4
Test Facility : ECBC
Chemical Code : SLS
2nd Chem. Code*: none

November 2006
Study Number.: ECBC-3T3 Ia 0#
96-Well Plate ID : 090602-2
Experiment ID : SLS-B(50ug NR/ml 1hr)

VC1

C1

C2

C3

C4

C5

C6

C7

C8

VC2

Conc. (µg/mL) :

0.0

100.0

68.0

46.3

31.5

21.4

14.6

9.9

6.7

0.0

Mean Corr. OD :
SD :

0.328
0.003

0.009
0.004

0.005
0.004

0.129
0.017

0.252
0.020

0.287
0.009

0.322
0.008

0.361
0.014

0.356
0.015

0.349
0.012

Mean Vehicle Control :Mean Blank :

0.338
0.056

% of Vehicle Control :SD :% CV :

128.5%
1.0%
0.81%

3.6%
1.6%
44.09%

2.2%
1.4%
65.56%

50.6%
6.9%
13.56%

98.7%
7.9%
8.04%

112.4%
3.6%
3.20%

126.2%
3.1%
2.47%

141.5%
5.5%
3.85%

139.8%
5.7%
4.09%

136.8%
4.5%
3.31%

Mean VC - VC1 (%) :Mean VC - VC2 (%) :3.11%-3.11%

Mean Absolute OD :0.387

Neutral Red Uptake% Viability

125%
100%
75%
50%
25%
0%
1

10

100
Concentration (µg/mL)

SLS

E-30

1000

In Vitro Cytotoxicity Test Methods BRD Appendix E4

November 2006

Test Facility : ECBC
Chemical Code : SLS
2nd Chem. Code*: none

Study Number.: ECBC-3T3 Ia 0#
96-Well Plate ID : 090602-2
Experiment ID : SLS-B(25ug NR/ml 3hr)

96-WELL PLATE MAP
A
B
C
D
E
F
G
H

1
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

2
Blank
VC1
VC1
VC1
VC1
VC1
VC1
Blank

3
Blank
C1
C1
C1
C1
C1
C1
Blank

A
B
C
D
E
F
G
H

1
0.052
0.049
0.052
0.052
0.000
0.000
0.000
0.000

2
0.047
0.559
0.613
0.554
0.000
0.000
0.000
0.000

3
0.050
0.047
0.051
0.052
0.000
0.100
0.000
0.000

A
B
C
D
E
F
G
H

1
0.003
0.000
0.003
0.003
-0.049-0.049-0.049-0.049

4
Blank
C2
C2
C2
C2
C2
C2
Blank

5
Blank
C3
C3
C3
C3
C3
C3
Blank

6
Blank
C4
C4
C4
C4
C4
C4
Blank

7
Blank
C5
C5
C5
C5
C5
C5
Blank

8
Blank
C6
C6
C6
C6
C6
C6
Blank

9
Blank
C7
C7
C7
C7
C7
C7
Blank

10
Blank
C8
C8
C8
C8
C8
C8
Blank

11
Blank
VC2
VC2
VC2
VC2
VC2
VC2
Blank

12
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

9
0.046
0.580
0.487
0.527
0.000
0.000
0.000
0.000

10
0.046
0.489
0.444
0.555
0.000
0.000
0.000
0.000

11
0.046
0.610
0.520
0.485
0.000
0.000
0.000
0.000

12
0.046
0.048
0.047
0.057
0.000
0.000
0.000
0.000

10
-0.003
0.441
0.396
0.507
-0.049-0.049-0.049-0.04911-0.003
0.562
0.472
0.437
-0.049-0.049-0.049-0.04912-0.003-0.001-0.0020.008-0.049-0.049-0.049-0.04912

RAW ABSORBANCE DATA (OD540)
4
0.048
0.050
0.061
0.052
0.000
0.000
0.000
0.000

5
0.046
0.175
0.183
0.195
0.000
0.000
0.000
0.000

6
0.048
0.387
0.414
0.364
0.000
0.000
0.000
0.000

7
0.046
0.506
0.525
0.507
0.000
0.000
0.000
0.000

8
0.048
0.474
0.518
0.523
0.000
0.000
0.000
0.000

CORRECTED ABSORBANCE (Sample OD540 - Mean Blank OD540)
2-0.002
0.511
0.565
0.506
-0.049-0.049-0.049
0.000

3
0.001
-0.0020.002
0.003-0.0490.052-0.049-0.049Mean Blank =

0.049

4
-0.001
0.001
0.013
0.003
-0.049-0.049-0.049-0.0495-0.003
0.127
0.135
0.147
-0.049-0.049-0.049-0.0496-0.001
0.339
0.366
0.316
-0.049-0.049-0.049-0.0497-0.003
0.458
0.477
0.459
-0.049-0.049-0.049-0.0498-0.001
0.426
0.470
0.475
-0.049-0.049-0.049-0.0499-0.003
0.532
0.439
0.479
-0.049-0.049-0.049-0.049

RELATIVE VIABILITY (% OF VEHICLE CONTROL)
1
A
B
C
D
E
F
G
H

2

3

4

5

6

7

8

9

10

11

200.3%
221.5%
198.4%-19.0%-19.0%-19.0%

-0.6%
1.0%
1.4%
-19.0%
20.2%-19.0%

0.6%
4.9%
1.4%
-19.0%-19.0%-19.0%

49.6%
52.8%
57.5%
-19.0%-19.0%-19.0%

132.8%
143.4%
123.8%-19.0%-19.0%-19.0%

179.5%
187.0%
179.9%-19.0%-19.0%-19.0%

167.0%
184.2%
186.2%-19.0%-19.0%-19.0%

208.6%
172.1%
187.8%-19.0%-19.0%-19.0%

172.9%
155.2%
198.8%-19.0%-19.0%-19.0%

220.3%
185.0%
171.3%-19.0%-19.0%-19.0%E-31

In Vitro Cytotoxicity Test Methods BRD Appendix E4
Test Facility : ECBC
Chemical Code : SLS
2nd Chem. Code*: none

November 2006
Study Number.: ECBC-3T3 Ia 0#
96-Well Plate ID : 090602-2
Experiment ID : SLS-B(25ug NR/ml 3hr)

VC1

C1

C2

C3

C4

C5

C6

C7

C8

VC2

Conc. (µg/mL) :

0.0

100.0

68.0

46.3

31.5

21.4

14.6

9.9

6.7

0.0

Mean Corr. OD :
SD :

0.527
0.033

0.001
0.003

0.006
0.006

0.136
0.010

0.340
0.025

0.464
0.011

0.457
0.027

0.483
0.047

0.448
0.056

0.490
0.064

Mean Vehicle Control :Mean Blank :

0.508
0.049

% of Vehicle Control :SD :% CV :

206.7%
12.8%
6.21%

0.6%
1.0%
176.38%2.3%
2.3%
100.45%

53.3%
4.0%
7.41%

133.4%
9.8%
7.36%

182.1%
4.2%
2.30%

179.1%
10.6%
5.91%

189.5%
18.3%
9.66%

175.6%
21.9%
12.48%

192.2%
25.3%
13.16%

Mean VC - VC1 (%) :Mean VC - VC2 (%) :-3.64%
3.64%

Mean Absolute OD :0.557

Neutral Red Uptake% Viability

125%
100%
75%
50%
25%
0%
1

10

100
Concentration (µg/mL)

SLS

E-32

1000

In Vitro Cytotoxicity Test Methods BRD Appendix E4

November 2006

Test Facility : ECBC
Chemical Code : SLS
2nd Chem. Code*: none

Study Number.: ECBC-3T3 Ia 0#
96-Well Plate ID : 090602-2
Experiment ID : SLS-B(50ug NR/ml 3hr)

96-WELL PLATE MAP
A
B
C
D
E
F
G
H

1
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

2
Blank
VC1
VC1
VC1
VC1
VC1
VC1
Blank

3
Blank
C1
C1
C1
C1
C1
C1
Blank

A
B
C
D
E
F
G
H

1
0.059
0.057
0.059
0.059
0.000
0.000
0.000
0.000

2
0.065
0.513
0.488
0.516
0.000
0.000
0.000
0.000

3
0.053
0.057
0.058
0.054
0.000
0.100
0.000
0.000

A
B
C
D
E
F
G
H

1
0.011
0.008
0.011
0.011
-0.049-0.049-0.049-0.049

4
Blank
C2
C2
C2
C2
C2
C2
Blank

5
Blank
C3
C3
C3
C3
C3
C3
Blank

6
Blank
C4
C4
C4
C4
C4
C4
Blank

7
Blank
C5
C5
C5
C5
C5
C5
Blank

8
Blank
C6
C6
C6
C6
C6
C6
Blank

9
Blank
C7
C7
C7
C7
C7
C7
Blank

10
Blank
C8
C8
C8
C8
C8
C8
Blank

11
Blank
VC2
VC2
VC2
VC2
VC2
VC2
Blank

12
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

9
0.056
0.473
0.500
0.478
0.000
0.000
0.000
0.000

10
0.053
0.457
0.438
0.455
0.000
0.000
0.000
0.000

11
0.054
0.485
0.562
0.508
0.000
0.000
0.000
0.000

12
0.051
0.050
0.059
0.051
0.000
0.000
0.000
0.000

10
0.004
0.409
0.390
0.407
-0.049-0.049-0.049-0.049

11
0.005
0.437
0.514
0.460
-0.049-0.049-0.049-0.049

12
0.002
0.001
0.011
0.002
-0.049-0.049-0.049-0.04912

RAW ABSORBANCE DATA (OD540)
4
0.052
0.056
0.056
0.056
0.000
0.000
0.000
0.000

5
0.054
0.154
0.152
0.146
0.000
0.000
0.000
0.000

6
0.052
0.302
0.326
0.326
0.000
0.000
0.000
0.000

7
0.054
0.416
0.420
0.496
0.000
0.000
0.000
0.000

8
0.053
0.485
0.460
0.447
0.000
0.000
0.000
0.000

CORRECTED ABSORBANCE (Sample OD540 - Mean Blank OD540)
2
0.017
0.465
0.440
0.468
-0.049-0.049-0.049
0.000

3
0.004
0.008
0.009
0.005
-0.0490.052-0.049-0.049Mean Blank =

0.055

4
0.003
0.007
0.007
0.007
-0.049-0.049-0.049-0.049

5
0.005
0.106
0.104
0.098
-0.049-0.049-0.049-0.049

6
0.003
0.254
0.278
0.278
-0.049-0.049-0.049-0.049

7
0.005
0.368
0.372
0.448
-0.049-0.049-0.049-0.049

8
0.004
0.437
0.412
0.399
-0.049-0.049-0.049-0.049

9
0.007
0.425
0.452
0.430
-0.049-0.049-0.049-0.049

RELATIVE VIABILITY (% OF VEHICLE CONTROL)
1
A
B
C
D
E
F
G
H

2

3

4

5

6

7

8

9

10

11

182.3%
172.5%
183.5%-19.0%-19.0%-19.0%

3.3%
3.7%
2.2%
-19.0%
20.2%-19.0%

2.9%
2.9%
2.9%
-19.0%-19.0%-19.0%

41.4%
40.6%
38.3%
-19.0%-19.0%-19.0%

99.5%
108.9%
108.9%-19.0%-19.0%-19.0%

144.2%
145.8%
175.6%-19.0%-19.0%-19.0%

171.3%
161.5%
156.4%-19.0%-19.0%-19.0%

166.6%
177.2%
168.5%-19.0%-19.0%-19.0%

160.3%
152.8%
159.5%-19.0%-19.0%-19.0%

171.3%
201.5%
180.3%-19.0%-19.0%-19.0%E-33

In Vitro Cytotoxicity Test Methods BRD Appendix E4
Test Facility : ECBC
Chemical Code : SLS
2nd Chem. Code*: none

November 2006
Study Number.: ECBC-3T3 Ia 0#
96-Well Plate ID : 090602-2
Experiment ID : SLS-B(50ug NR/ml 3hr)

VC1

C1

C2

C3

C4

C5

C6

C7

C8

VC2

Conc. (µg/mL) :

0.0

100.0

68.0

46.3

31.5

21.4

14.6

9.9

6.7

0.0

Mean Corr. OD :
SD :

0.457
0.015

0.008
0.002

0.007
0.000

0.102
0.004

0.270
0.014

0.396
0.045

0.416
0.019

0.435
0.014

0.402
0.010

0.470
0.040

Mean Vehicle Control :Mean Blank :

0.464
0.055

% of Vehicle Control :SD :% CV :

179.4%
6.0%
3.36%

3.1%
0.8%
26.57%

2.9%
0.0%
0.00%

40.1%
1.6%
4.08%

105.8%
5.4%
5.14%

155.2%
17.7%
11.40%

163.0%
7.6%
4.65%

170.8%
5.6%
3.30%

157.6%
4.1%
2.60%

184.4%
15.5%
8.41%

Mean VC - VC1 (%) :Mean VC - VC2 (%) :1.37%-1.37%

Mean Absolute OD :0.512

Neutral Red Uptake% Viability

125%
100%
75%
50%
25%
0%
1

10

100
Concentration (µg/mL)

SLS

E-34

1000

In Vitro Cytotoxicity Test Methods BRD Appendix F

November 2006

Appendix F

Reference Substance Information

F1

NRU Test Information for the 72 Reference Substances .................F-3

F2

Chemical, Physical, and Biological Information from the
Literature for the 72 Reference Substances.....................................F-11

F3

Candidate Reference Substances ......................................................F-27

F-1

In Vitro Cytotoxicity Test Methods BRD Appendix F

November 2006

[This Page Intentionally Left Blank]

F-2

In Vitro Cytotoxicity Test Methods BRD Appendix F1

November 2006

Appendix F1

NRU Test Information for the 72 Reference Substances

F-3

In Vitro Cytotoxicity Test Methods BRD Appendix F1

November 2006

[This Page Intentionally Left Blank]

F-4

In Vitro Cytotoxicity Test Methods BRD Appendix F1Table F-1

November 2006

NRU Test Information for the 72 Reference Substances

Chemical

CASRN

Purity(%)

Supplier

pH in 3T3 Concentrations Tested in pH in NHK Concentrations Tested in
MediumaMediumb
3T3 Assay (µg/mL)
NHK Assay (µg/mL)

Acetaminophen

103-90-2

99

Sigma

8.1

4.7-1000

7.7

11.8-4000

Acetonitrile

75-05-8

99.5

Sigma

8.4

118-100000

7.9

8.12-200000

Acetylsalicylic acid

50-78-2

99.5

Sigma

7.5

9.4-2500

6.9

11.8-2500

Aminopterin

54-62-6

100.3

Fluka

8.1

0.00005-0.1

7.2

67.4-1000

5-Aminosalicylic acid89-57-6

99

Sigma

6.7

169-2500

7.5

2.4-500

Amitriptyline HCl

549-18-8

100

Sigma

8.1

0.4-100

7.6

0.24-100

Arsenic III trioxide

1327-53-3

99.9

Sigma

7.9

0.169-100

7.5

0.46-100

Atropine sulfate
monohydrate

5908-99-6

100

Fluka

7.9

4.7-1000

7.5

3.8-10000

Boric aid

10043-35-3

101.1

Fluka

7.1

4.7-10000

7.4

28.3-10000

Busulfan

55-98-1

100.2

Fluka

8.1

2.4-500

7.8

2.35-800

10108-64-2

99.8

Fluka

8.1

0.135-5

7.7

0.337-100

Caffeine

58-08-2

99.9

Fluka

8.3

1.6-5000

7.8

3.25-10000

Carbamazepine

298-46-4

> 99

Sigma

8.0

0.3-1000

7.9

1.88-1000

Carbon tetrachloride

56-23-5> 99.5Sigma-AldrichNA

169-7000

7.7

11.8-7000

Chloral hydrate

302-17-0

100.1

Sigma

8.4

4.7-1000

7.6

4.7-1000

Chloramphenicol

56-75-7

> 99

Fluka

8.3

4.7-2500

7.8

9.15-2500

Citric acid

77-92-9

98

Sigma

2.9

23.5-10000

4.0

23.5-10000

Cadmium II chloride

F-5

In Vitro Cytotoxicity Test Methods BRD Appendix F1Table F-1

November 2006

NRU Test Information for the 72 Reference Substances

Chemical

CASRN

Purity(%)

Supplier

pH in 3T3 Concentrations Tested in pH in NHK Concentrations Tested in
MediumaMediumb
3T3 Assay (µg/mL)
NHK Assay (µg/mL)

Colchicine

64-86-8

> 98

Fluka

8.2

0

7.7

0.0014-0.10

Cupric sulfate
pentahydrate

7758-99-8

99.7

Sigma

7.8

0.0059-5.0

7.4

2.4-750

Cycloheximide

66-81-9

100

Sigma

8.0

0.01-507.8

0.0040-100

Dibutyl phthalate

84-74-2

> 99

Sigma

8.0

3.7-2500

7.7

0.9-1000

Dichlorvos

62-73-7

99.5

Chem Service, Inc.

8.1

0.5-100

7.7

0.235-500

Diethyl phthalate

84-66-2

99.5

Aldrich8.1

4.7-2000

7.8

2.35-2000

20830-75-5

98.6

Sigma

8.2

3.5-1000

7.8

0.0000047-0.100

Dimethylformamide

68-12-2

99.95

Sigma-Aldrich

8.1

236-50000

7.7

70.6-30000

Diquat dibromide
monohydrate

6385-62-2

99

Chem Service, Inc.

7.9

0.03-1007.7

0.47-500

Disulfoton

298-04-4

99.4

Chem Service, Inc.

8.0

2.4-2500

7.8

2.4-2500

Endosulfan

115-29-7

99.5

Chem Service, Inc.

8.3

0.1-100

7.8

0.67-50

Epinephrine bitartrate

51-42-3> 99

Sigma-Aldrich

7.9

6.74-200

7.6

4.7-1000

Ethanol

64-17-5100

Sigma-Aldrich

8.6

1011-50000

7.8

118-150000

Ethylene glycol

107-21-1

99.99

Sigma

8.4

1770-100000

7.8

1770-100000

Fenpropathrin

39515-41-8

91.8

Valent8.3

2.4-500

7.8

0.301-100

Gibberellic acid77-06-5

99

Acros

4.5

1348-100000

6.5

23.6-10000

Glutethimide

77-21-4> 99

Sigma-Aldrich

8.0

19-1000

7.7

4.7-1000DigoxinF-6

In Vitro Cytotoxicity Test Methods BRD Appendix F1Table F-1

November 2006

NRU Test Information for the 72 Reference Substances

Chemical

CASRN

Purity(%)

Supplier

pH in 3T3 Concentrations Tested in pH in NHK Concentrations Tested in
MediumaMediumb
3T3 Assay (µg/mL)
NHK Assay (µg/mL)

Glycerol

56-81-5

99.9

Sigma

8.2

4586-100000

7.8

47-101960

Haloperidol

52-86-8

99

Sigma

8.3

0.1-257.7

0.188-100

Hexachlorophene70-30-4

99.2

Sigma-Aldrich

8.1

0.5-100

7.5

0.002-1

Lactic acid50-21-5

88.6

Sigma

3.2

47.1-100003.0

47.1-10000

Lindane58-89-9

100

Sigma

8.1

0.8-2500

7.7

2.35-2000

Lithium I carbonate554-13-2

99.4

Sigma

9.3

74.3-1102.5

9.5

4.7-2000

Meprobamate

57-53-4

> 99

Sigma

8.1

9.4-2500

7.7

4.71-2500

7487-94-7

99.5

Sigma

8.1

0.05-10

7.6

0.67-10

Methanol

67-56-1

99.97

Sigma-Aldrich

8.0

398-3500 (no toxicity)

7.6

9.42-2500

Nicotine

54-11-5> 99.0Fluka

8.8

94.9-1000

8.5

8.02-5000

Paraquat

1910-42-5

100

Sigma

7.9

0.5-100

7.8

2.4-1000

Parathion

56-38-2

98

Supelco8.2

0.5-2500

7.7

0.47-1500

Phenobarbital

50-06-6100

Spectrum

7.7

11.8-2500

7.4

7.06-3000

Phenol108-95-2

> 99

Sigma

8.0

0.3-1500

7.7

4.7-1000

Phenylthiourea

103-85-5

98

Sigma

8.1

0.8-2500

7.7

9.42-2500

Physostigmine

57-47-6

100

Sigma

8.1

5.4-200

7.7

0.32-1000

7447-40-7

100

Sigma

8.3

163-15000

7.8

23.5-10000

Mercury II chloride

Potassium I chloride

F-7

In Vitro Cytotoxicity Test Methods BRD Appendix F1Table F-1

November 2006

NRU Test Information for the 72 Reference Substances
pH in 3T3 Concentrations Tested in pH in NHK Concentrations Tested in
MediumaMediumb
3T3 Assay (µg/mL)
NHK Assay (µg/mL)

CASRN

Purity(%)

Supplier

Potassium cyanide

151-50-8

99.4

Mallinckrodt Baker

9.0

0.5-1500

8.20.401-500

Procainamide HCl

51-06-999.7

Sigma-Aldrich

8.3

67-1000

7.5

47-10000

2-Propanol

67-63-0> 99.9Sigma

8.5

1011-50000

7.7

47.1-20000

Propranolol HCl

3506-09-0

100

Sigma

7.9

1.78-1000

7.4

1.8-350

Propylparaben

94-13-3

> 99

Fluka

8.1

2.4-1000

7.7

0.47-300

Sodium arsenite

7784-46-5> 99.0Fluka

8.0

0.05-10.0

7.7

0.038-30

Sodium chloride

7647-14-5

99.5

Sigma

8.2

94-20000

7.9

4.71-10000

Sodium dichromate
dihydrate7789-12-0

100.4

Sigma

8.0

0.03-10.0

7.7

0.0318-100

Sodium I fluoride

7681-49-4

100

Sigma

8.1

10.1-1000

7.7

0.3-1000

Sodium hypochlorite

7681-52-9 12.9% Cl

Sigma-Aldrich

8.0

24-10000

7.7

47.1-10000

ChemicalSodium oxalate

62-76-099.99

Sigma-Aldrich

8.1

1.2-500

7.7

40.5-2000

Sodium selenate

13413-01-0

100

Sigma-Aldrich

8.2

6.8-300

7.8

0.47-556

57-24-9

99

Sigma

8.4

9.5-800

7.8

1.18-500

Aldrich8.3

0.1-500

7.8

0.0047-2

Strychnine
Thallium I sulfate

7446-18-6 99.995

Trichloroacetic acid76-03-9> 99

Aldrich2.3

24-10000

1.933.0-10000

1,1,1-Trichloroethane

71-55-6

99.78

Sigma-Aldrich

8.4

1686-50000

8.0

674-10000

Triethylenemelamine

51-18-3

98

Acros

8.0

0.02-4

7.6

0.024-10

F-8

In Vitro Cytotoxicity Test Methods BRD Appendix F1Table F-1

November 2006

NRU Test Information for the 72 Reference Substances

Chemical

CASRN

Purity(%)

Supplier

pH in 3T3 Concentrations Tested in pH in NHK Concentrations Tested in
MediumaMediumb
3T3 Assay (µg/mL)
NHK Assay (µg/mL)

Triphenyltin hydroxide

76-87-9~ 99.5Sigma-Aldrich

8.0

0.0002-0.1

7.6

0.005-0.1

Valproic acid

99-66-1

100

Sigma

6.9

12-2500

6.0

11.8-2500

Verapamil HCl

152-11-4

98

Sigma-Aldrich

8.1

3.4-100

7.5

3.8-1500

Xylene1330-20-7

99.9

Mallinckrodt Baker

6.8

398-2500

7.5

190-2000

Abbreviations:NRU=Neutral red uptake; CASRN=Chemical Abstracts Service Registry Number; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes;
pH=Mean pH of the highest concentration tested (of all acceptable NRU tests)a
3T3 Medium - Dulbecco’s Modification of Eagle’s Medium, with supplements.
b
NHK medium - Keratinocyte Growth Medium (KGM from Cambrex).

.

F-9

In Vitro Cytotoxicity Test Methods BRD Appendix F1

November 2006

[This Page Intentionally Left Blank]

F-10

In Vitro Cytotoxicity Test Methods BRD Appendix F2

November 2006

Appendix F2

Chemical, Physical, and Biological Information fromthe Literature for the 72 Reference Substances

F-11

In Vitro Cytotoxicity Test Methods BRD Appendix F2

November 2006

[This Page Intentionally Left Blank]

F-12

In Vitro Cytotoxicity Test Methods BRD Appendix F2Table F-2
ChemicalNovember 2006

Chemical, Physical, and Biological Information from the Literature for the 72 Reference Substances
CASRN

LD50
MW
(mg/kg)a (g/mol)

ChemicalClassb

Water
Solubilityc

log
Kowc

Boiling
Point (oC)d

0.8

NA

Liver toxin

Free?

-0.34

81.6

CNS stimulant

Presumed

3.49@
25°C

1.19

NA

5.5

NA

NA

2 mg/mL; <1
mg/mL @ 21°C

3.25

1.32

NA

Kidney toxin

Yes

Not activated Unknown

0.0097 mg/mL@ 24°C/HCl isfreely soluble

9.4

5.04

NA

Cardiotoxin

Free

Nortriptyline, Hypothetical: Blocks
a metabolite, norepinephrine, 5also activehydroxytryptamine,and dopaminepresynaptic uptake;prevents reuptake ofheart norepinephrine.

Acetaminophen

103-90-22404

151.20 Organic
compound;
Amide

Acetonitrile

75-05-83798

41.05 Organic
compound;NitrileSlightly in cold, NAmuch more in
hot; 1-5 mg/mL@ 22°C
Miscible; >100 -4.30
mg/mL @
22.5°C

Acetylsalicylic acid

50-78-2

1000

180.20 Organic
compound;Carboxylic
acid;
Phenol3.3 mg/mL @25°C; 4.6
mg/mL @
25°C; <1
mg/mL @ 23°C

Aminopterin

54-62-6

3
476.45 OrganicNA(mouse)compound;Heterocyclic
compound

5-Aminosalicylic acid89-57-6

Amitriptyline HCl

7749
153.10 Organic(mouse)compound;Carboxylic
acid;
Phenol549-18-8 319
313.90 Organic
compound;Polycyclic
compoundF-13

Toxic EffectClassfPassage of
Metabolic
Blood: Brain Activation/
Barrierg
Inactivationf

pKd

More toxic
intracellular
metabolites

Mechanism of
Lethaliyf
Covalent NAPQI
binding and lipid
peroxidation.

Must beAssumed to be same as
metabolized to cyanide: Generalhydrogen
enzyme inhibition.
cyanide forHigh affinity for Fe+++.
Inhibits cell respiration
effect.
by inhibition ofcytochrome oxidase;
solvent
Gastric irritant, Restricted
Salicylic acid General cell poison,
CNS
is an active
works by uncoupling(encephalometabolite
oxidation
pathy), kidney
phosphorylation andtoxin
inhibition of Kreb's
cycle dehydrogenases.
Hematotoxin
Presumed to Not expected Hypothetical: Inhibits
be minimal
to require
folic acid utilization(likemetabolism
and thus cellmethotrexate) for toxicity
proliferation.

In Vitro Cytotoxicity Test Methods BRD Appendix F2Table F-2
ChemicalNovember 2006

Chemical, Physical, and Biological Information from the Literature for the 72 Reference Substances
CASRN

LD50
MW
(mg/kg)a (g/mol)

ChemicalClassb

Toxic EffectClassfPassage of
Metabolic
Blood: Brain Activation/
Barrierg
Inactivationf

Water
Solubilityc

pKd

log
Kowc

Boiling
Point (oC)dsparingly incold; in 15 partsboiling; 17
mg/mL @ 16°C

NA

NA

465

CNS toxin(encephalopathy)

RestrictedNo

Mechanism of
Lethaliyf

Arsenic III trioxide

1327-53-3

20

197.80 Inorganic
compound;Arsenical

Atropine sulfate
monohydrate

5908-99-6

623

694.80 Organic
2.2 mg/mL
compound;Heterocyclic
compound

NA

1.83

NA

CNS stimulant

Free

No

Boric aid

10043-35-3

2660

NA

NA

300

Skin, kidney,
liver, testicular
toxin

Yes

No

Busulfan

55-98-1

2

NA

-0.52

NA

Hematotoxin

Freely (similar Reactive
Hypothetical:
to plasmaintermediatesh Alkylation of sufhydrylhconcentrationgroupsi; antineoplastic

10108-64-2

88

61.83 Inorganic
56 mg/mL incompound; cold water; 10Boron
50 mg/mL @compound; 19°C
Acids
Decomposes
246.31 Organic
compound;Alcohol;
Acyclic
hydrocarbon;
Sulfurcompound183.31 Organic
1400 mg/mL @compound; 20°C; >100
Cadmium
mg/mL @ 20°C
compound

NA

NA

960

Kidney, liver
Yesj
toxin, corrosive

Cadmium II chloride

F-14

No

Cellular poison.
Multisystem failure due
to uncoupling oxidative
phosphorylation &inhibition of pyruvateand succinate oxidative
pathways; Apoptosis
induction; angiogenesis
inhibition; cellular
growth inhibition
Antimuscarinic,
anticholinergic action.
Competitive
antagonism ofanticholinesterase at
cardiac & CNS
receptor sites.
Inhibits enzymes
involved in metabolism
and RNA synthesis.g

Alters Ca++translocation, affects
membrane ATPase &mitochondrial
respiration.

In Vitro Cytotoxicity Test Methods BRD Appendix F2Table F-2
ChemicalNovember 2006

Chemical, Physical, and Biological Information from the Literature for the 72 Reference Substances
CASRN

LD50
MW
(mg/kg)a (g/mol)

ChemicalClassb

Water
Solubilityc

pKd

log
Kowc

Boiling
Point (oC)dToxic EffectClassf

14 @25°C;
pKb=14
.15 @
19°C

-0.07

17
(sublimes)

CNS stimulant

Passage of
Metabolic
Blood: Brain Activation/
Barrierg
Inactivationf

Caffeine

58-08-2

192

194.20 Organic
21 mg/mL @compound; 25°C; 10-50
Heterocyclic mg/mL @ 23°C
compoundCarbamazepine

298-46-4

1957

236.30 Organic
Practicallycompound; insoluble
Heterocyclic
compound

NA

2.45

NA

CNS depressant, Free
hematotoxin

Carbon tetrachloride

56-23-52799

153.82 Organic
0.793 mg/mL atcompound; 25°C; <1
Halogenated mg/mL @ 21°C
hydrocarbon

NA

2.83

76.8

Liver, kidney
toxin, CNS
depressant

Chloral hydrate

302-17-0479

165.40 Organic
compound;Alcohol

9310 mg/mL @25°C; >10mg/mL @
20.5°C

NA

0.99

96

CNS depressant Freely& cardiotoxin

Chloramphenicol

56-75-73393

2.5 mg/mL @323.14 Organic
compound; 25°C
Alcohol;
Cyclichydrocarbon;
Nitro
compound

NA

1.14

NA

Hematotoxin

F-15

Free

Free

Free

Mechanism of
LethaliyfNo

Hypothetical:
Inhibition ofphosphodiesterase
leading to AMP
accumulation.
Translocation of
intracellular Ca++?
Adenosine receptor
antagonism?;neurotoxic
10,11-epoxide Not known.
metabolite as Therapeutically
active as
decreases firing ofparentnoradrenergic neurons.
More toxic
intracellular
metabolites?

Hypothetical: Covalentbinding of toxic
intracellular
metabolites. Free
radicals inducing lipid
peroxidation?
Proposed: potentiation
Activeof GABAA receptor
metabolite
trichloroethan activity, inhibition ofol is partlyf or N-methyl-D-aspartate
totallyk
activity, & modulation
responsible for of 5CNS effecthydroxytryptamine3
receptor-mediateddepolarization of thevagas nerve.k
No
Hypothetical: Binds tomitochondrial
ribosomes & inhibits
enzyme syntheses (e.g.,
those necessary foroxidative
phosphorylation)

In Vitro Cytotoxicity Test Methods BRD Appendix F2Table F-2
Chemical
Citric acidColchicine

November 2006

Chemical, Physical, and Biological Information from the Literature for the 72 Reference Substances
CASRN
77-92-9LD50
MW(mg/kg)a (g/mol)
3000

ChemicalClassb7758-99-8

300

Cycloheximide

66-81-92

Dibutylphthalate

84-74-2 11998

Dichlorvos

62-73-7

17

pKd

log
Kowc

1=3.128 -1.72
2=4.7613=6.396@ 25°C
pK=12. 1.03
45 mg/mL;
35 @>100 mg/mL @
20°C;
21°C
pKa=1.
7&
12.4
NA
NA
249.70 Inorganic
148 & 316
compound; mg/mL @ 0°C;
Sulfur2033 mg/mL @compound; 100°C; 230.5
Metal
mg/mL @
25°C; 32
mg/mL @
20°C; >100
mg/mL @ 21°C
NA
0.55
281.40 Organic
21 mg/mL @compound; 2°C; 10-50°
Heterocyclic mg/mL @ 20 C
compound278.30 Organic
NA
4.9
0.013 mg/mL
compound; @ 25°C; 0.01
Carboxylic mg/mL @acid
20°C; <1
mg/mL @ 20°C

192.10 Organic
compound;Carboxylic
acid
64-86-8
6
399.45 Organic
compound;(mouse)Polycyclic
compoundCupric sulfate
pentahydrate

Water
Solubilityc
592 mg/mL @20°C; >100
mg/mL @ 22°C

220.98 Organic
10 mg/mL @compound; 20°C; 5 g/mL;
Organophos- 10-50 mg/mL
phorous@ 20°C
compoundNA

Boiling
Point (oC)dToxic EffectClassfdecomposes Acidosis

Passage of
Metabolic
Blood: Brain Activation/
Barrierg
Inactivationf
NAGI, liver, kidney, No
hemato-, PNS
toxin

decomposes Liver, kidney@ 150°C
toxin

NA

Mechanism of
LethaliyfNA

Not expected Depresses respiratory
center.

Restricted

No

Unknown

Metabolically Inhibition of protein
activatedsynthesis?; metabolic
inhibitor

NA

Liver toxin

340

CNS depressant; Yesp
pulmonary,
liver, testicular
toxin

Hypothetical: Copper isreduced by thiol groups
in cell membranes.
superoxide is formedby reoxidation ofcopper, inducing lipid
peroxidation.

Monobutyl
Peroxisome
metabolite has proliferatoru
greater
toxicity thanparent in rats

1.43, 245; 140 @ 20 CNS depressant Assumed due Rapidly
Inhibition of
1.45
mmHg
to CNS effects inactivated by acetylcholinesterase
hepatic
resulting in
metabolism
acetylcholine
accumulation in CNS& effector organs;irreversible
cholinesterase inhibitor

F-16

In Vitro Cytotoxicity Test Methods BRD Appendix F2Table F-2
ChemicalDiethyl phthalate

DigoxinNovember 2006

Chemical, Physical, and Biological Information from the Literature for the 72 Reference Substances
CASRN
84-66-2

20830-75-5LD50MW(mg/kg)a (g/mol)

Water
Solubilityc

pKd

log
Kowc

Boiling
Point (oC)d222.20 Organic
compound;Carboxylicacid<1 mg/mL @19°C and 25°C

NA

2.47

298

CNS depressant, Yesm
liver toxin

18780.90 Organic
0.0648 mg/mL
compound; @ 25°C
(mouse)Polycyclic
compound;Carbohydrate

NA

1.26

NA

Cardiotoxin

Restricted-0.01 @ -1.01
-20°C

153

Liver, kidney
toxin

NA

NA

GI, pulmonary, Freenliver, kidney
toxin

8602

Dimethylformamide

68-12-2

2800

Diquat dibromide
monohydrate

6385-62-2

231

Disulfoton

298-04-4

2

Endosulfan

115-29-7

18

73.10 Organic
compound;Amide
362.10 Organic
compound;Heterocyclic
compoundMiscible; >100
mg/mL @ 22°C

Toxic EffectClassfPassage of
Metabolic
Blood: Brain Activation/
Barrierg
Inactivationf

ChemicalClassbNA-3.05

0.012 mg/mL
274.42 Organic
compound; @ 20°C
Organophosphorouscompound;SulfurcompoundNA

4.02

132-33 @ 1.5 CNS depressant Yes
mmHg; 108
and 62 @ 0.01
mmHg

406.91 Organic
0.00053 mg/mL
compound; @ 25°C
Heterocyclic
compound;SulfurcompoundNA

3.83

106 @ 0.7mm, partial
decomposition

700 mg/mL @20°C; >100
mg/mL @ 20°C

F-17

CNS depressant Yeso

Mechanism of
Lethaliyf

Monoethyl
Peroxisome
metabolite has proliferatoru
greater
toxicity thanparent in rats
Also active
Impairs ion transport &metabolites
increases sarcoplasmic
calcium by binding to
Na+/K+ ATPase,
increasing automaticity
of cardiac cells.
NA
Hepatocellularnecrosisu
NonMore toxic
metabolites

Noo

Assumed to be same as
Paraquat; Hypothetical:
Multisystem failure due
to depletion ofsuperoxide dismutase,formation of free
radicals & lipid
peroxidation. Lung
fibrosis due toaccumulation.
Inhibition ofacetylcholinesterase
resulting inacetylcholine
accumulation in CNS& effector organs;irreversible
cholinesterase inhibitor
Affects brainneurotransmitterlevels.oIn Vitro Cytotoxicity Test Methods BRD Appendix F2Table F-2
ChemicalNovember 2006

Chemical, Physical, and Biological Information from the Literature for the 72 Reference Substances
CASRN

LD50
MW
(mg/kg)a (g/mol)

ChemicalClassb4
333.30 Organic
compound;(mouse)Alcohol;
Amine

Passage of
Metabolic
Blood: Brain Activation/
Barrierg
Inactivationf

Water
Solubilityc

pKd

log
Kowc

Boiling
Point (oC)dToxic EffectClassf

1 mg/mL @25°C; < 0.1
mg/mL @ 18°C
(for base)

NA

-1.52

NA

Cardiovascular
toxin

78.5

CNS depressant Free

Epinephrine bitartrate

51-42-3

Ethanol

64-17-5 14008

46.07 Organic
compound;Alcohol>10% why
include; > 100
mg/ml @ 23°C

15.9 @
25°C-0.31

Ethylene glycol

107-21-1

8567

62.07 Organic
compound;Alcohol

Miscible; > 100
mg/mL @
17.5°CNA-1.36

Fenpropathrin

39515-41-8

18

349.43 Organic
0.00033 mg/mL
compound; @ 25°C
Nitrile; Ester;
Ether

NA

6.0 @
20°C

377

PNS toxin

Yesp

Gibberellic acid77-06-5

6305

346.38 Organic
compound;Polycyclic
compound

5 mg/mL;slightly

4

0.24

NA

NA

NA

Glutethimide

77-21-4600

Practicallyinsoluble

4.2

1.9

NA

CNS depressant Presumed

Glycerol

56-81-5 12691

217.30 Organic
compound;Heterocyclic
compound
92.09 Organic
compound;Alcohol

Soluble in all
proportions; >100 mg/mL @
18 °C

14.4

-1.76

F-18

No

197.6 @ 760 CNS depressant, Free
mmHg
kidney toxin

182; 290 @ Body fluids
760 mmHg,
decomposes

No evidence
found

Mechanism of
Lethaliyf

Large first
Adrenergic receptor
passstimulation.
metabolism toinactive
metabolites
Acetaldehyde, Hypothetical:
activeInterferes with cell
metabolite
membrane fluidity,
perturbing proteinssuch as ion channels.
Depression of
postsynaptic potentials
in CNS; solvent
Glyoxalate,
Hypothetical:
glycolate, & Metabolites inhibit
oxalate, active mitochondria tometabolites
produce metabolic
acidosis. Oxalate
decreases sarcoplasmic
Ca++; affects kidney
function; oxalic acid is
toxic metabolite
Rapidly
Delays closure ofhydrolyzed to sodium channel
inactive
causing persistentproducts indepolarization ofe,pmammals
membrane.
NANA

2X active
CNS depression;
metabolite: 4- anticholinergic activityhydroxygluthethimide
No
Cellular dehydration;
osmotic effectIn Vitro Cytotoxicity Test Methods BRD Appendix F2Table F-2
ChemicalNovember 2006

Chemical, Physical, and Biological Information from the Literature for the 72 Reference Substances
CASRN

LD50
MW
(mg/kg)a (g/mol)

Haloperidol

52-86-8128

Hexachlorophene70-30-4

61

Lactic acid50-21-5

3730

Lindane58-89-976

Lithium I carbonate554-13-2

1187(sulfatesalt;mouse)

Meprobamate

57-53-4

794

ChemicalClassb

Water
Solubilityc

log
Kowc

Boiling
Point (oC)d

8.3

3.36

NA

CNS depressant Presumed

No

Blocks dopamine
receptors

4.95

6.91

NA

CNS depressant Restricted

No3.86 @
25°C-0.72

122 @ 14-15 Acidosis,
mmHg
corrosive

Yesg

Unknown

Hypothetical:
Uncoupling ofoxidative
phosphorylation.
Binding to proteins incytoplasmic membrane& cell organelles.
Disturbance of
metabolism (lactic
acidosis).

0.0073 mg/mL@ 25°C; < 1
mg/mL @ 24°C

NA

3.72

323.4 @ 760 CNS stimulant
mmHg

Free

No?

CNS depression
through inhibition of
GABA receptor linked
chloride channel at thepicrotoxin binding site,leading to blockade ofchloride influx into
neurons?

1.5 mg/mL @0°C; 1.3 mg/mL@ 20°C; 1.2
mg/mL @
40°C; 12.2
mg/mL cold; 7
mg/mL hot

NA

NA

NA

CNS depressant Restricted(assumedsame aslithium
sulfate)No

Unknown: Partial
substitution for normal
cations of cells may
disturb energy
processes?

3.4 mg/mL @20°C; 7.9
mg/mL @
37°C; < 1
mg/mL @ 20°C

9.2

NA

NA

CNS depressant NA
cardiotoxin

Rapidly
Unknown
inactivated byhepatic
metabolism

0.014 mg/mL
375.90 Organic
compound;Ketone406.91 Organic
0.140 mg/mL
compound; @ 25 °C; < 1
Cyclicmg/mL @ 20°C
hydrocarbon;
Phenol90.08 Organic
compound;Carboxylicacid
290.80 Organic
compound;
Halogenatedhydrocarbon

73.89 Inorganic
compound;Lithiumcompound;Alkalies;
Inorganiccarboncompound
218.30 Organic
compound;Carboxylic
acid

SolubleF-19

Toxic EffectClassfPassage of
Metabolic
Blood: Brain Activation/
Barrierg
Inactivationf

pKd

Mechanism of
Lethaliyf

In Vitro Cytotoxicity Test Methods BRD Appendix F2Table F-2
ChemicalMercury II chloride

November 2006

Chemical, Physical, and Biological Information from the Literature for the 72 Reference Substances
CASRN
7487-94-7

LD50MW(mg/kg)a (g/mol)
1

ChemicalClassb

Water
Solubilityc

pKd

log
Kowc

Boiling
Point (oC)d271.50 Inorganic
compound;
Mercury
compound;
Chlorinecompound69 mg/mL at
20°C; 5-50
mg/mL @ 22°C

NA

0.22

302

Completely
miscible at
20°C; >100
mg/mL @ 21°C

Methanol

67-56-1 13012

32.04 Organic
compound;AlcoholNicotine

54-11-550

162.20 Organic
Miscible below
compound; 60°C
Heterocyclic
compoundParaquat

1910-42-5

58

257.20 Organic
Soluble; >100
compound; mg/mL @ 19°C
Heterocyclic
compoundParathion

56-38-2

2

0.011 mg/mL
291.28 Organic
compound; @ 20°C; <1
Organomg/mL @ 23°C
phosphorouscompound;SulfurcompoundToxic EffectClassfRestrictedNo

Mechanism of
Lethaliyf

Hypothetical: Changes
membrane potentials &blocks enzyme
reactions in cells bytargeting the sulfhydryl
part of active sites ofsome enzymes.
15.3
-0.77
64.7 @ 760 CNS depressant Free
ActiveHypothetical:
mmHg
metabolites: Accumulation offormadehyde, formic acid leads toformic acidmetabolic acidosis.
Lactate inhibits
mitochondrial
respiration;formaldehyde
metabolite
pKb1=6. 1.17
247
CNS stimulant FreeNo
CNS nicotinic receptor;
16 @cholinergic block
15°C;
causing polarization of
pKb2=1
CNS and PNS
0.96
synapses.
NA -4.22 @ 175-180 @ Pulmonary toxin Free?
No
Multisystem failure due
pH 7.4 760 mmHg,
to depletion of
decomposes
superoxide dismutase,with formation of free
radicals & lipid
peroxidation. Lung
fibrosis due toaccumulation;interferes with ATP
synthesis.
NA
3.83
375 @ 760 CNS depressant Free (assumed Paraoxon is Inhibition ofmm Hg
the same asactiveacetylcholinesterase
malathion)
metabolite.
resulting inacetylcholine
accumulation in CNS& effector organs;irreversible
cholinesterase inhibitor

F-20

Corrosive,
kidney toxin

Passage of
Metabolic
Blood: Brain Activation/
Barrierg
Inactivationf

In Vitro Cytotoxicity Test Methods BRD Appendix F2Table F-2
ChemicalNovember 2006

Chemical, Physical, and Biological Information from the Literature for the 72 Reference Substances
CASRN

LD50
MW
(mg/kg)a (g/mol)

ChemicalClassb

Water
Solubilityc

pKd

log
Kowc

Boiling
Point (oC)dToxic EffectClassfPassage of
Metabolic
Blood: Brain Activation/
Barrierg
Inactivationf

Phenobarbital

50-06-6

163

232.23 Organic
compound;Heterocyclic
compound

1 mg/mL; 1.3
mg/mL at 25°C;<0.1 mg/mL @14°C

pK1=7.
3,
pK2=11
.8

1.47

NA

CNS depressant FreeNo

Phenol108-95-2

414

94.11 Organic
compound;PhenolNA

1.46

182 @ 760
mm Hg

Corrosive; CNS Free
depressantNo

Phenylthiourea

103-85-5

3.0

67 mg/mL; 82.8
mg/mL @
25°C; 93
mg/mL @
25°C; 50-100
mg/mL @ 19°C
2.5 mg/mL @25°C; <1
mg/mL @ 21°C

NA

0.71

NA

Pulmonary toxin NA

Physostigmine

57-47-6

4.5

152.20 Organic
compound;Sulfurcompound;
Urea
Slightly soluble
275.40 Organic
compound;Carboxylic
acid;
Heterocyclic
compound

NA

NA

NA

CNS depressant Easily

Humans &animals havehigh capacity
to detoxify
sulfidesNone known

Potassium I chloride

7447-40-7

2602

Potassium cyanide

151-50-8

10Procainamide HCl

51-06-91950

Mechanism of
Lethaliyf
Neurotoxic; CNS
depression throughinhibition of GABA
synapses? Inhibits
hepatic NADH
cytochromeoxidoreductase;
General protoplasmic
poison that denaturesproteins; depresses
vasomotor center

Destroys cytochromep450; interferes withpulmonary, thyroid
functions.
Inhibition ofacetylcholinesterase
resulting inacetylcholine
accumulation in CNS& effector organs.

74.55 Inorganic
compound;Potassiumcompound;
Chlorinecompound65.12 Inorganic
compound;Potassiumcompound;Nitrogencompound

342 mg/mL @20°C; >100
mg/mL @ 20°C

NA

NA

1500

Cardiotoxin

Free?

No

Essential cellularelectrolyte maintains
normal transmembranepotential, necessary forheart conduction.

500 mg/mL
cold; 1000
mg/mL hot

NA

NA

NA

CNS stimulant, Free
corrosiveNo271.79 Organic
compound;Carboxylic

Freely soluble

NA

NA

NA

CNS depressant, Some
cardiotoxin

Less potentr;
activemetabolitec

General enzyme
inhibition. Interfereswith ATP synthesis.
High affinity for Fe+++.
Inhibits cell respiration
by inhibition ofcytochrome oxidase.
Slows impulse
conduction in theheart?rF-21

In Vitro Cytotoxicity Test Methods BRD Appendix F2Table F-2
ChemicalNovember 2006

Chemical, Physical, and Biological Information from the Literature for the 72 Reference Substances
CASRN

LD50
MW
(mg/kg)a (g/mol)

ChemicalClassb

Toxic EffectClassfPassage of
Metabolic
Blood: Brain Activation/
Barrierg
Inactivationf

Water
Solubilityc

pKd

log
Kowc

Boiling
Point (oC)dMechanism of
Lethaliyf>100 mg/mL@ 22°C

NA

0.05

82.3

CNS depressant Free

No.

Soluble

NA

3.09

NA

Cardiotoxin

Free

No?

0.463 mg/mL@ 25°C; <1
mg/mL @ 12°C

NA

3.04

NA

CNS depressant NANA

Very to freely
soluble

NA

NA

NA

PNS, liver,hematotoxin

Yes

357 mg/mL @0°C; 391.2
mg/mL @
100°C

NA

NA

1413°C

Body fluids

Restricted

Not expected Assumed the same asarsenic trioxide causes multisystem
failure due touncoupling of oxidative
phosphorylation &inhibition of pyruvate& succinate oxidative
pathways.
No
Acute dehydration ofbrain cells caused byosmotic shift of water
to the outside of theblood:brain barrier.

2380 mg/mL @
0°C

NA

NA

acid; Amide
2-Propanol
Propranolol HCl

Propylparaben

Sodium arsenite

67-63-05843

60.10 Organic
compound;Alcohol
350-60-90 470
295.80 Organic
compound;(mouse)Alcohol;
Amine;
Polycyclic
compound
94-13-3 6326
180.20 Organic
compound;(mouse)Carboxylicacid; Phenol
7784-46-5
41129.90 Inorganic
compound;Arsenical;
Sodiumcompound

Sodium chloride

7647-14-5

2998

Sodium dichromate
dihydrate7789-12-050

58.44 Inorganic
compound;
Sodiumcompound;
Chlorinecompound
298.00 Inorganic
compound;
Sodiumcompound;
ChromiumcompoundF-22

decomposes Kidney, liver
@ 400
toxin

Yess

CNS depression
through membrane
effectsu
Unknown: Betaadrenergic blockade?

NA

Less active in Inhibition ofpresence of
respiratory chain
metabolizing activity; carcinogenic
systemIn Vitro Cytotoxicity Test Methods BRD Appendix F2Table F-2
ChemicalNovember 2006

Chemical, Physical, and Biological Information from the Literature for the 72 Reference Substances
CASRN

LD50
MW
(mg/kg)a (g/mol)

ChemicalClassb

Water
Solubilityc

pKd

log
Kowc

Boiling
Point (oC)dToxic EffectClassfPassage of
Metabolic
Blood: Brain Activation/
Barrierg
Inactivationf

Mechanism of
Lethaliyf

Sodium I fluoride

7681-49-4

180

41.99 Inorganic
compound;
Sodiumcompound;fluorinecompound43 mg/mL @25°C; 10-50
mg/mL @ 23°C

NA

NA

NA

GI irritant, CNS Restricted
depressantNo

Sodium hypochlorite

7681-52-9

8910

293 mg/mL @
0°C

NA

NA

111

Corrosive, body NA
fluidsNA

Sodium oxalate

62-76-015574.44 Inorganic
compound;
Sodiumcompound;Oxygencompound;chlorinecompound
134.00 Organic
compound;Carboxylicacid

220 mg/mL @
25°C

NA

NA

NA

Corrosive, body Restrictedfluids, kidney &cardiotoxin,
CNS depressantSodium selenate

13413-01-0

1.6> 100 mg/mL@ 21°C

NA

NA

NA

Liver, kidney
toxin

Yest

57-24-92

188.90 Inorganic
compound;
Sodiumcompound;Seleniumcompound334.40 Organic
compound;Heterocyclic
compoundHypothetical: Ca++complexing action,
depressing the level ofionized Ca++ in body
fluids, but doesn't
explain action on GI,
vasculature, & kidney.
Corrosivity not due toacidity.
Not expected Inactivates sulfhydryl
enzymes for oxidative
reactions in cellularrespiration.t

0.16 mg/mL @25°C

8.26 @
25°C

1.93

270 @ 5mmHg

CNS stimulant

Expected

Strychnine

F-23

Hypothetical:
Protoplasmic poison
interfering with many
enzymes. May lower
sarcoplasmic Ca++ &induce K+ efflux from
cells.
NA

No

No

Increases glutamic acidin the CNS. Alkaloid
poison.

In Vitro Cytotoxicity Test Methods BRD Appendix F2Table F-2

November 2006

Chemical, Physical, and Biological Information from the Literature for the 72 Reference Substances

Chemical

CASRN

LD50
MW
(mg/kg)a (g/mol)

pKd

log
Kowc

Boiling
Point (oC)d29504.80 Inorganic
48.7 mg/mL @compound; 15°C; 191.4(mouse)Metal; Sulfur mg/mL @compound
100°C

NA

NA

NA

GI irritant, CNS Restricted
toxin(encephalopathy)

No

4999

10 g/mL @25°C; 1200
mg/mL @
25°C; 13.06
g/mL @ 25°C;>100 mg/mL @22°C
133.41 Organic
4.4 mg/mL @compound; 20°C; <1
Halogenated mg/mL @ 20°C
hydrocarbon
204.23 Organic
400 mg/mL @compound; 26°C; <1
Heterocyclic mg/mL @ 16°C
compound
367.02 Organic
0.0012 mg/mL;
compound; <1 mg/mL @Organo21°C
metallic
compound

NA

1.33

196

GI corrosion,
acidosis

Not expected Corrosive; possible
carcinogen

NA

2.49

76

CNS depressant; Free
liver toxin

NA

-0.54

NA

NA

NA

CNS toxin(encephalopathy), skin &GI irritant

2 mg/mL @20°C; 1.27
mg/mL; <1
mg/mL @ 22°C

NA

2.75

220

CNS depressant, Yesliver toxin

7446-18-6

Trichloroacetic acid76-03-9

1,1,1-Trichloroethane

71-55-6 10298

Triethylenemelamine

51-18-3

1.0

Triphenyltin hydroxide

76-87-944

Valproic acid

99-66-1

163.40 Organic
compound;Carboxylic
acid

670
144.20 Organic
compound;(mouse)Carboxylicacid; Lipids

F-24

Toxic EffectClassfPassage of
Metabolic
Blood: Brain Activation/
Barrierg
Inactivationf

Water
Solubilityc

Thallium I sulfate

ChemicalClassb139
Hemato-, liver,(decomposes) kidney toxin

Expected

Mechanism of
Lethaliyf
Hypothetical: Enzyme
inhibition by binding
sulfhydryl groups of
mitochondrial
membranes. Interfereswith oxidative
phosphorylation by
inhibition of Na+/K+
ATPase.

No.

Arrhythmogenicu

Unknown

Expectedsince it's analkylator

Genotoxic; binds with
DNA; alkylating agent;alkylates proteins

RapidlyNo

Affects a number ofenzymes involved incellular energy
production and use.
Affects immune
system; causes
lymphopenia;
clastogenic
Increases GABA in the
CNS?

Some
metabolites
may be activeIn Vitro Cytotoxicity Test Methods BRD Appendix F2Table F-2
ChemicalNovember 2006

Chemical, Physical, and Biological Information from the Literature for the 72 Reference Substances
CASRN

LD50
MW
(mg/kg)a (g/mol)

ChemicalClassb

Water
Solubilityc
70 mg/mL

Verapamil HCl

152-11-4

108

491.08 Organic
compound;Amine

Xylene1330-20-74300

106.17 Organic
Practicallycompound; insoluble; <1Cyclicmg/mL @ 22°C
hydrocarbon

pKd

log
Kowc

Boiling
Point (oC)d

NA

3.79

NA

NA

3.12-3.2

136-140

Toxic EffectClassfCardiotoxin

Passage of
Metabolic
Blood: Brain Activation/
Barrierg
Inactivationf
Restricted?

CNS depressant Free

Also active
metabolites

No

Mechanism of
Lethaliyf
Inhibition oftransmembrane Ca++flux in excitatory
tissues. Cardiac-Ca++channel blocker. Alsoalpha-adrenergic
blockade.
Unknown: Heart failurecaused by sensitizationof heart tocatecholamines?;solvent

Abbreviations: MW=Molecular weight; NA=No information found; NADPQI=N-acetyl-p-benzoquinoneimine; CNS=Central nervous system; AMP=Adenosine monophosphate; GABA=Gamma aminobutyric acid;
GI=Gastrointestinal; PNS=Peripheral nervous system; NADH=Nicotine adenine dinucleotide (reduced).
LD50 data from Registry of Cytotoxicity (Halle 1998), Hazardous Substances Data Bank (NLM 2001, 2002), or Registry of Toxic Effects of Chemical Substances® (MDL information Systems 2001, 2002). Rat data unlessotherwise noted. Rounded to the nearest one.
b
Based on the Medical Subject Heading [MeSH] index (NLM 2005).
c
Hazardous Substances Data Bank (NLM 2001, 2002) and NTP Chemical Health and Safety Data (2001) at http://ntp-server.niehs.nih.gov/Main_Pages/Chem-HS.html. The NTP database is no longer available. NTP values
can be identified by the use of the following symbols: <, >, and ≥. Conditions are reported if available.
dHazardous Substances Data Bank (NLM 2001, 2002) unless otherwise specified. pK measured under the conditions specified. If no conditions were specified, none are reported.
eHazardous Substances Data Bank (NLM 2001, 2002) or Material Safety Data Sheets. Boiling point measured under the conditions specified. If no conditions were specified, none are given.
f
Ekwall et al. (1998) or Hazardous Substances Data Bank (NLM 2001, 2002) unless otherwise noted.
g
Cosmetic Ingredient Review Panel (1983).
hOrphan Medical (1999).
i
Glaxo Wellcome (2000).
j
ATSDR (1999).
k
EPA (2000b).
l
ATSDR (2001).
m
ATSDR (1995).
n
EPA (1995).
o
ATSDR (2000a).
p
ATSDR (2004a).
q
Ames (2000).
r
Hardman et al. (1996).
s
ATSDR (2000b).
t
ATSDR (2004b).
u
Casarett et al. (2001).
a

F-25

In Vitro Cytotoxicity Test Methods BRD Appendix F2

November 2006

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F-26

In Vitro Cytotoxicity Test Methods BRD Appendix F3

November 2006

Appendix F3

Candidate Reference Substances

F-27

In Vitro Cytotoxicity Test Methods BRD Appendix F3

November 2006

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F-28

In Vitro Cytotoxicity Test Methods BRD Appendix F3

November 2006

F.3

Candidate Reference Substances

F.3.1

Sources of Candidate Substances

The process of identifying the 72 reference substances started with the compilation of adatabase that ultimately contained 116 candidate substances. The intent of the SMT was tocompile a database with more than 12 substances in each toxicity category that also met theother criteria, and then to prioritize the substances in each category to select the 72 reference
substances to be tested. As recommended by the Workshop 2000 participants (ICCVAM
2001a), the following publicly available databases and other indicated sources were used to
identify candidate chemicals:•

The MEIC program, which collected human toxicity data and in vitro toxicity
data from 61 test methods for the first 50 chemicals (Ekwall et al. 1998). The
ECVAM members of the SMT preferred these chemicals since human acutetoxicity data had already been collected.

•

The RC (Halle 1998, 2003), which contains a compilation of in vitro
cytotoxicity and in vivo rodent LD50 data for 347 chemicals•

The Toxic Exposure Surveillance System (TESS) (Litovitz et al. 2000), which
compiles reports of toxic human exposures from poison control centersthroughout the United States•

Pesticides recommended for consideration by the EPA Office of Pesticide
Programs (OPP)•The Guidance Document (ICCVAM 2001b), which reported in vitro NRU
results for 11 RC chemicals using protocols similar to those used in the
NICEATM/ECVAM validation study•

The U.S. NTP test database, which contains information on the toxicity ofchemicals relevant to human exposure (NTP 2002)•

The EPA High Production Volume (HPV) Challenge Program, which is avoluntary testing program to provide the public with a complete set ofbaseline health and environmental effects data for each chemical that is
manufactured within or imported into the United States at amounts > 1 million
pounds/year (EPA 2000a)
F-29

In Vitro Cytotoxicity Test Methods BRD Appendix F3

F.3.2

November 2006

Selection of Candidate Substances

The 116 candidate substances consisted of the 72 reference substances selected for testing in
the NICEATM/ECVAM validation study (see Table 3-2) and the alternate substances thatwere not selected for testing (see Table F3-1). The alternate candidate substances in Table
F3-1 are grouped by GHS acute oral toxicity classification. For each reference substance, thetable provides the corresponding rat or mouse oral LD50 value, the database(s) or othersource(s) used to identify the chemical as a potential candidate, notes on volatility and/or
DEA restrictions, and the type of product and/or use for the substance. Product/use categories
were identified from HSDB (NLM 2001, 2002) or RTECS (MDL Information Systems
2001, 2002).
The final list of candidate substances, which includes the substances in Table 3-2 and Table
F3-1, included:•

Sixty-five MEIC chemicals. These include the first 50 chemicals evaluated by
MEIC as well as another 15 chemicals that were identified for futureevaluation (C. Clemedson, personal communication 2001). Twenty of thesechemicals were identified for the EDIT program, a follow-on project to the
MEIC study to develop supplementary toxicity and kinetic tests (to determine
distribution of chemicals in the body and biotransformation of chemicals tomore toxic metabolites) to improve the prediction of human toxicity by thebattery of tests identified as the best predictors in the MEIC program(Clemedson et al. 2002). The EDIT chemicals were selected by excluding
MEIC chemicals that were volatile, those that precipitated at the IC50 doselevel, and those with sparse or insufficient data on human toxicity ormechanism of acute toxicity.

•

Sixteen pesticides with extensive human exposure nominated by the EPA
OPP. These included fenpropathrin, endosulfan, bromoxynil (phenol),fipronil, carbaryl, rotenone, metaldehyde, molinate, 1,3-dichloropropene,dichlorvos, chlorpyrifos, sodium arsenite, triphenyltin hydroxide,
F-30

In Vitro Cytotoxicity Test Methods BRD Appendix F3

November 2006

cycloheximide, acrolein, and boric acid. Pentachlorophenol was also
nominated, but was already on the candidate list since it was a MEIC
chemical.
•Five substances associated with the highest incidence of toxic exposures
reported by U.S. poison control centers participating in the TESS (Litovitz etal. 2000): hypochlorite, acetaminophen, ethanol, diphenhydramine, andisopropanol. The five chemicals with the greatest incidence of toxic exposuresamong children were the same, except that oxalate replaced ethanol. Most ofthese chemicals were already identified as candidate substances due to their
inclusion in the MEIC study. Since hypochlorite (sodium salt) anddiphenhydramine, were not already included, they were added to the list of
candidates.

•

Eleven substances recommended in the Guidance Document (ICCVAM
2001b) for qualifying in vitro cytotoxicity assays for the prediction of starting
doses using the RC regression. These substances were recommended because
the IC50 and LD50 data for these substances fit the RC regression line
extremely well. These chemicals were sodium dichromate dihydrate, cadmium
chloride, p-phenylenediamine, DL-propranolol HCl, trichlorfon, ibuprofen,
nalidixic acid, salicylic acid, antipyrene, dimethylformamide, and glycerol•

Sixteen substances from the NTP databaseo Furfural, methyleugenol, and methylphenidate, scheduled for testing by
the NTP National Center for Toxicogenomics (NCT) (G. Boorman,
personal communication 2001), were added. Acetaminophen, anotherhepatotoxin to be tested by the NCT, was already a candidate substancebecause it was included in the MEIC study. Chromium (VI),recommended by the NTP for consideration due to the potential for human
exposure via drinking water (NTP 2002) was represented in the list of
candidate substances by sodium dichromate dihydrate, which was also
recommended in the Guidance Document (ICCVAM 2001b).

F-31

In Vitro Cytotoxicity Test Methods BRD Appendix F3

November 2006

o Dibutyl phthalate, 5-aminosalicylic acid, propylparaben, gibberellic acid,and diethyl phthalate were added to increase the number of chemicals with
LD50 values >5000 mg/kg.
o Trichloroacetic acid was added to increase the number of substances in the2000 < LD50 ≤5000 mg/kg category.
o Sodium selenate was added to increase the number of chemicals in the
LD50 ≤5 mg/kg category to 12.
o Six chemicals that were also on the HPV list were added. Lactic acid,citric acid, and acetonitrile were added to increase the number ofchemicals in the 2000 < LD50 ≤5000 mg/kg category. Tert-butylamine,
2,4-dinitrophenol, and acrolein were added to increase the number ofchemicals in the 5 < LD50 ≤50 mg/kg category.
•

Eight additional RC substances in the LD50 ≤5 mg/kg category. These were:triethylenemelamine, busulfan, disulfoton, parathion, aminopterin,
phenylthiourea, epinephrine bitartrate, and aflatoxin B1.

The goal to identify more than 12 candidate substances for each toxicity category was
unrealized for three toxicity categories. The most toxic category (LD50 ≤5 mg/kg), and least
toxic categories (2000 < LD50 ≤5000 mg/kg, LD50 >5000 mg/kg), contained only 12
candidate substances each. The intermediate toxicity categories (50 < LD50 ≤300 mg/kg, >300 < LD50 ≤2000 mg/kg), however, contained two to three times the minimum number ofcandidate chemicals.

F-32

In Vitro Cytotoxicity Test Methods BRD Appendix F3Table F3-1

November 2006

Alternate Candidate Substances

GHS
Category1/Chemical

RodentOral LD502(mg/kg)Source3

Notes5

Product/Use4

LD50 ≤5 mg/kg
Aflatoxin B1

Prohibitively
expensive

5.0

RC (outlier)

Food contaminant

2,4-Dinitrophenol30

RC (outlier), NTP,
HPV

Pesticide (fungicide/ insecticide)
manufacturing

t-Butylamine

44a

EPA, NTP, HPVManufacturing

Acrolein46

RC, TESS, EPA,
NTP, HPV

5< LD50 ≤50 mg/kg

Volatile(BP=52°C)

Pesticide (herbicide/ rodenticide/
algicide), manufacturing

50 < LD50 ≤300 mg/kg
Pentachlorophenol

51

MEIC, RC (outlier),
NTP

Disinfectant

Amphetamine sulfate

55

MEIC, EDIT, RC
DEA(outlier), TESS, NTP

Pharmaceutical (stimulant)

Rotenone

60

RC, TESS, EPA,
NTP

Pesticide (insecticide/ piscicide)

Furfural

65a

NTP, HPV

Solvent, food additive

p-Phenylenediamine

80

RC, GD, NTP, HPV

DyeingChlorpyrifos
Dextropropoxyphene
HCl

82a

TESS, EPA, NTP
MEIC, RC (outlier),
TESS

Pesticide (insecticide)

Methadone

86aMEIC,TESS, NTP

Fipronil

92a

EPA

83

Pharmaceutical (analgesic)
DEA

Pharmaceutical (analgesic)
Pesticide (insecticide)

Pentobarbital

125

MEIC, RC TESS

Bromoxynil (phenol)

190a

Diphenylhydantoin

199

Metaldehyde
Carbaryl

227a
230

EPA
MEIC, RC, TESS,NTPTESS, EPA
RC, EPA. NTP

DEA

Pharmaceutical (sedative)
Pesticide (herbicide)
Pharmaceutical (anticonvulsant)Pesticide (molluscicide)
Pesticide (insecticide)300 < LD50 ≤2000 mg/kg
Ferrous sulfate

319

MEIC, RC, TESS

Food additive

Warfarin

324

MEIC, RC, TESS,
EPA

Pharmaceutical (anticoagulant), pesticide

Disopyramide

333a

MEIC, TESS

Pharmaceutical (antiarrythmic)

Barium II nitrate

355

Thioridazine HCl

358

Methylphenidate

a

NTP

a

EPA, NTP

Molinate

367
369

MEIC, RC, TESS,NTPMEIC, RC, TESS

Pyrotechnic
Pharmaceutical (antipsychotic)
DEA

Pharmaceutical (stimulant)
Pesticide (herbicide)F-33

In Vitro Cytotoxicity Test Methods BRD Appendix F3Table F3-1

November 2006

Alternate Candidate Substances

GHS
Category1/Chemical

RodentOral LD502(mg/kg)Source3

2,4-Dichlorophenoxyacetic acid

369

MEIC, RC, TESS,EPA, NTP, HPV

Pesticide (herbicide)

Orphenadrine HCl

425

MEIC, RC, NTP

Pharmaceutical (analgesic)

Trichlorfon

451

RC, EPA, GD, NTP

Pesticide (insecticide)

Quinidine sulfate
1,3-Dichloropropene

456
a

470

b

Theophylline

600

Isoniazid

650

Diazepam709
a

Maprotiline
Methyleugenol

760
810a

Diphenhydramine HCl

855

Malathion

885

Salicylic acid891

Chloroform

908

Chloroquine diphosphate
Ibuprofen
Nalidixic acid

970
1009
1349

Dichloromethane

1597

Antipyrene

1800

Notes5

MEIC, RC, NTP(base)
TESS, EPA, NTP
MEIC, RC, TESS,NTPMEIC, RC, TESS,NTPMEIC, EDIT, RC,
DEA
TESS, NTP
MEIC, TESSNTPMEIC, RC, TESS,NTPMEIC, EDIT, RC,
TESS, EPA, NTP
RC, TESS, GD, NTP,
HPV
Volatile
MEIC, RC, NTP,
HPV
(BP=61°C)
MEIC, RC
RC, TESS, GD
RC, GD, NTP
MEIC, RC, TESS, Volatile
NTP, HPV(BP=40°C)
RC, GD

1

Product/Use4

Pharmaceutical (antiarrhythmic)
Pesticide (nematocide)
Pharmaceutical (antiasthmatic)
Pharmaceutical (antibiotic)
Pharmaceutical (anxiolytic)
Pharmaceutical (antidepressant)Food additive
Pharmaceutical (antihistamine)
Pesticide (insecticide)
Pharmaceutical (analgesic)
Solvent
Pharmaceutical (antimalarial))
Pharmaceutical (analgesic)
Pharmaceutical (antibiotic)
Solvent
Pharmaceutical (analgesic)

GHS=Globally Harmonized System of Classification and Labelling of Chemicals for acute oral toxicity (UN 2005).
2
LD50 data are from the Registry of Cytotoxicity (Halle 1998) and are for rats, the preferred species for oral acute toxicity studies, unlessotherwise noted. Data with decimal places are rounded to the nearest one.
3
Sources used to identify candidate chemicals: EDIT=Evaluation-guided Development of New In Vitro Test Batteries; EPA=Pesticides
registered with the Environmental Protection Agency; EHS=EPA’s Extremely Hazardous Substance list; HPV=High Production Volume
chemicals (i.e., those that are imported into or produced in the United States in amounts > 1,000,000 lbs/year; GD=Guidance Document(ICCVAM 2001b); MEIC=Multicentre Evaluation of In Vitro Cytotoxicity; NTP=National Toxicology Program; RC=Registry ofCytotoxicity with chemicals classified as regression outliers shown in parentheses; TESS=Toxic Exposure Surveillance System (Litovitz etal. 2000).
4
Product/use categories from Hazardous Substances Data Bank (NLM 2002) or Registry of Toxic Effects of Chemical Substances([RTECS®], MDL Information Systems 2002). Pharmaceutical uses from Gilman et al. (1985) or Thomson PDR® (2004).
5
Only chemicals expected to be too volatile for the cytotoxicity assay system have "volatile" notations. BP=Boiling point. DEA (U.S. Drug
Enforcement Agency) refers to Schedule II controlled substances. Chemicals with no "DEA" notation are expected to be under less strict
control.
a
RTECS® (MDL Information Systems 2002).
b
Mouse

F-34

In Vitro Cytotoxicity Test Methods BRD Appendix G

November 2006

Appendix G

STATEMENT OF WORK (SOW)G1

A Validation Study For In Vitro Basal Cytotoxicity Testing ..... G-3

G2

Procedures for Acquisition, Preparation, Solubility Testing,and Distribution of Test Chemicals for a Validation Study
for In Vitro Basal Cytotoxicity Testing ..................................... G-57

G-1

In Vitro Cytotoxicity Test Methods BRD Appendix G

November 2006

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G-2

In Vitro Cytotoxicity Test Methods BRD Appendix G1

November 2006

Appendix G1A Validation Study For In Vitro Basal Cytotoxicity Testing

G-3

In Vitro Cytotoxicity Test Methods BRD Appendix G1

November 2006

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G-4

In Vitro Cytotoxicity Test Methods BRD Appendix G1

November 2006

STATEMENT OF WORKA VALIDATION STUDY FOR IN VITRO BASAL CYTOTOXICITYTESTINGBALB/c 3T3 Neutral Red Uptake Cytotoxicity Assay
andNormal Human Keratinocyte Neutral Red Uptake Cytotoxicity Assay

June 21, 2002
Prepared by
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)

Based on Standard Operating Procedure Recommendations from anInternational Workshop Organized by the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM)

National Institute of Environmental Health Sciences (NIEHS)
National Institutes of Health (NIH)
U.S. Public Health Service
Department of Health and Human Services

G-5In Vitro Cytotoxicity Test Methods BRD Appendix G1

November 2006

Table of Contents
1.0 PROJECT OBJECTIVES AND GENERAL REQUIREMENTS......................................................... 9
1.1
Project Objectives............................................................................................................................... 9
1.2
Response to the Statement of Work ................................................................................................ 10
1.2.1
General Capabilities................................................................................................................. 10
1.3
Guidelines ......................................................................................................................................... 10
1.4
Definitions ........................................................................................................................................ 10
2.0 ORGANIZATION.................................................................................................................................. 12
2.1
Validation Study Sponsors............................................................................................................... 12
2.2Management Team ........................................................................................................................... 12
2.2.1
Study Management Team........................................................................................................ 122.2.1.1
NIEHS/NICEATM .......................................................................................................... 122.2.1.2
ECVAM ........................................................................................................................... 12
2.2.2
Project Management and Chemical Distribution Team ........................................................ 12
2.2.3
Contract Management.............................................................................................................. 13
3.0 TESTING FACILITY AND KEY PERSONNEL ............................................................................... 13
3.1
Testing Facility................................................................................................................................. 13
3.1.1
Personnel .................................................................................................................................. 13
3.1.1.1
Facility Management ....................................................................................................... 13
3.1.1.2
Study Director.................................................................................................................. 13
3.1.1.3
Quality Assurance (QA) Director................................................................................... 13
3.1.1.4
Scientific Advisor(s)........................................................................................................ 14
3.1.1.5
Laboratory Technician(s) ................................................................................................ 14
3.1.1.6
Safety Officer................................................................................................................... 14
3.1.2
Facilities, Equipment, and Supplies........................................................................................ 14
3.1.2.1
Cell Culture Laboratory .................................................................................................. 14
3.1.2.2
Equipment ........................................................................................................................ 14
3.1.2.3
Supplies ............................................................................................................................ 15
3.1.3
Health and Safety ..................................................................................................................... 15
3.1.4
Quality Assurance .................................................................................................................... 16
3.1.4.1
GLP-Compliant Laboratories.......................................................................................... 16
3.1.4.2
Non GLP-Compliant Laboratories ................................................................................. 16
4.0 TEST PHASES AND SCHEDULE ...................................................................................................... 17
4.1
Study Timeline and Deliverables .................................................................................................... 17
4.2
Phase I ............................................................................................................................................... 18
4.2.1
Study Procedures...................................................................................................................... 18
4.2.1.1
Phase Ia: Positive Control Database............................................................................... 18
4.2.1.2
Reporting Positive Control Data (Phase Ia)................................................................... 19
4.2.2
Criteria for Advancing to Phase Ib ......................................................................................... 20
4.2.3
Study Procedures...................................................................................................................... 20
4.2.3.1.
Phase Ib: Chemical Testing............................................................................................. 20
4.2.3.2
Reporting Test Chemical Data (Phase Ib)...................................................................... 21
4.2.4
Criteria for Advancing to Phase II .......................................................................................... 22
4.3
Phase II.............................................................................................................................................. 22
4.3.1
Study Procedures...................................................................................................................... 22
4.3.1.1
Reporting Test Chemical Data (Phase II) ...................................................................... 22
4.3.2
Criteria for Advancing to Phase III......................................................................................... 23
4.4
Phase III ............................................................................................................................................ 23
4.4.1
Study Procedures...................................................................................................................... 23
4.4.1.1
Reporting Data (Phase III) .............................................................................................. 24
G-6

In Vitro Cytotoxicity Test Methods BRD Appendix G1

November 2006

4.4.2
Criteria for Completion of Phase III ....................................................................................... 24
4.5
Report Submission Timelines .......................................................................................................... 24
4.5.1
Draft Reports ............................................................................................................................ 24
4.5.2
Final Report .............................................................................................................................. 25
5.0 IDENTIFICATION OF TEST CHEMICALS AND CONTROL SUBSTANCES ........................... 25
5.1
Test Chemicals.................................................................................................................................. 25
5.1.1
Range of Toxicities .................................................................................................................. 25
5.1.2
Receipt of Chemicals ............................................................................................................... 25
5.1.3
Test Chemical Information for the Study Director ................................................................ 26
5.2
Control Materials.............................................................................................................................. 26
5.2.1
Vehicle Control (VC)............................................................................................................... 26
5.2.1.1
3T3 NRU Assay (VC) ..................................................................................................... 26
5.2.1.2
NHK NRU Assay (VC) ................................................................................................... 26
5.2.2
Positive Control (PC) ............................................................................................................... 26
5.3
Inventory of Test Chemicals............................................................................................................ 26
5.4
Disposition of Test Chemicals......................................................................................................... 26
5.5
Handling of Test Chemicals............................................................................................................. 26
5.6
Determination of Purity, Composition, and Stability of Test Chemicals ..................................... 27
6.0 TEST SYSTEM ...................................................................................................................................... 27
6.1
Neutral Red Uptake (NRU) Cytotoxicity Assay ............................................................................ 27
6.1.1
Background............................................................................................................................... 27
6.1.2
Sterility of the Test System ..................................................................................................... 27
7.0 EXPERIMENTAL DESIGN AND METHODOLOGY – 3T3 NRU ASSAY................................... 28
7.1
Major Steps in the Performance of the Assay................................................................................. 28
7.2
Procedures for Conducting the Test ................................................................................................ 28
7.2.1
Cell Maintenance and Culture Procedures ............................................................................. 28
8.0 EXPERIMENTAL DESIGN AND METHODOLOGY – NHK NRU ASSAY ................................ 29
8.1
Major Steps in the Performance of the Assay................................................................................. 29
8.2
Procedures for Conducting the Test ................................................................................................ 29
8.2.1
Cell Maintenance and Culture Procedures ............................................................................. 30
9.0 PREPARATION AND DELIVERY OF TEST CHEMICAL ............................................................. 30
9.1
Preparation of Test Chemical .......................................................................................................... 30
9.1.1.
Dissolving the Test Chemical.................................................................................................. 30
9.1.1.1
Treatment Medium/Routine Culture Medium) .............................................................. 30
9.1.1.2
DMSO............................................................................................................................... 30
9.1.1.3
Ethanol.............................................................................................................................. 31
9.1.2.
Test Chemical Solubility ......................................................................................................... 31
9.1.3
pH of Dilutions......................................................................................................................... 31
9.2
Delivery of Test Chemical ........................................................................................................... 31
9.3
Range Finder Experiment ........................................................................................................ 32
9.4
Test Chemical Dilutions .......................................................................................................... 32
10.0 DATA COLLECTION ........................................................................................................................... 32
10.1
Nature of Data to be Collected .................................................................................................... 32
10.2Type of Media Used for Data Storage ........................................................................................ 33
10.3
Documentation ............................................................................................................................. 33
11.0 ACCEPTANCE CRITERIA FOR NRU ASSAYS .............................................................................. 33
11.1
Test Acceptance Criteria.............................................................................................................. 33
11.2
IC50 Acceptance Criteria .............................................................................................................. 33
12.0 EVALUATION OF TEST RESULTS .................................................................................................. 34
12.1
Cell Viability Determination ....................................................................................................... 34
12.2
ICX Determination ........................................................................................................................ 34
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13.0 DRAFT AND FINAL REPORTS ......................................................................................................... 35
14.0 RECORDS AND ARCHIVES .............................................................................................................. 35
15.0 ALTERATIONS OF THE STATEMENT OF WORK ....................................................................... 35
16.0 REFERENCES ....................................................................................................................................... 35
17.0 APPROVAL OF STATEMENT OF WORK ....................................................................................... 37
ADDENDUM I ................................................................................................................................................. 38
ADDENDUM II................................................................................................................................................ 49
ADDENDUM III .............................................................................................................................................. 50
ADDENDUM IV .............................................................................................................................................. 51
ADDENDUM V ............................................................................................................................................... 54
ADDENDUM VI .............................................................................................................................................. 55

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STATEMENT OF WORKA VALIDATION STUDY FOR IN VITRO BASAL CYTOTOXICITY TESTINGBALB/c 3T3 Neutral Red Uptake Cytotoxicity Assay
andNormal Human Keratinocyte Neutral Red Uptake Cytotoxicity Assay

1.0

PROJECT OBJECTIVES AND GENERAL REQUIREMENTS
1.1 Project Objectives
This Statement of Work outlines and supports the procedures for performing two in vitro basal
cytotoxicity assays (the BALB/c 3T3 Neutral Red Uptake [NRU] assay and the Normal Human
Keratinocyte [NHK] Neutral Red Uptake [NRU] assay) for analysis of test chemicals for a
multi-laboratory in vitro Validation Study. These in vitro assays, recommended in GuidanceDocument On Using In Vitro Data To Estimate In Vivo Starting Doses For Acute Toxicity(ICCVAM, 2001a) use mammalian cell culture techniques to assess the basal cytotoxicity ofchemicals.
A primary goal of this Validation Study is to evaluate the usefulness and effectiveness of invitro basal cytotoxicity assays for reducing and refining animal use for acute oral toxicity
determinations of chemicals by predicting starting doses for in vivo rodent acute lethality
assays. Participants at an international workshop (ICCVAM, 2001b) suggested that avalidation study for in vitro assays is needed to continue the development of alternative tests as
replacements for animal testing. This is the first step to further standardization and evaluationof two test methods that may be used in conjunction with other methods as components of a test
battery which may eventually replace the rodent acute oral toxicity tests.
Data will be used to:1) Develop standardized in vitro basal cytotoxicity protocols with sufficient detail andinstruction for distribution to other laboratories (e.g., Federal regulatory agencies) for their
immediate use,2) Evaluate the intra- and inter-laboratory reproducibility of the assays (i.e., to access test
reproducibility and optimize to further enhance reproducibility),3) Determine the reduction in the number of animals that would be used and/or killed inlethality assays compared with the conventional method of predicting starting doses, and
4) Assess the relevance of the two standardized in vitro cytotoxicity assays for estimatingrodent oral LD50 values across the six Globally Harmonised System (GHS; OECD, 2001)
categories of acute oral toxicity and estimating human lethal concentrations.
This study will test the hypothesis of the Registry of Cytotoxicity (RC) prediction model(Halle, 1998) by comparing the NRU regressions that are developed from the two assays to the
RC regression. The hypothesis is that the two NRU assays will provide the same regression as
the RC (i.e., comparison of IC50 data vs. LD50 data).
The proposed Validation Study will provide the means to determine IC20, IC50, and IC80 values
for a test set of 72 chemicals with varying degrees of toxicity. This set of chemicals was
selected separate and prior to this Statement of Work by the Study Management Team. The
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basis for selection of this test set is discussed in the Study Design document prepared by the
Study Management Team.
1.2 Response to the Statement of Work
The proposals submitted in response to the Statement of Work to the designated contacts shall
include:a) A timetable for project milestonesb) A cost estimate for performing all testing (both assays) in all phases of the Validation
Study.
c) Cost estimates for repeating Phases Ia, Ib, and II as options, if necessary (see Sections
4.2.2, 4.2.4, and 4.3.2).
d) Cost for a third replicate of Phase III testing as an option, if necessarye) Cost of software for data analysis (e.g., GraphPad PRISM® 3.0) not to exceed $500.
1.2.1 General Capabilities
The contracted laboratories (Testing Facilities) shall be capable of performing thefollowing:a) The Testing Facilities shall prepare Standard Operating Procedures (SOPs) for the
3T3 NRU assay and the NHK NRU assay (see Section 1.4 – Definitions - SOPs)b) The Testing Facilities shall perform the 3T3 NRU assay and the NHK NRU assay(under aseptic in vitro laboratory conditions) for the three phase Validation Study as
identified in Section 4.0.
c) The Testing Facilities shall provide IC20, IC50, and IC80 values for each tested
chemical and other information addressed in this document (e.g., phase reports) to the
Study Management Team through the designated contacts (Section 2.2).
d) Testing Facilities that are compliant with Good Laboratory Practices (GLP) shallperform all aspects of the Validation Study in accordance with GLPs.
e) Testing Facilities that are not GLP-compliant shall perform all aspects of the
Validation Study “in the spirit” of GLP which is defined in Section 1.4 and addressed
throughout this Statement of Work.
f) All Testing Facilities shall adhere to this Statement of Work throughout the
Validation Study.
1.3 Guidelines
The Management Team and/or its representatives may inspect and audit the Testing Facilities
used for this study to ensure that the Study Management Team’s minimum requirements andguidelines are being followed. The contractor shall notify the Study Management Team of any
changes in Key Personnel (identified in Section 3.1.1)
1.4 Definitions
Blinded/Coded Chemicals: Test chemicals supplied to the Testing Facilities that are coded (byan NIEHS/NTP-designated contractor) such that the Testing Facilities do not know the identityof the chemicals. Only the Project Officer, Management Team, and contractor know thecontents of each test chemical vessel. The test chemicals will be purchased, aliquoted, coded,and distributed by a contractor under the guidance of the NIEHS Project Officer and theManagement Team.
Good Laboratory Practices (GLPs): Regulations governing the conduct, procedures, andoperations of toxicology laboratories; regulations to assure the quality and integrity of the data
and to address such matters as organization and personnel, facilities, equipment, facility
operations, test and control articles, and Validation Study protocol (Statement of Work) and
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conduct (U.S. Food and Drug Administration, Title 21 CFR Part 58; Environmental Protection
Agency, Title 40 CFR Part 160).
ICX: Inhibitory concentration estimated to affect endpoint in question by X % (IC20 = 20 %affected; IC50 = 50 % affected; IC80 = 80 % affected).
Lead Laboratory (Protocols): A designated laboratory (identified by the Study Management
Team and different from the lead laboratory for data analysis) with experience in eachcytotoxicity method. The laboratory will assist the Study Management Team withtroubleshooting laboratory challenges; the lead laboratory shall develop a study protocol fromthe Statement of Work and the Test Method Protocols that shall be used by all laboratories inthe Validation Study.
Lead Laboratory (Data Analysis): A designated laboratory (identified by the StudyManagement Team and different from the lead laboratory for protocols) with experience in data
analysis specific to the software that will be used in the study; The laboratory will assist the
Study Management Team with troubleshooting data analysis challenges.
Replicate: An independent test run on different days (e.g., duplicate 96-well plates for aparticular test chemical, each plate a replicate assay); replicate wells within the 96-well plate(e.g., six wells of one test chemical concentration equals six replicate wells).
Spirit of GLP: Laboratories that are non GLP-compliant shall adhere to GLP principles andother method parameters as put forth in this Statement of Work and the Test Method Protocols(provided by NIEHS/NICEATM); documentation and accountability shall be equal to GLP
requirements; laboratories must make assurances that they are equal in performance criteria andthat there is parity amongst the laboratories.
Standard Operating Procedures (SOPs): Written documents that describe, in great detail, theroutine procedures to be followed for a specific operation, analysis, or action; consistent use of
an approved SOP ensures conformance with organizational practices, reduced work effort,
reduction in error occurrences, and improved data comparability, credibility, and defensibility;
SOPs also serve as resources for training and for ready reference and documentation of proper
procedures; each Testing Facility involved in the Validation Study shall draft SOPs specificallyfor its laboratories based on: protocols supplied by commercial sources specifically for cell
culture products and cell lines; this Statement of Work and the Test Method Protocols provided
by NIEHS/NICEATM, and the study protocol developed by the lead laboratory.
Statement of Work: A description of testing required for the in vitro Validation Study; defines
all phases of the Validation Study and the purpose of the procedures; provides the details of theexperimental design, data acquisition, data analysis, and preparation of reports; supports Test
Method Protocols (equivalent to GLP protocols) and acts as a study plan.
Study Protocol: A description of the objectives and all methods for the conduct of the study(i.e., same as “protocol” according to GLP guidelines, 40 CFR 792, at
http://www.ovpr.uga.edu/qau/tscatoc.html. The Study Protocol shall be developed from the
Test Method Protocols for NHK and 3T3 NRU assays, which accompany this Statement ofWork. The Study Protocol shall contain information such as the title and purpose of the study,
name and address of the sponsor, the name and address of the testing facility at which the study
is being conducted, proposed experimental start and termination dates, and other items
specified in 40 CFR 792.
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Test Method Protocols: Specific and detailed guides for performing the 3T3 NRU and NHK
NRU cytotoxicity assays; adapted by NICEATM from protocols included in ICCVAM (2001a);
equivalent to GLP protocols; protocols shall be incorporated into the SOPs specific to each Test
Facility in the Validation Study.
2.0

ORGANIZATION
2.1
•
•
•
•
2.2

Validation Study Sponsors
National Institute of Environmental Health Sciences (NIEHS)
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)
U.S. Environmental Protection Agency (U.S. EPA)The European Centre for the Validation of Alternative Methods (ECVAM).
Management Team2.2.1 Study Management Team2.2.1.1 NIEHS/NICEATM
Dr. William S. Stokes (NICEATM/NIEHS) – Co-chair – Study Management TeamDr. Judy Strickland (NICEATM/ILS) – Project Coordinator
Mr. Michael Paris (NICEATM/ILS) – Assistant Project Coordinator
Dr. Ray Tice (NICEATM/ILS) – Technical Advisor
NICEATM79 T.W. Alexander Drive
Bldg. 4401, MD-EC-17
3rd Floor, Room 3126
P.O. Box 12233
Research Triangle Park, NC 27709
2.2.1.2 ECVAM
Professor Michael Balls – Co-chair – Study Management TeamDr. Silvia Casati
Dr. Andrew WorthEuropean Commission
Joint Research Centre
Institute for Health and Consumer Protection
Management Support Unit - TP 202
I-21020 Ispra (VA) - Italy
2.2.2 Project Management and Chemical Distribution Team
Ms. Molly Vallant (NIEHS) – NIEHS Project Officer for BioReliance, Inc.
Dr. Martin L. Wenk (BioReliance, Inc., Rockville, MD) – Principal
Investigator/Chemical Distribution

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2.2.3 Contract Management
Ms. Jackie Osgood (NIEHS) – Contracting Officer
Mr. Don Gula (NIEHS) – Contracting Officer
3.0

TESTING FACILITY AND KEY PERSONNEL
3.1 Testing Facility
The Testing Facility shall have competence in performing in vitro cytotoxicity assays underaseptic laboratory conditions and shall provide competent personnel, adequate facilities,
equipment, supplies, proper health and safety guidelines, and satisfactory quality assurance
procedures.
3.1.1 Personnel
3.1.1.1 Facility Management
The facility management is responsible for establishing scientific guidelines andprocedures, training and supervision of professional and technical staff, andevaluation of results and performance within their discipline area relative to the
Study Management Team requirements. The manager must maintain records of thequalifications, training and experience, and a job description for each professional
and technical individual involved in the Validation Study.
3.1.1.2 Study Director
A scientist or other professional of appropriate education, training, and experience
in in vitro cytotoxicity assay performance, or combination thereof, shall be theStudy Director. The Study Director has the overall responsibility for the technicalconduct of the Validation Study (e.g., GLP adherence or implementation of spiritof GLP) at the Testing Facility and shall be responsible for determining test
acceptance. The Study Director shall be responsible for providing SOPs for the
Validation Study and incorporating pertinent information obtained from theStatement of Work and the Test Method Protocols. Other duties include theinterpretation and analysis of data, documentation of all Validation Study aspects(including maintenance of a Study Workbook), and production of all draft and final
written Validation Study reports.
3.1.1.3 Quality Assurance (QA) Director
For Testing Facilities that are GLP-compliant, the Quality Assurance Director shallmonitor the Validation Study to assure conformance with GLP requirements for all
aspects of the Validation Study (i.e., facilities, equipment, personnel, methods,
practices, records, controls, transference of data into software, SOPs). The Quality
Assurance Director or unit can be any person or organizational element, except the
Study Director, designated by Testing Facility management to perform the duties
relating to quality assurance of the studies. The Quality Assurance duties are not a
substitute for the Study Director duties.
For Testing Facilities performing the Validation Study in the spirit of GLP,
management shall appoint an individual to assure that all records, documents, raw
data, reports, and specimens are available to the Management Team through thedesignated contacts if an inspection is requested.

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3.1.1.4 Scientific Advisor(s)Scientists or other professionals of appropriate education, training, and experience
in in vitro laboratory methods and techniques who provide scientific guidance to
the Study Director and other laboratory personnel.
3.1.1.5 Laboratory Technician(s)In vitro cytotoxicity assays require personnel trained in sterile tissue culture
techniques and general laboratory procedures. At least two individuals must be
capable of performing the in vitro assays for the Validation Study. Performance of
the assays requires a relatively moderate degree of technical capability and a highdegree of technical accuracy. Each individual engaged in the conduct of or
responsible for the supervision of a Validation Study shall have education, training,and experience, or combination thereof, to enable that individual to perform theassigned duties. The individuals in a GLP-compliant laboratory must be trained in
GLP requirements and technical ability must be documented as per GLP
requirements. Non GLP-compliant laboratory personnel must be able to perform
all aspects of the Validation Study in the spirit of GLP.
3.1.1.6 Safety Officer
A designated Safety Officer (someone not involved in the actual conduct of the
Validation Study) at each participating laboratory will receive the blinded (coded)
test chemicals from an NIEHS/NTP-designated contractor (BioReliance) and shalltransfer the test chemicals to the Study Director without revealing the contents ofthe test chemical containers. A sealed health and safety information package willaccompany the test chemicals and the Safety Officer shall retain the package until
the completion of the Validation Study. At the end of the Validation Study, theSafety Officer shall return the unopened package to the contractor (BioReliance).
If any Test Facility personnel should open the package at any time during the
Validation Study, the Safety Officer shall notify the Management Team throughthe designated contacts.
3.1.2 Facilities, Equipment, and Supplies
3.1.2.1 Cell Culture Laboratory
Each Testing Facility must provide a designated cell culture laboratory to ensurethat in vitro cytotoxicity assays can be performed under clean and proper aseptic
conditions. The laboratory must be located such that there is minimal throughtraffic to reduce possible disturbances that may compromise the cell culture assays.
Room temperature of the laboratory must be easily regulated, monitored, anddocumented. Access to the Validation Study assays and test chemicals shall be
restricted to appropriate personnel as determined by facility management.
3.1.2.2 Equipment
Each Testing Facility must provide at a minimum the following equipment:a) Laminar flow hood (biohazard type and restricted to cell culture assays)b) Cell culture incubators• 37oC ± 1oC, 5 % ± 1 % CO2, 90 ± 5 % humidifiedc) Low-speed centrifuged) Water bath (37oC)e) Inverse phase microscopef) Pippettors (multichannel pipettor, micropipettors, multichannel pipette units)
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November 2006g) Spectrophotometric plate reader (equipped with a 540 nm ± 10 nm filter)h) Computer (for data transformation and analysis)i) Liquid nitrogen freezer (for storage of cryopreserved cells)j) Refrigerator (4oC)k) Freezers (-20oC and -70oC to -80oC)l) Autoclave (for instruments and for biohazardous waste materials)m) Balancen) pH metero) Cell counting system (e.g., hemocytometer, Coulter counter)p) General cell culture laboratory equipment (e.g., glassware, filtration systems,
cell culture plasticware, etc.)q) pH paper (wide and narrow range)
All equipment maintenance and calibration shall be routinely performed anddocumented as per GLP guidelines (or spirit of GLP for non GLP-compliant
laboratories) and Testing Facility procedures. Additional detail is provided in
Section 10.3 and Addendum IV.
3.1.2.3 Suppliesa) General cell culture materials and supplies are needed and are specificallydescribed in the provided Test Method Protocols and in the GuidanceDocument (ICCVAM, 2001a). All cell culture reagents must be labeled so as
to indicate source, identity, concentration, stability, preparation and expiration
dates, and storage conditions.
b) BALB/c 3T3 mouse cells, clone 31• Cryopreserved (5 vials, same lot)
• CCL-163, LGC Reference Materials, Customer Service, Queens Road,
Teddington, Middlesex, TW110LY, UK (http://www.lgc.co.uk/atcc/)• CCL-163, American Type Culture Collection (ATCC), Manassas, VA,
USA (http://www.atcc.org/)c) Normal Human Epidermal Keratinocytes (NHK)• Cryopreserved (20 vials, same lot, first passage)• Non-transformed cells; from cryopreserved primary cells (Clonetics #CC2507 [pooled neo-natal keratinocytes])• Clonetics/BioWhittaker [BioWhittaker, 8830 Biggs Ford Road,
Walkersville, MD 21793-0127(http://www.cambrex.com/subsidiaries/s%2Dbw%5Finc/s%2Dbiowhittake
r%2Dinc%2Dcontact2.htm)• BioWhittaker Europe [BioWhittaker Europe, S.P.R.L. Parc Industriel dePetit Rechain, B-4800 Verviers, BELGIUM](http://www.biowhittaker.be/index.htm)
3.1.3
Health and Safety
Each Testing Facility shall conform to all local, state, and federal statutes in effect at thetime of this Validation Study. The designated Safety Officer shall be the point of contact
for health and safety issues.

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3.1.4

November 2006

Quality Assurance
3.1.4.1 GLP-Compliant Laboratories
GLP-compliant laboratories shall conduct this Validation Study in compliance with
Good Laboratory Practice (GLP) Standards (U.S. Food and Drug Administration,
Title 21 CFR Part 58; Environmental Protection Agency, Title 40 CFR Part 160).
The appropriate QA unit (as per GLPs) shall review the protocol and audit the inlife phase, laboratory notebooks, and final report data.
The Final Reports for all phases of the Validation Study shall be audited by the
Quality Assurance unit of the Testing Facility for GLP compliance and a QA
Statement shall be provided by the Testing Facility. Each Final Report shallidentify: 1) the phases and data inspected, 2) dates of inspection, and 3) dates
findings were reported to the Study Director and Testing Facility management.
The QA Statement shall identify whether the methods and results described in the
Final Report accurately reflect the raw data produced during the Validation Study.
3.1.4.2 Non GLP-Compliant Laboratories
Non GLP-compliant laboratories shall use GLP standards referenced in the
ECVAM Workshop 37 Report (Cooper-Hannan, 1999) and the OECD Principles of
GLP (OECD, 1998) as guidelines for conducting the Validation Study in the spiritof GLP.
At a minimum, the following laboratory parameters and equipment must be
routinely documented (e.g., log books; see Addendum IV). The documents shallbe archived such that they can be available to the Study Management Teamthrough the designated contacts upon request.
Daily Documentation (value, time, and date)• Laboratory: room temperature• Incubators: temperature, %CO2, %humidity• Water bath: temperature• Refrigerators and freezers: temperature• Cell cultures: visual observations (see Test Method Protocols)
Per Use Documentation (value, time, and date)• Cryogenic storage unit: amount of liquid N2 in container; when liquid N2 added• Balance: standard weight used to calibrate• pH meter: values for standards used to determine slope• Cell counter: standard used
• Media: identification of all media and components used
Periodic Documentation• Media and components: date of receipt; lot numbers; expiration dates• 3T3 and NHK cells: date of receipt; lot number; storage conditions• Plastic tissue-culture ware (sterile, disposable): stock and lot numbers• Computer software: identification and description• Calibration of Instruments: SOPs for laboratory equipment
Incubators
Laminar flow hoods
Autoclaves
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Micropipettors
Balances
pH meters
Cell counters
Refrigerators
Freezers
Water baths
Spectrophotometer plate readers
A statement from the Testing Facility shall be included with each Final Report andshall identify whether the methods and results described in the Final Reportaccurately reflect the raw data produced during the Validation Study and provide
assurance that all testing was done in the spirit of GLP.
4.0

TEST PHASES AND SCHEDULE

See Addendum VI for Gantt Chart of study timelines and deliverables.
4.1

Study Timeline and Deliverables

TASK
Statement of Work issued by NIEHS
to the Testing Facility
Response /Proposal received fromthe Testing FacilityAward of Contracts
Submission of Study Protocol, CVs of
Key Personnel and SOPs
Start Testing – Phase I (Phase Ia)
End Phase Ia
Begin Phase Ib
End Phase Ib
Begin Phase II
End Phase II
Begin Phase III
Final Report (Phase III) to SMT

WEEK
0

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ESTIMATED DATEMarch 29, 2002

6

May 10, 2002

13
15

June 28, 2002
July 12, 2002

18
22
26
31
36
46
52
89

July 29, 2002
August 26, 2002
September 26, 2002
October 29, 2002
December 2, 2002
February 10, 2003
March 26, 2003
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4.1.1

November 2006

Deliverables

REPORTS
Biweekly
Draft
Final
Study
Workbook(Draft)Study
Workbook(Final)PHASE 1a*
Week 24
Sept. 9, 2002
Week 33
Nov. 11, 2002
Week 24
Sept. 9, 2002

ESTIMATED DUE DATESPHASE Ib
PHASE II**
Week 33
Week 48
Nov. 11, 2002
Feb. 25, 2003
Week 42
Week 57
Jan. 13, 2003
April 28, 2003
Week 33
Week 48
Nov. 11, 2002
Feb. 25, 2003

PHASE III*
Week 82
Oct. 24, 2003
Week 89
Dec.9, 2003
Week 82
Oct. 24, 2003

Week 33
Nov. 11, 2002

Week 42Jan. 13, 2003

Week 89
Dec.9, 2003

Week 57
April 28, 2003

* Biweekly reports shall begin at the time of implementation of the contracts andcontinue until the final report is submitted.
4.2 Phase I
Phase I will be the training phase for laboratory personnel. This phase includes developing a
positive control database (Phase Ia) and testing three unknown chemicals (Phase Ib). SOPs forthe two NRU cytotoxicity assays shall be developed by the appropriate laboratory personnel
prior to implementation of test procedures (See Section 1.4 – Definitions – SOPs). They will
be submitted along with the signed protocols to the designated contacts before initiation of
Phase I.
4.2.1 Study Procedures
4.2.1.1 Phase Ia: Positive Control DatabaseAn historical database of IC50 values for the positive control chemical (Sodium
Lauryl Sulfate [SLS]) will be established and maintained for each NRU assay by
performing 10 concentration-response assays (10 microtiter plates, one plate per
assay) on both cell types. A range finder experiment will be performed before
initiating the 10 concentration-response assays (Section 9.3). The Test Facility
personnel shall prepare and test eight concentrations (per microtiter plate) of thepositive control chemical by diluting the stock solution with a constant factor forthe range finder experiment (e.g., log dilutions [1:10, 1:00, 1:1000, etc.]). For thedefinitive concentration-response assays, the Study Director shall use a 6√10 = 1.47
dilution scheme centered on the IC50 identified in the range-finding assay.
Once a range has been determined that satisfies the criteria in Section 11.2, thenthe Test Facility shall perform two tests per day (each assay) on five different days.
Control limits for the positive control chemical shall be established and a draft
report (including range finding data) shall be provided to the designated contacts.
After evaluation of the data, the Management Team will decide when to advance tothe next phase of the Validation Study.
The 95 % confidence interval (CI) of the IC50 of SLS will be established anddefined as an acceptance criterion for test sensitivity for the 3T3 NRU and NHK
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NRU assays. The confidence intervals shall be calculated using the average of theindividual IC50 values from each positive control assay performed. An example ofan historical mean IC50 of SLS in mammalian cultures is 93 µg /ml and the 95 %CI is 70 - 116 µg /ml (Spielmann et. al., 1991). An example of an historical mean
IC50 of SLS in NHK cultures is 4.4 µg/ml ± 0.97 µg/ml [two standard deviations](Triglia, 1989).
The following 96-well plate configuration will be used for the positive control
assays.
Figure 1. 96-Well Plate Configuration for Positive Control Assays (Phase Ia)
1

2

3

4

5

6

7

8

9

10

11

12

A

b

b

b

b

b

b

b

b

b

b

b

b

B

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

C

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

D

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

E

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

F

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

G

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

H

b

b

b

b

b

b

b

b

b

b

b

b

VC
=
C1 – C8 =b=untreated VEHICLE CONTROL (mean viability set to 100 %)POSITIVE CONTROL (SLS) at eight concentrations (C1 = highest,
C8 = lowest)
BLANKS (contain no cells)

4.2.1.2 Reporting Positive Control Data (Phase Ia)
Biweekly Reports: Each testing facility will provide a biweekly progress report tothe designated contacts. These reports will be provided in electronic format (i.e.,email with attachments) and will include raw and interim data as the study
progresses. The Management Team will in turn provide a weekly progress reportaddressing the Validation Study as a whole to all of the Testing Facilities.
Draft Report: At the conclusion of Phase Ia, a draft report of the positive control
data shall be provided by the Study Director to the designated contacts. The draft
report (entitled: In Vitro Validation Study – Phase Ia: Development of a Positive
Control Database in Rodent and Human Cell Systems) shall include everything
noted in Addendum I (Draft Report – Phase Ia). If the Phase Ia data does not meet
test acceptance criteria, then the Management Team (through the designatedcontacts) will work with the Test Facility and lead laboratory to identify problems
and make corrections as needed. Once unresolved issues have been resolved, the
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Validation Study will proceed. The Validation Study will advance to Phase Ib
once all participating laboratories have submitted acceptable draft reports. The
draft report shall be submitted in email and five (5) hard copy formats. Fileattachments in email shall be submitted in Microsoft® Word (or equivalent) andExcel format and all email correspondence shall be copied to the designatedcontacts. Copies of the unaudited Study Workbook pages may be submitted asattachments in hard copy format.
Final Report: Once the draft report provides data that meets test acceptancecriteria, then the Project Coordinator shall inform the Study Director to prepare a
Quality Assurance audited final report for Phase Ia. The final report shall be
submitted in email and five (5) hard copy formats. File attachments in email shallbe submitted in Microsoft® Word (or equivalent) and Excel format and all email
correspondence shall be copied to the designated contacts. Copies of the audited
Study Workbook pages may be submitted in hard copy format as an attachment to
the report. The final report will not need to be completed to continue to Phase Ib.
(See Validation Study timelines in Section 4.1 and Report submission timelines in
Section 4.5.)
4.2.2 Criteria for Advancing to Phase Ib
If there is excessive variation of ICx data within or among laboratories involved in the
Validation Study, the lead laboratory for each method shall assist the Management Team
(through the designated contacts) to determine the cause and recommend appropriate
actions needed to reduce the variation. The Statement of Work, Test Method Protocols,and SOPs shall be revised if necessary, and testing repeated until acceptable proficiency
is achieved. The Management Team will decide when all laboratories will advance to thenext phase of the Validation Study. A teleconference shall be held with all of theappropriate participants of the Validation Study and the Management Team will relate
information concerning the advancement of the Validation Study.
4.2.3 Study Procedures
4.2.3.1. Phase Ib: Chemical TestingThree blinded/coded chemicals with varying cytotoxicity (high, medium, and low)
will be tested in both NRU assays. Eight concentrations of each chemical will betested in a 96-well plate (six wells per concentration) with at least four replicates
per concentration required for data analysis (Section 12.0). Only one test chemical
will be tested on each plate. The assay setup will follow the 96-well (microtiter)
plate configuration in Figure 2. A range finder experiment will be performed
before initiating concentration-response assays (Section 9.3). After the range
finding assay is completed, the concentration-response experiment shall be
performed three times on three different days for each assay and each chemical.
Laboratories will calculate IC20, IC50, and IC80 values in µg/ml, calculateconfidence limits for each value, and report this and all raw data to theManagement Team through the designated contacts.

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Figure 2. Plate Configuration for 3T3 NRU and NHK NRU Assays (Phase Ib)
1

2

3

4

5

6

7

8

9

10

11

12

A

b

b

b

b

b

b

b

b

b

b

b

b

B

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

C

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

D

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

E

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

F

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

G

b

VC

C1

C2

C3

C4

C5

C6

C7

C8

VC

b

H

b

b

b

b

b

b

b

b

b

b

b

b

VC
=
C1 – C8 =b
4.2.3.2=

untreated VEHICLE CONTROL (mean viability set to 100 %)TEST CHEMICAL at eight concentrations (C1 = highest,
C8 = lowest)
BLANKS (contain no cells)
Reporting Test Chemical Data (Phase Ib)

Biweekly Reports: Each testing facility will provide a biweekly progress report tothe designated contacts (See Addendum I). These reports will be in electronic
format (i.e., email with attachments) and will include raw and interim data as thestudy progresses. The Management Team will in turn provide a weekly progress
report addressing the Validation Study as a whole to all of the Testing Facilities.
Problems and issues shall be resolved in this manner.
Draft Report: At the conclusion of Phase Ib, a draft report of the Phase Ib test
chemical data shall be provided by the Study Director to the designated contacts.
The draft report (entitled: In Vitro Validation Study – Phase Ib: Training Phase for
Cytotoxicity Study of Three Coded Chemicals in Rodent and Human Cell Systems)
shall include everything noted in Addendum I (Draft Report – Phase Ib). If thePhase Ib data does not meet test acceptance criteria, then the Management Team
(through the designated contacts) will work with the Test Facility and leadlaboratory to identify problems and make corrections as needed. Once unresolved
issues have been resolved, the Validation Study will proceed. The Validation
Study will advance to Phase II once all participating laboratories have submitted
acceptable draft reports. The draft report shall be submitted in email and five (5)hard copy formats. File attachments in email shall be submitted in Microsoft®
Word (or equivalent) and Excel format and all email correspondence shall be

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copied to the designated contacts. Copies of the unaudited Study Workbook pages
may be submitted as attachments in hard copy format.
Final Report: Once the draft report provides data that meets test acceptancecriteria, then the Project Coordinator shall inform the Study Director to prepare a
Quality Assurance audited final report for Phase Ib. The final report shall be
submitted in email and five (5) hard copy formats. File attachments in email shallbe submitted in Microsoft® Word (or equivalent) and Excel format and all email
correspondence shall be copied to the designated contacts. Copies of the audited
Study Workbook pages may be submitted in hard copy format as an attachment to
the report. The final report will not need to be completed to continue to Phase II.
(See Validation Study timelines in Section 4.1 and Report submission timelines in
Section 4.5.)
4.2.4 Criteria for Advancing to Phase II
If there is excessive variation of ICx data within or among laboratories involved in the
Validation Study, the lead laboratory for each method shall assist the Management Team
(through the designated contacts) to determine the cause and recommend appropriate
actions needed to reduce the variation. The Statement of Work, Test Method Protocols,and SOPs shall be revised if necessary, and testing repeated until acceptable proficiency
is achieved. The Management Team will decide when all laboratories will advance to thenext phase of the Validation Study. A teleconference shall be held with all of theappropriate participants of the Validation Study and the Management Team will relate
information concerning the advancement of the Validation Study.
4.3

Phase II
4.3.1 Study Procedures
Phase II of this Validation Study is the qualification phase. This phase requires testingnine blinded/coded chemicals in the same in vitro cytotoxicity assays and in the same
concentration-response fashion as in Phase Ib. After a range-finding assay is completed,the concentration-response experiment for each chemical shall be performed three times,
once each on three different days. Laboratories will calculate IC20, IC50, and IC80 values
in µg/ml, calculate confidence limits for each value, and report this and all raw data to
the Study Management Team through the designated contacts.
4.3.1.1

Reporting Test Chemical Data (Phase II)

Biweekly Reports: Each testing facility will provide a biweekly progress report tothe designated contacts (See Addendum I). These reports will be in electronic
format (i.e., email with attachments) and will include raw and interim data as thestudy progresses. The Management Team will in turn provide a weekly progress
report addressing the Validation Study as a whole to all of the Testing Facilities.
Problems and issues shall be resolved in this manner.
Draft Report: At the conclusion of Phase II, a draft report of the Phase II test
chemical data shall be provided by the Study Director to the designated contacts.
The draft report (entitled: In Vitro Validation Study – Phase II: Qualification Phase
for Cytotoxicity Study of Nine Coded Chemicals) shall include everything noted in
Addendum I (Draft Report – Phase II). If the Phase II data does not meet test
acceptance criteria, then the Management Team (through the designated contacts)
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will work with the Test Facility and lead laboratory to identify problems and makecorrections as needed. Once unresolved issues have been resolved, the Validation
Study will proceed. The Validation Study will advance to Phase III once allparticipating laboratories have submitted acceptable draft reports. The draft reportshall be submitted in email and five (5) hard copy formats. File attachments inemail shall be submitted in Microsoft® Word (or equivalent) and Excel format andall email correspondence shall be copied to the designated contacts. Copies of theunaudited Study Workbook pages may be submitted as attachments in hard copy
format.
Final Report: Once the draft report provides data that meets test acceptancecriteria, then the Project Coordinator shall inform the Study Director to prepare a
Quality Assurance audited final report for Phase II. The final report shall be
submitted in email and five (5) hard copy formats. File attachments in email shallbe submitted in Microsoft® Word (or equivalent) and Excel format and all email
correspondence shall be copied to the designated contacts. Copies of the audited
Study Workbook pages may be submitted in hard copy format as an attachment to
the report. The final report will not need to be completed to continue to Phase III.
(See Validation Study timelines in Section 4.1 and Report submission timelines in
Section 4.5.)
Any solubility problems/issues with the test chemicals shall be addressed by thelead laboratory and Management Team (through the designated contacts) and
resolved at the end of Phase II before proceeding to Phase III.
4.3.2 Criteria for Advancing to Phase III
If there is excessive variation of ICx data within or among laboratories in the Validation
Study, the lead laboratory/testing facility shall assist the Management Team (through thedesignated contacts) to determine the cause and recommend appropriate actions needed
to reduce the variation. The Statement of Work, Test Method Protocols, and SOPs shallbe revised if necessary and testing repeated until acceptable proficiency andreproducibility is achieved in all participating laboratories. The Management Team will
decide when all laboratories will advance to the next phase of the Validation Study. A
teleconference shall be held with all of the appropriate participants of the Validation
Study and the Management Team will relate information concerning the advancement of
the Validation Study.
4.4

Phase III4.4.1 Study Procedures
Phase III of this Validation Study requires testing 60 blinded/coded chemicals in thesame manner as in Phases I and II (i.e., in the in vitro cytotoxicity assays in aconcentration-response fashion with two - three replicate assays [see Figure 2] after
completing a range-finding assay for each chemical). The definitive number of replicate
assays will be determined based on recommendations of the Management Team andprojected costs for doing replicates (see Section 1.4). Laboratories will calculate IC20,
IC50, and IC80 values in µg/ml, calculate confidence limits for each value, and report thisand all raw data to the Study Management Team through the designated contacts.

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November 2006

Reporting Data (Phase III)

Biweekly Reports: Each testing facility will provide a biweekly progress report tothe designated contacts of the Management Team (See Addendum I). These
reports will be in electronic format (i.e., email with attachments) and will include
raw and interim data as the study progresses. The Management Team will in turnprovide a weekly progress report addressing the Validation Study as a whole to allof the Testing Facilities. Problems and issues shall be resolved in this manner.
Draft Report: At the conclusion of Phase III, a draft report of the Phase III test
chemical data shall be provided by the Study Director to the designated contacts.
The draft report (entitled: In Vitro Validation Study – Phase III: Cytotoxicity Studyof 60 Coded Chemicals in Rodent and Human Cell Systems) must include
everything noted in Addendum I Draft Report – Phase III). If the Phase III data
does not meet test acceptance criteria, then the Management Team (through thedesignated contacts) will work with the Test Facility and lead laboratory to identify
problems and make corrections as needed. Once unresolved issues have been
resolved, the Validation Study will proceed. The draft report shall be submitted inemail and five (5) hard copy formats. File attachments in email shall be submitted
in Microsoft® Word (or equivalent) and Excel format and all email correspondence
shall be copied to the designated contacts. Copies of the unaudited Study
Workbook pages may be submitted as attachments in hard copy format.
Final Report: Once the draft report provides data that meets test acceptancecriteria, then the Project Coordinator shall inform the Study Director to prepare a
Quality Assurance audited final report for Phase III. The final report shall be
submitted in email and five (5) hard copy formats. File attachments in email shallbe submitted in Microsoft® Word (or equivalent) and Excel format and all email
correspondence shall be copied to the designated contacts. Copies of the audited
Study Workbook pages may be submitted in hard copy format as an attachment to
the report. (See Validation Study timelines in Section 4.1 and Report submission
timelines in Section 4.5.)
4.4.2 Criteria for Completion of Phase III
Phase III will be complete once all of the test chemicals (60) have been tested and the
Study Director provides a final report to the designated contacts. The Validation Study
will be complete (for all Testing Facilities) after the Study Management Team hasreceived final reports from each Testing Facility and has statistically analyzed all of thedata provided by all Testing Facilities.
4.5

Report Submission Timelines
4.5.1 Draft Reports
Draft reports for each phase shall be submitted to the Management Team through thedesignated contacts as per Section 4.1.1. The Management Team will respond to the
Test Facility within two – four weeks after receipt of the report. If data are acceptable,
then the Management Team (through the designated contacts) will instruct the Test
Facility to continue to the next phase (teleconference with all participants). If the data do
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not meet the criteria and adjustments to the Validation Study are needed, a new timeline
will be created and relayed to the Test Facility.
4.5.2 Final Report
Once the Management Team (through the designated contacts) declares to a Test Facility
that the Validation Study testing phase is complete, then the Test Facility shall provide a
final report (electronic and hard copy) for the identified phase of the Validation Study to
the Management Team through the designated contacts as per Section 4.1.1.
5.0

IDENTIFICATION OF TEST CHEMICALS AND CONTROL SUBSTANCESThe NIEHS/NTP designated contractor (BioReliance) will supply all test chemicals and the positive
control to all Testing Facilities. Phase I chemicals will be shipped as a unit as will the Phase II
chemicals. Phase III chemicals will be shipped as one unit of 60 chemicals. The Management Teamwill have all pertinent information for each chemical (e.g., purity, CAS #, supplier, etc.) and will
make all decisions concerning any questions about or problems/issues with the chemicals.
5.1

Test Chemicals
5.1.1 Range of Toxicities
The chemicals proposed for the Validation Study are representative of a range of
toxicities and are relevant with regard to human exposure potential. The test chemicals
will represent each of the Globally Harmonized System (GHS) classification groups for
rat oral LD50s: ≤ 5 mg/kg, >5 ≤ 50 mg/kg, >50 ≤ 300 mg/kg, >300 ≤ 2000 mg/kg, >2000≤ 5000 mg/kg, and >5000 mg/kg (OECD, 2001).
5.1.2 Receipt of Chemicals
Test chemicals will be packaged so as to minimize damage during transit and will beshipped to the Testing Facility according to proper regulatory procedures. Chemicals areto be packaged and shipped so as to conceal their identities. The Study Management
Team and the Testing Facility shall be notified by the contractor (BioReliance) when thetest chemicals are shipped so as to prepare for receipt.
Upon receipt at the facility, the test chemicals shall be stored in appropriate storage
conditions as per recommendations provided by the contractor (BioReliance). The
Testing Facility shall immediately notify the Project Coordinator and the contractor about
receipt of chemicals. The blinded/coded test chemicals as well as a sealed health andsafety information package will be shipped to the Safety Officer. The Safety Officer
shall retain the package and pass the test chemicals to the Study Director. The package
will contain necessary information about the chemical hazards and provide instructions
for emergency actions. A disclosure key for identifying test chemicals by code will also
be included. At the end of the Validation Study, the Safety Officer shall return theunopened health and safety package to the contractor (BioReliance) who supplied thechemicals (through the designated contacts). If the health and safety package must be
opened by the laboratory, the Safety Officer shall immediately notify the designatedcontacts.
If regulatory transportation requirements dictate that each package must display a list of
the chemicals it contains on the outside of the package, the list can be removed byshippers before delivery to the participating Testing Facility. If shippers have not
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removed this information, the Safety Officer shall remove it prior to passing thechemicals to the Study Director.
5.1.3 Test Chemical Information for the Study Director
Each test chemical will be accompanied by data sheets giving a minimum of essential
information, including color, odor, physical state, weight or volume of sample, specificdensity for liquid test chemicals, and storage instructions (which will be the same foreach chemical). The Study Director shall receive this information.
5.2

Control Materials
5.2.1 Vehicle Control (VC)
5.2.1.1 3T3 NRU Assay (VC)
Dulbecco’s Modification of Eagle’s Medium (DMEM) buffered with sodium
bicarbonate and supplemented with (final concentrations in DMEM are quoted): 5% NBCS, 4 mM Glutamine, 100 IU Penicillin, 100 µg/ml Streptomycin. (Seespecifics in Test Method Protocol) [Note: Vehicle control may also be known as
negative control.]
5.2.1.2 NHK NRU Assay (VC)
A modified MCDB 153 formulation such as Clonetics® Keratinocyte Basal
Medium (KBM®) supplemented with: 0.1 ng/ml Human recombinant epidermal
growth factor, 5 g/ml Insulin, 0.5 g/ml Hydrocortisone, 50 g/ml Gentamicin, 50
ng/ml Amphotericin B, 0.1 mM Calcium, 2 ml 7.5 mg/ml Bovine pituitary extract.
(See specifics in Test Method Protocol) [Note: Vehicle control may also be known
as negative control.]
5.2.2 Positive Control (PC)
Sodium Lauryl Sulfate ([SLS], CAS # 151-21-3) will be the positive control for bothassays. A dose-response assay of SLS dilutions will be run in one plate for each set oftest chemical assays. There will be no PC in the test chemical assay plates.

5.3 Inventory of Test Chemicals
The amount of test chemical received, the amount used for specific tests, and the amountremaining shall be documented by the Testing Facility.
5.4 Disposition of Test Chemicals
After the studies are completed, the remaining test chemicals will be returned to the contractor(BioReliance) or appropriately disposed of by the Testing Facility.
5.5 Handling of Test Chemicals
Appropriate routine safety procedures shall be followed in handling the test chemicals unlessthe contractor (BioReliance) otherwise specifies more cautious procedures. Test Facility
personnel shall be instructed to treat all blinded/coded test chemicals as very hazardous andpotentially carcinogenic and to dispose of laboratory wastes as toxic wastes. The health andsafety information package provided to the Testing Facility Safety Officer shall be examined by
the Testing Facility only in an emergency/need-to-know situation.

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5.6 Determination of Purity, Composition, and Stability of Test Chemicals
The contractor (BioReliance) will be responsible for collecting information on the analyticalpurity, composition, and stability of the test chemicals and the positive control material frommanufacturer and supplier documentation. The contractor will provide information on
chemical homogeneity in the vehicle via solubility studies. Chemicals shall be stored in an
appropriate manner as stated by the contractor.
6.0

TEST SYSTEM
All testing procedures and data analyses shall follow the Test Method Protocols and Statementof Work provided by the Management Team which are based on the NIEHS Publication # 014500, Guidance Document on Using In Vitro Data to Estimate In Vivo Starting Doses for Acute
Toxicity (ICCVAM, 2001a).
6.1

Neutral Red Uptake (NRU) Cytotoxicity Assay
6.1.1 Background
The NRU cytotoxicity assay procedure is a cell survival/viability chemosensitivity assay
based on the ability of viable cells to incorporate and bind neutral red (NR), a supravitaldye. NR is a weak cationic dye that readily penetrates cell membranes by non-ionic
diffusion and accumulates intracellularly in lysosomes. Alterations of the cell surface or
the sensitive lysosomal membrane lead to lysosomal fragility and other changes that
gradually become irreversible. Such changes brought about by the action of xenobiotics
result in a decreased uptake and binding of NR. It is thus possible to distinguish between
viable, damaged, or dead cells, which is the basis of this assay.
Healthy mammalian cells, when maintained in culture, continuously divide and multiply
over time. A toxic chemical, regardless of site or mechanism of action, will interferewith this process and result in a reduction of the growth rate as reflected by cell number.
Cytotoxicity is expressed as a concentration dependent reduction of the uptake of the NR
after chemical exposure, thus providing a sensitive, integrated signal of both cell integrity
and growth inhibition.
6.1.2 Sterility of the Test System
All cell culture applications shall be conducted under aseptic conditions. The test systemshall be deemed free of mycoplasmal, fungal, and/or bacterial contamination. The cell
suppliers ship cryopreserved cells that have been tested for mycoplasma and are deemed
mycoplasma-free. If mycoplasma contamination is suspected, then the Testing Facility
shall have the cells tested in an appropriate manner. If mycoplasma is present, all oldcells of the specific lot of cells shall be eliminated and new cell stocks shall be preparedor purchased. The presence of bacterial or fungal contamination in the cultures shall be
determined by gross visual inspection during and at the conclusion of each assay. If
bacterial or fungal contamination is present in the cultures, the Study Director shalldetermine the course of action.

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EXPERIMENTAL DESIGN AND METHODOLOGY – 3T3 NRU ASSAY
7.1

Major Steps in the Performance of the Assay

BALB/c 3T3 cells are seeded into 96-well plates and maintained in culture for 24 hours(h) (~ 1 doubling period) to form a semi-confluent monolayer⇓
Remove culture medium⇓
Cells are then exposed to the test chemical in treatment medium over a range of 8concentrations for 48 h exposure⇓
Microscopic evaluation of morphological alterations⇓
Remove treatment medium; wash once with PBS; add Neutral Red (NR) medium;incubate for 3 h.
⇓
Discard NR medium; wash once with PBS; add NR desorbing fixative⇓
Shake plate for 20 minutes⇓
Detect NR Absorption at optical density (OD) 540nm⇓
Perform Neutral Red Uptake data calculations (% viability; calculations of IC20, IC50,
and IC80 values)
7.2

Procedures for Conducting the Test
All testing procedures and data analyses shall follow the Test Method Protocols andStatement of Work provided by the Management Team and SOPs produced by the
Testing Facility. All deviations from Statement of Work or SOPs shall be documented in
the Study Workbook. The following abbreviated descriptions of the SOPs provide an
overview of the assay, but must not be used in place of the formal SOPs.
7.2.1 Cell Maintenance and Culture Procedures
Ampules of cryopreserved BALB/c 3T3 cells are quickly thawed in a 37°C water bath.
The cells are resuspended in cell culture medium and transferred to cell culture flasks.
The thawed cells are incubated at 37°C in a 90 % humidified 5.0 % CO2 atmosphere.
Cells are passaged two to three times before using them in a cytotoxicity test. A freshbatch of cryopreserved cells should be thawed out approximately every two months (See
Section 7.2.1.1). This period resembles a sequence of about 18 passages.
The cells are routinely grown as a monolayer in tissue culture grade flasks, at 37°C in a
90 % humidified atmosphere of 5.0 % CO2 and are examined on a daily basis under aphase contrast microscope.
When cells approach a predetermined confluency, they must be detached from the flask
by trypsinization, resuspended in culture medium, and counted using a hemocytometer orcell counter. After determination of cell number, the cell culture must be sub-cultured
into other flasks or seeded into 96-well microtiter plates. Stocks of BALB/c 3T3 cells are
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prepared in a medium with DMSO as a cryoprotective agent and stored in sterile,
freezing tubes in a liquid nitrogen freezer for long-term storage.
7.2.1.1 Cryopreserved Lots of Cells
After the initial establishment of the 3T3 cells in culture from an ampule ofcryopreserved cells (from the cell supplier), laboratory personnel shall growenough cells for cryopreservation in a number of freeze tubes (e.g., 10 – 20 tubes).
These tubes will form the stock pool from which subsequent cultures will beestablished for use in the assays (See Section 7.2.1).
7.2.1.2 Determination of Cell Doubling TimeA cell doubling time procedure shall be performed on the initial lot of cells thatwill be used in the first cell culture assays of Phase Ia of the Validation Study. The
doubling time only needs to be determined again if there is a change in the lot ofcells used. The Test Method Protocol will provide the basic procedures for thisdetermination.
8.0

EXPERIMENTAL DESIGN AND METHODOLOGY – NHK NRU ASSAY
8.1

Major Steps in the Performance of the Assay

NHK cells are seeded into 96-well plates and maintained in culture for 24 – 72 hours(h) to form a semi-confluent (30 – 50 %) monolayer⇓
Remove culture medium⇓
Cells are then exposed to the test chemical in treatment medium over a range of 8concentrations for 48 h exposure⇓
Microscopic evaluation of morphological alterations⇓
Remove treatment medium; wash once with PBS; add Neutral Red (NR) medium;incubate for 3 h.
⇓
Discard NR medium; wash once with PBS; add NR desorbing fixative⇓
Shake plate for 20 minutes⇓
Detect NR Absorption at optical density (OD) 540nm⇓
Perform Neutral Red Uptake data calculations (% viability; calculations of IC20, IC50,
and IC80 values)
8.2 Procedures for Conducting the Test
All testing procedures and data analyses shall follow the Test Method Protocols and Statementof Work provided by the Management Team and SOPs produced by the Testing Facility. All
deviations from the Statement of Work or SOPs shall be documented in the Study Workbook.
The following abbreviated descriptions of the SOPs provide an overview of the assay, but mustnot be used in place of the formal SOPs. Information specific to the keratinocytes as provided
by the supplier (e.g., Clonetics) shall be considered when preparing SOPs.
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8.2.1 Cell Maintenance and Culture Procedures
Ampules of cryopreserved NHK cells are quickly thawed in a 37°C water bath. The cells
are resuspended in cell culture medium and transferred to cell culture flasks. The thawed
cells are incubated at 37°C in a 90 % humidified 5.0 % CO2 atmosphere. NHK cells will
be sustained in culture through only one passage after establishing cells in culture.
The cells are routinely grown as a monolayer in tissue culture grade flasks, at 37°C in a
90 % humidified atmosphere of 5.0 % CO2 and are examined on a daily basis under aphase contrast microscope.
When cells approach a predetermined confluency, they must be detached from the flask
by trypsinization, resuspended in culture medium, and counted using a hemocytometer orcell counter. After determination of cell number, the cell culture must be seeded into 96well microtiter plates.
8.2.1.1 Determination of Cell Doubling TimeA cell doubling time procedure shall be performed on the initial lot of cells thatwill be used in the first cell culture assays of Phase Ia of the Validation Study. The
doubling time only needs to be determined again at the initiation of the cells in
culture if there is a change in the lot of cells used. The Test Method Protocol will
provide the basic procedures for this determination.
9.0

PREPARATION AND DELIVERY OF TEST CHEMICAL
9.1 Preparation of Test Chemical
The test chemical must be freshly prepared immediately prior to use. All chemicals shall beweighed on a calibrated balance (including liquid test chemicals) and added to the appropriate
solvent (Section 9.1.1). Test chemicals must be at room temperature before dissolving anddiluting. Preparation under red or yellow light may be necessary, if rapid photodegradation is
likely to occur. The solutions must not be cloudy nor have noticeable precipitate.
The following hierarchy (culture medium, DMSO, ethanol) shall be followed for dissolving thetest chemical.
9.1.1. Dissolving the Test Chemical
9.1.1.1 Treatment Medium/Routine Culture Medium)a) Dissolve test chemical in Treatment Medium [3T3] or Routine Culture
Medium [NHK] (See Test Method Protocols).
b) Gently mix. Vortex (1 –2 minutes).
c) If test chemical hasn’t dissolved, use sonication (up to five minutes).
d) If sonication doesn’t work, then warm solution to 37°C.
9.1.1.2 DMSO
If the test chemical doesn’t dissolve in the Treatment Medium/Routine Culture
Medium, then follow steps a) through d) in Section 9.1.1.1 using DMSO instead ofTreatment Medium/Routine Culture Medium.

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9.1.1.3 Ethanol
If the test chemical doesn’t dissolve in DMSO, then follow steps a) through d) in
Section 9.1.1.1 using ethanol instead of DMSO.
9.1.2. Test Chemical Solubility
Each test chemical will be prepared such that the highest test concentration in each range
finding experiment is 100 mg/ml (100,000 µg/ml) in culture medium (10 mg/ml [10,000µg/ml] in culture medium if DMSO or ethanol is used as a solvent). If the range finding
experiment shows that 100,000 µg/ml is not high enough for the IC50 values in the range
to meet the acceptance criteria, then higher concentrations will be used for the definitive
experiment.
Solubility of the test chemical will be determined by following a modified version of
EPA Product Properties Test Guidelines OPPTS 830.7840 (EPA, 1998). (See Test
Method Protocols).
Dissolve the test chemical (at 200-fold the desired final concentration in the case ofsolvents) in an appropriate solvent. The final solvent (i.e., DMSO or ethanol)
concentration should be kept at a constant level of no more than 0.5 % (v/v) in the vehicle
controls and in all of the eight test concentrations (i.e., each concentration shall have thesame amount of solvent). This means the test chemical is dissolved in the vehicle first,
and then 1 part of this stock solution is added to 199 parts of sterile pre-warmed (37°C)
medium. Check carefully to determine whether the chemical is still dissolved after thetransfer from solvent stock solution to medium, and reduce the highest test concentration,
if necessary.
The test chemicals selected for the Validation Study will be soluble. If an appropriate
concentration cannot be achieved for the range finding experiments, then the Study
Director shall contact the Study Management Team through the designated contacts.
Prior to initiating any test chemical assay (and after performing solubility tests on thechemicals), the Study Director shall contact the Study Management Team (through thedesignated contacts) for discussion of the solvent to be used for test chemical application.
The Management Team will provide direct guidance to the Study Director as to whichsolvent will be used for the assay.
9.1.3 pH of Dilutions
Measure the pH (using pH paper) of the highest concentration of the test chemical to betested in the assay. Document the pH and note the color of the medium. Do not adjust
the pH of the test chemical solutions.
9.2 Delivery of Test Chemical
The test chemical will be administered by direct addition (pipetting) to the 96-well microtiter platewith a vehicle compatible with the test system. The cells will be exposed to the test chemical for
approximately 48 hours..
[Note: The 3T3 and NHK cells in the 96-well plate will have freshculture medium on the cells
immediately prior to dosing with the test chemical. Each well will receive a volume of test
chemical concentration therefore diluting the concentration by a factor of two.]

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9.3 Range Finder Experiment
Test eight concentrations of the test chemical by diluting the stock solution with a constantfactor. The initial dilution series will be log dilutions (i.e., 1:10, 1:100, 1:1000, etc.). If thisdilution series meets test acceptance criteria (Section 11.0), then the range finding experiment
dilutions can be used as the actual dilutions in the separate definitive test chemical experiment.
If the dilution factor needs to be adjusted for the actual definitive experiment, then follow
dilution schemes provided in Section 9.4.
9.4 Test Chemical Dilutionsa) A factor of 2√10 = 3.16 could be used for covering a large range:(e.g., 1 ⇒3.16 ⇒10 ⇒31.6 ⇒100 ⇒316 ⇒1000 ⇒3160 µg/ml).
b) The simplest geometric concentration series (i.e., constant dilution / progression factor) aredual geometric series (e.g., a factor of 2). These series have the disadvantage of numericalvalues that permanently change between logs of the series:(e.g., log0-2, 4, 8; log1- 16, 32, 64; log2- 128, 256, 512; log3- 1024, 2048,).
c) The decimal geometric series, first described by Hackenberg and Bartling (1959) for use
in toxicological and pharmacological studies, has the advantage that independent
experiments with wide or narrow dose factors can be easily compared because they shareidentical concentrations. Furthermore, under certain circumstances, experiments can evenbe merged together:
EXAMPLE:
10
10
10
10

31.6

12.1

14.7
14.7

17.8

21.5
21.5
21.5

26.1

31.6
31.6

38.3

46.4
46.4
46.4

56.2

68.1
68.1

82.5

100
100
100
100

The dosing factor of 3.16 (= 2√10) divides a log into two equidistant steps, a factor of 2.15 (=3√10) divides a decade into three steps. The factor of 1.47 (= 6√10) divides a log into six
equidistant steps, and the factor of 1.21 (= 12√10) divides the log into 12 steps.
For an easier biometrical evaluation of several related concentration response experiments
use decimal geometric concentration series rather than dual geometric series. The technicalproduction of decimal geometric concentration series is simple. An example is given forfactor 1.47:
Dilute 1 volume of the highest concentration by adding 0.47 volumes of diluent. Afterequilibration dilute 1 volume of this solution by adding 0.47 volumes of diluent...(etc.).
d) Determine which test chemical concentration is closest to the IC50 value (e.g., 50 %cytotoxicity). Use that value as the central concentration and adjust dilutions higher andlower in equal steps for the definitive assay.
10.0 DATA COLLECTION
10.1 Nature of Data to be Collected
After the test is performed and the NR is desorbed from the cells, measure the absorption of theresulting colored solution at 540 nm in a microtiter spectrophotometric plate reader, using theblanks as a reference. Save raw data in the file format provided by the Study Management
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Team (Microsoft® Excel template [Addendum II]) for further analysis of the concentrationresponse (% viability calculations). Data from the OD analyses will be used for the calculationof IC20, IC50, and IC80 values (µg/ml).
10.2 Type of Media Used for Data Storage
Originals of the raw data (the Study Workbook and computer printouts of absorbance readings
from the plate reader) and copies of other raw data such as instrument logs shall be collected
and archived at the end of the Validation Study (under the direction of the Study Director),
according to GLP-compliant procedures. The electronic files of plate reader data and any
derived data shall be saved, and a backup of these electronic files shall be produced andmaintained. Calculations to convert the raw data to derived data shall be performed using
Microsoft® Excel (Addendum II). The derived assay data that are stored electronically shall be
periodically copied, and backup files shall be produced and maintained.
10.3 Documentation
Original raw data that shall be collected shall include but are not limited to the following:• Data recorded in the Study Workbook, which shall consist of all recordings of all activities
related to preparing the 3T3 and NHK cultures and test chemicals and performing the NRU
assay;• Computer printouts of absorbance readings from the plate reader spectrophotometer;• Other data collected as part of GLP compliance− Equipment logs
− Equipment calibration records− Test chemical logs
− Cryogenic freezer inventory logs
− Cell culture media preparation logs
Addendum IV provides examples of equipment logs.
11.0 ACCEPTANCE CRITERIA FOR NRU ASSAYS
11.1 Test Acceptance Criteria
The test method protocols provide the definitive test acceptance criteria which include a
specific mean OD540 of all vehicle controls, a set percent difference of the mean OD540 between
two sets of vehicle controls, and a set range of the IC50 for SLS.
The Study Director shall decide if a test meets acceptance criteria and the Study Management
Team will make decisions concerning re-testing of test chemicals.
11.2 IC50 Acceptance Criteria
The IC50 derived from the concentration-response assays shall be based on at least three
responses that are ≥ 10 % and ≤ 90 % inhibition of NRU. If this is not the case, and theconcentration progression factor can be easily reduced, the experiment shall be rejected and aretest shall be performed with a smaller progression factor.
The raw data output from the plate reader shall be converted into the derived data using
Microsoft® Excel (Addendum II). The PC and VC from each assay shall be compared to theacceptable historical ranges as noted. If the assay is found to be valid by these criteria, then thedata from that assay is considered to be acceptable. If the PC or VC values are not acceptable,

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the assay shall be repeated. Results of all assays, acceptable and failed, shall be forwarded tothe designated contacts via the previously identified reports.
12.0 EVALUATION OF TEST RESULTS
12.1 Cell Viability Determination
A calculation of cell viability expressed as NRU is made for each concentration of the test
chemical by using the mean NRU of the six replicate values (minimum of four acceptable
replicate wells) per test concentration. The Study Director shall determine if any wells do notmeet expected performance criteria through visual microscopic evaluation (i.e., experimental
conditions within the wells are compromised due to situations such as insufficient cell
population, mechanical disruption of the monolayer, etc.). The Study Director shall decide if
any of the wells of the plate need to be excluded from data analyses. If a concentration does
not have a minimum of four replicate wells, then data from that concentration will not used.
The test may still be acceptable if all criteria in Section 11.1 are met (e.g., the IC50 derived
from the concentration-response assays is backed by at least three responses ≥ 10 % and ≤ 90 %inhibition of NRU.) If any wells have bacterial or fungal contamination, the entire plate must
be repeated.
The cell viability value is compared with the mean NRU of all VC values (provided VC values
have met the VC acceptance criteria). Relative cell viability is then expressed as percent of
untreated VC. If achievable, the eight concentrations of each chemical tested will span therange of no effect up to total inhibition of cell viability.
12.2 ICX Determination
The concentration of a test chemical reflecting a 20 %, 50 %, and 80 % inhibition of cell
viability (i.e., the IC20, IC50, and IC80) is determined from the concentration-response and shallbe done by applying a Hill function to the concentration-response data. It will not be necessary
for the Testing Facilities to derive the equation. The Testing Facility shall calculate the IC20,
IC50, and IC80 values for each test chemical and the confidence limits for each value using
statistical software (e.g., GraphPad PRISM® 3.0) specified by the Study Management Team.
In addition, the Study Management Team shall provide guidelines for calculating ICx values
and confidence limits. The Testing Facility shall report data using at least three (3) significant
figures and shall forward the results from each assay to the Study Management
Team/biostatistician through the designated contacts in electronic format and hard copy uponcompletion of all testing. The Study Management Team will be directly responsible for thestatistical analyses of the Validation Study data.
Hill function: a four-parameter logistic mathematical model relating the concentration of test
chemical to the response being measured in a sigmoidal shape.

Y = Bottom +Top ! Bottom
1 + 10(logIC50! X)HillSlope

where Y= response, X is the logarithm of dose (or concentration), Bottom is the minimum
response, Top is the maximum response, logIC50 is logarithm of X at the response midway
between Top and Bottom, and HillSlope describes the steepness of the curve.

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13.0 DRAFT AND FINAL REPORTS
A draft report shall be submitted to the Management Team through the designated contacts atthe completion of each study phase (Ia, Ib, II, III). A Final Report for each phase of the
Validation Study shall be prepared by the Testing Facility, signed by the Study Director, andprovided to the Management Team through the designated contacts upon acceptance of data
provided in the corresponding draft report. The submitted results shall accurately describe all
methods used for generation and analysis of the data, provide a complete record of the
preparation of test chemicals, and present any relevant data necessary for the assessment of theresults (See Addendum I).
14.0 RECORDS AND ARCHIVESAt the end of the Validation Study, the original raw and derived assay data, as well as copies ofother raw data not exclusive to this Validation Study (instrument logs, calibration records,
facility logs, etc.), shall be submitted to NIEHS/NICEATM for storing and archiving according
to the facility's SOP and in compliance with GLP Standards.
Originals of all raw and derived data, or copies where applicable, shall be stored and archived
at NIEHS/NICEATM.
Copies of all raw and derived data shall be stored and archived at the participating Testing
Facility for at least five years after completion of the Validation Study.
15.0 ALTERATIONS OF THE STATEMENT OF WORKNo changes in the Statement of Work shall be made without the consent of the Management
Team. A Statement of Work Amendment detailing any change(s) and the basis for thechange(s) shall be approved and prepared by the Study Director, and the amendment shall besigned and dated by the Study Director and the NIEHS representative. The amendment shall be
retained with the original Statement of Work.
16.0 REFERENCES
Clonetics Normal Human Keratinocyte Systems Instructions for Use, AA-1000-4-Rev.03/00.
(http://www.clonetics.com).
Cooper-Hannan, R., J.W. Harbell, S. Coecke, M. Balls, G. Bowe, M., Cervinka, R. Clothier, F.
Hermann, L.K. Klahm, J. de Lange, M. Liebsch and P. Vanparys. 1999. The Principles ofGood Laboratory Practice: application to in vitro toxicology studies. The Report andRecommendations of the ECVAM Workshop 37. ATLA. 27 (4). July/August. Pp. 539 – 577.
EPA Product Properties Test Guidelines. OPPTS 830.7840. Water Solubility: Column Elution
Method; Shake Flask Method. United States Environmental Protection Agency. Prevention,
Pesticides and Toxic Substances (7101). EPA 712-C-98-041. March 1998.
Halle, W. 1998. Toxizitätsprüfungen in Zellkulturen für eine Vorhersage der akuten Toxizität(LD50) zur Einsparung von Tierversuchen. Life Sciences/ Lebenswissenschaften, Volume 1, 94
pp., Jülich: Forschungszentrum Jülich.

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ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods).
2001a. Guidance document on using in vitro data to estimate in vivo starting doses for acutetoxicity NIH publication 01-4500. NIEHS, Research Triangle Park, North Carolina.
http://iccvam.niehs.nih.gov/methods/invidocs/guidance/iv_guide.pdf
ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods).
2001b. Report of the international workshop on in vitro methods for assessing acute systemic
toxicity. NIH Publication 01-4499. NIEHS, Research Triangle Park, North Carolina.
http://iccvam.niehs.nih.gov/methods/invidocs/finalall.pdf
National Toxicological Program, September 2000, Attachment 2 revised. Specifications for the
Conduct of Studies to Evaluate the Toxic and Carcinogenic Potential of Chemical, Biologicaland Physical Agents in Laboratory Animals for the National Toxicology Program (NTP).
NICEATM (The National Toxicology Program [NTP] Interagency Center for the Evaluation of
Alternative Toxicological Methods). 2001. Test Method Protocol for the BALB/c 3T3 Neutral
Red Uptake Cytotoxicity Test. A Test for Basal Cytotoxicity for an In Vitro Validation Study.
NICEATM (The National Toxicology Program [NTP] Interagency Center for the Evaluation of
Alternative Toxicological Methods). 2001. Test Method Protocol for the Normal Human
Keratinocyte [NHK] Neutral Red Uptake Cytotoxicity Test. A Test for Basal Cytotoxicity for
an In Vitro Validation Study.
OECD (Organisation for Economic Co-operation and Development). 2001. Harmonised
Integrated Classification System for Human Health and Environmental Hazards of Chemical
Substances and Mixtures as Endorsed by the 28th Joint Meeting of the Chemicals Committee
and the Working Party on Chemicals in November 1998, Part 2, p.21. OECD, Paris.
http://www.oecd.org/ehs/class/HCL6htm.
OECD (Organisation for Economic Co-operation and Development). 1998. Organisation for
Economic Cooperation and Development series on Principles of Good Laboratory Practice andcompliance monitoring. Number 1. OECD Principles on Good Laboratory Practice (as reusedin 1997), OECD. Environmental Health and Safety Publications, Environment Directorate:
ENV/MC/CHEM(98) 17. Paris, France: OECD.
OECD (Organisation for Economic Co-operation and Development). 1992. Environment
Monograph No 50, Series on Principles of Good Laboratory Practice and Compliance
Monitoring: Number 6, The Application of the GLP Principles to Field Studies. Paris, France:
OECD.
Spielmann, H., S. Gerner, S. Kalweit, R. Moog, T. Wirnserberger, K. Krauser, R. Kreiling, H.
Kreuzer, N.P. Luepke, H.G. Miltenburger, N. Müller, P. Murmann, W. Pape, B. Siegmund, J.
Spengler, W. Steiling, and F.J. Wiebel. 1991. Interlaboratory assessment of alternatives to the
Draize eye irritation test in Germany. Toxicol. In Vitro 5: 539-542.
Triglia, D., P.T. Wegener, J. Harbell, K. Wallace, D. Matheson, and C. Shopsis. 1989.
Interlaboratory validation study of the keratinocyte neutral red bioassay from Clonetics
Corporation. In Alternative Methods in Toxicology, Volume 7. A.M. Goldberg, ed., pp. 357365. Mary Ann Liebert, Inc., New York.

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17.0 APPROVAL OF STATEMENT OF WORK____________________________________________
Sponsor Representative_________________
Date____________________________________________
Testing Facility Management_________________
Date

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ADDENDUM I
SUGGESTED REPORT FORMATTITLE PAGE•

Study Title
Draft/Final Report 1:
Draft/Final Report 2:
Draft/Final Report 3:
Draft/Final Report 4:

In Vitro Validation Study – Phase Ia: Development of a Positive
Control Database in Rodent and Human Cell Systems
In Vitro Validation Study – Phase Ib: Training Phase for
Cytotoxicity Study of Three Coded Chemicals in Rodent and Human
Cell Systems
In Vitro Validation Study – Phase II: Qualification Phase for
Cytotoxicity Study of Nine Coded Chemicals in Rodent and Human
Cell Systems
In Vitro Validation Study – Phase III: Cytotoxicity Study of 60 Coded
Chemicals in Rodent and Human Cell Systems

In Vitro Assay
Identify the assays: 3T3 NRU and NHK NRUc) Test Articles
Draft/Final Report 1: (Phase Ia) identify the positive control chemical
Draft/Final Report 2: (Phase Ib) identify the three (3) test chemicals
Draft/Final Report 3: (Phase II) identify the nine (9) test chemicals
Draft/Final Report 4: (Phase III) identify the sixty (60) test chemicals• Authors• Study Completion Date
• Testing Facility• Validation Study Number/Identification•

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ADDENDUM I (cont.)
SUGGESTED REPORT FORMATSIGNATURE PAGE
•
•
•
•

•

•
•
•
•
•
•

Validation Study Initiation Date
Date Protocol was signed by Study Director
Initiation Date of Laboratory Studies
Actual laboratory start date
Validation Study Completion Date
Date report signed by Study Director
Sponsor Representative
The National Institute of Environmental Health Sciences (NIEHS)
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)NICEATM79 T.W. Alexander Drive
Bldg. 4401, MD-EC-17
3rd Floor, Room 3126
P.O. Box 12233
Research Triangle Park, NC 27709
Study Management Team Representatives
Judy Strickland, Ph.D. (Project Coordinator)
Michael Paris (Assistant Project Coordinator)
Testing Facility
Name and address
Archive Location
Name and address
Study Director
Name and signature and date
Key Personnel
Laboratory technicians, QA Director, Safety Officer
Facility Management
Name
Scientific Advisor
Name

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ADDENDUM I (cont.)
SUGGESTED REPORT FORMATTEST CHEMICAL RECEIPT PAGE
Test Chemical Receipt Reporting Template for In Vitro Validation Study
Test Facility
Test Chemical
Identification
NumberSponsor
Test Chemical
Identification
NumberTest Chemical
PhysicalDescription

Storage
Conditions

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Test
ChemicalReceipt
Date

Test
ChemicalReceived By

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In Vitro Cytotoxicity Test Methods BRD Appendix G1

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ADDENDUM I (cont.)
SUGGESTED REPORT FORMAT
DRAFT/FINAL REPORT 1
In Vitro Validation Study – Phase Ia: Development of a Positive Control Database in Rodent andHuman Cell Systems•

Table of Contents•

Objectives: The reports shall provide specific objectives•

Description of the Test System Used: Description of 3T3 NRU assay and the NHK NRU assay•

Summary of the Findings: Referenced to the raw data where appropriate; Include all
information for range finding experiments•

Narrative Description of the Assays: Describe any problems that were encountered and how
such problems were solved. Justifications for solvents used for each test chemical will be
included in the description. Deviations from the protocols, SOPs, and/or the Statement of Workshall be addressed in this section. Copies of appropriate sections of the Study Workbook shall be
included with the report as attachments. The draft report will include unaudited Study Workbook
pages. The final report will include a copy of the audited Study Workbook with a statement(signed and dated by the Study Director) on the front of it stating that it is an exact copy of theoriginal audited workbook.

•

Statement Signed by the Study Director: Confirm that the Validation Study was conducted incompliance with GLP (or indicating where the Study deviated from GLP), or for non GLPcompliant laboratories, confirm that the Validation Study adhered to the spirit of GLP. Confirm thatthe report fully and accurately reflects the raw data generated in the Validation Study.

••

Quality Assurance Statement: (For Final Report only)
For GLP-Compliant Laboratories: QA Statement identifying: 1) the phases and data inspected, 2)
dates of inspection, and 3) dates findings were reported to the Study Director and Testing Facility
management. The QA Statement shall identify whether the methods and results described in the
Final Report accurately reflect the raw data produced during the Validation Study.
For Non GLP-Compliant Laboratories: A statement from the Testing Facility shall be included
with the Final Report of Phase III. This statement shall identify whether the methods and results
described in the Final Report accurately reflect the raw data produced during the Validation Studyand provide assurance that all testing was done in the spirit of GLP.

••

Data Analysis: (for each NRU assay) calculate the % viability for each positive control chemical
concentration (eight concentrations per assay); determine the IC50 values for the positive control
in each assay; follow guidelines/procedures in Statement of Work and Test Method Protocols.

•

Other Information: (All copies of printouts, documents, and spreadsheets will be noted asexact duplicates of the data.)• Copies of spectrometric plate reader raw data
• Copies of the completed Microsoft® Excel spreadsheets (Addendum II) used for calculationof cytotoxicity values• Copies of data pages showing IC50 calculations for the positive control• Copy of the protocols• Deviations to the protocols, SOPs, and Statement of Work
• Revisions/amendments to the protocols, SOPs, and Statement of Work
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ADDENDUM I (cont.)
SUGGESTED REPORT FORMAT
DRAFT/FINAL REPORT 2
In Vitro Validation Study – Phase Ib: Training Phase for Cytotoxicity Study of Three Coded
Chemicals in Rodent and Human Cell Systems•

Table of Contents•

Objectives: The reports shall provide specific objectives•

Description of the Test System Used: Description of 3T3 NRU assay and the NHK NRU assay•

Summary of the Findings: Referenced to the raw data where appropriate; Include all
information for range finding experiments•

Narrative Description of the Assays: Describe any problems that were encountered and how
such problems were solved. Justifications for solvents used for each test chemical shall be
included in the description. Deviations from the protocols, SOPs, and/or the Statement of Workshall be addressed in this section. Copies of appropriate sections of the Study Workbook shall be
included with the report as attachments. The draft report will include unaudited Study Workbook
pages. The final report will include a copy of the audited Study Workbook with a statement(signed and dated by the Study Director) on the front of it stating that it is an exact copy of theoriginal audited workbook.

•

Statement Signed by the Study Director: Confirm that the Validation Study was conducted incompliance with GLP (or indicating where the Study deviated from GLP), or for non GLPcompliant laboratories, confirm that the Validation Study adhered to the spirit of GLP. Confirm
that the report fully and accurately reflects the raw data generated in the Validation Study.

••

Quality Assurance Statement: (For Final Report only)
For GLP-Compliant Laboratories: QA Statement identifying: 1) the phases and data inspected, 2)
dates of inspection, and 3) dates findings were reported to the Study Director and Testing Facility
management. The QA Statement shall identify whether the methods and results described in the
Final Report accurately reflect the raw data produced during the Validation Study.
For Non GLP-Compliant Laboratories: A statement from the Testing Facility shall be included
with the Final Report of Phase III. This statement shall identify whether the methods and results
described in the Final Report accurately reflect the raw data produced during the Validation Studyand provide assurance that all testing was done in the spirit of GLP.

••

Data Analysis: (for each assay) calculate the % viability for the positive control and each test
chemical concentration (eight concentrations per assay); determine the IC50 value for the positive
control; determine the IC20, IC50, and IC80 values (and confidence limits) for each of the three (3)test chemicals.

•

Other Information: (All copies of printouts, documents, and spreadsheets shall be noted asexact duplicates of the data.)• Copies of spectrometric plate reader raw data
• Copies of the completed Microsoft® Excel spreadsheets (Addendum II) used for calculationof cytotoxicity values
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•
•
•
•

November 2006

Copies of data pages showing IC50 calculations for the positive control and the IC20, IC50, andIC80 values (and confidence limits) for each test chemical
Copy of the protocols
Deviations to the protocols, SOPs, and Statement of Work
Revisions/amendments to the protocols, SOPs, and Statement of WorkG-43

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ADDENDUM I (cont.)
SUGGESTED REPORT FORMAT
DRAFT/FINAL REPORT 3
In Vitro Validation Study – Phase II: Qualification Phase for Cytotoxicity Study of Nine Coded
Chemicals in Rodent and Human Cell Systems•

Table of Contents•

Objectives: The reports shall provide specific objectives•

Description of the Test System Used: Description of 3T3 NRU assay and the NHK NRU assay•

Summary of the Findings: Referenced to the raw data where appropriate; Include all
information for range finding experiments•

Narrative Description of the Assays: Describe any problems that were encountered and how
such problems were solved. Justifications for solvents used for each test chemical will be
included in the description. Deviations from the protocols, SOPs, and/or the Statement of Workshall be addressed in this section. Copies of appropriate sections of the Study Workbook shall be
included with the report as attachments. The draft report will include unaudited Study Workbook
pages. The final report will include a copy of the audited Study Workbook with a statement(signed and dated by the Study Director) on the front of it stating that it is an exact copy of theoriginal audited workbook.

•

Statement Signed by the Study Director: Confirm that the Validation Study was conducted incompliance with GLP (or indicating where the Study deviated from GLP), or for non GLPcompliant laboratories, confirm that the Validation Study adhered to the spirit of GLP. Confirm
that the report fully and accurately reflects the raw data generated in the Validation Study.

••

Quality Assurance Statement: (For Final Report only)
For GLP-Compliant Laboratories: QA Statement identifying: 1) the phases and data inspected, 2)
dates of inspection, and 3) dates findings were reported to the Study Director and Testing Facility
management. The QA Statement shall identify whether the methods and results described in the
Final Report accurately reflect the raw data produced during the Validation Study.
For Non GLP-Compliant Laboratories: A statement from the Testing Facility shall be included
with the Final Report of Phase III. This statement shall identify whether the methods and results
described in the Final Report accurately reflect the raw data produced during the Validation Studyand provide assurance that all testing was done in the spirit of GLP.

••

Data Analysis: (for each assay) calculate the % viability for the positive control and each test
chemical concentration (eight concentrations per assay); determine the IC50 value for the positive
control; determine the IC20, IC50, and IC80 values (and confidence limits) for each of the nine (9)test chemicals.

•

Other Information: (All copies of printouts, documents, and spreadsheets shall be noted asexact duplicates of the data.)• Copies of spectrometric plate reader raw data
• Copies of the completed Microsoft® Excel spreadsheets (Addendum II) used for calculationof cytotoxicity values
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•
•
•
•

November 2006

Copies of data pages showing IC50 calculations for the positive control and the IC20, IC50, andIC80 values (and confidence limits) for each test chemical
Copy of the protocols
Deviations to the protocols, SOPs, and Statement of Work
Revisions/amendments to the protocols, SOPs, and Statement of Work

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ADDENDUM I (cont.)
SUGGESTED REPORT FORMAT•

DRAFT/FINAL REPORT 4
In Vitro Validation Study – Phase III: Cytotoxicity Study of 60Coded Chemicals in Rodent andHuman Cell Systems
Table of Contents•

Objectives: The draft report shall provide specific objectives•

Description of the Test System Used: Description of 3T3 NRU assay and the NHK NRU assay•

Summary of the Findings: Referenced to the raw data where appropriate; Include all
information for range finding experiments•

Narrative Description of the Assays: Describe any problems that were encountered and how
such problems were solved. Justifications for solvents used for each test chemical shall be
included in the description. Deviations from the protocols, SOPs, and/or the Statement of Workshall be addressed in this section. Copies of appropriate sections of the Study Workbook shall be
included with the report as attachments. The draft report will include unaudited Study Workbook
pages. The final report will include a copy of the audited Study Workbook with a statement(signed and dated by the Study Director) on the front of it stating that it is an exact copy of theoriginal audited workbook.

•

Statement Signed by the Study Director: Confirm that the Validation Study was conducted incompliance with GLP (or indicating where the Study deviated from GLP), or for non GLPcompliant laboratories, confirm that the Validation Study adhered to the spirit of GLP. Confirm thatthe report fully and accurately reflects the raw data generated in the Validation Study.

••

Quality Assurance Statement: (For Final Report only)
For GLP-Compliant Laboratories: QA Statement identifying: 1) the phases and data inspected, 2)
dates of inspection, and 3) dates findings were reported to the Study Director and Testing Facility
management. The QA Statement shall identify whether the methods and results described in the
Final Report accurately reflect the raw data produced during the Validation Study.
For Non GLP-Compliant Laboratories: A statement from the Testing Facility shall be included
with the Final Report of Phase III. This statement shall identify whether the methods and results
described in the Final Report accurately reflect the raw data produced during the Validation Studyand provide assurance that all testing was done in the spirit of GLP.

•

•

•

Data Analysis: (for each assay) calculate the % viability for the positive control and each test
chemical concentration (eight concentrations per assay); determine the IC50 value for the positive
control; determine the IC20, IC50, and IC80 values (and confidence limits) for each of the 60 (or 30)
test chemicals.

•

Other Information: (All copies of printouts, documents, and spreadsheets shall be noted asexact duplicates of the data.)• Copies of spectrometric plate reader raw data
• Copies of the completed Microsoft® Excel spreadsheets (Addendum II) used for calculationof cytotoxicity values
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•
•
•
•
•

November 2006

Copies of data pages showing IC50 calculations for the positive control and the IC20, IC50, andIC80 values (and confidence limits) for each test chemical
Deviations to the protocols, SOPs, and Statement of Work
Copy of the protocols
A list of all SOPs used by the laboratory for the assays (SOP title and laboratory
identification code)
The Statement of Work and The Test Method Protocols

G-47

In Vitro Cytotoxicity Test Methods BRD Appendix G1

November 2006

ADDENDUM I (cont.)
SUGGESTED REPORT FORMATBIWEEKLY REPORTS

Testing Facility:
Chemicals Received:
Chemicals Tested:
3T3 NRU Assay:
NHK NRU Assay:
Solubility Determinations: (solvents used and concentrations obtained)
Range Finding Experiments: (number performed; outcomes)
Successful Tests: (number of tests and calculated IC20, IC50, and IC80 values; include Excel®
spreadsheets)
Failed Tests: (number of failed tests and reasons for failure)
Problems Encountered/Resolutions:
Projected Testing Schedule:

G-48

In Vitro Cytotoxicity Test Methods BRD Appendix G1

November 2006

ADDENDUM II
EXCEL SPREADSHEET TEMPLATE FOR ASSAY DATA
Test Facility

dfdgs

Chemical Code

4567

Plate ID
Date Read

Cell Line/Type

3T3

Vehicle Contol

0.5% DMSO

qa789######
Plate Map
1

2

3

4

5

6

7

8

9

A

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

B

Blank

VC 1

Conc 1

Conc 2Conc 3

Conc 4Conc 5

Conc 6

Conc 7

Conc 8

VC2

Blank

C
D

Blank
Blank

VC 1
VC 1

Conc 1
Conc 1

Conc 2Conc 2Conc 3
Conc 3

Conc 4Conc 4Conc 5
Conc 5

Conc 6
Conc 6

Conc 7
Conc 7

Conc 8Conc 8

VC2
VC2

Blank
Blank

E

Blank

VC 1

Conc 1

Conc 2Conc 3

Conc 4Conc 5

Conc 6

Conc 7

Conc 8

VC2

Blank

F
G

Blank
Blank

VC 1
VC 1

Conc 1
Conc 1

Conc 2Conc 2Conc 3
Conc 3

Conc 4Conc 4Conc 5
Conc 5

Conc 6
Conc 6

Conc 7
Conc 7

Conc 8Conc 8

VC2
VC2

Blank
Blank

H

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Blank

Plate Data
1

10

11

12

2

3

4

5

6

7

8

9

10

A

0.004

0.006

0.036

0.004

0.028

0.019

0.023

0.029

0.012

0.003

0.004

0.011

B

0.009

0.832

0.832

0.855

0.780

0.755

0.693

0.419

0.265

0.052

0.832

0.008

C
D

0.014
-0.006

0.894
0.918

0.894
0.918

0.916
0.87

0.884
0.914

0.83
0.835

0.73
0.806

0.368
0.450

0.213
0.270

0.105
0.098

0.935
0.918

0.012
0.009

E
F

-0.004-0.004

0.915
1.098

0.915
1.098

0.826
0.984

0.903
0.814

0.879
0.952

0.73
0.746

0.591
0.436

0.295
0.201

0.086
0.151

0.915
1.098

0.015
0.014

G

0.016

0.948

0.948

0.845

0.842

0.832

0.663

0.431

0.319

0.09

0.89

0.015

H

-0.001-0.0060.017-0.005

0.009

0.004

0.002

0.014

-0.013-0.003-0.061

0.012

5

6

7

8

9

10

11

12

Mean blank OD

11

12

0.0068Corrected OD = OD- mean blank OD
1
2
3
4

A
B
C

0.825
0.887

0.825
0.887

0.848
0.909

0.773
0.877

0.748
0.823

0.686
0.723

0.412
0.361

0.258
0.206

0.045
0.098

0.825
0.928

D

0.911

0.911

0.863

0.907

0.828

0.799

0.443

0.263

0.091

0.911

E
F

0.908
1.091

0.908
1.091

0.819
0.977

0.896
0.807

0.872
0.945

0.723
0.739

0.584
0.429

0.288
0.194

0.079
0.144

0.908
1.091

G

0.941

0.941

0.838

0.835

0.825

0.656

0.420

0.312

0.083

0.883

VehicleControl 1 Conc 1

Conc 2Conc 5 Conc 6

Conc 7H

blanksConcentration[µg/ml]Mean

OD

Corrected

SD of Mean OD
Corrected Mean -----% Viability = Mean
Corrected OD/Mean
Corrected VC

Conc 3

Conc 4Conc 8

VehicleControl 2

0

1000

500

250

125

62.5

31.25

15.625

7.2

0

0.927

0.927

0.876

0.849

0.840

0.721

0.442

0.254

0.090

0.925

0.0158All VCs

0.089
0.926

0.089

0.058

0.053

0.065

0.049

0.075

0.046

0.032

0.089

100%

100%

100%

95%

92%

91%

78%

48%

27%

10%

100%

10%

10%

6%

6%

7%

5%

8%

5%

3%

10%

9.6%

9.6%

6.6%

6.3%

7.7%

6.8%

17.0%

18.1%

35.7%

9.7%

SD (% Viability) = SD
OD/Mean OD All VCs%CV = SD/mean OD*100
Mean Vehicle Control - VC1 (%)
Mean Vehicle Control - VC2 (%)

9%-0.15%
0.15%

Concentration-response
125%% Viability

100%

75%

50%

25%

0%
1

10

100
Concentration

G-49(ug/ml)1000

blanksIn Vitro Cytotoxicity Test Methods BRD Appendix G1

November 2006

ADDENDUM III
SUGGESTED STANDARD TEST REPORTING TEMPLATE FOR IN VITRO VALIDATIONSTUDY WORKBOOKTEST CHEMICAL
Test Facility

96-Well Plate ID _______________

Chemical CodeExperiment ID ________________

PREPARATION OF TEST CHEMICAL
Solvent_____Culture Medium_____DMSOHighest Percent Solvent (v/v) in Dilutions _______%
Aids Used to Dissolve_____Vortex

pH (Highest Test Concentration)________________Ethanol

Highest Concentration Tested_______µg/ml_____Ultra-sonicaton_____Heat to 37oC

Media color of test chemical solutions:Concentration Series (µg/ml)

C1

C2

C3

C4

C5

C6

Positive Control [SLS]_________µg/ml

Vehicle Control __________% solvent

C7

C8

CELL LINE/TYPE
NameSupplier

From Cell Lot No.________

Total Passage No.

No. of Passages after Thawing

From:____ proliferating ____frozen

CELL CULTURE CONDITIONSName of Medium

Supplier/ID

Lot No./Lab I.D.

Name of Serum

Supplier/ID

Lot No.

Serum Concentration

During Growth: _________%

During Exposure: __________%TEST ACCEPTANCE CRITERIA
VC: Mean Absolute OD540

Mean OD =__________Accept____Reject

VC: Difference Between Col.2 and Col. 10

Difference =_____%____Accept____Reject

PC: IC50 of Concurrent SLS Test

IC50 =________µg/ml____Accept____Reject

TIMELINE
Assay Start Date (cells to plates)

Application of Test Chemical Date

G-50

NRU/OD540 Measurement Date

In Vitro Cytotoxicity Test Methods BRD Appendix G1

November 2006

ADDENDUM IV
EXAMPLES OF LABORATORY EQUIPMENT LOGSINCUBATOR
INCUBATOR I.D.________________________MONTH:___________
DATE

TIME

YEAR:___________INITIALS

CO2 %

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
FYRITE CHECK OF CO2:
ADDITION OF WATER:TOTAL INCUBATOR DISINFECTION:

G-51

LOCATION:________________
RH %

TEMP.
(OC.)

CO2 TANK(PSI)

CO2 TANK(NEW)In Vitro Cytotoxicity Test Methods BRD Appendix G1

November 2006

ADDENDUM IV (cont.)
EXAMPLES OF LABORATORY EQUIPMENT LOGS
pH METER
pH METER I.D.________________________MONTH:___________
DATE

TIME

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
pH STANDARDSSUPPLIER/I.D.
LOT NUMBER
EXPIRATION DATENOTES:

YEAR:___________

LOCATION:________________INITIALS

pH STD.
7.00

pH STD.
10.00

pH STD.
4.00

7.00

10.00

4.00

7.40

G-52

pH STD.
7.40

SLOPE

In Vitro Cytotoxicity Test Methods BRD Appendix G1

November 2006

ADDENDUM IV (cont.)
EXAMPLES OF LABORATORY EQUIPMENT LOGSMONTH________________
YEAR__________________
DATE

TIME

INITIALSRERIGERATORFREEZER

I.D. NUMBER______________
LOCATION________________

I.D. NUMBER______________
LOCATION________________TEMPERATURE (OC.)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
NOTES:G-53

TEMPERATURE (OC.)

In Vitro Cytotoxicity Test Methods BRD Appendix G1

November 2006

ADDENDUM V
SUGGESTED STANDARD TEST REPORTING TEMPLATE FOR STUDY WORKBOOK
1SOLUBILITY TESTING
Test Chemicals for the In Vitro Validation Study
Study No.___________________
Test Chemical_________________#____________

Test Chemical Code__________

CAS

Physical Description_______________________________________ Liquid Density_________
Solubility Determined by__________________________
Date______________
Solvent

Amountof TestChemicalVolume
Added

Total
VolumepH andmediumcolor

Vortex (V)Sonication (S)Heating-37oC (H)

0.1ml
Treatment
Medium(3T3 NRU)

0.5ml
1.0ml
0.1ml

RoutineCultureMedium(NHK NRU)

0.5ml
1.0ml
0.1ml

DMSO

0.1ml
Ethanol

Reference Color of Treatment Medium________________________
Reference Color of Routine Culture Medium____________________
Balance I.D.______________
Treatment Medium and Routine Culture Medium: minimum concentration of 100mg/ml.
DMSO and Ethanol: minimum concentration of 1000mg/ml.
1

Adaptation of Institute of In Vitro Sciences (IIVS) form – 350 [2/2002]
G-54

Comments

In Vitro Cytotoxicity Test Methods BRD Appendix G1

November 2006

ADDENDUM VI
GANTT CHART OF STUDY TIMELINES AND DELIVERABLES

1
2

July 29
Aug. 26

Sept. 9Nov. 11
Sept. 26
Oct. 29
Nov. 11
Jan. 13

2/10/03

Dec. 2Feb. 10
Feb. 25

4/28/03

April 28

12/9/03
10/24/03

Mar. 26
Dec. 9Oct. 24

12/9/03

Dec. 912/9/03

July 10, 2002 – December 9, 2003

G-55

DECEMBER 2003

2
9

NOVEMBER 2003
OCTOBER 2003

10

SEPTEMBER 2003

AUGUST 2003

JULY 2003

JUNE 2003

29

2/25/03

3/26/03

MAY 2003

12/2/02

APRIL 2003

1111/02
1/13/03

MARCH 2003

10/29/02

FEBRUARY 2003

9/26/02

JANUARY 2003

11/11/02

DECEMBER 2002

9/9/02

NOVEMBER 2002

8/26/02

OCTOBER 2002

7/29/02

SEPTEMBER 2002

7/12/02

AUGUST 2002

6/29/02

JULY 2002

7/10/02

5/10/02

JUNE 2002

Biweekly
Reports

FINISH
3/29/02

MAY 2002

START

APRIL 2002

TASK
Statement ofWork Issued
by NIEHS
Proposalreceived
ContractsAwardedSubmissionof Study
Protocol,CVs of Key
Personnel,
and SOPs
Phase Ia
Positive
control
Phase Ia
Draft Report
Phase Ia
Final Report
Phase Ib
3 chemicals
Phase Ib
Draft Report
Phase Ib
Final Report
Phase II
9 chemicals
Phase II
Draft Report
Phase II
Final Report
Phase III60 chemicals
Phase III
Draft Report
Phase III
Final Report

MARCH 2002

In Vitro Cytotoxicity
Validation StudyIn Vitro Cytotoxicity Test Methods BRD Appendix G1[This Page Intentionally Left Blank]

G-56

November 2006

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

Appendix G2Procedures for Acquisition, Preparation, Solubility Testing,and Distribution of Test Chemicals for a Validation Study
for In Vitro Basal Cytotoxicity Testing

G-57

In Vitro Cytotoxicity Test Methods BRD Appendix G2[This Page Intentionally Left Blank]

G-58

November 2006

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

NOTE: This Statement of Work shall not be cited, quoted, nor distributed to any
Testing Facility participating in the In Vitro Validation Study. Confidentiality
must be maintained to ensure that test chemicals remain unknown to the Testing
Facilities.

STATEMENT OF WORK
Procedures for Acquisition, Preparation, Solubility Testing, andDistribution of Test Chemicals for a Validation Study for In Vitro Basal
Cytotoxicity Testing

April 26, 2002
Revision 1: May 8, 2002
Revision 2: June 21, 2002
Revision 3: September 17, 2002
Revision 4: October 11, 2002
Prepared by
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)

National Institute of Environmental Health Sciences (NIEHS)
National Institutes of Health (NIH)
U.S. Public Health Service
Department of Health and Human Services

G-59

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

Table of Contents
1.0 PROJECT OBJECTIVES AND GENERAL REQUIREMENTS....................................................... 62
1.1 Project Objectives ................................................................................................................................... 62
1.2 Response to the Statement of Work ...................................................................................................... 62
1.2.1 General Capabilities ..................................................................................................................... 62
1.3 Guidelines ............................................................................................................................................... 63
1.4 Definitions............................................................................................................................................... 63
2.0 ORGANIZATION.................................................................................................................................. 63
2.1 Validation Study Sponsors ..................................................................................................................... 63
2.2 Management Team ................................................................................................................................. 64
2.2.1 Project Management and Chemical Distribution Team ............................................................. 64
2.2.2 Contract Management .................................................................................................................. 64
2.2.3 Study Management Team ............................................................................................................ 64
2.2.3.1 NIEHS/NICEATM ......................................................................................................... 64
2.2.3.2 ECVAM........................................................................................................................... 64
2.2.4 Testing Facilities........................................................................................................................... 64
3.0 CONTRACTOR AND KEY PERSONNEL......................................................................................... 65
3.1 Contractor................................................................................................................................................ 65
3.1.1 Personnel ....................................................................................................................................... 65
3.1.1.1 Facility Management ...................................................................................................... 65
3.1.1.2 Study Director ................................................................................................................. 65
3.1.1.3 Quality Assurance (QA) Director .................................................................................. 65
3.1.1.4 Scientific Advisor(s) ....................................................................................................... 66
3.1.1.5 Laboratory Technician(s) ............................................................................................... 66
3.1.1.6 Safety Officer .................................................................................................................. 66
3.1.2 Facilities, Equipment, and Supplies ............................................................................................ 66
3.1.2.1 Laboratory ....................................................................................................................... 66
3.1.2.2 Equipment ....................................................................................................................... 66
3.1.2.3 Supplies ........................................................................................................................... 66
3.1.3 Health and Safety.......................................................................................................................... 67
3.1.4 Quality Assurance ........................................................................................................................ 67
4.0 TEST PHASES AND SCHEDULE ...................................................................................................... 67
4.1 Study Timeline........................................................................................................................................ 67
4.2 Deliverables ............................................................................................................................................ 68
4.3 In Vitro Validation Study Phases........................................................................................................... 69
4.4 Report Submission Timelines ................................................................................................................ 69
4.4.1 Draft Reports................................................................................................................................. 69
4.4.2 Final Report .................................................................................................................................. 69
5.0 ACQUISITION, PREPARATION, AND DISTRIBUTION OF TEST CHEMICALS .................... 69
5.1 Test Chemicals........................................................................................................................................ 69
5.1.1 Range of Toxicities....................................................................................................................... 69
5.1.2 Procurement of Test Chemicals................................................................................................... 70
5.1.3 Dispensing Chemicals .................................................................................................................. 70
5.1.4 Shipment of Chemicals ................................................................................................................ 70
5.1.5 Receipt of Chemicals by the Testing Facilities .......................................................................... 71
5.1.6 Test Chemical Information for the Study Director..................................................................... 71
5.2 Handling of Test Chemicals................................................................................................................... 71
5.3 Determination of Purity, Composition, and Stability of Test Chemicals ........................................... 71
6.0 SOLUBILITY DETERMINATION OF TEST CHEMICALS ........................................................... 71
6.1 Cell Culture Media and Control Material ............................................................................................. 72
6.1.1 Test Chemical Medium Solvents................................................................................................. 72
G-60

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

6.1.1.1 Treatment Chemical Dilution3 Medium (BALB/c 3T3 NRU)..................................... 72
6.1.1.2 Routine Culture Medium (NHK NRU) ......................................................................... 72
6.1.2 Positive Control (PC) ................................................................................................................... 73
6.2 Preparation of Test Chemical................................................................................................................. 73
6.2.1 Dissolving the Test Chemical ...................................................................................................... 73
6.2.1.1 Treatment Chemical Dilution3 Medium/Routine Culture Medium) ............................ 75
6.2.1.2 DMSO .............................................................................................................................. 75
6.2.1.3 Ethanol ............................................................................................................................. 75
6.2.2 pH of Solutions ............................................................................................................................. 76
7.0 DATA COLLECTION ........................................................................................................................... 76
7.1 Nature of Data to be Collected............................................................................................................... 76
7.1.1 Solubility Studies.......................................................................................................................... 76
7.1.2 Chemical Information .................................................................................................................. 76
7.2 Type of Media Used for Data Storage................................................................................................... 76
7.3 Documentation ........................................................................................................................................ 77
8.0 DRAFT AND FINAL REPORTS ......................................................................................................... 77
9.0 RECORDS AND ARCHIVES............................................................................................................... 77
10.0 ALTERATIONS OF THE STATEMENT OF WORK........................................................................ 77
11.0 REFERENCES........................................................................................................................................ 77
12.0 APPROVAL OF STATEMENT OF WORK ....................................................................................... 78
ADDENDUM I ................................................................................................................................................. 79
ADDENDUM II ................................................................................................................................................ 83
ADDENDUM III .............................................................................................................................................. 84
ADDENDUM IV ............................................................................................................................................869
ADDENDUM V............................................................................................................................................8891

NOTE: Revisions in this document are identified by footnotes, strike-out text (i.e.,deleted), and added verbiage (i.e., italicized text).

3

Revised 9/17/02

G-61

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

STATEMENT OF WORK
Procedures for Acquisition, Preparation, Solubility Testing, andDistribution of Test Chemicals for a Validation Study for In Vitro Basal
Cytotoxicity Testing
1.0

PROJECT OBJECTIVES AND GENERAL REQUIREMENTS
1.1 Project Objectives
This Statement of Work outlines and supports the procedures that the Contractor will initiate
for the acquisition, preparation, solubility testing, and distribution of the test chemicals needed
to perform two in vitro basal cytotoxicity assays (the BALB/c 3T3 Neutral Red Uptake [NRU]
assay and the Normal Human Keratinocyte [NHK] Neutral Red Uptake [NRU] assay) for a
multi-laboratory Validation Study. These assays, recommended in Guidance Document On
Using In Vitro Data To Estimate In Vivo Starting Doses For Acute Toxicity (ICCVAM, 2001),
use mammalian cell culture techniques to assess the basal cytotoxicity of chemicals.
A primary goal of this Validation Study is to evaluate the usefulness of the BALB/c 3T3
Neutral Red Uptake (NRU) and the Normal Human Keratinocyte (NHK) NRU assays for
reducing and refining animal use for acute oral toxicity determinations of chemicals by
predicting starting doses for in vivo rodent acute lethality assays.
The proposed Validation Study will determine IC20, IC50, and IC80 values for a test set of 72
chemicals with varying degrees of toxicity. This set of chemicals was selected separate andprior to this Statement of Work by the Study Management Team. The basis for selection of thistest set is discussed in the Study Design document prepared by the Study Management Team.
The Contractor shall perform the following activities:− Acquire 73 high quality and high purity (99% or greater when economically feasible)
chemicals from reputable commercial sources− Perform solubility tests on all chemicals using solvents and procedures that have been
recommended to the test laboratories− Repackage chemicals into multiple smaller units− Code chemicals with a unique identification number so that chemicals can be provided totesting laboratories in a blinded fashion− Distribute chemicals and health and safety information to the Testing Facilities− Provide draft and final reports of these activities.
1.2 Response to the Statement of Work
Proposals submitted in response to this Statement of Work shall include:a) A Work Planb) A timetable for project milestonesc) A cost estimate based on chemical acquisition, performance of solubility tests for all test
chemicals, chemical coding, repackaging, and distribution to two U. S labs and one U. K.
lab.
1.2.1 General Capabilities
The Contractor shall be capable of performing the following:a) Prepare/provide Standard Operating Procedures (SOPs) for the performance of theactivities outlined in Section 1.1 (see Section 1.4 – Definitions - SOPs)
G-62

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006b) Perform all aspects of the Test Chemical Preparation in accordance with Good
Laboratory Practices (GLP).
c) Adhere to this Statement of Work throughout the Validation Study.
1.3 Guidelines
The Project Officer and/or her/his representatives (e.g., Study Management Team) may inspect
and audit the Contractor to ensure that the Project Officer’s minimum requirements andguidelines are being followed.
1.4 Definitions
Blinded/Coded Chemicals: Test chemicals supplied to the Testing Facilities that are codedand distributed by the Contractor such that only the Project Officer, Management Team, and theContractor have knowledge of the contents of each test chemical vessel. The test chemicals
will be purchased, aliquoted, coded, and distributed by the Contractor under the guidance of the
NIEHS/NTP Project Officer and the Management Team.
Contractor: Facility that will initiate the acquisition, preparation, solubility testing, anddistribution of the test chemicals needed to perform two in vitro basal cytotoxicity assays for a
multi-laboratory in vitro Validation Study.
Good Laboratory Practices (GLPs): Regulations governing the conduct, procedures, andoperations of toxicology laboratories; regulations to assure the quality and integrity of the data
and to address such matters as organization and personnel, facilities, equipment, facility
operations, test chemicals, and study protocol (Statement of Work) and conduct (U.S. Food andDrug Administration, Title 21 CFR Part 58; Environmental Protection Agency, Title 40 CFRPart 160).
Standard Operating Procedures (SOPs): Written documents that describe, in great detail, theroutine procedures to be followed for a specific operation, analysis, or action; consistent use of
an approved SOP ensures conformance with organizational practices, reduced work effort,
reduction in error occurrences, and improved data comparability, credibility, and defensibility;
SOPs also serve as resources for training and for ready reference and documentation of proper
procedures;
Statement of Work: A description of test chemical preparation required for the in vitro
Validation Study; defines all phases of the Validation Study and the purpose of the procedures;provides the details of test chemical acquisition, preparation, solubility testing, and distribution;
provides guidance for the preparation of reports
Testing Facility: A laboratory that has been designated to participate in the In Vitro Validation
Study; facilities identified in Section 2.2.4.
2.0

ORGANIZATION
2.1
•
•
•
•

Validation Study Sponsors
National Institute of Environmental Health Sciences (NIEHS)
The National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)
U.S. Environmental Protection Agency (U.S. EPA)The European Centre for the Validation of Alternative Methods (ECVAM).

G-63

In Vitro Cytotoxicity Test Methods BRD Appendix G2

2.2

November 2006

Management Team2.2.1 Project Management and Chemical Distribution Team
Ms. Molly Vallant (NIEHS) – NIEHS Project Officer for BioReliance, Inc.
NIEHS
MD E1-03
P.O. BOX 12233
RTP, NC 27709
Dr. Martin L. Wenk (BioReliance, Inc.) – Chemical acquisition, preparation,solubility testing, and distribution
BioReliance Corporation
14920 Broschart Road
Rockville, Maryland 20850-3349
2.2.2 Contract Management
Ms. Jackie Osgood (NIEHS) – Contracting Officer
Mr. Don Gula (NIEHS) – Contracting Officer
2.2.3 Study Management Team2.2.3.1 NIEHS/NICEATM
Dr. William S. Stokes (NICEATM/NIEHS) – Co-chair – Study Management TeamDr. Judy Strickland (NICEATM/ILS) – Project Coordinator
Mr. Michael Paris (NICEATM/ILS) – Assistant Project Coordinator
Dr. Ray Tice (NICEATM/ILS) – Technical Advisor
NICEATM79 T.W. Alexander Drive
Bldg. 4401, MD-EC-17
3rd Floor, Room 3126
P.O. Box 12233
Research Triangle Park, NC 27709
2.2.3.2 ECVAM
Professor Michael Balls – Co-chair – Study Management TeamDr. Silvia Casati
Dr. Andrew WorthEuropean Commission
Joint Research Centre
Institute for Health and Consumer Protection
Management Support Unit - TP 202
I-21020 Ispra (VA) - Italy
2.2.4 Testing Facilities
XXX, Safety Officer
Institute for In Vitro Sciences (IIVS)
21 Firstfield Road
Suite 220
Gaithersburg, MD 20878
G-64

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

Bill Cappuccio, Safety Officer
5183 Blackhawk Rd
E3330/Room 278
Aberdeen Proving Ground-EA, MD 21010
410-436-7462
Rodger Dainty, Safety Officer
School of Biomedical Sciences
University of Nottingham Medical School
Queen's Medical Centre
Nottingham, NG7 2UH UK
3.0

CONTRACTOR AND KEY PERSONNEL
3.1 Contractor
The Contractor shall have competence in chemical acquisition, preparation, solubility testing,and distribution and shall provide competent personnel, adequate facilities, equipment,
supplies, proper health and safety guidelines, and satisfactory quality assurance procedures.
3.1.1 Personnel
3.1.1.1 Facility Management
The facility management is responsible for establishing scientific guidelines andprocedures, training and supervision of professional and technical staff, andevaluation of results and performance within their discipline area relative to theProject Officer’s stated requirements. The manager must maintain records of thequalifications, training and experience, and a job description for each professional
and technical individual involved in test chemical acquisition, preparation,solubility testing, and distribution.
3.1.1.2 Study Director
A scientist or other professional of appropriate education, training, and experiencein chemical acquisition, preparation, solubility testing, and distribution, or
combination thereof, shall be the Study Director. The Study Director has theoverall responsibility for the technical conduct of chemical acquisition, preparation,solubility testing, and distribution for the Validation Study (e.g., GLP adherence)and shall be responsible for determining test acceptance. The Study Director shallbe responsible for providing SOPs that incorporate pertinent information obtained
from the Statement of Work. Other duties include the interpretation and analysis oftest chemical solubility data, documentation of all study aspects (including
maintenance of a Study Workbook), and production of all draft and final written
reports.
3.1.1.3 Quality Assurance (QA) Director
The Quality Assurance Director shall monitor all tasks and assure conformancewith GLP requirements (i.e., facilities, equipment, personnel, methods, practices,
records, controls, transference of data into software, SOPs). Quality Assurance
Director or unit can be any person or organizational element, except the Study
Director, designated by Contractor management to perform the duties relating toquality assurance of the studies and tasks. The Quality Assurance duties are not a
substitute for the Study Director duties.
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3.1.1.4 Scientific Advisor(s)Scientists or other professionals of appropriate education, training, and experiencein chemical acquisition, preparation, solubility testing, and distribution who
provide scientific guidance to the Study Director and other laboratory personnel.
3.1.1.5 Laboratory Technician(s)Each individual engaged in the conduct of or responsible for the supervision of astudy shall have education, training, and experience, or combination thereof, toenable that individual to perform the assigned duties. The individuals must be
trained in GLP requirements and technical ability must be documented as per GLP
requirements.
3.1.1.6 Safety Officer
The Contractor shall designate a Safety Officer who will provide a sealed healthand safety information package that will accompany the test chemicals to the Test
Facilities. A duplicate package will be provided to the Project Officer andManagement Team.
3.1.2 Facilities, Equipment, and Supplies
3.1.2.1 Laboratory
The Contractor must provide a designated laboratory/area to ensure that test
chemical preparation and solubility testing can be performed under clean
conditions. Potential for cross-contamination of chemicals should be minimal.
3.1.2.2 Equipment
The Contractor must provide at a minimum the following equipment:a) Water bath (37oC)b) Sonication unitc) Vortex unitd) Pippettors (micropipettors,)e) Computer (for data transformation and analysis)f) Balanceg) pH meterAll equipment maintenance and calibration shall be routinely performed anddocumented as per GLP guidelines and Contractor procedures
3.1.2.3 Supplies
All cell culture reagents must be labeled so as to indicate source, identity,
concentration, stability, preparation and expiration dates, and storage conditions.
a) Dulbecco’s Modification of Eagle’s Medium (DMEM) without L-Glutamine;should have Hanks’ salts and high glucose [4.5gm/l] (e.g., ICN-Flow Cat. No.
12-332-54)b) L-Glutamine 200 mM (e.g., ICN-Flow # 16-801-49)c) New Born Calf Serum (NBCS) (e.g., Biochrom # SO 125)d) Dimethyl sulfoxide (DMSO), U.S.P. analytical grade. DMSO shall be stored
under nitrogen at –20oC.
e) Ethanol (ETOH), U.S.P. analytical grade (100%, non-denatured)

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Keratinocyte Basal Medium without Ca++ (KBM®, Clonetics CC-3104) that iscompleted by adding the KBM® SingleQuots® Bullet Kit®2 (Clonetics CC4131) to achieve the proper concentrations of epidermal growth factor, insulin,hydrocortisone, antimicrobial agents, bovine pituitary extract, and calcium (e.g.,
Clonetics Calcium SingleQuots®, CC-4202)*.
g) Penicillin/streptomycin solution (e.g. ICN-Flow # 16-700-49)
f)* BioWhittaker, 8830 Biggs Ford Road, Walkersville, MD 21793(http://www.cambrex.com/subsidiaries/s%2Dbw%5Finc/s%2Dbiowhittaker%2Dinc%2Dcontact2.htm)
3.1.3 Health and Safety
The Contractor shall conform to all local, state, and federal statutes in effect at the time ofthis study.
3.1.4 Quality Assurance
The Contractor shall conduct the acquisition, preparation, solubility testing, anddistribution of test chemicals in compliance with Good Laboratory Practice (GLP)Standards (U.S. Food and Drug Administration, Title 21 CFR Part 58; Environmental
Protection Agency, Title 40 CFR Part 160). The appropriate QA unit (as per GLPs) shallaudit the procedures and final report.
The Final Report shall be audited by the Quality Assurance unit of the Contractor for GLP
compliance and a QA Statement shall be provided by the Contractor. The Final Reportshall identify: 1) the phases and data inspected, 2) dates of inspection, and 3) dates
findings were reported to the Study Director and Contractor management. The QA
Statement shall identify whether the methods and results described in the Final Reportaccurately reflect the raw data produced during the study.
4.0

TEST PHASES AND SCHEDULE
4.1 Study Timeline
The following timeline is for the laboratory testing aspect of the In Vitro Validation Study.
The Contractor shall provide the required chemicals in a timely fashion so that each phase of
the study can start on the appointed date.

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TASKStatement of Work issued by NIEHS
to the Testing Facility
Response /Proposal received fromthe Testing FacilityAward of Contracts2
Submission of Study Protocol, CVs of
Key Personnel, SOPs2
Award of Contracts2
Start Testing – Phase I (Phase Ia)
End Phase Ia
Begin Phase Ib
End Phase Ib
Begin Phase II
End Phase II
Begin Phase III
Final Report (Phase III) to SMT

November 2006

WEEK
0

ESTIMATED DATEMarch 29, 2002

6

May 10, 2002

92
11

May 29, 20022
June 12, 2002

132
14182
18222
22262
27312
31362
42462
48522
85892

June 28, 20022
July 1292, 2002
July August 2692, 2002
August September 29262, 2002
October 1292, 2002
October December 292, 2002
January February 13102, 2003
February March2 26, 2003
November December 1192, 2003

4.2 Deliverables
The following schedule of deliverables is for the acquisition, preparation, solubility testingand distribution of test chemicals.
Submission of SOPs
for Section 1.1
activities
REPORTS
Biweekly Reports
Draft Phase Reports

ESTIMATED DUE DATES (to Project Officer)
Week 11
June 12, 2002
PHASE Ia
PHASE Ibaa
Week 1317
June July 2462, 2002 b

Draft Final Report(all phasescombined)
Final Report(all phasescombined)
a
b
c
d

2

PHASE IIa
Week 2933
OctNov. 16132,
2002 bPHASE IIIa
Week 4448
JanFeb. 29262,
2003 b

Week 4852
MarchFeb.2 26, 2003 c
Week 5054
March April 9122, 2003 d

Biweekly reports shall begin at the time of implementation of the contracts and continue
until the final report is submitted.
Draft Phase Reports shall be submitted to the Project Officer no later than the dates
provided (at least two weeks before shipment of chemicals to the Test Facilities).
Draft Final Report shall be submitted to the Project Officer no later than the date provided(at the most one month after final shipment of chemicals to the Test Facilities).
Final Report shall be submitted to the Project Officer no later than the date provided (at themost one month after the Project Officer receives the Draft Final Report.

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The following schedule is for the distribution of test chemicals to the Testing Facilities.
CHEMICALSHIPPING TOTESTING
FACILITIESa
Positive Control(SLS)
Phase Ib
(3 chemicals)a

ESTIMATED DUE DATES (to Testing Facilities)
PHASE Ia
PHASE Ib
PHASE II
PHASE III

BeforeJuly 1292, 2002
---

---

---

---

---

---

BeforeOctober
December 292,
2002
---

---

Phase II
(9 chemicals)---

BeforeAugustSeptember
29262, 2002---

Phase III
(60 chemicals)

---

---

BeforeFebruary March2
26, 2003

Dates for chemical shipments are to ensure that the Testing Facilities receive Test
Chemicals prior to the start dates of each lab testing phase. Phase III chemicals shall beshipped as one group of 60 chemicals. Chemicals for each phase are identified in
Addendum IV.

4.3 In Vitro Validation Study Phases
Phase I: The training phase for laboratory personnel. This phase includes developing a
positive control database (Phase Ia) and testing three unknown chemicals (Phase Ib).
Phase II: The qualification phase. This phase requires testing nine blinded/coded chemicals inthe same in vitro cytotoxicity assays and in the same concentration-response fashion as in Phase
Ib.
Phase III: Testing 60 blinded/coded chemicals in the same manner as in Phases I and II.
4.4

Report Submission Timelines
4.4.1 Draft Reports
Draft reports for each phase shall be submitted to the Project Officer as per Section 4.2.
4.4.2 Final Report
The Final report shall be submitted to the Project Officer as per Section 4.2.

5.0

ACQUISITION, PREPARATION, AND DISTRIBUTION OF TEST CHEMICALS
5.1

Test Chemicals
5.1.1 Range of Toxicities
The chemicals proposed for the Validation Study are representative of a range of
toxicities and are relevant with regard to human exposure potential. The test chemicals

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will represent each of the Globally Harmonized System (GHS) classification groups for
rat oral LD50s: ≤ 5 mg/kg, >5 ≤ 50 mg/kg, >50 ≤ 300 mg/kg, >300 ≤ 2000 mg/kg, >2000≤ 5000 mg/kg, and >5000 mg/kg (OECD, 2001). Addenda III and IV provide the list of
test chemicals for the In Vitro Validation Study.
5.1.2 Procurement of Test Chemicals
The Contractor shall purchase 73 chemicals specified in Addenda III and IV (72 “testchemicals” and one “positive control”) from commercial manufacturers. Chemical purity
shall be 99% or greater when economically feasible. Chemical information from the
manufacturers shall be collected as specified in Section 7.1.2 and reported as indicated in
Addendum I. Chemicals shall be stored as recommended by the manufacturer.
5.1.3 Dispensing Chemicals
While preparing the purchased chemicals for distribution to the Testing Facilities, only
one bulk substance shall be dispensed at any time. All test samples shall be sealed andlabeled before dispensing the next substance. Once test samples have been dispensed
into aliquots, they shall be returned to appropriate storage conditions until they aredispatched.
During dispensing, all test chemicals, with the exception of the positive control, will berandomly blinded/coded so that testing by the Testing Facilities will be conducted on
chemicals with a masked identity. Each chemical shall have a code that is unique foreach Testing Facility (i.e., no chemical shall have the same code in any Testing Facility).
The Contractor shall dispense 4 g of test chemical/Testing Facility (see Addendum V forassumptions used to determine the amount of chemical/Testing Facility) into clean, sterile
containers, and assign unique code identifiers, and archive two additional samples.
About 100 g of the positive control shall be distributed to each lab and one additional
sample shall be archived.
5.1.4 Shipment of Chemicals
After dispensing and labeling chemical aliquots with unique codes, the Contractor shallship a set of the test chemicals, including the positive control, to the each of three Testing
Facilities. Two Facilities will be in the US and one will be in the United Kingdom. The
Contractor will package test chemicals so as to minimize damage during transit and willship them to each Testing Facility according to proper regulatory procedures. Except forthe positive control in Phase Ia, chemicals are to be packaged and shipped so as toconceal their identities. Test chemicals shall be shipped under conditions that will
preserve the integrity of the chemicals. The Contractor shall notify the Testing Facilities(and the Project Officer) when the test chemicals are shipped so as to prepare for receipt.
The Contractor will retain the archived chemicals, which may be required for retesting or
purity analysis, until the completion of the Validation Study.
5.1.4.1 Distribution Phases
Phase Ia: For Phase I, the positive control chemical identified in Addendum III
shall be distributed to all three Testing Facilities.
Phase Ia: For Phase Ib, the three (3) blinded/coded chemicals identified in
Addendum III shall be distributed to all three Testing Facilities.
Phase II: Nine (9) blinded/coded chemicals identified in Addendum III shallbe distributed to all three Testing Facilities.

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Phase III: Sixty (60) blinded/coded chemicals identified in Addendum III shallbe distributed to the Test Facilities. Chemicals will be shipped –as a group of
60 chemicals.
5.1.5 Receipt of Chemicals by the Testing Facilities
With the exception of the positive control shipment, which shall be shipped directly to
the Study Director, the chemical shipments shall be addressed to the Testing Facility
Safety Officers and accompanied by a sealed information packet containing theappropriate health and safety procedures for use (i.e., Material Safety Data Sheets(MSDS) or equivalent documentation with proper protection, procedures for accidental
ingestion or contact with skin or eyes, and procedures for containing and recovering
spills) and a disclosure key for identifying test chemicals by code. The shipment shall
include instructions for the Testing Facility Safety Officer to:1) Immediately notify the Contractor and Study Project Coordinator upon receipt ofchemicals,2) Retain the health and safety package and pass the test chemicals to the Study Director
without revealing the identities of the test chemicals,3) Notify the Management Team if Test Facility personnel open the health and safety
packet at any time during the Validation Study, and
4) Return the unopened health and safety package to the Contractor after testing iscomplete. The Contractor shall immediately notify the Project Officer regarding
chemical receipt.
If regulatory transportation requirements dictate that each package must display a list of
the chemicals it contains on the outside of the package, the Contractor shall direct theTesting Facility Safety Officer to remove it prior to passing the chemicals to the Study
Director.
5.1.6 Test Chemical Information for the Study Director
The Contractor shall supply, with each test chemical, data sheets giving a minimum ofessential information, including color, odor, physical state, weight or volume of sample,
specific density for liquid test chemicals, and storage instructions. The Study Director
shall receive this information from the Safety Officer.
5.2 Handling of Test Chemicals
Appropriate routine safety procedures shall be followed in handling the test chemicals. The
Contractor shall include instructions to the Test Facilities to treat all blinded/coded test
chemicals as very hazardous and potentially carcinogenic. After the studies are completed,
the remaining test chemicals will be returned by the Testing Facilities to the Contractor.
5.3 Determination of Purity, Composition, and Stability of Test Chemicals
As indicated in Section 7.1.2, the Contractor will be directly responsible for collecting
information (from manufacturer and supplier documentation) on the analytical purity,
composition, and stability of the test chemicals and the positive control material, and their
homogeneity (via Contractor solubility studies) in the vehicle.
6.0

SOLUBILITY DETERMINATION OF TEST CHEMICALSThe Contractor shall determine solubility of the test chemicals in the same manner as recommended
to the Testing Facilities (i.e., by following the hierarchy below).

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6.1

November 2006

Cell Culture Media and Control Material
6.1.1 Test Chemical Medium Solvents
6.1.1.1 Treatment Chemical Dilution3 Medium (BALB/c 3T3 NRU)
Serum-free3 Dulbecco’s Modification of Eagle’s Medium (DMEM) [see Section
3.1.2.3.a] buffered with sodium bicarbonate and supplemented with (final
concentrations in DMEM are quoted):
5%
NBCS3
4 mM
Glutamine
100 200 IU/mL3
Penicillin
100 200 µg/ml3
Streptomycin
This serum-free3 medium is used in the assay for application ofdissolving3 test
chemicals prior to application3 to the 3T3 cells.
6.1.1.2 Routine Culture Medium (NHK NRU)
KBM® (Clonetics CC-3104) supplemented with KBM® SingleQuots® (Clonetics
CC-4131) and Clonetics Calcium SingleQuots® (CC-4202) to make 500ml of
medium. Final concentration of supplements in medium are:A modified MCDB
153 formulation such as Clonetics® Keratinocyte Basal Medium (KBM®)
supplemented with (final concentrations in KBM® are quoted):2
0.0001 ng/ml2
Human recombinant epidermal growth factor
5 µg/ml2
Insulin
0.5 g/ml2
Hydrocortisone
50 30 µg/ml2
Gentamicin
2
50 15 ng/ml
Amphotericin B
0.10 mM
Calcium
2 ml 7.5 mg/ml30 µg/ml2
Bovine pituitary extract.
This medium is used in the assay as the routine culture medium and for applicationof test chemicals to the NHK cells. Complete media should be kept at 4°C andstored for no longer than two weeks.2
NOTE: KBM® SingleQuots® contain the following stock concentrations andvolumes:2
0.1 ng/ml
hEGF
0.5 ml2
5.0 mg/ml
Insulin
0.5 ml2
0.5 mg/ml
Hydrocortisone
0.5 ml2
30 mg/ml
Gentamicin, 15 ug/ml Amphotericin-B 0.5 ml2
7.5 mg/ml
Bovine Pituitary Extract (BPE)
2.0 ml2
Clonetics Calcium SingleQuots® are 2 ml of 300mM concentration of calcium. 2
165 ul of solution per 500 ml calcium-free medium equals 0.10 mM calcium in themedium.2

3
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6.1.2 Positive Control (PC)
Sodium Lauryl Sulfate ([SLS], CAS # 151-21-3) will be the positive control material for
the In Vitro Validation Study.
6.2 Preparation of Test Chemical
All chemicals (including the positive control [SLS]) shall be weighed on a calibrated balance(including liquid test chemicals) and added to the appropriate solvent (Section 6.2.1). Test
chemicals must be at room temperature before dissolving. Preparation under red light or
yellow light may be necessary, if rapid photodegradation is likely to occur. The solutions mustnot be cloudy nor have noticeable precipitate.
6.2.1 Dissolving the Test Chemical3
The hierarchy specified in Sections 6.2.1.1 to 6.2.1.3 (i.e., culture medium, DMSO,
ethanol) shall be followed for dissolving the test chemicals and positive control. Bothassay-specific culture media specified in Section 6.1.1 (i.e., Chemical Dilution Medium
for 3T3 cells and Routine Culture Medium for NHK cells) must be tested.
Approximately 100 mg (100,000 µg) of the test chemical will be weighed into a glass tube
and the weight will be documented. Assay-specific media will be added to the vessel sothat the concentration is 200,000 µg/ml (200 mg/mL) (i.e., approximately 0.5 mL). The
solution is mixed as specified in Section 6.2.1.1. If complete solubility is achieved, thenadditional solubility procedures are not needed. If only partial solubility is achieved,
follow the test chemical dissolving steps in Table 1, derived from EPA (1998), to add
additional medium in steps until the concentration is a minimum of 2,000 µg/mL (2mg/mL). If complete solubility at 2,000 µg/mL in medium can’t be attained, then repeat
the solubility steps using the other solvent(s) in the solubility hierarchy. Test chemicals
that are only soluble in DMSO or ethanol will be prepared at 500,000 µg/mL as thehighest concentration of stock solution.
Table 1: Determination of Solubility in Media
STEP

1

2

3

4

5

Total Volume of Medium
0.5 mL
2.5 mL
5.0 mL
2.0 mL10.0 mL
Concentration of Test Chemical
200,000
40,00020,000(Add 100 mg to a tube. Add the
µg/mL
µg/mL
µg/mL
first volume of medium. Dilutewith subsequent volumes if(200 mg/mL) (40 mg/mL) (20 mg/mL)
necessary.)
Concentration of Test Chemical
10,0002,000(Add 20 mg to a large tube. Add
µg/mL
µg/mL
the first volume of medium.
Dilute with subsequent volume if(10 mg/mL)(2.0 mg/mL)
necessary.)
If test chemical is insoluble in medium at 2000 µg/mL, then attempt to dissolve chemical in DMSO. Actual volume
of solution can be determined after test chemical is dissolved and solution is measured using a calibrated instrument(e.g., micropipettor, or serological pipette). The actual stock concentration can be calculated accordingly.

Example: If complete solubility is not achieved in 0.5 mL medium (Step 1) using the mixing
procedures specified in Section 6.2.1.1, b-d, then 2.0 mL must be added to obtain a total volume of
2.5 mL (Step 2). Chemical and medium are again mixed as prescribed in Section 6.2.1.1 in an
attempt to dissolve. If solubility is not achieved at Step 2, then 2.5 mL medium is added in Step 3.
3

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Chemical and medium are again mixed as prescribed in Section 6.2.1.1 in an attempt to dissolve. No
additional weighing of the chemical is required until Step 4.
6.2.1.1 Chemical Dilution Medium/Routine Culture Mediuma) Dissolve test chemical in Chemical Dilution Medium and Routine Culture
Medium as in Step 1 of Table 1.
b) Gently mix. Vortex for 1-2 minutes.
c) If test chemical hasn’t dissolved, use sonication for up to five minutes.
d) If sonication doesn’t work, then warm solution to 37°C.
e) Proceed to Step 2 (and Steps 3-5, if necessary) of Table 1 and repeat
procedures b-d.
6.2.1.2 DMSO
If the test chemical doesn’t dissolve in the Chemical Dilution Medium or Routine
Culture Medium, then follow the dilution steps in Table 1A and mixing steps a)
through e) in Section 6.2.1.1 using DMSO instead of Chemical Dilution
Medium/Routine Culture Medium.
6.2.1.3 Ethanol
If the test chemical doesn’t dissolve in DMSO, then follow the dilution steps in
Table 1A and mixing steps a) through e) in Section 6.2.1.1 using ethanol instead of
DMSO.
Table 1A: Determination of Solubility in DMSO and Ethanol
Steps

1

2

3

4

5

6

Total Volume of
0.2 mL
0.5 mL
2.5 mL
5.0 mL
2.0 mL10.0 mLDMSO or Ethanol
Concentration of Test
500,000
200,000
40,00020,000Chemical (Add 100 mgµg/mL
to a tube. Add the first
µg/mL
µg/mL
µg/mL
volume of solvent.
Dilute with subsequent(200 mg/mL)(40 mg/mL)(20 mg/mL)(500 mg/mL)volumes if necessary.)
Concentration of Test
10,0002,000
Chemical (Add 20 mg
µ
g/mL
µ
g/mL
to a tube. Add the first
volume of solvent. .
(10(2.0
Dilute with subsequent
mg/mL)
mg/mL)
volume if necessary.)
If test chemical is insoluble in DMSO at 2000 µg/mL, then attempt to dissolve chemical in ethanol. Actual volume
of solution can be determined after test chemical is dissolved and solution is measured using a calibrated
instrument (e.g., micropipettor, or serological pipette). The actual stock concentration can be calculated
accordingly.

If the test chemical does not dissolve in Chemical Dilution Medium/Routine
Culture Medium, DMSO, or ethanol, at 2 mg/mL, then repeat the entire solubility
procedure with each solvent (in the order of Chemical Dilution Medium/Routine
Culture Medium, DMSO, and ethanol) using the dilution steps in Table 1B andmixing steps a) through e) in Section 6.2.1.1.4
4

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Table 1B: Further Determination of Solubility in Chemical Dilution Medium/Routine Culture
Medium, DMSO, or Ethanol4
STEP
6
7
8
9
10
Total Volume of Solvent
5 mL
10 mL
20 mL
40 mL
100 mL
Concentration of Test Chemical
500 µg/mL
125 µg/mL
50 µg/mL
250 µg/mL(Add 5 mg to a tube. Add the first 1,000 µg/mL
volume of solvent. Dilute withsubsequent volumes if necessary.)(0.5
mg/mL)(0.125(0.05
mg/mL)
mg/mL)
If test chemical is insoluble in medium at 50 µg/mL, then attempt to dissolve chemical in DMSO and then ethanol.
Actual volume of solution can be determined after test chemical is dissolved and solution is measured using acalibrated instrument. The concentration can be calculated accordingly.

(1 mg/mL)(0.25 mg/mL)

Approximately 100 200 mg (100200,000 µg)2 of the test chemical will be weighed into a
glass tube and the weight will be documented. Assay-specific culture media will be
added to the vessel so that the concentration is 12,000,000 µg/ml (1000 2000 mg/ml)2(i.e., approximately 0.1 ml). If complete solubility is achieved, then additional solubility
procedures are not needed. If only partial solubility is achieved, follow the test chemicaldissolving steps in Table 1, derived from EPA (1998), to add additional medium in steps
until the concentration is a minimum of 100200,000 µg/ml (100 200 mg/ml)2. Ifcomplete solubility at 100,000 µg/ml in culture medium can’t be attained, then repeat thesolubility steps using the other solvent(s) in the solubility hierarchy. Test chemicals thatare only soluble in DMSO or ethanol will be prepared at 12,000,000 µg/ml2 as the highest
concentration of stock solution.
Table 1: Determination of Solubility
Solubility Data
Total volume of medium added (ml)Total volume of DMSO or ethanol added (ml)Approximate solubility (µg/ml)

Step 10.1
0.1≥
12,000,0002

Step 20.5***0.52
200400,0002

Step 31.0***1.02100200,0002

6.2.1.1 Treatment Medium/Routine Culture Medium)a)f) Dissolve test chemical in Treatment Medium and Routine Culture Mediumb)g)Gently mix. Vortex for 5-10 seconds1-2 minutes.2c)h) If test chemical hasn’t dissolved, use sonication (up to five minutes).
d)i) If sonication doesn’t work, then warm solution to 37°C.
6.2.1.2 DMSO
If the test chemical doesn’t dissolve in the Treatment Medium/Routine Culture
Medium, then follow steps a) through d) in Section 6.2.1.1 using DMSO instead ofTreatment Medium/Routine Culture Medium.
6.2.1.3 Ethanol
If the test chemical doesn’t dissolve in DMSO, then follow steps a) through d) in
Section 6.2.1.1 using ethanol instead of DMSO.

2
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6.2.2 pH of Solutions
Measure the pH (using pH paper) of the highest concentration of test chemical dissolvedin the culture media. Document the pH and note the color of each test chemical
concentration in medium.
7.0

DATA COLLECTION
7.1

Nature of Data to be Collected
7.1.1 Solubility Studies
The Contractor shall record all information pertinent to the solubility of the test chemical:a) Approximate tT3est chemical solubility in all solvents tested (i.e., media, DMSO,
and/or ethanol) in weight per unit volume (i.e. mg/mL) estimated by following thestep-wise solubility protocol culture medium at a minimum of 100200,0002 µg/ml3b) pH of test chemical in culture medium; color of culture mediumc) Test chemical solubility in DMSO or ethanol at 12,000,0002 µg/ml3d) Need of vortexing, sonication, and/or heating
The Contractor shall provide this information to the Study Management Team via the
Project Officer by the avenues described in Section 8. This information shall NOT be
provided to the Testing Facilities. Information to be provided to the Testing Facilities
is specified in Sections 5.1.5 and 5.1.6.
7.1.2 Chemical Information
The Contractor shall supply at a minimum the following information about each test
chemical and report as specified in Addendum I.
a) Purityb) CAS #c) Supplierd) Specification sheetse) Certificates of analysisf) Material Safety Data Sheet (MSDS)g) Colorh) Odori) Physical statej) Weight or volume of sample distributed to the Testing Facilityk) Specific density for liquid test chemicalsl) Storage instructionsm) Chemical hazardsn) Special handling instructionso) Amount of material archived[Note: Much of the information will be in the MSDS.]

7.2 Type of Media Used for Data Storage
Originals of the raw data (the Study Workbook) and copies of other raw data such as instrument
logs shall be collected and archived at the end of the study (under the direction of the Study
Director), according to GLP-compliant procedures. Data that are stored electronically shall be
periodically copied, and backup files shall be produced and maintained.
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7.3 Documentation
Original raw data that shall be collected shall include but are not limited to the following:• Data recorded in the Study Workbook, which shall consist of all recordings of all activities
related to acquisition, preparation, solubility testing, and distribution of the test chemicals;• Other data collected as part of GLP compliance− Equipment logs
− Equipment calibration records
8.0

DRAFT AND FINAL REPORTS

Biweekly Reports: The Contractor will provide a biweekly progress report to the Project Officer andcopied to the Project Coordinators of the Study Management Team (See Section 4.2 and Addendum
I). These reports will include raw and interim data as the study progresses. These reports will be inelectronic format (i.e., email with Microsoft Word (or equivalent) or Excel attachments).
Draft Reports: A draft report shall be submitted to the Project Officer for each Validation Study
phase (See Section 4.2 and Addendum I). A Draft Final Report detailing the Contractor’s
involvement in all phases of the Validation Study shall be prepared by the Contractor, signed by the
Study Director, and provided to the Project Officer. The submitted results shall accurately describe all
methods used for generation and analysis of the data, provide a complete record of the preparation oftest chemicals, and present any relevant data necessary for the assessment of the results (SeeAddendum I).
Final Report: The Draft Final Report shall be revised according to comments from the Project Officer
and submitted as the Final Report (See Section 4.2 and Addendum I).
9.0 RECORDS AND ARCHIVESAt the conclusion of the Contractor’s participation in the distribution of chemicals for the Validation
Study, the original raw and derived data, as well as copies of other raw data not exclusive to this
Validation Study (instrument logs, calibration records, facility logs, etc.), shall be submitted to
NIEHS/NICEATM (via the Project Officer) for storing and archiving according to the facility's SOP
and in compliance with GLP Standards.
Originals of all raw and derived data, or copies where applicable, shall be stored and archived at
NIEHS/NICEATM.
10.0 ALTERATIONS OF THE STATEMENT OF WORKNo changes in the Statement of Work shall be made without the consent of the Project Officer andStudy Management Team. A Statement of Work Amendment detailing any change(s) and the basis
for the change(s) shall be approved and prepared by the Study Director, and the amendment shall besigned and dated by the Study Director and the NIEHS representative. The amendment shall be
retained with the original Statement of Work.
11.0 REFERENCES
Clonetics Normal Human Keratinocyte Systems Instructions for Use, AA-1000-4-Rev.03/00.
(http://www.clonetics.com).
EPA Product Properties Test Guidelines. OPPTS 830.7840. 1998. Water Solubility: Column Elution
Method; Shake Flask Method. United States Environmental Protection Agency. Prevention,
Pesticides and Toxic Substances (7101). EPA 712-C-98-041. March 1998.
G-77

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

National Toxicological Program, September 2000, Attachment 2 revised. Specifications for the
Conduct of Studies to Evaluate the Toxic and Carcinogenic Potential of Chemical, Biological andPhysical Agents in Laboratory Animals for the National Toxicology Program (NTP).
NICEATM (The National Toxicology Program [NTP] Interagency Center for the Evaluation of
Alternative Toxicological Methods). 2001. Test Method Protocol for the BALB/c 3T3 Neutral Red
Uptake Cytotoxicity Test. A Test for Basal Cytotoxicity for an In Vitro Validation Study.
NICEATM (The National Toxicology Program [NTP] Interagency Center for the Evaluation of
Alternative Toxicological Methods). 2001. Test Method Protocol for the Normal Human
Keratinocyte [NHK] Neutral Red Uptake Cytotoxicity Test. A Test for Basal Cytotoxicity for an In
Vitro Validation Study.
ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods). 2001.
Guidance document on using in vitro data to estimate in vivo starting doses for acute toxicity NIH
publication 01-4500. NIEHS, Research Triangle Park, North Carolina.
OECD (Organisation for Economic Co-operation and Development). 2001. Harmonised Integrated
Classification System for Human Health and Environmental Hazards of Chemical Substances and
Mixtures as Endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Partyon Chemicals in November 1998, Part 2, p. 21. OECD, Paris.
http://www.oecd.org/ehs/class/HCL6htm.

12.0 APPROVAL OF STATEMENT OF WORK____________________________________________
Sponsor Representative_________________
Date____________________________________________
Testing Facility Management_________________
Date

G-78

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

ADDENDUM I
SUGGESTED REPORT FORMATTITLE PAGE•

Study Title
Draft Report 1:•

Test Articles
Draft Report 1:

•
•
•
•

Acquisition, Preparation, Solubility Testing, and Distribution of Test
Chemicals: Phase I of the In Vitro Validation Study
Draft Report 2:
Acquisition, Preparation, Solubility Testing, and Distribution of Test
Chemicals: Phase II of the In Vitro Validation Study
Draft Report 3:
Acquisition, Preparation, Solubility Testing, and Distribution of Test
Chemicals: Phase III of the In Vitro Validation Study
Draft/Final Report: Acquisition, Preparation, Solubility Testing, and Distribution of Test
Chemicals: Final Report for the In Vitro Validation Study
Identify the positive control chemical of Phase Ia and the three (3) test
chemicals of Phase Ib
Draft Report 2:
Identify the nine (9) test chemicals of Phase II
Draft Report 3:
Identify the sixty (60) test chemicals of Phase III
Draft/Final Report: Identify all seventy-two (72) test chemicals and positive control of the In
Vitro Validation Studies
Authors
Study Completion Date
Contract Facility
Study Number/Identification

SIGNATURE PAGE
•
•
•
•

•

•
•
•
•
•
•

Study Initiation Date: Date Statement of Work was signed
Initiation Date of Laboratory Studies: Actual laboratory start dateStudy Completion Date: Date report signed by Study Director
Sponsor Representative:
Ms. Molly Vallant – Project Officer
The National Institute of Environmental Health Sciences (NIEHS)
Study Management Team Representatives
Judy Strickland, Ph.D. (Project Coordinator)
Michael Paris (Assistant Project Coordinator)
Contractor Facility: Name and address
Archive Location: Name and address
Study Director: Name and signature and date
Key Personnel: Laboratory technicians, QA Director, Safety Officer
Facility Management: Name
Scientific Advisor: Name

G-79

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

ADDENDUM I (cont.)
DRAFT REPORT 1
Acquisition, Preparation, Solubility Testing, and Distribution of Test Chemicals: Phase I of the In
Vitro Validation Study• Table of Contents• Objectives :The report shall provide specific objectives• Summary of the Findings: Referenced to the raw data where appropriate; Include all
information for the positive control (SLS) and the three (3) Phase Ib chemicals.
• Narrative Description of the Solubility Studies: Describe any problems that were encounteredand how such problems were solved. Justifications for solvents used for each test chemical will
be included in the description. Provide the information requested in Section 7.1.1. Deviations
from the protocols, SOPs, and/or the Statement of Work shall be addressed in this section. Copies
of appropriate sections of the Study Workbook shall be included with the report (as attachments).
The draft report will include unaudited Study Workbook pages. The final report will include a
copy of the audited Study Workbook with a statement (signed and dated by the Study Director)
on the front of it stating that it is an exact copy of the original audited workbook.
• Statement Signed by the Study Director: Confirm that the solubility studies, acquisition,
preparation, and distribution of the test chemicals were conducted in compliance with GLP (orindicating where the Study deviated from GLP). Confirm that the report fully and accuratelyreflects the raw data generated in the Study.
• Other Information: (All copies of documents will be noted as exact duplicates of the data.)• Information requested in Section 7.1.2• Deviations to the protocols, SOPs, and Statement of Work
• Revisions/amendments to the protocols, SOPs, and Statement of WorkDRAFT REPORT 2
Acquisition, Preparation, Solubility Testing, and Distribution of Test Chemicals: Phase II of the In
Vitro Validation Study
•
•
•
•

•

•

Table of Contents
Objectives: The report shall provide specific objectives
Summary of the Findings: Referenced to the raw data where appropriate; Include all
information for the nine (9) Phase II chemicals.
Narrative Description of the Solubility Studies: Describe any problems that were encounteredand how such problems were solved. Justifications for solvents used for each test chemical shallbe included in the description. Provide the information requested in Section 7.1.1. Deviations
from the protocols, SOPs, and/or the Statement of Work shall be addressed in this section. Copies
of appropriate sections of the Study Workbook shall be included with the report (as attachments).
The draft report will include unaudited Study Workbook pages. The final report will include a
copy of the audited Study Workbook with a statement (signed and dated by the Study Director)
on the front of it stating that it is an exact copy of the original audited workbook.
Statement Signed by the Study Director: Confirm that the solubility studies, acquisition,
preparation, and distribution of the test chemicals were conducted in compliance with GLP (orindicating where the Study deviated from GLP). Confirm that the report fully and accuratelyreflects the raw data generated in the Study.
Other Information: (All copies of printouts, documents, and spreadsheets shall be noted as exact
duplicates of the data.)• Information requested in Section 7.1.2• Deviations to the protocols, SOPs, and Statement of Work
• Revisions/amendments to the protocols, SOPs, and Statement of Work
G-80

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

ADDENDUM I (cont.)
DRAFT REPORT 3
Acquisition, Preparation, Solubility Testing, and Distribution of Test Chemicals: Phase III of the In
Vitro Validation Study
•
•
•
•

•

•

Table of Contents
Objectives: The report shall provide specific objectives
Summary of the Findings: Referenced to the raw data where appropriate; Include all
information for sixty (60) Phase III chemicals.
Narrative Description of the Solubility Studies: Describe any problems that were encounteredand how such problems were solved. Justifications for solvents used for each test chemical will
be included in the description. Provide the information requested in Section 7.1.1. Deviations
from the protocols, SOPs, and/or the Statement of Work shall be addressed in this section. Copies
of appropriate sections of the Study Workbook shall be included with the report (as attachments).
The draft report will include unaudited Study Workbook pages. The final report will include a
copy of the audited Study Workbook with a statement (signed and dated by the Study Director)
on the front of it stating that it is an exact copy of the original audited workbook.
Statement Signed by the Study Director: Confirm that the solubility studies, acquisition,
preparation, and distribution of the test chemicals were conducted in compliance with GLP (orindicating where the Study deviated from GLP). Confirm that the report fully and accuratelyreflects the raw data generated in the Study.
Other Information: (All copies of printouts, documents, and spreadsheets shall be noted as exact
duplicates of the data.)• Information requested in Section 7.1.2• Deviations to the protocols, SOPs, and Statement of Work
• Revisions/amendments to the protocols, SOPs, and Statement of WorkDRAFT/FINAL REPORT
Acquisition, Preparation, Solubility Testing, and Distribution of Test Chemicals: Draft/Final Reportfor the In Vitro Validation Study
•
•
•
•

•

•

Table of Contents
Objectives: The draft/final report shall provide specific objectives
Summary of the Findings: Referenced to the raw data where appropriate; Include all
information for the seventy-two (72) test chemicals and the positive control (SLS).
Narrative Description of the Solubility Studies: Describe any problems that were encounteredand how such problems were solved. Justifications for solvents used for each test chemical shallbe included in the description. Provide the information requested in Section 10.1.1. Deviations
from the protocols, SOPs, and/or the Statement of Work shall be addressed in this section.
Copies of appropriate sections of the Study Workbook shall be included with the report (asattachments). The draft report will include unaudited Study Workbook pages. The final reportwill include a copy of the audited Study Workbook with a statement (signed and dated by the
Study Director) on the front of it stating that it is an exact copy of the original audited workbook.
Statement Signed by the Study Director: Confirm that the acquisition, preparation, solubility
studies, and distribution of the test chemicals were conducted in compliance with GLP (orindicating where the Study deviated from GLP). Confirm that the report fully and accuratelyreflects the raw data generated in the Study.
Quality Assurance Statement: (For Final Report only)
QA Statement identifying: 1) the phases and data inspected, 2) dates of inspection, and 3) dates
findings were reported to the Study Director and Testing Facility management. The QA
G-81

In Vitro Cytotoxicity Test Methods BRD Appendix G2•

November 2006

Statement shall identify whether the methods and results described in the Final Report accuratelyreflect the raw data produced during the Study.
Other Information: (All copies of printouts, documents, and spreadsheets shall be noted as exact
duplicates of the data.)• Deviations to the protocols, SOPs, and Statement of Work
• A list of all SOPs used by the laboratory (SOP title and laboratory identification code)• The Statement of WorkBIWEEKLY REPORTS

Contract Facility:
Chemicals Acquired:Chemicals Tested for Solubility:
Results of Solubility Tests:
Chemicals Shipped to Testing Facilities:
Date of Shipping:
Problems Encountered/Resolutions:
Projected Shipping Schedule:

G-82

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

ADDENDUM II
SUGGESTED STANDARD TEST REPORTING TEMPLATE FOR STUDY WORKBOOK
1SOLUBILITY TESTING

Test Chemicals for the In Vitro Validation Study
Study No.___________________
Test Chemical_________________#____________

Test Chemical Code__________

CAS

Physical Description_______________________________________ Liquid Density_________
Solubility Determined by__________________________
Date______________
Solvent

Amountof TestChemicalVolume
Added

Total
VolumepH andmediumcolor

Vortex (V)Sonication (S)Heating-37oC (H)

0.1ml
Treatment
Medium(3T3 NRU)

0.5ml
1.0ml
0.1ml

RoutineCultureMedium(NHK NRU)

0.5ml
1.0ml
0.1ml

DMSO

0.1ml
Ethanol

Reference Color of Treatment Medium________________________
Reference Color of Routine Culture Medium____________________
Balance I.D.______________
Treatment Medium and Routine Culture Medium: minimum concentration of 100mg/ml.
DMSO and Ethanol: minimum concentration of 1000mg/ml.
1

Adaptation of Institute of In Vitro Sciences (IIVS) form – 350 [2/2002]

G-83

Comments

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

ADDENDUM III
TEST CHEMICALS FOR THE IN VITRO VALIDATION STUDY (ALPHABETICAL)[NOTE: TESTING FACILITIES MUST NOT SEE THIS LIST OF CHEMICALS]CHEMICAL
1,1,1-Trichloroethane
2-Propanol
5-Aminosalicylic acidAcetaminophen
Acetonitrile
Acetylsalicylic acid
To be determined1
Aminopterin
Amitriptyline HCl3
Arsenic III trioxide
Atropine sulfate monohydrate3
Boric aid
Busulphan
Cadmium II chloride
Caffeine
Carbamazepine
Carbon tetrachloride
Chloral hydrate
Chloramphenicol
Citric Acid
Colchicine
Cupric sulfate * 5 H2OCycloheximide
Dibutylphthalate
Dichlorvos (DDVP)
Diethyl phthalate
DigoxinDimethylformamide
DiquatDisulfoton
Endosulfan
Epinephrine bitartrate
Ethanol
Ethylene glycol
Fenpropathrin
Gibberellic acid
Glutethimide
GlycerolHaloperidol
Hexachlorophene
Lactic acid
Lindane
1
3

CAS NO.
71-55-6
67-63-0
89-57-6
103-90-2
75-05-8
50-78-2
54-62-6
50-48-6549-18-83
1327-53-3
55-48-1, (17108-73-5)73791-47-63
10043-35-3
55-98-1
10108-64-2
58-08-2
298-46-4
56-23-5
302-17-0
56-75-7
77-92-964-86-87758-99-8
66-81-984-74-2
62-73-7
84-66-2
20830-75-5
68-12-22764-72-9
298-04-4
115-29-7
51-42-3
64-17-5
107-21-1
39515-41-8
77-06-5
77-21-4
56-81-5
52-86-870-30-4
50-21-5
58-89-9

Revised 5/23/02
Revised 9/17/02

G-84

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

ADDENDUM III (CONT.)CHEMICAL

Lithium I sulfatecarbonate3
Meprobamate
Mercury II chloride
MethanolNicotine
Paraquat
Parathion
Phenobarbital
PhenolPhenylthiourea
Physostigmine1
Potassium cyanide
Potassium I chloride
Procainamide HCl3
Propranolol HCl
Propylparaben
Sodium arsenite
Sodium chloride
Sodium dichromate dihydrate
Sodium hypochlorite
Sodium I fluoride
Sodium oxalate
Sodium selenate*10 H201
Strychnine
Thallium I sulfate
Trichloroacetic acid
Triethylene melamine
Triphenyltin hydroxide
Valproic acid
Verapamil HCl
Xylene3
1

CAS NO.

554-13-210377-48-73
57-53-4
7487-94-7
67-56-1
54-11-5
1910-42-5, (3765-78-4,57593-74-5,65982-505,136338-65-3,205105-68-6,247050-57-3)3
56-38-2
50-06-6
108-95-2
103-85-5
57-47-61
151-50-87447-40-7
51-06-9614-39-13
318-98-9, (3506-09-0, 146874-86-4)1
94-13-37784-46-5
7647-14-5
7789-12-0
8007-59-8, (7681-52-9)3
7681-49-4
62-76-0
1341313410-01-01
57-24-97446-18-6
76-03-951-18-3
76-87-9
99-66-1
152-11-4
1330-20-7

Revised 9/17/02
Revised 5/23/02

G-85

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

ADDENDUM IV
TEST CHEMICALS FOR THE IN VITRO VALIDATION STUDYBY STUDY PHASE
PHASE Ia
Sodium laurel sulfate
PHASE Ib
Arsenic III trioxide
Ethylene glycol
Propranolol HCl
PHASE II
Aminopterin
Chloramphenicol
Colchicine
Cupric sulfate * 5 H2OLithium I sulfatecarbonate3
Potassium I chloride
2-Propanol
Sodium I fluoride
Sodium selenate*10 H201
PHASE III1,1,1-Trichloroethane
5-Aminosalicylic acidAcetaminophen
Acetonitrile
Acetylsalicylic acid
To be determined1
Amitriptyline HCl3
Atropine sulfate monohydrate3
Boric aid
Busulphan
Cadmium II chloride
Caffeine
Carbamazepine
Carbon tetrachloride
Chloral hydrate
Citric Acid
Cycloheximide
Dibutylphthalate
Dichlorvos (DDVP)
Diethyl phthalate
DigoxinDimethylformamide
DiquatDisulfoton
Endosulfan
Epinephrine bitartrate3
1

151-21-3
1327-53-3
107-21-1
318-98-9, (3506-09-0, 146874-86-4)1
54-62-6
56-75-7
64-86-87758-99-8
554-13-210377-48-73
7447-40-7
67-63-07681-49-4
1341313410-01-01
71-55-6
89-57-6
103-90-2
75-05-8
50-78-2
549-18-850-48-63
73791-47-655-48-1, (17108-73-5)310043-35-3
55-98-1
10108-64-2
58-08-2
298-46-4
56-23-5
302-17-0
77-92-966-81-984-74-2
62-73-7
84-66-2
20830-75-5
68-12-22764-72-9
298-04-4
115-29-7
51-42-3

Revised 9/17/02
Revised 5/23/02

G-86

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

ADDENDUM IV (CONT.)
PHASE III (cont.)
Ethanol
Fenpropathrin
Gibberellic acid
Glutethimide
GlycerolHaloperidol
Hexachlorophene
Lactic acid
LindaneMeprobamate
Mercury II chloride
MethanolNicotine
Paraquat
Parathion
Phenobarbital
PhenolPhysostigmine1
Phenylthiourea
Potassium cyanide
Procainamide HCl3
Propylparaben
Sodium arsenite
Sodium chloride
Sodium dichromate dihydrate
Sodium hypochlorite
Sodium oxalate
Strychnine
Thallium I sulfate
Trichloroacetic acid
Triethylene melamine
Triphenyltin hydroxide
Valproic acid
Verapamil HCl
Xylene1364-17-5
39515-41-8
77-06-5
77-21-4
56-81-5
52-86-870-30-4
50-21-5
58-89-9
57-53-4
7487-94-7
67-56-1
54-11-5
1910-42-5, (3765-78-4,57593-74-5,65982-505,136338-65-3,205105-68-6,247050-57-3)3
56-38-2
50-06-6
108-95-2
57-47-61
103-85-5
151-50-8
51-06-9614-39-13
94-13-37784-46-5
7647-14-5
7789-12-0
8007-59-8, (7681-52-9)3
62-76-0
57-24-97446-18-6
76-03-951-18-3
76-87-9
99-66-1
152-11-4
1330-20-7

Revised 5/23/02
Revised 9/17/02

G-87

In Vitro Cytotoxicity Test Methods BRD Appendix G2

November 2006

ADDENDUM V
ASSUMPTIONS FOR CALCULATION OF AMOUNT OF TEST MATERIAL NEEDED FOR
EACH TESTING FACILITY
ChemicalAmountPhase I
Test in 3 solvents
Test in 3 replicate assays
Repeat 3 times
Phase I Amount/Testing Facility
x 3 Testing Facilities
2 Archive samples (3 solubility + 3
assays)Total Phase I Amount
Phase II
Test in 3 solvents
Test in 3 replicate assays
Repeat 2 times
Phase II Amount/Testing Facility
x 3 Testing Facilities
2 Archive samples (3 solubility + 3
assays)Total Phase II Amount
Phase III
Test in 3 solvents
Test in 3 replicate assays
Phase III Amount/Testing Facility
x 3 Testing Facilities
2 Archive samples (3 solubility + 3
assays)Total Phase III Amount

Assumption

300 mg300
300
900 mgChemical must be tested in all 3 solvents
3 replicate assays must be performed
3 replicate assays must be repeated 3 times

2700
1200

Assumes 3 labs participate in study
Archive samples use same amount of chemical
as testing sample

3900 mg

300 mg300
200
800 mgChemical must be tested in all 3 solvents
3 replicate assays must be performed
2 replicate assays must be repeated 3 times

2400
1200

Assumes 3 labs participate in study
Archive samples use same amount of chemical
as testing sample

3600 mg

300 mg300
600 mgChemical must be tested in all 3 solvents
3 replicate assays must be performed

1800
1200

Assumes 3 labs participate in study
Archive samples use same amount of chemical
as testing sample

3000 mg

Specification of 4 g of chemical per Testing Facility in Section 5.1.3 was chosen to allow a generous
amount of error (in the direction of the Testing Facilities being provided with more chemical thannecessary) in the calculations and assumptions made here.

G-88

In Vitro Cytotoxicity Test Methods BRD Appendix H

November 2006

Appendix H

Candidate Reference Oral LD50 Data

H1

Rat and Mouse Oral LD50 Database .................................................. H-3

H2

Evaluation of the Candidate Reference Oral LD50 Data ............... H-39

H-1

In Vitro Cytotoxicity Test Methods BRD Appendix H

November 2006

[This Page Intentionally Left Blank]

H-2

In Vitro Cytotoxicity Test Methods BRD Appendix H1

November 2006

Appendix H1

Rat and Mouse Oral LD50 Database

H-3

In Vitro Cytotoxicity Test Methods BRD Appendix H1

November 2006

[This Page Intentionally Left Blank]

H-4

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Notes

Reference
Substance Source

Primary Reference

reference in Russian

NA

Paligov VI, Khananaev LI, Goinatskii MG, Gavrilyuk VM. 1990. Hygienic substantiation of content
of methylchloroform in water bodies. Gigiena Naselennykh Mest 29:45-49. (RTECSREFERENCE)

single dose; undiluted; no more
all surviving rats observed up to 2 weeks; 35 rats usedthan 7 cc administered

NA

NA

Torkelson TR, Oyen F, McCollister DD, Rowe VK. 1958. Toxicity of 1,1,1-trichloroethane asdetermined on laboratory animals and human subjects. Am Ind Hyg Assoc J 19:353-362.
The Dow Chemical Company, Midland, MI

oral; stomach tube

single dose; undiluted; no more
all surviving rats observed up to 2 weeks; 35 rats usedthan 7 cc administered

this compound is an inhibited form

NA

Torkelson TR, Oyen F, McCollister DD, Rowe VK. 1958. Toxicity of 1,1,1-trichloroethane asdetermined on laboratory animals and human subjects. Am Ind Hyg Assoc J 19:353-362.
The Dow Chemical Company, Midland, MI

Holtzman, Sprague-Dawleymale
albino rats; 215-330 g; adultoral; gastricintubation

single dose; undiluted; 464,
1000, 2150, 4660, 10000,
21500 mg/kg doses

observations recorded at 1, 4, 24 hours, daily thereafter for 7 days; 53-4 hour fasting period; stabilized
dead at highest dose; depression, ataxia, labored respiration, salivation, 1,1,1-trichloroethane; inhibited
ptosis, excessive urination, diarrhea
formulation

NAfrom EPA TSCATS database; Acute Oral Administration-Rats Acute Dermal Application-Rabbits
Acute Eye Irritation-Rabbits Primary Skin Irritation-Rabbits Subacute (Four-Week) Inhalation;
1969. EPA Doc. No. 878210366, Fiche No. OTS0205891; Ethyl Corp.

Litchfield andWilcoxon

Holtzman, Sprague-Dawleymale
albino rats; 215-330 g; adultoral; gastricintubation

single dose; undiluted; 464,
1000, 2150, 4660, 10000,
21500 mg/kg doses

observations recorded at 1, 4, 24 hours, daily thereafter for 7 days; 53-4 hour fasting period; stabilized
dead at highest dose; depression, ataxia, labored respiration, salivation, 1,1,1-trichloroethane; inhibited
ptosisformulation

NAfrom EPA TSCATS database; Acute Oral Administration-Rats Acute Dermal Application-Rabbits
Acute Eye Irritation-Rabbits Primary Skin Irritation-Rabbits Subacute (Four-Week) Inhalation;
1969. EPA Doc. No. 878210366, Fiche No. OTS0205891; Ethyl Corp.

no CL ("all-or-none" Litchfield andresponse)
Wilcoxon

Holtzman, Sprague-Dawleymale
albino rats; 215-330 g; adultoral; gastricintubation

single dose; undiluted; 464,
1000, 2150, 4660, 10000,
21500 mg/kg doses

observations recorded at 1, 4, 24 hours, daily thereafter for 7 days; 43-4 hour fasting period; unstabilized
dead at highest dose; depression, ataxia, labored respiration, excessiveNA
1,1,1-trichloroethane
urination, diarrhea, ruffled fur, salivation, piloerection

from EPA TSCATS database; Acute Oral Administration-Rats Acute Dermal Application-Rabbits
Acute Eye Irritation-Rabbits Primary Skin Irritation-Rabbits Subacute (Four-Week) Inhalation;
1969. EPA Doc. No. 878210366, Fiche No. OTS0205891; Ethyl Corp.

Wistar rats; 90-120 g; 3-4
weeks oldoral; stomach
intubation

doses differ by a factor of 2 in a 14 day observation; dose, number of dead/total: 16 mL/kg -- 3/3; 8geometric series
mL/kg -- 5/5; 4 mL/kg -- 1/5

non-fasted; tested in 1975; 13 rats
used

NA

from EPA TSCATS database; Range Finding Toxicity Studies With Isopropanol Recovery Column,
Side Stream Decanter Make With Cover Letter Dated 020987; EPA Document No. 86870000097
Fiche No. OTS0513282; Union Carbide Corp.; Carnegi Mellon 1976

oral

solvent used in undiluted form

nonfasted rats; 6-12 rats of both
sexes used for studies; solvent usedin undiluted form

analytical grade
meeting A.C.S.
specifications

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, IL

oral gavage

clinical observations: soft stools, diarrhea, decreased limb tone,
hypoactivity, hypothermia, lacrimation, pinna and pain reflex absent,
undiluted dose (g/kg) 3.5, 4.5,
red-stained nose, mouth, and eyes, dyspnea, brown-stained urogenital
5.8, 7.5
or anal region, bradypnea and piloerection, ataxia; dose (g/kg), rats
dead: 3.5-0/2; 4.5-2/4; 5.8-2/2; 7.5-1/1

18-20 hour fasted rats; 1-4 rats per
dose; GLP study

NAfrom EPA TSCATS database; Acute Oral Toxicity (Up/Down Method) Report with Cover Letter
Dated 020987; 1983. EPA Document No. 86870000160, Fiche No. OTS0513345;
Hazelton Labs; Hazelton 1983

oral

solvent used in undiluted form

animals observed for a week after medication

nonfasted rats; groups of 6 rats used analytical grade
for the studies; solvent used inmeeting A.C.S.
undiluted form
specifications

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, IL

female?

oral

NA

NA

reference in RussianNA

Antonova VI, Salmina ZH. 1978. The maximal permissible concentration of isopropyl alcohol inwater bodies with due regards for its action on the gonads and the progeny. Gigiena i Sanitariya
43(1):9-11. (RTECS REFERENCE)

Wistar rats; 90-120 g; 3-4
weeks oldmale

oral; stomach
intubation

doses differ by a factor of 2 in a 14 day observation;dose, number of dead/total: 10mL/kg - 5/5; 5geometric series
mL/kg - 1/5

non-fasted; tested in 1971; 10 rats
used

NA

from EPA TSCATS database; Isopropanol, Anhydrous Range Finding Toxicity Studies with CoverLetter Dated 020987, (1971), EPA Document No. 86870000102, Fiche No. OTS0513287;
Carnegie-Mellon Inst. of Res. 1971

Sprague-Dawley rats; ~ 7
weeks

male

oral gavage

undiluted dose (g/kg) 4.5, 5.8,
7.5, 9.8

clinical observations: soft stools, diarrhea, ataxia, decreased limb tone,
hypoactivity, hypothermia, lacrimation, pinna and pain reflex absent,
18-20 hour fasted rats; 1-3 rats per
red-stained nose, mouth and eyes, brown-stained urogenital or anal
dose; GLP study
region, dyspnea, bradypnea and piloerection; dose (g/kg), rats dead:4.50/2; 5.8-2/3; 7.5-3/3; 9.8-1/1

NAfrom EPA TSCATS database; Acute Oral Toxicity (Up/Down Method) Report with Cover Letter
Dated 020987, (1983), EPA Document No. 86870000160, Fiche No. OTS0513345;
Hazelton Labs; Hazelton 1983

oral

solvent used in undiluted form

animals observed for a week after medication

1,1,1-Trichloroethane

9600

9600

7384 - 12480

NA

oral

NA

1,1,1-Trichloroethane

9600

10300

8270 - 12800(95% CL)

Thompson method of Wistar white rats; 175 - 250
femalemoving averagesgoral; stomach tube

1,1,1-Trichloroethane

9600

12300

11000 - 13700(95% CL)

Thompson method of Wistar white rats; 175 - 250
malemoving averagesg

1,1,1-Trichloroethane

9600

12600

926 - 17100(CL)

Litchfield andWilcoxon

1,1,1-Trichloroethane

9600

12627

5356 - 29765(CL)

1,1,1-Trichloroethane

9600

16000

2-Propanol

5045

4074
(5.19 mL/kg; sp.
density =0.78505; convert
LD50 to mg/kg)

2-Propanol

5045

4396
3297 - 5809
(5.6 mL/kg; sp. (95% CL; 4.2 - 7.4 Litchfield andSprague-Dawley rats; 16-50 male anddensity =mL/kg; sp. density = Wilcoxon method andg; 14 days
female0.78505; convert 0.78505; convert probit analysis
LD50 to mg/kg) LD50 to mg/kg )2-Propanol

5045

2-Propanol

5045

2-Propanol

5045

5045

4650 - 5400

NA

rats

2-Propanol

5045

5087
(6.48 mL/kg; sp.
density =0.78505; convert
LD50 to mg/kg)

3768 - 6877

moving averagemethod2-Propanol

5045

5300

4100 - 7000
(95% CL)

UDP

2-Propanol

5045

4500

Observations

3015 - 5503

3500 - 5800
(95% CL)moving averagemethodUDP

rats

Sprague-Dawley rats; ~ 7
weeks

NA

male

female4710
4082 - 5495
(6.0 mL/kg; sp. (95% CL; 5.2 - 7.0 Litchfield andSprague-Dawley rats; 80-160
density =mL/kg; sp. density = Wilcoxon method and g; young adult (4-6 weeks
male
0.78505; convert 0.78505; convert probit analysis
according to Taconic Farms)
LD50 to mg/kg)
LD50 to mg/kg)

5338
4161 - 6908
Sprague-Dawley rats; 300(6.8 mL/kg;(95% CL; 5.3 - 8.8 Litchfield and
470 g; older adult (9-18
sp.density is mL/kg; sp. density = Wilcoxon method andmaleweeks according to Taconic
0.78505; convert 0.78505; convert probit analysis
Farms)
LD50 to mg/kg)
LD50 to mg/kg)

NAanimals observed for a week after medication

H-5

nonfasted rats; groups of 6 rats used analytical grade
for the studies; solvent used inmeeting A.C.S.
undiluted form
specifications

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, IL

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

2-Propanol

LD501
mg/kg

5045

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

5840

NA

Animal Information(stock, weight, age)

Genderbased on assumptionthat probit mortality
Sherman rats; 90 -120 g; 4-5
vs log dose has same
male
weeks
slope as similar
chemicalRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

oral gastricintubation

in aqueous solution; doses (ing/kg) differ by 1 log to bracket
LD50, then refine LD50 with
LD50 based on mortalities during a 14 day period
doses in a series of antilog 1.1,
1.3, 1.5, etc.

6 rats/dose at doses that differ by 1log to bracket LD50 (given 1 week
apart); then refined LD50 with 10
rats/dose in a dose series of antilog
reagent grade
1.1, 1.3, 1.5, etc.; assumed to usematerials/methods of Smyth &Carpenter (1944) except for reportedchangesPrimary Reference

Smyth HF Jr, Carpenter CP. 1948. Further experience with the range finding test in the industrial
toxicology laboratory. J Ind Hyg Toxicol 30: 63-68. (LD50 value)
Smyth HF Jr, Carpenter CP. 1944. The place of the range-finding test in the industrial toxicology
laborotory. J Ind Hyg Toxicol 26:269-273. (most materials/methods).

5-Aminosalicylic acid

2800

2800

1781 - 3819
(95% CL)

Miller and Tainter(1944)CDR Sprague-Dawley albino
rats; male 288-346 g; 9-12 maleweeks oldoral; intubation

single dose; 2500, 3500, 5000
mg/kg doses; conc. 250, 350,
500 mg/mL; 10 mL dose vol.;methylcellulose vehicle

14 day observation; initial checks at 1, 2, and 4 hours afteradministration; 2 daily thereafter

from EPA TSCATS database; Acute Toxicity Study in Rats Administered 10 Materials (final report)
15 rats used (five/dose level); fasted
Monsanto Company with Cover Letter dated 062669, (1969), EPA Doc. No. 40-6942188, Fiche No. OTS0519234;
overnight; GLP
Monsanto Co./Bio/dynamics

5-Aminosalicylic acid

2800

3450

2513 - 4387
(95% CL)

Miller and Tainter(1944)CDR Sprague-Dawley albino male andrats; male 288-346 g; female female (equal oral; intubation
225-267 g; 9-12 weeks old numbers)

single dose; 2500, 3500, 5000
mg/kg doses; conc. 250, 350,
500 mg/mL; 10 mL dose vol.;methylcellulose vehicle

14 day observation; initial checks at 1, 2, and 4 hours afteradministration; 2 daily thereafter

30 rats used (five/sex/dose level);fasted overnight; GLP; used same
animals as 2800 and 4200 mg/kg
values from Monsanto 1969

5-Aminosalicylic acid

2800

4200

2863 - 5537
(95% CL)

Miller and Tainter(1944)CDR Sprague-Dawley albino
rats; female 225-267 g; 9-12 female
weeks oldoral; intubation

single dose; 2500, 3500, 5000
mg/kg doses; conc. 250, 350,
500 mg/mL; 10 mL dose vol.;methylcellulose vehicle

14 day observation; initial checks at 1, 2, and 4 hours afteradministration; 2 daily thereafter; toxicologic signs: soft stool,
hyponea, hypoactivity; urinary and fecal staining

from EPA TSCATS database; Acute Toxicity Study in Rats Administered 10 Materials (final report)
15 rats used (five/dose level); fasted
Monsanto Company with Cover Letter dated 062669, (1969), EPA Doc. No. 40-6942188, Fiche No. OTS0519234;
overnight; GLP
Monsanto Co./Bio/dynamics

Acetaminophen

1944

1944

NA

Litchfield andWilcoxon

Wistar rats; 130-150 g

stomach tube

5 mL/kg bw in 1%
carboxymethyl-cellulose

observed 3 weeks

fasted 18 hours before dosing

Acetaminophen

1944

2404

+/- 95(S.E.)

Miller and Tainter(1944)Charles River CD andSprague-Dawley rat strains; NA>100 g; adultoral intubation

up to 50 mL/kg

rats observed for 7 days; observed up to 14 days when heavy metals or
fasted overnight
other compounds that produce latent death were investigatedAcetonitrile

2460

157
79 - 236(0.2 mL/kg; sp. (95% CL; 0.1 - 0.3 Litchfield andSprague-Dawley rats; 16-50 male anddensity = 0.7857; mL/kg; sp. density = Wilcoxon method andg; 14 days
femaleconvert LD50 to
0.7857; convert probit analysis
mg/kg
LD50 to mg/kg)

oral

solvent used in undiluted form

animals observed for a week after medication

Acetonitrile

2460

1320
972 - 1799
(1.68 mL/kg; sp. (1.24 - 2.27 mL/kg;
density = 0.7839; sp. density = 0.7839; NA
convert LD50 to convert LD50 to
mg/kg)
mg/kg)

oral gastricintubation

undiluted cmpd; single dose

Acetonitrile

2460

male

oral gavage

Acetonitrile

2460

Carworth Farms Wistar or
male
Nelson albino rats; 90-112g

Acetonitrile

2460

1730

1100 - 2720

Carworth Farms Wistar or
femaleNelson albino rats; 90-112g

Acetonitrile2460> 2000NA

Acetonitrile

2460

2230

1900 - 2620

1453

1123 - 1879
(95% CL)NA

Kammerer F-J, Schleyerbach R. 1987. U.S. Patent 4,636,513. Isoxazole derivatives and medicaments
containing these compounds (January 13, 1987). (RTECS REFERENCE)

NA

Yeary RA, Benish RA, Finkelstein M. 1966. Acute Toxicity of Drugs in Newborn Animals. Journalof Pediatrics 69(4):663-667.
Dept. of Veterinary Preventive Medicine, Ohio State University, Columbus, OH

nonfasted rats; 6-12 rats of both
sexes used for studies; solvent usedin undiluted form

analytical grade
meeting A.C.S.
specifications

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, ILNA

non-fasted

Union Carbide
Pozzani UC, Carpenter CP, Palm PE, Weil CS, Nair JH. 1959. An investigation of the mammalian
Chemicals Company toxicity of acetonitrile. J Occup Med 1: 634-642. Mellon Institute, Pittsburgh, PA

single dose

14 day observation; toxicity symptoms: ptosis, posture, respiratory
effects, lethargy, ataxia, convulsions; time to onset of signs < 1 day;duration of signs 5 days; 5 rats dead (average per test)

3 dose levels (5 male each); 15 rats
used; OECD TG401 (1981) followed NAfor experimental proceduresoral gastricintubation

undiluted cmpd; single doseNA

non-fasted

Union Carbide
Pozzani UC, Carpenter CP, Palm PE, Weil CS, Nair JH. 1959. An investigation of the mammalian
Chemicals Company toxicity of acetonitrile. J Occup Med 1: 634-642. Mellon Institute, Pittsburgh, PA

oral gastricintubation

0.1 in corn oil; single doseNA

non-fasted

Union Carbide
Pozzani UC, Carpenter CP, Palm PE, Weil CS, Nair JH. 1959. An investigation of the mammalian
Chemicals Company toxicity of acetonitrile. J Occup Med 1: 634-642. Mellon Institute, Pittsburgh, PA

femaleoral gavage

single dose

14 day observation; toxicity symptoms: Ptosis, posture, respiratory
effects, lethargy, ataxia, convulsions; time to onset of signs < 1day;
duration of signs 5 days; 5 rats dead (average per test)

3 dose levels (5 female each); 15 rats
used; OECD TG401 (1981) followed
for experimental procedures

Carworth Farms Wistar or
Nelson albino rats; 30-54 g; female
weanlings

oral gastricintubation

0.1 in 1% aqueous Tergitol 7;
single doseNA

non-fasted

Carworth Farms Wistar or
male
Nelson albino rats; 90-112g

acceptable methods(e.g., Bliss, Litchfield
Sprague-Dawley ratsand Wilcoxon, Weil,
Thompson, etc.)

1623
1050 - 2524
(2.07 mL/kg; sp. (1.34 - 3.22 mL/kg;
density = 0.7839; sp. density = 0.7839; NA
convert LD50 to convert LD50 to
mg/kg
mg/kg

NA

acceptable methods(e.g., Bliss, Litchfield
Sprague-Dawley ratsand Wilcoxon, Weil,
Thompson, etc.)

NAmale andfemalefrom EPA TSCATS database; Acute Toxicity Study in Rats Administered 10 Materials (final report)
Monsanto Company with Cover Letter dated 062669, (1969), EPA Doc. No. 40-6942188, Fiche No. OTS0519234;
Monsanto Co./Bio/dynamics

H-6

Vandenheuvel MJ, Clark DG, Fielder RJ, Koundakjian PP, Oliver GJA, Pelling D, Tomlinson NJ,
Walker AP. 1990. Jul. The International Validation Of A Fixed-Dose Procedure As An Alternative To
The Classical LD50 Test Food And Chemical Toxicology 28(7):469-482.

Vandenheuvel MJ, Clark DG, Fielder RJ, Koundakjian PP, Oliver GJA, Pelling D, Tomlinson NJ,
Walker AP. 1990. Jul. The International Validation Of A Fixed-Dose Procedure As An Alternative
To The Classical LD50 Test Food And Chemical Toxicology 28: (7) 469-482.

Union Carbide
Pozzani UC, Carpenter CP, Palm PE, Weil CS, Nair JH. 1959. An investigation of the mammalian
Chemicals Company toxicity of acetonitrile. J Occup Med 1: 634-642. Mellon Institute, Pittsburgh, PA

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

Acetonitrile

2460

2340

2030 - 2700

NA

Carworth Farms Wistar or
femaleNelson albino rats; 90-112g

oral gastricintubation

0.1 in 1% aqueous Tergitol 7;
single doseNA

non-fasted

Union Carbide
Pozzani UC, Carpenter CP, Palm PE, Weil CS, Nair JH. 1959. An investigation of the mammalian
Chemicals Company toxicity of acetonitrile. J Occup Med 1: 634-642. Mellon Institute, Pittsburgh, PA

Acetonitrile

2460

2460

1600 - 2780

NA

Carworth Farms Wistar or
male
Nelson albino rats; 90-120g

oral gastricintubation

0.1 in water; single doseNA

non-fasted

Union Carbide
Pozzani UC, Carpenter CP, Palm PE, Weil CS, Nair JH. 1959. An investigation of the mammalian
Chemicals Company toxicity of acetonitrile. J Occup Med 1: 634-642. Mellon Institute, Pittsburgh, PA

Acetonitrile

2460

2460

NA

NA

rat

NA

oral

NA

NA

Duplicate record. Assumed to be thesame values from Pozzani et al.
NA(1959), Mellon Institute and Union
Carbide.

UCDS** Bibliographic Data: Union Carbide Data Sheet. (Union Carbide Corp., 39 Old RidgeburyRd., Danbury, CT 06817) (see Pozzani et al. 1959) (RTECS REFERENCE)

Acetonitrile

2460

2830

NA

Thompson (1947) andManor farms Wistar; 150Weil (1952); moving
200 g
average tables

male

oral; stomach
intubation

single dose; geometric factor
between dosage levels=2;
undiluted

14 day observation

5 rats per dosage level; fasted
overnight

Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388.
Mellon Institute, Pittsburgh, PA and The Dow Chemical Company, Midland, MI

Acetonitrile

2460

oral

solvent used in undiluted form

animals observed for a week after medication

nonfasted rats; groups of 6 rats used analytical grade
for the studies; solvent used inmeeting A.C.S.
undiluted form
specifications

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, IL

Acetonitrile

2460

Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388.
Mellon Institute, Pittsburgh, PA and The Dow Chemical Company, Midland, MI

Acetonitrile

3064
2593 - 3614
(3.9 mL/kg; sp. (95% CL; 3.3 - 4.6 Litchfield andSprague-Dawley rats; 80-160
density = 0.7857; mL/kg; sp. density = Wilcoxon method andmaleg; young adultconvert LD50 to
0.7857; convert probit analysis
mg/kg)
LD50 to mg/kg)

NA

Thompson (1947) andManor farms Wistar rats;
Weil (1952); moving
150-200 g
average tables

male

oral; stomach
intubation

single dose; geometric factor
between dosage levels=2;
undiluted

14 day observation

5 rats per dosage level; fasted
overnight

2460

3457
2200 - 5343
(4.4 mL/kg; sp. (95% CL; 2.8 - 6.8 Litchfield andSprague-Dawley rats; 300density = 0.7857; mL/kg; sp. density = Wilcoxon method and
470 g; older adultconvert LD50 to
0.7857; convert probit analysis
mg/kg)
LD50 to mg/kg)

male

oral

solvent used in undiluted form

animals observed for a week after medication

nonfasted rats; groups of 6 rats used analytical grade
for the studies; solvent used inmeeting A.C.S.
undiluted form
specifications

Acetonitrile

2460

3504
2187 - 5613
(4.47 mL/kg; sp. (2.79 - 7.16 mL/kg;
density = 0.7839; sp. density is 0.7839; NA
convert LD50 to convert LD50 to
mg/kg)
mg/kg)

Carworth Farms Wistar or
male
Nelson albino rats; 84-114 goral gastricintubation

undiluted cmpd; single doseNA

fastedUnion Carbide
Pozzani UC, Carpenter CP, Palm PE, Weil CS, Nair JH. 1959. An investigation of the mammalian
Chemicals Company toxicity of acetonitrile. J Occup Med 1: 634-642. Mellon Institute, Pittsburgh, PA

Acetonitrile

2460

3520
1419 - 8748
(4.49 mL/kg; sp. (1.81 - 11.16 mL/kg;
density = 0.7839; sp. density = 0.7839; NA
convert LD50 to convert LD50 to
mg/kg)
mg/kg)

Carworth Farms Wistar or
male
Nelson albino rats; 90-120g

oral gastricintubation

undiluted cmpd; single doseNA

non-fasted

Union Carbide
Pozzani UC, Carpenter CP, Palm PE, Weil CS, Nair JH. 1959. An investigation of the mammalian
Chemicals Company toxicity of acetonitrile. J Occup Med 1: 634-642. Mellon Institute, Pittsburgh, PA

Acetonitrile

2460

oral; stomach
intubation

single dose; geometric factor
between dosage levels=2;
undiluted

14 day observation

5 rats per dosage level; fasted
overnight

NA

Acetonitrile

2460

oral gastricintubation

undiluted cmpd; single doseNA

non-fasted

Union Carbide
Pozzani UC, Carpenter CP, Palm PE, Weil CS, Nair JH. 1959. An investigation of the mammalian
Chemicals Company toxicity of acetonitrile. J Occup Med 1: 634-642. Mellon Institute, Pittsburgh, PA

based on assumptionthat probit mortality
Sherman rats; 90 -120 g; 4-5
vs log dose has same
male
weeks
slope as similar
chemicaloral gastricintubation

in aqueous solution; doses (ing/kg) differ by 1 log to bracket
LD50, then refine LD50 with
LD50 based on mortalities during a 14 day period
doses in a series of antilog 1.1,
1.3, 1.5, etc

Litchfield andWilcoxon (1948)

oral

undiluted; 3220 - 4970 mg/kg
doses

observatons recorded frequently on the day of dosing, daily thereafterfor 14 days; tremors, clonic/tonic convulsions, weight loss; clinicalovernight fasted; groups of at least 5 99+%; Aldrichsigns appeared within 3 hour after dosing and progessed to death in 24- rats per dose
Chemical Co.
72 houroral; stomach
intubation

single dose; geometric factor
between dosage levels = 2;
undiluted

14 day observation

Acetonitrile

2460

3360

3570

NA

NA

Thompson (1947) andManor farms Wistar; 150Weil (1952); moving
200 g
average tables

3717
1921 - 6436
(4.49 mL/kg; sp. (2.45 - 8.21mL/kg;
density = 0.7839; sp. density = 0.7839; NA
convert LD50 to convert LD50 to
mg/kg)
mg/kg)

3800

Acetonitrile

2460

4050

Acetonitrile

2460

4240

NA

NA

male

Carworth Farms Wistar or
Nelson albino rats; 250 - 318 femaleg; yearlings

Sprague-Dawley rats; 175260 g

Thompson (1947) andManor farms Wistar rats;
Weil (1952); moving
150-200 g
average tables

male

H-7

NA

6 rats/dose at doses (in g/kg) thatdiffer by 1 log to bracket LD50
(given 1 week apart); then refined
LD50 with 10 rats/dose in a dosereagent grade
series of antilog 1.1, 1.3, 1.5, etc.;
assumed to use materials/methods ofSmyth & Carpenter (1944) except forreported changes. RC reference

5 rats per dosage level; fasted
overnight

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, IL

Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388.
Mellon Institute, Pittsburgh, PA and The Dow Chemical Company, Midland, MI

Smyth HF Jr, Carpenter CP. 1948. Further experience with the range finding test in the industrial
toxicology laboratory. J Ind Hyg Toxicol 30:63-68. (RC and 1983/84 RTECS LD50 value)
Smyth HF Jr, Carpenter CP. 1944. The place of the range-finding test in the industrial toxicology
laborotory. J Ind Hyg Toxicol 26:269-273. (most materials/methods)

Freeman JJ, Hayes EP. 1985. Acetone potentiation of acute acetonitrile toxicity in rats. J Toxicol
Environ Hlth 15:609-621. Rutgers University, Piscataway, NJ

Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388.
Mellon Institute, Pittsburgh, PA and The Dow Chemical Company, Midland, MI

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Acetonitrile

2460

4490

2460 - 8210

Acetonitrile

2460

4850

NA

Acetonitrile

2460

Acetonitrile

2460

5450

NA

Acetonitrile

2460

5900

4580 - 7220

2460

6498
(8.27 mL/kg; sp.
density = 0.7857;
convert LD50 to
mg/kg)

NA

6500

NA

NA

Animal Information(stock, weight, age)

Carworth Farms Wistar or
Nelson albino rats; 240-425 femaleg; yearlings

Thompson (1947) andManor farms Wistar rats;
Weil (1952); moving
150-200 g
average tables

5244
3222 - 8545
(6.69 mL/kg; sp. (1.34 - 3.22 mL/kg;
density = 0.7839; sp. density = 0.7839; NA
convert LD50 to convert LD50 to
mg/kg)
mg/kg)

Gendermale

Carworth Farms Wistar or
male
Nelson albino rats; 82-109 g

Route and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

oral gastricintubation

0.1 in 1% aqueous Tergitol 7;
single dose

oral; stomach
intubation

single dose; geometric factor
between dosage levels = 2;
undiluted

14 day observation

5 rats per dosage level; fasted
overnight

oral gastricintubation

undiluted cmpd; single doseNA

fastedUnion Carbide
Pozzani UC, Carpenter CP, Palm PE, Weil CS, Nair JH. 1959. An investigation of the mammalian
Chemicals Company toxicity of acetonitrile. J Occup Med 1: 634-642. Mellon Institute, Pittsburgh, PANA

non-fasted

Union Carbide
Pozzani UC, Carpenter CP, Palm PE, Weil CS, Nair JH. 1959. An investigation of the mammalian
Chemicals Company toxicity of acetonitrile. J Occup Med 1: 634-642. Mellon Institute, Pittsburgh, PA
Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388.
Mellon Institute, Pittsburgh, PA and The Dow Chemical Company, Midland, MI

Thompson (1947) andManor farms Wistar rats;
Weil (1952); moving
150-200 g
average tables

male

oral; stomach
intubation

single dose; geometric factor
between dosage levels = 2;
undiluted

14 day observation

5 rats per dosage level; fasted
overnight

NANA

oral; intragastricNANA(source of information not provided) NAoral gastricintubation

in aqueous solution;
concentration intubated = 10
mg/mL; dosages arranged in a LD50 based on mortalities during a 14 day period
logarithmic series differing by afactor of 2

non-fasted; groups of 5 rats; single
oral dose toxicity

oral; stomach
intubation

single dose; geometric factor
between dosage levels = 2;
undiluted

14 day observation

5 rats per dosage level; fasted
overnightNAoral gastricintubation

undiluted cmpd; single doseNA

non-fasted

Union Carbide
Pozzani UC, Carpenter CP, Palm PE, Weil CS, Nair JH. 1959. An investigation of the mammalian
Chemicals Company toxicity of acetonitrile. J Occup Med 1: 634-642. Mellon Institute, Pittsburgh, PANArats; 220 +/- 40 g

Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388.
Mellon Institute, Pittsburgh, PA and The Dow Chemical Company, Midland, MI

Izmerov NF, Sanotsky IV, Sidorov KK. 1982. Toxicometric Parameters of Industrial Toxic Chemicals
under Single Exposure. International Register of Potentially Toxic Chemicals (IRPTC). United
Nations Environment Programme (UNEP). Centre of International Projects, GKNT. Moscow, Russia.
Smyth HF, Weil CS, West JS, Carpenter CP. 1970. An exploration of joint toxic action:II. Equitoxic
versus equivolume mixtures. Toxicol Appl Pharmacol 17:498-503. (LD50 value)

Acetonitrile

Thompson method;
Weil tables

Carworth-Wistar rats; 90 120 g; 4 - 5 weeks

Thompson (1947) andManor farms Wistar; 150Weil (1952); moving
200 g
average tables

male

Smyth HF Jr., Carpenter CP., Weil CS., Pozzani, UC., Striegel, JA. And Nycum, JS. 1969. Rangefinding toxicity data: List VII. Am Ind Hyg Assoc J 30:470-476.
Carnegie-Mellon University, Pittsburgh, PA.
Smyth HF Jr., Carpenter CP., Weil CS., Pozzani, UC., and Striegel, JA. 1962. Range-finding toxicity
data: List VI. Am Ind Hyg Assoc J 23:95-107.
Mellon Institute of Industrial Research, Pittsburg, PA (experimental parameters).
Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388.
Mellon Institute, Pittsburgh, PA and The Dow Chemical Company, Midland, MI

Acetonitrile

2460

Acetonitrile

2460

Acetonitrile

2460

8120

NA

Thompson (1947) andManor farms Wistar rats;
Weil (1952); moving
150-200 g
average tables

male

oral; stomach
intubation

single dose; geometric factor
between dosage levels = 2;
undiluted

14 day observation

5 rats per dosage level; fasted
overnight

Acetylsalicylic acid

200

200

NA

NA

NA

NA

NA

NA

NA

RTECS reference for 200 mg/kg(from Deichman 1969) is a typo; thisis a secondary reference which cites NA
Caldwell and Boyd 1966; the value
should be 920 mg/kg.

Toxicology of Drugs and Chemicals. Deichmann, W.B., New York, Academic Press, Inc., 1969
(RTECS REFERENCE)

Acetylsalicylic acid

200

616

+/- 46(S.E.)

Miller and Tainter(1944)

FDRL-strain rats; 100 days

femaleoral

NAobserved for 7 days post-treatment

NA

NA

Weinberg MS, Goldhamer RE, Carson S. 1966. Acute oral toxicity of various drugs in newborn rats
after treatment of the dam during gestation. Toxic Appl Pharmac 9:234-239.
Food and Drug Research Laboratories, Inc., Maspeth, NY

Acetylsalicylic acid

200

920

+/- 43(S.E.)

Croxton (1953) and
Waugh (1952)

Wistar albino rats; 213 +/16 g; 3-5 months

femalesingle dose; suspension of cmpd
in 0.2% gum tragcanth solution
in distilled water; 15 mL/kg
within 1 hour of dosing rats were drowsy, withdrawn, hearing andoral; stomach tube dose; dose (mg/kg), rats per
vision impared, confused, tense, liquid stool, nasal bleeding,dose: 0-14; 750-10; 875-10;
convulsionsrespiratory failure, cardiovascular shock
1000-10;1125-10; 1250-2; 15002; 2000-2

fasted overnight (16 hour); 60 rats
used; 26/46 rats dead fromcompound

USP grade

Boyd EM. 1959. The acute oral toxicity of acetylsalicylic acid. Toxic Appl Pharmac 1: 229-239.
Queen's University, Ontario, Canada

Acetylsalicylic acid

200

1360

NA

Reed and Muench(1938)

Wistar albino rats

male andsingle dose; solution in 2%
female (75% oral; stomach tube acaia in physiological saline;
male)
volume of dose is 10 mL/kg

Acetylsalicylic acid

200

1430

1065 - 1921
(95% CL)

Litchfield andWilcoxon method(1949)

HLA strain albino rats; 95180 g (mean wt. 122 g)maleoral intubation

10-20 mL/kg in 10% gum
acacia suspension; 4 doses

toxic effects included neurological abnormality; LD50 at 168 hour
(7days); same result at 96 hour; observed at 24 & 48 hour with higher
LD50

rats fasted 15-17 hours before dosing
and for 6 hours after intubation; 40 NA
rats used (10/dose)

Boxill GC, Nash CB, Wheeler AG. 1958. Comparative pharmacological and toxicological evaluationof N-acetyl-p-aminophenol, salicylamide, and acetylsalicylic acid. J Am Pharm Assoc 47:479-487.

Acetylsalicylic acid

200

1430

1065 - 1921
(95% CL)

Litchfield andWilcoxon method(1949)

HLA strain albino rats; 95180 g (mean wt. 122 g)maleoral intubation

10-20 mL/kg in 10% gum
acacia suspension; 4 doses

toxic effects included neurological abnormality; this LD50 at 96 hour(same as 158 hour); observed at 24 & 48 hour with higher LD50

rats fasted 15-17 hours before dosing
and for 6 hours after intubation; 40
rats used (10/dose)

Boxill GC, Nash CB, Wheeler AG. 1958. Comparative pharmacological and toxicological evaluationof N-acetyl-p-aminophenol, salicylamide, and acetylsalicylic acid. J Am Pharm Assoc 47:479-487.

6687
4797 - 9328
(8.53 mL/kg; sp. (6.12 - 11.9 mL/kg;
density = 0.7839; sp. density = 0.7839; NA
convert LD50 to convert LD50 to
mg/kg)
mg/kg)

malereagent grade

Carworth Farms Wistar or
femaleNelson albino rats; 90-114 gobserved for one week; more than 80% of fatalities occurred within 48
182 rats used; fasted for 18 hour
hour

H-8

Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388.
Mellon Institute, Pittsburgh, PA and The Dow Chemical Company, Midland, MI

Eagle E, Carlson AJ. 1950. Toxicity, antipyretic and analgesic studies on 39 compounds including
G.D. Searle and Co. aspirin, phenacetin and 27 derivatives of carbazole and tetrahydrocarbazole. J Pharm Exp Ther
99:450-457. University of Chicago, Chicago, IL

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

Acetylsalicylic acid

LD501
mg/kg

200

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference1459(value converted
from mM/kg to
mg/kg)

1009 - 2108
(95% CL)

Weil (1952)

Animal Information(stock, weight, age)

Homozygous Gunn rat(Gunn strain bred frommutant Wistar stock); 137230 g

Gendermale

Route and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

single dose; solution in 0.5 1.0% (w/v) aqueous methyl
low dose experiment observed for 3 days; high dose observed for 7
cellulose; 10 mL/kg dose vol.;days; LD50 determined at 7 days; dose (mg/kg), rats dead per dose:
fasted overnight (16 hour); 6 rats peroral; gastric lavage low dose (mg/kg): 176.6, 281.1,
NA
176.6-0/6; 281.1-0/6; 450.4-0/12; 720.7-1/12; 1153.1-1/12; 1844.9-5/6; dose; 60 rats used
450.4, 720.7, 1153.1; high dose
2951.2-5/6(mg/kg): 450.4, 720.7, 1153.1,

Primary Reference

Axelsen RA. 1976. Analgesic-induced renal papillary necrosis in the Gunn rat: the comparative
nephrotoxicity of aspirin and phenacetin. J Path 120:145-150.
University of Queensland, Queensland, Australia

1844.9, 2951.2

Acetylsalicylic acid

200

1500

NA

determinedgraphically

rats

NA

oral; stomach tube

aqueous with gum ragacanth(cmpd at 5 - 10% concen)

rats dead within 48 hours considered for determination of LD50

15 rats used

NA

Hart ER. 1947. The toxicity and analgetic potency of saliccylamide and certain of its derivatives ascompared with established analgetic-antipyretic drugs. J Pharmacol Exp Ther 89:205-209.
Jefferson Medical College, Philadelphia, PA

Acetylsalicylic acid

200

1500

NA

Litchfield andWilcoxon

Wistar rats; 130-150 g

male andfemalestomach tube

5 mL/kg bw in 1%
carboxymethylcellulose

observed 3 weeks

Fasted 18 hour before dosing

NA

Kammerer F-J, Schleyerbach R. 1987. U.S. Patent 4,636,513. Isoxazole derivatives and medicaments
containing these compounds (January 13, 1987). (RTECS REFERENCE)

Acetylsalicylic acid

200

1523

NA

NA

Upjohn Sprague-Dawley
strain albino rats; ~140 g;
young

male

oral

fasted overnight (12+ hour); 5 rats
per dose; 65 rats used

NA

Gray JE, Jones PM, Feeenstra ES. 1960. Comparative effect of acetylsalicylic acid and acetylsalicylic
acid anhydride on the non-glandular portion of the stomach. Toxic Appl Pharmac 2:514-522.
The Upjohn Company, Kalamazoo, MI

NAYeary RA, Benish RA, Finkelstein M. 1966. Acute Toxicity of Drugs in Newborn Animals. Journalof Pediatrics. 69 (4):663-667.
Dept. of Veterinary Preventive Medicine, Ohio State University, Columbus, OH

single dose; cmpd suspended in
1% aqueous
observed for 7 days post-treatment; most deaths occurred during thecarboxymethylcellulose; 13
first day; frequently, animals observed in convulsions prior to deathdose groups from 400 - 2500
mg/kg

Acetylsalicylic acid

200

1528

+/- 156(S.E.)

Miller and Tainter(1944)Charles River CD andSprague-Dawley rat strains; NA> 100 goral intubationdose up to 50 mL/kg

rats observed for 7 days; observed up to 14 days when heavy metals or
fasted overnight
other compounds that produce latent death were investigatedAcetylsalicylic acid

200

1600

1194 - 2144
(95% CL)

Litchfield andWilcoxon method(1949)

HLA strain albino rats; 95180 g (mean wt. 122 g)maleoral intubation

10-20 mL/kg in 10% gum
acacia suspension; 4 doses

toxic effects included neurological abnormality; this LD50 at 24 hour(same as 48 hour); observed at 96 & 168 hour with lower LD50

rats fasted 15-17 hours before dosing
and for 6 hours after intubation; 40 NA
rats used (10/dose)

Boxill GC, Nash CB, Wheeler AG. 1958. Comparative pharmacological and toxicological evaluationof N-acetyl-p-aminophenol, salicylamide, and acetylsalicylic acid. J Am Pharm Assoc 47:479-487.

Acetylsalicylic acid

200

1600

1194 - 2144
(95% CL)

Litchfield andWilcoxon method(1949)

HLA strain albino rats; 95180 g (mean wt. 122 g)maleoral intubation

10-20 mL/kg in 10% gum
acacia suspension; 4 doses

toxic effects included neurological abnormality; this LD50 at 48 hour(same as 24 hour); observed at 96 & 168 hour with lower LD50

rats fasted 15-16 hours before dosing
and for 6 hours after intubation; 10 NA
rats used

Boxill GC, Nash CB, Wheeler AG. 1958. Comparative pharmacological and toxicological evaluationof N-acetyl-p-aminophenol, salicylamide, and acetylsalicylic acid. J Am Pharm Assoc 47:479-487.

Acetylsalicylic acid

200

1761

+/- 162(S.E.)

Miller and Tainter(1944)

FDRL-strain rats; 100 days

male

oral

NA

observed for 7 days post-treatment

NA

NA

Weinberg MS, Goldhamer RE, Carson S. 1966. Acute oral toxicity of various drugs in newborn rats
after treatment of the dam during gestation. Toxic Appl Pharmac 9:234-239.
Food and Drug Research Laboratories, Inc., Maspeth, NY

Acetylsalicylic acid

200

1880

1528 - 2312
(95% CL; slope =1.27)

Litchfield andWilcoxon method(1949)

Wistar SPF rats; 150-200 g

femaleoral

cmpd suspended in a solution of
10% gum arabic in distilled
observed for 7 days post-treatment
water

10 animals per dose

NA

Zapatero J, Sanfeliu C, Bruseghini L. 1981. Toxicological studies of Plafibride Part 1: Acute toxicity
and its determination after several administrations of plafibride. Arsneim Forsch 31:1816-1819.
Chemical Pharmaceutical Research Centre, Barcelona, Spain

Acetylsalicylic acid

200

1960

1441 - 2666
(95% CL; slope =1.64)

Litchfield andWilcoxon method(1949)

Wistar SPF rats; 150-200 g; maleoral

cmpd suspended in a solution of
10% gum arabic in distilled
observed for 7 dayswater

10 animals per dose

NA

Zapatero J, Sanfeliu C, Bruseghini L. 1981. Toxicological studies of Plafibride Part 1: Acute toxicity
and its determination after several administrations of plafibride. Arsneim Forsch 31:1816-1819.
Chemical Pharmaceutical Research Centre, Barcelona, Spain

Acetylsalicylic acid

200

1992

1692 - 2345
(95% CL; slope =1.45)

Litchfield andWilcoxon method(1949)

Wistar SPF rats; 150-200 g;

oral

cmpd suspended in a solution of
10% gum arabic in distilled
observed for 7 days post-treatment
water

10 animals per dose

NA

Zapatero J, Sanfeliu C, Bruseghini L. 1981. Toxicological studies of Plafibride Part 1: Acute toxicity
and its determination after several administrations of plafibride. Arsneim Forsch 31:1816-1819.
Chemical Pharmaceutical Research Centre, Barcelona, Spain

Acetylsalicylic acid200

> 2000Litchfield andWilcoxon method(1949)

Sprague-Dawley SPF rats
(Charles River, France); 100- male
110 g

oral

suspended in 0.25%
carboxymethylcellulose with
0.2% polysorbate 80; doses ingeometrical progression

observed for 7 days post-treatment; rats presented no signs

10 animals per dose; fasted 6 h priorNA
to dosing

Glomot R, Chevalier B, Vannier B. 1976. Toxicological studies on floctafenine. Toxicol Appl
Pharmac 36:173-185.

Acetylsalicylic acid200

> 2000Litchfield andWilcoxon method(1949)

Sprague-Dawley SPF rats
(Charles River, France); 100- female
110 g

oral

suspended in 0.25%
carboxymethylcellulose with
0.2% polysorbate 80; doses ingeometrical progression

observed for 7 days post-treatment; rats presented no signs

10 animals per dose; fasted 6 h priorNA
to dosing

Glomot R, Chevalier B, Vannier B. 1976. Toxicological studies on floctafenine. Toxicol Appl
Pharmac 36:173-185.

Acetylsalicylic acid

200

2840

2075 - 3890
(95% CL)

Litchfield andWilcoxon method(1949)

Sprague-Dawley CD strain
albino rats

oral; gavage

single dose; 5 mL/kg dose; min.
of 3 dose levels; cmpd
observed for 7 days post-treatment; LD50 based on number of deaths at 20 animals per dose level; 60 animals Aldrich Chemicalsuspended in solution of 1%
7 daysused; not fasted
Company
gum acacia vehicle

Aminopterin

NA

7

NA

Maximum likelihood
estimation using log
male andWistar albino rats; 100-200 g
probit model (BMDS
femaleby US EPA)

oral

observed for 14 days; deaths delayed until 3rd day; moderate weightused measured samples
loss by 1st day; intoxicated animals lost 20% by 3rd day; severe,
neutralized before drying or
watery diarrhea after 48 hour; yellowish brown feces, terminally,
LD50 calculated by NICEATM; 36
added 2 molar eq NaHCO3 to grossly stained with blood; deaths/dose: 40 mg/kg-5/6 (3 at 3-4 days, 2
rats usedweighed amounts of free acid; at 5-7 days), 20 mg/kg-5/6 (2 at 3-4 days, 2 at 5-7 days, 1 at 8-14 days),in 09% NaCl at 1 mL/100 g bw 10 mg/kg-4/6 (3 at 3-4 days, 1 at 5-7 days); 5 mg/kg-2/6 (1 at 3-4 days,
1 at 8-14 days), 2.5 mg/kg-2/6 (2 at 3-4 days), 1.25 mg/kg-0/6

Amitriptyline

320

320

286 - 359

Litchfield andWilcoxon method(1949)

oral

NA

rats

male andfemalemaleNAlethality counted after 7 days

H-9

Sofia RD. 1977. Alteration of hepatic microsomal enzyme systems and the lethal action of nonsteroidal anti-arthritic drugs in acute and chronic models of inflammation. Agents and Actions 7: 289297. Wallace Laboratories, Cranbury, NJ

ampuled and bulk
Philips FS, Thiersch JB. 1949. Studies of the actions of 4-amino-pteroylglutamic acid in rats andsamples frommice. J Pharmacol Exp Ther 95:303-311.
Lederle Laboraotries

40-50 rats used; reference in German NA

Ribbentrop VA, Schaumann W. 1965. Pharmakologische Untersuchungen mit Doxepin, einem
Antidepressivum mit zentral anticholinerger und sedierender Wirkung. Arzneimittel-Forschung
15:863-868. Aus den Pharmakologischen Laboratorien der Firma C.F. Boehringer & Soehne
Gmbh, Mannheim, Germany (RTECS REFERENCE)In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Litchfield andWilcoxon method(1949)

Wistar strain rats; 200 -300 g male

oral

NA

NA

rats

NA

NA

rats

Amitriptyline

320

380

300 - 486
(95% CL)

Arsenic III trioxide

14.6

13

Arsenic III trioxide

14.6

14.6

Arsenic III trioxide

14.6

Arsenic III trioxide

14.6

32.6

28.4 - 36.7
(95% CI)

Finney (1971). Probit
NA
Analysis.

Arsenic III trioxide

14.6

81.5

70.5 - 94.3

Bliss-Probit method

Sprague-Dawley rats; 5
weeks+/- 2.4
19.9(reported as +/- 1.8 de Beer (1945)(15.1 mg As/kg)
mg As/kg)

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

72 hour observations

8 rats per group used; hydrochlorideNA
salt

Tobe A, Yoshida Y, Ikoma H, Tonomura S, Kikumoto R. 1981. Pharmacological evaluation of 2-(4methylaminobutoxy)diphenylmethane hydrochloride (MCI-2016), a new psychotropic drug withantidepressant activity. Arzneimittelforschung 31(8):1278-85.

oral; stomach tube NA

violent gastroenteritis, diarrhea, rice water stools

information from the laboratories ofDivision of Pharmacology, U.S.
NA
FDA.; fasted animals

Lehman AJ. 1951. Chemicals in Foods: a report to the association of food and drug officials on
current developments. Part II. Pesticides. Quarterly Bulletine (Association of Food and Drug Officialsof the United States). Vol.15:122-133. U.S. FDA

oral

NA

no clinical picture givenreference is in Russian; not translated NA

Tulakino NV, Novikov JV. 1987. On the question of reglameutation of arsenic in drinking water ofdifferent hardness. Gigiena i Sanitariya. 52 (1):21 -24. (RTECS REFERENCE)

oral; intraesophageallypure arsenic trioxide dissolved observed over 96 hours for LD50; experiment lasted 2 weeks; norats fasted 24 hour before dosing; 5
in distilled water; 0.03 mL per g significance between male or female; 95 dead at 24 hour; No ofgroups of 30 rats each (150 total);of bw; max volume 8 mL; dose deaths/dose at 96 hour (male): 10 mg As/kg - 9/30; 20 mg As/kg male and female rats tested; results
range 10 - 50 mg As/kg
20/30; 30 mg As/kg - 27/30; 40 mg As/kg - 28/30; 50 mg As/kg - 30/30 and information given for male

male

intubated; single
dose

dissolved in distilled water;
administered by gavage involume of 2mL/kg

rats dosed with one of 5 or 6 doses of chemical; deaths recorded dailyfor 7 days

maleoral gavage

dissolved in saline; range(mg/kg) of doses 51.2, 66.5,
86.5, 112.5, 146.2

rats observed at 6 hours after dosing and a once a day for 1 - 2 weeks; animals acclimated to environment
most rats found dead within 3 days; 27 of 50 rats died; toxic symptoms: for 1 week before testing; 5 groups
Kishida Chemical
vomiting and diarrhea; No of deaths/dose (mg As/kg) at 14 days: 51.2 of10 rats each; fasted 16 hours before
Co., Ltd.
mg - 0/10; 66.5 mg - 2/10; 86.5 mg - 6/10; 112.5 mg - 9/10; 146.2 mg - dosing; 100% lethal dose = 143.2
10/10
mg/kg; 0% lethal dose = 51.2 mg/kg

oral gavage

chemical suspended in 10%
acacia solution; received
appropriate doses in 1mL per
100 g bw

a number of individual doses oforal, stomach tube; a cmpd, each dose at a different
enteritis and neuritis
single doseconc level are given to an equal
number of test animals

male

Sprague-Dawley Albino rats;male
125 - 200 g

Arsenic III trioxide

14.6

138

+/- 13(standard error)

Litchfield and Fetig(1941)

wild Norway rats (trapped inmale and
Baltimore, MD); 148-493 g
female(ave = 253 g), adult

Arsenic III trioxide

14.6

140

NA

statistical formulabased on mortality
rates

wild Norway rats

Arsenic III trioxide

14.6

191.8
(145.2 mg
As/kg)

Arsenic III trioxide

14.6

385

350 - 424
(95% CL)

Atropine sulfate

600

600

Atropine sulfate

600

Atropine sulfate

Atropine sulfate

unknownNArats survived from 6 - 72 hours

99.999% pure

Harrison JWE, Packman EW, Abbott DD. 1958. Acute oral toxicity and chemical and physicalproperties of arsenic trioxides. AMA Arch ind Health, 17:118-123. LaWall and Harrison ResearchLaboratories

animals acclimated to environmentfor 2 weeks before testing; used only
Mallinckrodt
healthy rats; all rats assigned to oneof 5 to 6 groups of 8 to 10 rats eachPryor GT, Uyeno ET, et al. 1983. "Assessment of chemicals using a battery of neurobehavioral tests:
a comparative study." Neurobehav Toxicol Teratol 5(1): 91-117.
SRI International, Menlo Park, CA; NIEHS, Research Triangle Park, NC

Kitagawa H, Saito H, Sugimoto T, Yanaura S, Kitagawa H, Hosokawa T, Sakamoto K. 1982. Effects
of diiospropyl-1,3-dithiol-2-ylidene malonate (NKK-105) on acute toxicity of various drugs andheavy metals. J Toxicol Sci 7(2):123-34.
Chiba University; Hoshi College of Pharmacy; Showa University -- Japan

41 rats used (approx. equal numberof male and female); overnight
fasting before dosing; assays
performed in winter, repeated in
Merck U.S.P.
summer; LD50s from combined
information; final LD50 higher thanwinter LD50; attributed to not having
enough rats in winter.

Dieke SH, Richter CP. 1946. Comparative assays of rodenticides on wild Norway rats. I. Toxicity.
Publ. Health Rep 61:672-679. Johns Hopkins Hospital, Baltimore, MD

NA

NA

Peardon DL, Kilbourn E, et al. 1972. "New selective rodenticides." Soap Cosmet Chem Spec
48(12):6.
Rohm and Haas Company, Philadelphia, PA

99.999% pure

Harrison JWE, Packman EW, Abbott DD. 1958. Acute oral toxicity and chemical and physicalproperties of arsenic trioxides. AMA Arch ind Health 17:118-123.
LaWall and Harrison Research Laboratories

Sprague-Dawley Albino rats;male
125- 200 g

oralpure arsenic trioxide
incorporated into 3 g rat Purina
chow; rats consumed meal in 1
hour; dose range 301 - 338 mg
As/kg

observed over 96 hours for LD50; 2 week experiment; no significancebetween male or female; 76 dead at 24 hour; No of deaths/dose (mgAs/kg) at 96 hour: 301 mg - 0/20; 91 mg - 2/20; 1281 mg - 6/20; 1809
mg -12/20; 2078 mg -18/20; 269 mg - 20/20; 338 mg - 20/20

rats fasted 24 hour before dosing; 7groups of 20 rats each (140 total);
male and female rats tested; results
and information given for male

Litchfield andWilcoxon method

Holtsman rats; 300 -500 g;
100-300 days old (13 - 41
weeks)

male andfemaleoral; gelatin
capsules20, 50, 100, 250, 500, 750,
1000 (all in mg/kg)

rats dosed under light anesthesia; death occurred within 4 days

Baker Analyzed
approximately 70 rats used; 24 hour
Done AK and Peart AJ. 1971. Acute Toxicities of Arsenical Herbicides. Cinical Toxicology,
Reagent with 0.02%
fasting before dosing
4(3):343 - 355. University of Utah, Salt Lake City, UT
impurities

530 - 675

Litchfield andWilcoxon method

rats

NA

oral

NA

NA

reference in German622+/- 36NA

Sprague-Dawley rats; fromCharles River; adult

male

oral

NA

NA

information from: drug applications
from pharmaceutical manufacturers, NA
the literature, and FDA labs

600

698.7

629.2 - 776.0

Bliss-Probit method

Sprague-Dawley rats; 5
weeks

male

oral gavage

rats observed at 6 hours after dosing and a once a day for 1 - 2 weeks; animals acclimated to environment
most rats dead within 3 days; 20 of 40 rats died; toxic symptoms:
for 1 week before testing; 4 groups
dissolved in saline; range
decrease of spontaneous movement, myasthenia and coma observed at of 10 rats each; fasted 16 hours(mg/kg) of doses 500, 625, 781,
10 minutes; stretching of the limbs, abdominal posture, anaerosis and before dosing; 100% lethal dose =977
cardiac arrest after convulsions; dose (mg/kg), dead rats per dose: 500 -- 977 mg/kg; 0% lethal dose = 500
1/10; 625 -- 4/10; 781 -- 6/10; 977 -- 10/10
mg/kg

600

840

NA

Rosiello (1979) andBliss (1938)

rats

male

oral

200, 400, 800, 1000, 1600
mg/kg+/- 11.5(reported as +/- 8.7 de Beer (1945)
mg As/kg)

NA

Goldenthal EI. 1971. A compilation of LD50 values in newborn and adult animals. Toxicology andApplied Pharamacology 18:185 -207. Bureau of Drugs, Food and Drug Administration, Dept. of
Health, Education, and Welfare, Rockville, MD.

Kitagawa H, Saito H, Sugimoto T, Yanaura S, Kitagawa H, Hosokawa T, Sakamoto K. 1982. Effects
Tokyo Kasei Kogyo of diiospropyl-1,3-dithiol-2-ylidene malonate (NKK-105) on acute toxicity of various drugs andCo.
heavy metals. J Toxicol Sci 7(2):123-34.
Chiba University; Hoshi College of Pharmacy; Showa University -- Japanrats acclimated for 5 days; rats
200 mg/kg: 0/3 dead; 400 mg/kg: 0/3 dead; 800 mg/kg: 1/3 dead; 1600 observed for 14 days; 4 groups of
mg/kg: 3/3 dead; 4 of 12 rats dead; LD50 based on 12 rats used; LD50 rats used for each dose (1, 2, 3, 5 rats
recalculated using US EPA Benchmark Dose software; Lorke used data per group; 11 rats per dose); 9 rats
NAfrom 1000 mg/kg in range finder for all animal groups; omitted thisfor initial range finding;10 mg/kg data in recalculation; orginial LD50 from Lorke = 900 mg/kg
0/3 dead; 100 mg/kg - 0/3 dead; 1000
mg/kg - 2/3 deadH-10

Wirth W, Gosswald R. 1965. Pharmakologische Untersuchungen in der Reihe derDiphenylcarbamidsaurethioester. Arch Int Pharmacodyn 155 (2):393 - 417. (RTECSREFERENCE)

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

oral

200, 400, 800, 1000, 1600
mg/kg

rats acclimated for 5 days; rats
200 mg/kg: 0/5 dead; 400 mg/kg: 0/5 dead; 800 mg/kg: 1/5 dead; 1600 observed for 14 days; 4 groups of
mg/kg: 5/5 dead; 6 of 20 rats dead; LD50 based on 20 rats used; LD50 rats used for each dose (1, 2, 3, 5 rats
recalculated using US EPA Benchmark Dose software; Lorke used data per group; 11 rats per dose); 9 rats
NAfrom 1000 mg/kg in range finder for all animal groups; omitted thisfor initial range finding;10 mg/kg data in recalculation; orginial LD50 from Lorke = 950 mg/kg
0/3 dead; 100 mg/kg - 0/3 dead; 1000
mg/kg - 2/3 dead

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

oral

200, 400, 800, 1000, 1600
mg/kg

rats acclimated for 5 days; rats
200 mg/kg: 0/11 dead; 400 mg/kg: 0/11 dead; 800 mg/kg: 2/11 dead;observed for 14 days; 4 groups of1600 mg/kg: 11/11 dead; 13 of 44 rats dead; LD50 based on 44 rats
rats used for each dose (1, 2, 3, 5 rats
used; LD50 recalculated using US EPA Benchmark Dose software;
per group; 11 rats per dose); 9 rats
NA
Lorke used data from 1000 mg/kg in range finder for all animal groups;
for initial range finding;10 mg/kg omitted this data in recalculation; orginial LD50 from Lorke = 900
0/3 dead; 100 mg/kg - 0/3 dead; 1000
mg/kg
mg/kg - 2/3 dead

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

oral

200, 400, 800, 1000, 1600
mg/kg

rats acclimated for 5 days; rats
200 mg/kg: 0/1 dead; 400 mg/kg: 0/1 dead; 800 mg/kg: 0/1 dead; 1600 observed for 14 days; 4 groups of
mg/kg: 1/1 dead; 1 of 4 rats dead; LD50 based on 4 rats used; LD50
rats used for each dose (1, 2, 3, 5 rats
recalculated using US EPA Benchmark Dose software; Lorke used data per group; 11 rats per dose); 9 rats
NAfrom 1000 mg/kg in range finder for all animal groups; omitted thisfor initial range finding;10 mg/kg data in recalculation; orginial LD50 from Lorke = 950 mg/kg
0/3 dead; 100 mg/kg - 0/3 dead; 1000
mg/kg - 2/3 dead

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

rats acclimated for 5 days; rats
200 mg/kg: 0/2 dead; 400 mg/kg: 0/2 dead; 800 mg/kg: 0/2 dead; 1600 observed for 14 days; 4 groups of
mg/kg: 2/2 dead; 2 of 8 rats dead; LD50 based on 8 rats used; LD50
rats used for each dose (1, 2, 3, 5 rats
recalculated using US EPA Benchmark Dose software; Lorke used data per group; 11 rats per dose); 9 rats
NAfrom 1000 mg/kg in range finder for all animal groups; omitted thisfor initial range finding;10 mg/kg data in recalculation; orginial LD50 from Lorke = 950 mg/kg
0/3 dead; 100 mg/kg - 0/3 dead; 1000
mg/kg - 2/3 dead

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

Pfeiffer CC, Hallman LF, Gersh IG. 1945. Boric Acid Ointment. A study of possible intoxication in
the treatment of burns. Journal of the American Medical Association 128:266 - 274.
National Naval Medical Center, Bethesda, MD (RTECS REFERENCE)

Atropine sulfate

600

874

NA

Rosiello (1979) andBliss (1938)

Atropine sulfate

600

878

NA

Rosiello (1979) andBliss (1938)

NA

Rosiello (1979) andBliss (1938)

NA

Rosiello (1979) andBliss (1938)

rats

male

oral

200, 400, 800, 1000, 1600
mg/kg+/- 220
Litchfield and Fetig(S.E.; slope = 7.7) (1941)

rats

NA

oral

NA

NA

45 rats used(source of information not provided);NAreference in Russian;Atropine sulfate

600

1135

rats

male

rats

male

rats

male

Primary Reference

Atropine sulfate

600

1136

Boric acid

2662

2660

Boric acid

2662

2660

+/- 200(S.E.)

NA

rats; 220 +/- 40 goral; intragastric

NA

NA

Boric acid

2662

3160
(estimate)

NA

NALong Evans rats from Diablo
male
Laboratories; 85-118 goral; stomach
intubation

50% w/v in distilled watersuspension

observed for 14 days; signs included depression, ataxia, convulsion and fasted rats; 6 groups of 5 rats each;death
total of 30 rats

NA

Weir RJ Jr, Fisher RS. 1972. Toxicologic studies on borax and boric acid. Toxicol Appl Pharmac
23:351-364.

Boric acid

2662

3450

2950-4040(CL)

NA

Albino Sprague-Dawley rats
(Charles River SPF); 267male
310 goral; stomach
intubation

50% w/v in 0.5% aqueous
methylcellulose suspension

observed for 14 days; signs included depression, ataxia, convulsion and fasted rats; 6 groups of 5 rats each;death
total of 30 rats

NA

Weir RJ Jr, Fisher RS. 1972. Toxicologic studies on borax and boric acid. Toxicol Appl Pharmac
23:351-364.

Boric acid

2662

4080

3640-4560(CL)

NA

Albino Sprague-Dawley rats
(Charles River SPF); 206female
248 goral; stomach
intubation

50% w/v in 0.5% aqueous
methylcellulose suspension

observed for 14 days; signs included depression, ataxia, convulsion and fasted rats; 6 groups of 5 rats each;death
total of 30 rats

NA

Weir RJ Jr, Fisher RS. 1972. Toxicologic studies on borax and boric acid. Toxicol Appl Pharmac
23:351-364.

Boric acid

2662

5140

4750 - 5580
(range is +/- 1.96
S.D.)

Thompson method;
Weil tables

Carworth-Wistar rats; 90120 g; 4-5 weeks

maleoral gastricintubation

in aqueous solution;
concentration intubated = 200
mg/mL; dosages arranged in a LD50 based on mortalities during a 14 day period
logarithmic series differing by afactor of 2

non-fasted; groups of 5 rats; single
oral dose toxicity

NA

NA

NA

NA

NA

Value used by RC from 1983/84
RTECS. No rat oral LD50 in current
RTECS. This study treated rats with NA
0.13 mg/kg busulfan, which was 7%
LD50. LD50 = 1.9 mg/kg

Schmahl D, Osswald H. 1970. Experimental studies on the carcinogenic effects of anticancer
chemotherapeutics and immunosuppressive agents. Arzneimittelforschung. Oct;20(10):1461-1467.

NA

Izmerov NF, Sanotsky IV, Sidorov KK. 1982. Toxicometric Parameters of Industrial Toxic Chemicals
under Single Exposure. International Register of Potentially Toxic Chemicals (IRPTC). United
Nations Environment Programme (UNEP). Centre of International Projects, GKNT. Moscow,
Russia.

Smyth HF Jr, Carpenter CP, Weil CS, Pozzani UC, Striegel JA, Nycum, JS. 1969. Range-finding
toxicity data: List VII. Am Ind Hyg Assoc J 30: 470-476.
Carnegie-Mellon University, Pittsburgh, PA (LD50 value)
reagent grade
Smyth HF Jr, Carpenter CP, Weil CS, Pozzani UC, Striegel JA. 1962. Range-finding toxicity data:
List VI. Am Ind Hyg Assoc J 23:95-107.
Mellon Institute of Industrial Research, Pittsburg, PA (experimental parameters)

Busufan110(mouse) no
rat oral
value

2

NA

NA

Busufan110(mouse) no
rat oral
value

14

6
(SE)

probit method Finney JO13 strain rats; 170-250 g; male and(1962)
10-12 weeks
femaleoral

as aqueous emulsion withtragacanth powder

30 day observation

fasted rats; rats from CEN Breeding
Centre Mol, Belgium from former L NA
strain of Institute of Cancer

Dunjic A, Cuvelier A-M. 1973. Survival of rat bone marrow cells after treatment with Myleran andEndoxan. Experimental Hematology 1:11-21.

Busufan110(mouse) no
rat oralvalue

28

21 - 38(95% CL)NA

Sprague-Dawley strain rats

male

oral

doses (mg/kg): 20, 30, 40, 50,
100, 150, 200

observed for 14 days; doses (mg/kg, deaths at 14 days: 20 -- 1/5; 30 -2/5; 40, 50, 100, 150, and 200 -- 5/5

5 rats per dose; 35 rats used

NA

Kiso to Rinsho. Clinical Report. 1971. (Yubunsha Co., Ltd., 1-5, Kanda Suda-Cho, Chiyoda-ku, KS
Bldg., Tokyo 101, Japan. 5(12): 1894. (RTECS REFERENCE)

Busufan110(mouse) no
rat oralvalue

29

23 - 38(95% CL)NA

Sprague-Dawley strain rats

femaleoral

doses (mg/kg): 10, 30, 40, 50,
100, 150, 200

observed for 14 days; doses (mg/kg, deaths at 14 days: 10 -- 1/5; 30 -2/5; 40 -- 4/5; 50, 100, 150, and 200 -- 5/5

5 rats per dose; 35 rats used

NA

Kiso to Rinsho. Clinical Report. 1971. (Yubunsha Co., Ltd., 1-5, Kanda Suda-Cho, Chiyoda-ku, KS
Bldg., Tokyo 101, Japan. 5(12): 1894. (RTECS REFERENCE)

H-11

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

Thompson and Weil
(1952); method ofmoving averages

albino rats; 2 weeks

male andfemaleoral; stomach tube 1 mL/200 g bw

observed after 8 days after single oral administration

6 dose levels per group, 6 rats pergroup; 36 rats used

NA

rats

NA

oral; stomach tube NA

salivation, vomiting, diarrhea; onset within 30 minutes

information from the laboratories ofDivision of Pharmacology, U.S.
NA
FDA.; fasted animals

Quarterly Bulletin--Association of Food and Drug Officials of the United States. (Denver, CO) V.338, 1939-74.
Lehman AJ. 1951. Chemicals in Foods: a report to the association of food and drug officials on
current developments. Part II. Pesticides. Quarterly Bulletine (Association of Food and Drug Officialsof the United States). Vol. 15:122 - 133. U.S. Food and Drug Administration (RTECSREFERENCE)

86 - 136(95% CL)

Thompson and Weil
(1952); method ofmoving averages

albino rats; 54 weeks

femaleoral; stomach tube 1 mL/200g bw

observed after 8 days after single oral administration

6 dose levels per group, 6 rats pergroup; 36 rats used;

Kostial K, Kello D, Jugo S, Rabar I, Maljkovic T. 1978. Influence of age on metal metabolism andtoxicity. Environ Health Perspect 25:81-86.
Yugoslav Academy of Sciences and Art, Zagreb, Yugoslavia

132

109.4 - 159.3(95% CL)

Bliss-Probit method

Sprague-Dawley rats; 5
weeks

male

oral gavage

88

170

140 - 206
(95% CL)

Thompson and Weil
(1952); method ofmoving averages

albino rats; 18 weeks

femaleoral; stomach tube 1 mL/200 g bw

Cadmium II chloride

88

211

182 - 252
(95% CL)

Thompson and Weil
(1952); method ofmoving averages

albino rats; 6 weeks

femaleCadmium II chloride

88

240

198 - 291
(95% CL)

Thompson and Weil;
1952; method ofalbino rats; 3 weeks
moving averages

Caffeine

192

192+/- 18(S.E.)

NA

Cadmium II chloride

88

47

43 - 51(95% CL)

Cadmium II chloride

88

88

NA

Cadmium II chloride

88

109

Cadmium II chloride

88

Cadmium II chloride+/- 18(S.E.)

Caffeine

192

192

Caffeine

192

247

Caffeine

192

264+/- 10(S.E.)

Caffeine

192279

259 - 302
(95% CI)

Caffeine

Caffeine

192

192

288

300

NA

albino rats+/- 6
(S.E.)+/- 29(S.E.)

Linear regression.
Boyd (1965)

Linear regression.
Boyd (1965)

NA

animals acclimated to environment
rats observed at 6 hours after dosing and a once a day for 1 - 2 weeks;
for 1 week before testing; 6 groups
most rats found dead within 3 days; 29 of 60 rats died; toxic symptoms:
MITSUWAof10 rats each; fasted 16 hours before
drooling, diarrhea, nasal bleeding; dose (mg/kg), rats dead per dose:
Chemical Co., Ltd.
dosing; 100% lethal dose = 247.1
66.5-0/10; 86.5-1/10; 112.5-3/10; 146.2-6/10; 190.1-9/10; 247.1-10/10
mg/kg; 0% lethal dose = 66.5 mg/kg

Kostial K, Kello D, Jugo S, Rabar I, Maljkovic T. 1978. Influence of age on metal metabolism andtoxicity. Environ Health Perspect 25:81-86.
Yugoslav Academy of Sciences and Art, Zagreb, Yugoslavia

Kitagawa H, Saito H, Sugimoto T, Yanaura S, Kitagawa H, Hosokawa T, Sakamoto K. 1982. Effects
of diiospropyl-1,3-dithiol-2-ylidene malonate (NKK-105) on acute toxicity of various drugs andheavy metals. J Toxicol Sci 7(2):123-34.
Chiba University; Hoshi College of Pharmacy; Showa University -- Japanobserved after 8 days after single oral administration

6 dose levels per group, 6 rats pergroup; 36 rats used

NA

Kostial K, Kello D, Jugo S, Rabar I, Maljkovic T. 1978. Influence of age on metal metabolism andtoxicity. Environ Health Perspect 25:81-86.
Yugoslav Academy of Sciences and Art, Zagreb, Yugoslavia

oral; stomach tube

1 mL/200 g bw; 6 dose levels in
observed after 8 days after single oral administrationeach group

6 dose levels per group, 6 rats pergroup; 36 rats used

NA

Kostial K, Kello D, Jugo S, Rabar I, Maljkovic, T. 1978. Influence of age on metal metabolism andtoxicity. Environ Health Perspect 25:81-86.
Yugoslav Academy of Sciences and Art, Zagreb, Yugoslavia

male andfemaleoral; stomach tube

1 mL/200 g bw; 6 dose levels in
observed after 8 days after single oral administrationeach group

6 dose levels per group, 6 rats pergroup; 36 rats used

NA

Kostial K, Kello D, Jugo S, Rabar I, Maljkovic, T. 1978. Influence of age on metal metabolism andtoxicity. Environ Health Perspect 25:81-86.
Yugoslav Academy of Sciences and Art, Zagreb, Yugoslavia

NA

oral

NA

see Boyd 1959

NA

Boyd EM. 1965. Caffeine addiction and drug toxicity. The Journal of New Drugs 5:252. (secondaryreference) Queen's University, Canada(RTECS REFERENCE)

NA

Boyd EM. 1959. The acute oral toxicity of caffeine. Toxic Appl Pharmac 1: 250-257.
Queen's University, Ontario, CanadaNAaqueous solution; 2 mL/kg
dose; 0 mg/kg-20 rats; 160
oral; stomach tube mg/kg-8 rats; 180 mg/kg-16
rats; 200 mg/kg-8 rats;
220 mg/kg-8 rats

19 rats survived; 21 rats died; death time 300 +/- 96 hours after dosing;survivors: lack of curiosity, weak, tense, hyperreflexia, ataxic,
cataleptic stances, swollen and inflammed eyelids,loose stools, tremors,anorexia, loss of body weight, fluctuation in body temperature; normal
clinical appearance at 72 hours; dead rats: similar clinical signs as
fasted for 16 hours; 60 rats usedsurvivors, clinical deterioration progressive from 10th hour till death,
didn't eat or drink, diarrhea, loss of body weight, anuric, drop in body
temp.; two-thirds died of respiratory failure following tetanic
convulsions; remainder died of cardiovascular collapse

oral intubation

single dose

15 rats per dose level; 16 hour fasting Schwarz/Mann observed for 15 days; death usually 1-2 days after dosing; diarrhea, wt
before dosing; 5 -6 dose levels; 75-90 Becton Dickinson
loss/gain; 40% of female rats died
rats
Co.

CBL Wistar albino rats; 150 female
200 g

intragastricsingle dose; range of 200 - 350
mg/kg; dissolved in distilled
observed for 5 days
water; 20 mL/kg volume toeach ratno overnight fasting; 50 rats used;groups of 10 rats

Merck Reagent

Crl-CD rats; Charles River
Breeding lab; 220 -280 g; 60 maledays oldoral; intragastricintubation

0.5 - 3.9% suspension;
observed daily for 14 days; death within 2 days; toxic symptoms:
dissolved/suspended in corn oil;staining of the face, wet perineal area, slight weight loss, lacrimation,
single dose; 100, 200, 250, 300,
lethargy, diarrhea
500 mg/kg doses

fasted 24 hours before dosing; 5groups of 10; 50 rats used; 19 ratsdied

99+% pure; Aldrich Dashiell OL, Kennedy GL Jr. 1984. The effects of fasting on the acute oral toxicity of nine chemicals
Chemical Co.
in the rat. J Appl Toxicol 4(6): 320-325. E.I. Du Pont de Nemours & Co., Newark, DE

oral; intragastricdosingobservations recorded hourly 1st day then at 24 hour intervals; ave time
to death is 14 hours; 1 - 40 hours range; cause of early deaths: tonicclonic convulsions followed by resipiratory failure; for delayed death,dissolved in distilled water; 20
immediate cause was hypothermic coma and respiratory failuremL/kg dose; 14 doses ranging
fasted for 16 hours; 84 - 140 rats
following loss of corneal reflexes, impaired respiration, pallor,from 162 to 354 mg/kg; each
used; unanethetized rats
cyanosis, anuria; drop in colonic temperature; hypothermia appeared
dose given to 6 - 10 rats
within 2 hours, peaked at 8 - 24 hour at which time it was dose
dependent; hypothermia associated with stupor, anorexia, oligodipsia,
loss of body weight, oliguria, aciduria, proteinuria

U.S.P. grade

Boyd EM, Liu SJ, Singh J. 1968. The toxicity of aspirin, phenacetin, and caffeine following rectal
administration. Clin Toxicol 1:425 - 430. Queen's University, Ontario, Canada

oral; intragastricdosingobservations recorded hourly 1st day then at 24 hour intervals; ave time
to death is 14 hours; 1 - 40 hours range; cause of early deaths: tonicclonic convulsions followed by resipiratory failure; for delayed death,dissolved in distilled water; 20
fasted for 16 hours; 84 - 140 rats
immediate cause was hypothermic coma and respiratory failuremL/kg dose; 14 doses ranging
used; rats used; rats given thiopental
following loss of corneal reflexes, impaired respiration, pallor,
U.S.P. gradefrom 162 to 354 mg/kg; eachbefore dosing (anesthetized rats
cyanosis, anuria; drop in colonic temperature; hypothermia appeared
dose given to 6 - 10 rats
before dosing)
within 2 hours, peaked at 8 - 24 hour at which time it was dose
dependent; hypothermia associated with stupor, anorexia, oligodipsia,
loss of body weight, oliguria, aciduria, proteinuria

Boyd EM, Liu SJ, Singh J. 1968. The toxicity of aspirin, phenacetin, and caffeine following rectal
administration. Clin Toxicol 1:425 - 430. Queen's University, Ontario, Canada

albino rats; 203 +/-28 g; 3-6
femalemonthsSprague-Dawley CD rats;
220 - 277
Cornfield and Mantel
mean wt. of 164 g; young(95% CL; slope=7.7) (1950)
adult

Probit analysis

dissolved in saline; range(mg/kg) of doses 66.5, 86.5,
112.5, 146.2, 190.1, 247.1

NA

femaleWistar albino rats; 125-200 g male

Wistar albino rats; 125-200 g male

H-12

Palm PE, Arnold EP, Rachwall PC, Leyczech JC, Teague KW, Kensler CJ. 1978. Evaluation of theteratogenic potential of fresh brewed coffee and caffeine in the rat. Toxic Appl Pharmac 44:1 - 16.
Arthur D. Little, Inc., Cambridge, MA

Boyd EM, Dolman M, Knight LM, Sheppard EP. 1965. The chronic oral toxicity of caffeine. Canad J
Physiol Pharm 43:995 - 1007. Queen's University, Ontario, Canada

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

NA

Notes

Reference
Substance Source

Primary Reference(source of information not provided) NA

Izmerov NF, Sanotsky IV, Sidorov KK. 1982. Toxicometric Parameters of Industrial Toxic Chemicals
under Single Exposure. International Register of Potentially Toxic Chemicals (IRPTC). United
Nations Environment Programme (UNEP). Centre of International Projects, GKNT. Moscow, Russia.

fasted 18 - 20 hours before dosing;conventional LD50 method; groupsof 10; 40 rats used

Bruce RD. 1987. A confirmatory study of the up-and-down method for acute oral toxicity testing.
Fundam Appl Toxicol 8(1): 97-100. The Proctor and Gamble Co., Cincinnati, OH

Caffeine

192

310+/- 33NA

rats; 220 +/- 40 gNAoral; intragastricNA

Caffeine

192

344

307 - 383
(95% CI)

Probit analysis

Sprague-Dawley rats; 190300 g

maleoral gavage

geometric progression of 14 forobserved for 14 days after dosing;
dosingCaffeine

192

355

Sprague Dawley CD rats;
312 - 403
Cornfield and Mantel
mean wt. of 210 g; young(95% CL; slope=5.1) (1950)
adult

male

oral intubation

single dose; dose in water

15 rats per dose level; 16 hour fasting Schwarz/Mann observed for 15 days; death usually 1-2 days after dosing; diarrhea, wt
before dosing; 5 -6 dose levels; 75-90 Becton Dickinson
loss/gain; 21% of male mice died
rats
Co.

Caffeine

192

421

320 - 553
(95% CI)

Probit analysis

Sprague-Dawley rats; 190300 g

maleoral gavageNAobserved for 7 days

Caffeine

192

483

433 -562(95% CI)

Probit analysis

Crl-CD rats; Charles River
Breeding lab; 220 -280 g; 60 maledays oldoral; intragastricintubation

Carbamazepine

1957

1957

NA

NA

rats

NA

Carbamazepine

1957

4025

NA

NA

rats; 120-140 g

femaleCarbon tetrachloride

2350

1020

861 - 1211
(95% CL)

Weil (1952)

Carbon tetrachloride

2350

2343

2136 - 2566
(95% CL)

Carbon tetrachloride

2350

2350

Carbon tetrachloride

2350

2500

NA

Palm PE, Arnold EP, Rachwall PC, Leyczech JC, Teague KW, Kensler CJ. 1978. Evaluation of theteratogenic potential of fresh brewed coffee and caffeine in the rat. Toxic Appl Pharmac 44:1 - 16.
Arthur D. Little, Inc., Cambridge, MA

fasted 18 - 20 hours before dosing;Up-and-down LD50 method; 9 rats
used

NA

0.5 - 3.9% suspens; dissolved orobserved daily for 14 days; death within 3 days; toxic symptoms:
suspended in corn oil; single
staining of the face, wet perineal area, slight weight loss, lacrimation,
dose; 300, 400, 450, 650 mg/kg
lethargy, diarrhea
doses

non fasted; 4 groups of 10; 40 rats
used; 15 rats diedDashiell OL, Kennedy GL Jr. 1984. The effects of fasting on the acute oral toxicity of nine chemicals
99+% pure; Aldrichin the rat. J Appl Toxicol 4(6): 320-325.
Chemical Co.
E.I. Du Pont de Nemours & Co., Newark, DE

oral

NA

NA

reference in Japanese

NA

Japanese Kokai Tokyo Koho Patents. 54-163823 (U.S. Patent and Trademark Office. 79-163823)(RTECS REFERENCE)

oral

suspension in arabica gum

observed for 8 days

reference paper in German; 20
animals per doseNA

Stenger Von EG, Roulet FC. 1964. Zur Toxikologie des Antiepilepticum Tegretol. Medicina
Experimentalis 11:191-201.

Wistar-derived Porton strain
male
rats (SPF); 100 - 160 goral gastricintubation

1:1 (v/v) mixture in liquidparaffin; lightly anesthetizedw/ether; geometric doses byfactor of 12 or 144

deaths observed for 1 week

18 hour fasting before dosing; 20 25 rats used; groups of 5 rats; normal NA
stock diet

McLean AEM, McLean EK. 1966. The effect of diet and 1,1,1-trichloro-2,2-bis (p-chlorophenyl)
ethane (DDT) on microsomal hydroxilating enzymes and on sensitivity of rats to carbon tetrachloride
poisoning. Biochem J 100:564-571. Royal Free Hospital, London, UK

Weil (1952)

Wistar-derived Porton strain
male
rats (SPF); 100 - 160 goral gastricintubation

1:1 (v/v) mixture in liquidparaffin; lightly anesthetizedw/ether; geometric doses byfactor of 1.2 or 1.44

deaths observed for 1 week

18 hour fasting before dosing; 20 25 rats used; groups of 5 rats; proteinfree diet; rats fed protein-free diet 1 NA
3 weeks before dosing; continued
protein-free diet through out
observation period

McLean AEM, McLean EK. 1966. The effect of diet and 1,1,1-trichloro-2,2-bis (p-chlorophenyl)
ethane (DDT) on microsomal hydroxilating enzymes and on sensitivity of rats to carbon tetrachloride
poisoning. Biochem J 100:564-571. Royal Free Hospital, London, UK

NA

Rosiello (1979) andBliss (1938)

rats

male

oral

1500, 2000, 2800, 3900 mg/kg

1500 mg/kg: 0/1 dead; 2000 mg/kg: 0/1 dead; 2800 mg/kg: 1/1 dead;
3900 mg/kg: 1/1 dead; 2 of 4 rats dead; LD50 based on 4 rats used

rats acclimated for 5 days; rats
observed for 14 days; 4 groups ofrats used for each dose (1, 2, 3, 5 rats
per group; 11 rats per dose); 9 ratsNA
for initial range finding;10 mg/kg 0/3 dead; 100 mg/kg - 0/3 dead; 1000
mg/kg - 2/3 dead

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

NA

Rosiello (1979) andBliss (1938)

rats

male

oral

1500, 2000, 2800, 3900 mg/kg

1500 mg/kg: 0/2 dead; 2000 mg/kg: 2/2 dead; 2800 mg/kg: 1/2 dead;
3900 mg/kg: 2/2 dead; 5 of 8 rats dead; LD50 based on 8 rats used

rats acclimated for 5 days; rats
observed for 14 days; 4 groups ofrats used for each dose (1, 2, 3, 5 rats
per group; 11 rats per dose); 9 ratsNA
for initial range finding;10 mg/kg 0/3 dead; 100 mg/kg - 0/3 dead; 1000
mg/kg - 2/3 dead

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

rats

male

oral

rats acclimated for 5 days; rats
observed for 14 days; 4 groups ofrats used for each dose (1, 2, 3, 5 rats
1500 mg/kg: 0/5 dead; 2000 mg/kg: 3/5 dead; 2800 mg/kg: 3/5 dead;
1500, 2000, 2800, 3900 mg/kg
per group; 11 rats per dose); 9 rats
NA
3900 mg/kg: 5/5 dead; 11 of 20 rats dead; LD50 based on 20 rats used
for initial range finding;10 mg/kg 0/3 dead; 100 mg/kg - 0/3 dead; 1000
mg/kg - 2/3 dead

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

rats

male

oral

rats acclimated for 5 days; rats
observed for 14 days; 4 groups of1500 mg/kg: 0/11 dead; 2000 mg/kg: 5/11 dead; 2800 mg/kg: 6/11
rats used for each dose (1, 2, 3, 5 rats
1500, 2000, 2800, 3900 mg/kg dead; 3900 mg/kg: 11/11 dead; 22 of 44 rats dead; LD50 based on same per group; 11 rats per dose); 9 rats
NA
rats used for other Lorke (1983) values
for initial range finding;10 mg/kg 0/3 dead; 100 mg/kg - 0/3 dead; 1000
mg/kg - 2/3 deadArchives of Toxicology. (Springer-Verlag, Heidelberger Pl. 3, D-1000 Berlin 33, Fed. Rep. Ger.)
V.32- 1974.
Lorke D. 1983. "A new approach to practical acute toxicity testing." Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany(RTECS REFERENCE)

Carbon tetrachloride

2350

2500

NA

Rosiello (1979) andBliss (1938)

Carbon tetrachloride

2350

2500

NA

Rosiello (1979) andBliss (1938)

H-13

Bruce RD. 1987. A confirmatory study of the up-and-down method for acute oral toxicity testing.
Fundam Appl Toxicol 8(1): 97-100. The Proctor and Gamble Co., Cincinnati, OH

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

Smyth HF, Weil CS, West JS, Carpenter CP. (1970). An exploration of joint toxic action:II.
Equitoxic versus equivolume mixtures. Toxicol Appl Pharmacol. 17:498-503. (LD50 value)

Carbon tetrachloride

2350

2821
(1.77 mL/kg; sp.
density is 1.594;
convert LD50 to
mg/kg

NA

Thompson method;
Weil tables

Carworth-Wistar rats; 90120 g; 4-5 weeks

maleoral gastricintubation

in aqueous solution;
concentration intubated = 10
mg/mL; dosages arranged in a LD50 based on mortalities during a 14 day period
logarithmic series differing by afactor of 2

non-fasted; groups of 5 rats; single
oral dose toxicity

reagent grade

Smyth HF Jr, Carpenter CP, Weil CS, Pozzani UC, Striegel JA, Nycum JS. 1969. Range-finding
toxicity data: List VII. Am Ind Hyg Assoc J 30:470-476. Carnegie-Mellon University, Pittsburgh,
PA
Smyth HF Jr, Carpenter CP, Weil CS, Pozzani UC, Striegel JA. 1962. Range-finding toxicity data:
List VI. Am Ind Hyg Assoc J 23:95-107.
Mellon Institute of Industrial Research, Pittsburg, PA (experimental parameters)

rats acclimated for 5 days; rats
observed for 14 days; 4 groups ofrats used for each dose (1, 2, 3, 5 rats
1500 mg/kg: 0/3 dead; 2000 mg/kg: 0/3 dead; 2800 mg/kg: 1/3 dead;per group; 11 rats per dose); 9 rats
NA
3900 mg/kg: 3/3 dead; 4 of 412 rats dead; LD50 based on 12 rats used
for initial range finding;10 mg/kg 0/3 dead; 100 mg/kg - 0/3 dead; 1000
mg/kg - 2/3 deadRosiello (1979) andBliss (1938)

rats

male

oral

1500, 2000, 2800, 3900 mg/kg

2450 - 3470
(95% CL)NA

ratsmale andfemaleoral; stomach
intubation

10 dosage levels; suspended incorn oilk with acacia; single
190 rats used
dose

NA

NA

McCollister DD, Hollingsworth RL, Oyen F, Rowe VK. 1955. Comparative inhalation toxicity of
fumigant mixtures. Arch Ind Health pp.1 - 7. Dow Chemical, Midland, MI

2981 (1.87
mL/kg; sp.
density is 1.594;
convert LD50 to
mg/kg)

slope = 1.62

Litchfield andWilcoxon method(1949)

Scho:Wistar C rats; 150-180
femaleg; 56 +/- 2 days

oral

single dose; 50 mg/kg bw
carbon tetrachloride in 5 mL
peanut oil/kg bw

48 hour observation; LD50 determined on rats monthly for a year andaverage reported for whole year

reference in German; year 4

NA

Von Schmidt P, Wolff DL, Burck D, Wilhelm M. 1979. Sensitivity of female Wistar rats to carbon
tetrachloride, determined by the LD50, and the hexobarbital sleeping time after a single oral dose. Z
Versuchstierkd. 21(3):153-162. Zentralinstitut fur Arbeitsmedizin der DDR, Berlin, Germany

2350

3682 (2.31
mL/kg; sp.
density is 1.594;
convert LD50 to
mg/kg)

slope = 1.83

Litchfield andWilcoxon method(1949)

Scho:Wistar C rats; 150-180
femaleg; 56 +/- 2 days

oral

single dose; 50 mg/kg bw
carbon tetrachloride in 5 mL
peanut oil/kg bw

48 hour observation; LD50 determined on rats monthly for a year andaverage reported for whole year

reference in German; year 3

NA

Von Schmidt P, Wolff DL, Burck D, Wilhelm M. 1979. Sensitivity of female Wistar rats to carbon
tetrachloride, determined by the LD50, and the hexobarbital sleeping time after a single oral dose. Z
Versuchstierkd. 21(3):153-162. Zentralinstitut fur Arbeitsmedizin der DDR, Berlin, Germany

Carbon tetrachloride

2350

4081
(2.56 mL/kg; sp.
density is 1.594;
convert LD50 to
mg/kg)

slope = 1.60

Litchfield andWilcoxon method(1949)

Zam:Wistar C rats; 150-180
femaleg; 56 +/- 2 days

oral

single dose; 50 mg/kg bw
carbon tetrachloride in 5 mL
peanut oil/kg bw

48 hour observation; LD50 determined on rats monthly for a year andaverage reported for whole year

reference in German; year 4

NA

Von Schmidt P, Wolff DL, Burck D, Wilhelm M. 1979. Sensitivity of female Wistar rats to carbon
tetrachloride, determined by the LD50, and the hexobarbital sleeping time after a single oral dose. Z
Versuchstierkd. 21(3):153-162. Zentralinstitut fur Arbeitsmedizin der DDR, Berlin, Germany

Carbon tetrachloride

2350

4288
(2.69 ml/kg;
sp.density is
1.594; converted
LD50 to mg/kg

slope = 1.59

Litchfield andWilcoxon method(1949)

Zam:Wistar C rats; 150-180
femaleg; 56 +/- 2 days

oral

single dose; 50 mg/kg bw
carbon tetrachloride in 5 mL
peanut oil/kg bw

48 hour observation; LD50 determined on rats monthly for a year andaverage reported for whole year

reference in German; year 3

NA

Von Schmidt P, Wolff DL, Burck D, Wilhelm M. 1979. Sensitivity of female Wistar rats to carbon
tetrachloride, determined by the LD50, and the hexobarbital sleeping time after a single oral dose. Z
Versuchstierkd. 21(3):153-162. Zentralinstitut fur Arbeitsmedizin der DDR, Berlin, Germany

Carbon tetrachloride

2350

4336
(2.72 mL/kg; sp.
density is 1.594;
convert LD50 to
mg/kg)

slope = 1.44

Litchfield andWilcoxon method(1949)

Zam:Wistar C rats; 150-180
femaleg; 56 +/- 2 days

oral

single dose; 50 mg/kg bw
carbon tetrachloride in 5 mL
peanut oil/kg bw

48 hour observation; LD50 determined on rats monthly for a year andaverage reported for whole year

reference in German; year 2

NA

Von Schmidt P, Wolff DL, Burck D, Wilhelm M. 1979. Sensitivity of female Wistar rats to carbon
tetrachloride, determined by the LD50, and the hexobarbital sleeping time after a single oral dose. Z
Versuchstierkd. 21(3):153-162. Zentralinstitut fur Arbeitsmedizin der DDR, Berlin, Germany

Carbon tetrachloride

2350

4670
(2.93 mL/kg; sp.
density is 1.594;
convert LD50 to
mg/kg)

slope = 1.57

Litchfield andWilcoxon method(1949)

Zam:Wistar C rats; 150-180
femaleg; 56 +/- 2 days

oral

single dose; 50 mg/kg bw
carbon tetrachloride in 5 mL
peanut oil/kg bw

48 hour observation; LD50 determined on rats monthly for a year andaverage reported for whole year

reference in German; year 1

NA

Von Schmidt P, Wolff DL, Burck D, Wilhelm M. 1979. Sensitivity of female Wistar rats to carbon
tetrachloride, determined by the LD50, and the hexobarbital sleeping time after a single oral dose. Z
Versuchstierkd. 21(3):153-162. Zentralinstitut fur Arbeitsmedizin der DDR, Berlin, Germany

Carbon tetrachloride

2350> 5000NA

Dixon (1965) and
Bruce (1985)

Fischer 344 rats; 77 days old
femaleat testoral gavage

in deionized water; maximum
volume dose 10 mL/kg; 5 dose
7 day survival timelevels: 0, 150, 500, 1500, 5000
mg/kg; single dose

Carbon tetrachloride

2350

2850

NA

Carbon tetrachloride

2350

2920

Carbon tetrachloride

2350

Carbon tetrachloride

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

fasted overnight; initial dose levels =100, 1000, and 5000 mg/kg;
analytical grad_;Berman E, Schlicht M, Moser VC, MacPhail RC. 1995. A multidisciplinary approach to toxicologicalsubsequent doses selected by up-and99+% pure; Aldrich screening: I. Systemic toxicity. J Toxicol Environ Health 45(2): 127-43.
down method (Bruce, 1985, 1987); 5
Chemical Co.
Health Effects Res. Lab., U.S.EPA, Research Triangle Park, NC
groups of 8 rats each; 40 rats used; 7 15 rats used in first LD50 estimate

Carbon tetrachloride

2350

5453

4660 - 6404
(95% CI)

Probit analysis

Crl-CD rats from Charles
River Breeding lab; 220-280 maleg; 60 days oldoral; intragastricintubation

15 - 45% solution dissolved orsuspended in corn oil; single
dose; 2500, 3000, 4000, 5000,
8000, 10000, 11000 mg/kg
doses

observed daily for 14 days; death within 2 days; toxic symptoms:
salivation, weakness, pallor, lethargy, diarrhea, weight loss

H-14

24 hour fast before dosing; 7 groupsof 10; 70 rats used; 35 rats died;
99+% pure; E.I. Du Dashiell OL, Kennedy GL Jr. 1984. The effects of fasting on the acute oral toxicity of nine chemicals
doses of 10000 mg/kg or greater
Pont de Nemoursin the rat. J Appl Toxicol 4(6): 320-325. E.I. Du Pont de Nemours & Co., Newark, DE
administered in 2 portions at 15
minutes apart

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposureoral; intragastric

Dose

Notes

Reference
Substance Source(source of information not provided) NA

observed for 48 hour; doses (mL/kg), dead animals: 3.6 -- 0/4; 4.5 -1/4; 5.4 -- 3/4; 6.4 -- 4/4

16 rats used

Izmerov NF, Sanotsky IV, Sidorov KK. 1982. Toxicometric Parameters of Industrial Toxic Chemicals
under Single Exposure. International Register of Potentially Toxic Chemicals (IRPTC). United
Nations Environment Programme (UNEP). Centre of International Projects, GKNT. Moscow, Russia.

2350

6200

5082 - 7564

NArats; 220 +/- 40 g

Carbon tetrachloride

2350

7540
(4.73 mL/kg; sp.
density is 1.594;
convert LD50 to
mg/kg)

6631 - 8576
(95% CL)

Weil (1952)

Sprague-Dawley rats; 260360 g; 12-16 weeks

Carbon tetrachloride

2350

10054

8758 - 11009(95% CI; slope =9.2)

Finney (1971)
Probit Analysis

Crl-CD rats from Charles
River Breeding lab; 220-280 maleg; 60 days oldoral; intragastricintubation

0.5 - 3.9% suspension;
dissolved or suspended in cornoil; single dose; 2000, 2700,
observed daily for 14 days; death within 3 days; toxic symptoms:
3500, 4500, 8000, 10000,
salivation, weakness, pallor, lethargy, diarrhea, weight loss
11000, 12000, 14000, 15000,
17000 mg/kg doses

Dashiell OL, Kennedy GL Jr. 1984. The effects of fasting on the acute oral toxicity of nine chemicals
non fasted; 11 groups of 10; 110 rats
in the rat. J Appl Toxicol 4(6): 320-325.
E.I. Du Pont de Nemours & Co., Newark, DE
used; 49 rats died; doses of 10000
99+% pure; E.I. Du
data from EPA TSCATS database; Oral LD50 test in rats with methane,tetrachloro-* with cover letter
mg/kg or greater were administered Pont de Nemours
dated 081092; (1981) EPA Document No. 88-920010018 Fiche No. OTS0571676;
in 2 portions at 15 minutes apart
E.I Dupont DeNemours & Co., Inc./Haskell Labs

Chloral hydrate

479

285

+/- 21(S.E.)NA

Charles River SpragueDawley rats; 1-2 days

NA

oral

NA

NA

data is from Yeary et al.1966

NA

Goldenthal EI. 1971. A compilation of LD50 values in newborn and adult animals. Toxicology andApplied Pharamacology 18:185-207.

Chloral hydrate

479

479

+/- 42(S.E.)NA

Charles River SpragueDawley rats; adult

NA

oral

NA

NA

data is from Yeary et al.1966

NA

Toxicology and Applied Pharmacology. (Academic Press, Inc., 1 E. First St., Duluth, MN 55802) V.11959.
Goldenthal EI. 1971. A compilation of LD50 values in newborn and adult animals. Toxicology andApplied Pharamacology 18:185-207

Chloral hydrate

479

479

+/- 42(S.E.)

Miller and Tainter(1944)Charles River CD andSprague-Dawley rat strains; NA> 100 g; adultoral intubation; up
NAto 50 mL/kg

NA

Yeary RA, Benish RA, Finkelstein M. 1966. Acute Toxicity of Drugs in Newborn Animals. Journalof Pediatrics 69 (4):663-667.
Dept. of Veterinary Preventive Medicine, Ohio State University, Columbus, OH

Chloral hydrate

479

500

NA

NA

NA

rat

oral

aqueous solution or suspension produced degree of CNS depression

NA

NA

Finnegan JK, Larson PS, Haag HB, Page SG Jr. 1951. March. Sedative and toxic effects of several
chloral derivatives. Federation Proceedings v. 10:294. Medical College of Virginia, Richmond, VA

Chloral hydrate

479

800

NA

graphicallywhite rats; 125-250 g

male andfemaleoral;stomach tube

single dose; 4% solutions in
distilled water; dose is mg/kg,
rats per dose: 700-25; 800-34;
900-22; 1000-32; 1100-24

fasted for 16 hour; 137 rats used; firstNA
report for chloral hydrate LD50

Adams WL. 1943. The comparative toxicity if chloral alcoholate and chloral hydrate. J Pharm Exp
Ther 78:340-345. Union University, Albany, NY

Chloral hydrate

479

863

622.9 - 832.1

Bliss-Probit method

Sprague-Dawley rats; 5
weeks

male

oral gavage

animals acclimated to environmentfor 1 week before testing; 5 groups
dissolved in saline; range
rats observed at 6 hours after dosing and a once a day for 1 - 2 weeks;of 10 rats each; fasted 16 hours(mg/kg) of doses 417, 583, 816, most rats found dead within 3 days; 29 of 50 rats died; toxic symptoms:
before dosing; 100% mortality =1143, 1600
sleep to coma
1600 mg/kg; 0% mortality = 417
mg/kg

Wako Pure
Chemicals Co.

Kitagawa H, Saito H, Sugimoto T, Yanaura S, Kitagawa H, Hosokawa T, Sakamoto K. 1982. Effects
of diiospropyl-1,3-dithiol-2-ylidene malonate (NKK-105) on acute toxicity of various drugs andheavy metals. J Toxicol Sci 7(2):123-34.
Chiba University; Hoshi College of Pharmacy; Showa University -- Japan

Chloramphenicol

2500

692.9

-/+ 70(SEM)

Bliss (1938)

Harlan rats; < 4 days; 6-9 g

NAintragastric

cmpd suspended in 4% acacia
saline solution; 2% solution
doses at 400, 500, 620, 800
mg/kg

observed for 7 days; death within 24 h; 400 mg/kg-0/5, 500 mg/kg-0/5,NA620 mg/kg-3/5, 800 mg/kg-3/5

NAWorth HM, Kachman C, Anderson RC. 1963. Inartistric injection for toxicity studies with newborn
rats. Toxic Appl Pharmac 5:719-727. Eli Lilly and Company, Indianapolis, IN

Chloramphenicol

2500

1040

776 - 1394

NA

MJ rats; 1-2 days

NA

oral

NA

NA

information from: drug applications
from pharmaceutical manufacturers, NA
the literature, and FDA labs

Goldenthal EI. 1971. A compilation of LD50 values in newborn and adult animals. Toxicology andApplied Pharamacology. 18. Pp. 185 -207. Bureau of Drugs, Food and Drug Administration,
Dept. of Health, Education, and Welfare, Rockville, MD.

Chloramphenicol

2500

2188

NA

Bliss (1938)

Harlan rats; 30-40 g; 21-25
days; weanlingNA

gavagecmpd suspended in 4% acacia
saline solution; 20% solution
administ; 1800, 2500, 3300
mg/kg doses

observed for 7 days; death within 3 days; 1800 mg/kg-0/5, 2500 mg/kgNA
4/5, 3300 mg/kg-5/5

Chloramphenicol

2500

2500

NA

NA

albino rats

NA

oral

NA

NA

reference paper in Italian; 1983/84
RTECS used the same reference butNA
RC had a different LD50 and ZEBET
did not provide the reference)

Farmaco, Edizione Scientifica. (Casella Postale 227, 27100 Pavia, Italy) V.8-43 1953-88 ---Almirante L, Caprio L, de Carneri I, Defranceschi A, Zamboni V. 1955. Studi sul cloroamfenicolo:(1) nuove sintesi della d-treo-2-dichlorometil-4-[(4'-nitrofenil)Ossimetil] Ossazolina (2) E dati sur
potere antibiotico della stessa. Farmaco, Edizione Scientifica 10(1):3-13. (RTECS REFERENCE)

Chloramphenicol

2500

3400

2252 - 5139

NA

MJ rats; adult

NA

oral

NA

NA

information from: drug applications
from pharmaceutical manufacturers, NA
the literature, and FDA labs

Goldenthal EI. 1971. A compilation of LD50 values in newborn and adult animals. Toxicology andApplied Pharamacology 18:185-207. Bureau of Drugs, Food and Drug Administration, Dept. of
Health, Education, and Welfare, Rockville, MD. This value used by RC (1977 RTECS).

Chloramphenicol

2500

5000

NA

NA

Harlan Wistar rats

NA

oral

NA

NA

information from: drug applications
from pharmaceutical manufacturers, NA
the literature, and FDA labs

Goldenthal EI. 1971. A compilation of LD50 values in newborn and adult animals. Toxicology andApplied Pharamacology 18:185-207. Bureau of Drugs, Food and Drug Administration, Dept. of
Health, Education, and Welfare, Rockville, MD.

solution in 1.5 mL peanut oil;oral; stomach tube light anesthesia; doses(mL/kg)=3.6, 4.5, 5.4, 6.4

NA

Primary Reference

Carbon tetrachloride

male

NA

Observations

rats observed for 7 days; observed up to 14 days when heavy metals or
fasted overnight
other compounds that produce latent death were investigatedacute toxicity same for male and female;

H-15

British Drug Houses Pound AW, Horn L, Lawson TA. 1973. Decreased toxicity of dimethylnitrosamine in rats afterLtd, Pool, Great
treatment with carbon tetrachloride. Pathology 5:233-242.
Britain
University of Queensland, Brisbane, Australia

NA

Worth HM, Kachman C, Anderson RC. 1963. Inartistric injection for toxicity studies with newborn
rats. Toxic Appl Pharmac 5:719-727. Eli Lilly and Company, Indianapolis, IN

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)oral gavage

2500 - 5000mg/kg doses; cmpd
NAin hydroxyethylcellulose

SD-JCL rats; 110-140 g; 5
weeks

oral

2 mL/100 g bw

NA

Bliss (1938) method

Harlan rats; 150 g; adult

Citric acid

3000

3000

NA

approximative

Citric acid

3000

11700

10080 - 13570(95% CL)

Litchfield andWilcoxon method

3.901 - 7.508

ColchicineNA

18(mouse)Colchicine

NA

Cupric sulfate pentahydrate

Observations

THOM (SPF) rats; 151-213
male andg; 48 days-males; 62 daysfemale
female> 50005.886(mouse)Dose

cmpd suspended in 4% acacia
saline solution; 30% solutiondose at 5000 mg/kg

2500

NA

Route and/or
Method ofExposure

gavageChloramphenicol

Colchicine

Gender

NA

male

Notes

observed for either 7 or 14 days; 10 rats used; 2 dead; death on 1st day NA

32 male and 32 female rats; 64 rats
used; performed under GLPs

Reference
Substance Source

Primary Reference

NA

Worth HM, Kachman C, Anderson RC. 1963. Inartistric injection for toxicity studies with newborn
rats. Toxic Appl Pharmac 5:719-727. Eli Lilly and Company, Indianapolis, IN

NA

Schneider PM, Bauer A, Eckenfels C, Hohbach L, Lutzen H, Puschner R, Serbedija J, Wiegleb P,
Lehmann H. 1992. Acute, subacute and chronic toxicity studies of pimobendan in laboratory animals.
Oyo Yakuri/Pharmacometrics 43(6):561-578. (RTECS REFERENCE)

observed for 7 days; stimulation within several minutes, then ataxia andprostration at 50 minutes; mydriasis, decreased heart rate and6 rats/dose; number of doses not
respiration; death at 12500 and 18000 mg/kg in 20-180 minutes byreported
resp. failure; 1 rat at 10420 mg/kg died at 20 hours; autopsy showed

TAKEDA-citric acid(refined productYokotani H, Usui T, Nakaguchi T, Kanabayashi T, Tanda M, Aramaki Y. 1971. Acute and subacuteproduced by yeast
toxicological studies of TAKEDA-citric acid in mice and rats. J Takeda Res Lab 30(1):25-31.
fermention ofhemorrage of gastric mucosa

paraffins)

B6D2F1 (BDF1) mice

NA

Oral

in saline

NA

Lorke (1983)

MS/Ae mice from Hitachi
Medical Laboratories
male(Sanwa, Japan); 317-346 g;
7 weeks

oral

1.0, 10.0, 14.0, 22.5, 37.5, 60.0,
Wako Pure Chemical
Dose and Deaths: 1.0 - 0/3; 10.0 - 0/3; 14.0 - 0/1; 22.5 -1/1; 37.5 - 1/1; 13 mice used; acclimated for 1 week
Asano N, Morita T, Watanabe Y. 1989. Micronucleus test with colchicine given by intraperitoneal
100.0 mg/kg in physiologicalIndustries Ltd.
60.0 - 1/1;100.0 - 3/3
before test
injection and oral gavage. Mutat Res 223:391-394.
saline(Osaka, Japan)

29(mouse)NA

Lorke (1983)

CD-1 mice from Charles
River Japan Inc (Hino,
male
Japan); 312-382 g; 7 weeks

oral

1.0, 10.0, 14.0, 22.5, 37.5, 60.0,
Wako Pure Chemical
Dose and Deaths: 1.0 - 0/3; 10.0 - 0/3; 14.0 - 0/1; 22.5 -0/1; 37.5 - 1/1; 13 mice used; acclimated for 1 week
Asano N, Morita T, Watanabe Y. 1989. Micronucleus test with colchicine given by intraperitoneal
100.0 mg/kg in physiologicalIndustries Ltd.
60.0 - 1/1; 100.0 - 3/3
before test
injection and oral gavage. Mutat Res 223:391-394.
saline(Osaka, Japan)

300

236.2

NA

NA

Sprague-Dawley rats

NA

oral

200, 500, 1000, 2000

NA

NA

T.C. copper sulfate
powdered (50% inwater)

U.S. EPA, Office of Pesticide Programs; Health Effects Division; Tox Oneliners; EPA Chem. Code:024401; Core Grade/Tox Record No. 002705

Cupric sulfate pentahydrate

300

300

NA

NA

rats

NA

oral

NA

NA

value assumed to be from Lehman
1951

NA

Agricultural Chemicals. Thomson, W.T., 4 vols., Fresno, CA, Thomson Publications, 1976/77
revision (RTECS REFERENCE)

Cupric sulfate pentahydrate

300

300

NA

NA

rats

NA

oral; stomach tube NA

violent retching, muscular spasms and collapse; onset within minutes

information from the laboratories ofDivision of Pharmacology, U.S.
NA
FDA.; fasted animals

Lehman AJ. 1951. Chemicals in Foods: a report to the association of food and drug officials on
current developments. Part II. Pesticides. Quarterly Bulletine (Association of Food and Drug
Officials of the United States). v15:22 - 133. U.S. FDA (RTECS SOURCE)

Cupric sulfate pentahydrate

300

450

346 - 585
(95% CL)

Litchfield andWilcoxon method(1949)

Sprague-Dawley rats; 155175 g

femaleoral gavage

animals observed daily and survivors killed 14 days post-dose; allsingle dose; 9 dose levels from deaths within first week of dosing; weight loss, lethargy and death;
100 - 5000mg/kg
dose (mg/kg), no dead/no dosed: 100 - 0/5; 200 - 0/5; 300 - 3/10; 500 0/5; 625 - 0/10; 750 - 4/5; 5000 - 5/5

tested under GLPs; groups of rats
(5/sex/dose group) were administeredpowder 99% purevehicle (10 mL/kg) or test article; 45
animals used

Deenihan MJ.1987; Fine 20 Copper Sulfate Pentahydrate - Acute Toxicology Testing: (A) Acute Oral
Toxicity. Northview Pacific laboratories, Inc. U.S. EPA, Office of Pesticide Programs; Health Effects
Division; Tox Oneliners; MRID No.433962-01A; EPA Chem. Code: 024401; Core Grade/Tox
Record No. acceptable; 011521; Apr. 20, 1995

Cupric sulfate pentahydrate

300

472.5

NA

NA

rat

NA

oral

NA

NA

Cupric sulfate pentahydrate

300

790

416 - 1501
(95% CL)

Litchfield andWilcoxon method(1949)

Sprague-Dawley rats; 225250 g

maleoral gavage

animals observed daily and survivors killed 14 days post-dose; allsingle dose; 9 dose levels from deaths within first week of dosing; weight loss, lethargy and death;
100 - 5000 mg/kg
dose (mg/kg), no dead/no dosed: 100 - 0/5; 300 - 2/5; 750 - 1/5; 1000 3/5; 1250 - 2/5; 5000 - 5/5

tested under GLPs; groups of rats
(5/sex/dose group) were administeredpowder 99% purevehicle (10 ml/kg) or test article; 30
animals used

710 - 1300
Thompson method;(these limits are +/Weil tables
1.96 S.D.)

Carworth-Wistar rats; 90120 g; 4-5 weeks

maleoral gastricintubation

in aqueous solution;
concentration intubated = 50
mg/mL; dosages arranged in a LD50 based on mortalities during a 14 day period
logarithmic series differing by afactor of 2

non-fasted; groups of 5 rats; single
oral dose toxicity

reagent grade

Cupric sulfate pentahydrate

300

960

NA

Mice fasted prior to dosing

NA

NA

National Cancer Institute Screening Program Data Summary, Developmental Therapeutics Program.
(Bethesda, MD 20205) JAN1986. (RTECS REFERENCE)

NA

copper sulfate(powder)

WARF Institute, Inc.; WARF No. 5032161; Jan. 1, 1975; U.S. EPA, Office of Pesticide Programs;
Health Effects Division; Tox Oneliners; MRID No.00058839; EPA Chem. Code: 024401; Core
Grade/Tox Record No. supplementary 004457
Deenihan MJ.1987; Fine 20 Copper Sulfate Pentahydrate - Acute Toxicology Testing: (A) Acute Oral
Toxicity. Northview Pacific laboratories, Inc. U.S. EPA, Office of Pesticide Programs; Health Effects
Division; Tox Oneliners; MRID No.433962-01A; EPA Chem. Code: 024401; Core Grade/Tox
Record No. acceptable; 011521; Apr. 20, 1995
Smyth HF Jr, Carpenter CP, Weil CS, Pozzani UC, Striegel JA, Nycum JS. 1969. Range-finding
toxicity data: List VII. Am Ind Hyg Assoc J 30:470-476.
Carnegie-Mellon University, Pittsburgh, PA (LD50 value)
Smyth HF Jr, Carpenter CP, Weil CS, Pozzani UC, Striegel JA. 1962. Range-finding toxicity data:
List VI. Am Ind Hyg Assoc J 23:95-107.
Mellon Institute of Industrial Research, Pittsburg, PA (experimental parameters)

Cupric sulfate pentahydrate

300

1570

1030 - 2400

NA

rat

NA

oral

NA

NA

low purity (20%)

copper sulfate
Hazleton Laboratories America, Inc.; HLA B1100274; Feb.27, 1989; U.S. EPA, Office of Pesticidepentahydrate 20%
Programs; Health Effects Division; Tox Oneliners; MRID No. 41043001; EPA Chem. Code:(Odor
024401; Core Grade/Tox Record No. Guideline 009092; Feb. 5, 1992
inhibitor/bactericide)

Cupric sulfate pentahydrate

300

2300

1150 - 3390

NA

rat

female

oral

NA

NA

low purity (11%)

BASF; 82/168; Aug. 11, 1986; U.S. EPA, Office of Pesticide Programs; Health Effects Division;copper sulfate 11% Tox Oneliners; MRID No. 00149179; EPA Chem. Code: 024401; Core Grade/Tox Record No.
Guideline 006197

Cupric sulfate pentahydrate

300

2530

2010 - 3170

NA

rat

male andfemale

oral

NA

NA

low purity (11%)

BASF; 82/168; Aug. 11, 1986; U.S. EPA, Office of Pesticide Programs; Health Effects Division;copper sulfate 11% Tox Oneliners; MRID No. 00149179; EPA Chem. Code: 024401; Core Grade/Tox Record No.
Guideline 006197

Cupric sulfate pentahydrate

300

2610

1890 - 4140

NA

rat

male

oral

NA

NA

low purity (11%)

BASF; 82/168; Aug. 11, 1986; U.S. EPA, Office of Pesticide Programs; Health Effects Division;copper sulfate 11% Tox Oneliners; MRID No. 00149179; EPA Chem. Code: 024401; Core Grade/Tox Record No.
Guideline 006197

H-16

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

Cupric sulfate pentahydrate

LD501
mg/kg

300

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference> 0.5mL/kg <2.0 mL/kg

NA

NA

Animal Information(stock, weight, age)

Sprague-Dawley rats

Gendermale

Route and/or
Method ofExposure

oral

Dose

0.5, 2.0, 5.0 mL/kg

Observations

no toxic signs

Notes

NA

Reference
Substance Source

Primary Reference

WARF Institute, Inc.; WARF No. 1052198; Mar. 20, 1978; U.S. EPA, Office of Pesticide Programs;
Cutrine (28% copper
Health Effects Division; Tox Oneliners; MRID No.00157309; EPA Chem. Code: 024401; Coresulfate)Grade/Tox Record No. supplementary 002707

Cycloheximide2

1(calculated by
NICEATM)

NA

NA

rats

NA

aqueous solutions orsuspensions; 0.5, 0.75, 1.0, 1.5,
oral; stomach tube 2.0, 2.5, 5.0, 7.5, 10, 15, 25, 50, rats at higher doses had bloody urine and profuse watery feces
75, 100, 150, 200 mg/kg doserange

2 rats/dose; 32 rats used; 27/32 rats
dead; 75-200 mg/kg: all dead within
5 hour; 10-50 mg/kg: all deadovernight; 7.5 mg/kg: 1 deadovernight, other at 26 hour; 5.0
mg/kg: 1 dead overnight, other at 24
Upjohn Company
hour; 2.5 mg/kg: all dead at 24 and25 hour; 2.0 mg/kg: all deadovernight and 23 hour; 1.5 mg/kg: alldead at 25 hour; 1.0 mg/kg: 1 dead at
25 hour, 1 survived; 0.5 - 0.75
mg/kg: all survived

Cycloheximide

2

1.8

NA

NA

rats

NA

oral

NA

NA

NA

NA

Compounds Available for Fundamental Research, Volume II-6, Antibiotics, A Program of Upjohn
Company Research Labo (Kalamazoo, MI 49001).1971. (RTECS REFERENCE)

Cycloheximide

2

2.5

NA

NA

rats

NA

oral

NA

excessive salivation, diarrhea, nervousness, depression

NA

Upjohn Company

Ford JH, Klomparens W. 1960. Cycloheximide (Acti-dione) and its non agricultural uses. Antibiotics
and Chemotherapy 10:682 - 687. The Upjohn Co., Kalamazoo, MI

Dibutyl phthalate

7499

7499

7072 - 8006
(95% CL)

NA

rats

NA

oral

NA

NA

NA

NA

Weisheng Dulixue Zazhi. Journal of Health Toxicology. (Weisheng Dulixue Zazhi Bianjibu,
Dongdaqiao, Chaoyang Menwai, Beijing, Peop. Rep. China) V.1- 1987, 1991. (RTECSREFERENCE)

Dibutyl phthalate

7499

8000

NA

NA

Sprague-Dawley rats; 60-75
maleg; 5-6 weeks

oral

single undiluted doses; 4000,
8000, 16000, 32000 mg/kg
doses

7 day observation

4000 mg/kg - 0/3 dead; 8000 mg/kg 4/9 dead; 16000 mg/kg - 6/6 dead;NA
32000 mg/kg - 6/6 dead; 24 rats used

Smith CC. 1953. Toxicity of butyl stearate, dibutyl sebacate, dibutyl phthalate, and methoxyyethyl
oleate. Arch Ind Hyg 7:310-318.

Dibutyl phthalate

7499

8380

6860 - 10230

NA

Sherman strain rats; 120 gNANA

dosage series when expressed in/kg constitutes theNAantilogarithms of 1.0, 1.1, 1.2,
etc

NA

NA

Smyth HF, Carpenter CP. 1948. Further experience with the range finding test in the industrial
toxicology laboratory. J Ind Hyg Toxicol 30:63-68. Melon Institute, Pittsburgh, PA

Dibutyl phthalate

7499

12436
(11.9 mL/kg)

NA

Karber's method

white rats; 60-75 g; 6 weeks NA

oral

NA

degenerative liver changes notedreference is untranslated Russianwith English abstract; NICEATM
converted 11.9 mL/kg LD50 to
mg/kg using provided density of
1.045 g/mL

NA

Homrowski S, Nikonorow M. 1959. Toksycznosc ostra ftalanu dwubutylu oraz ftalanu dwu-2etyloheksylu produkcji krajowej. Roczniki Panstwowego Zakladu Higieny 10:321-327.

Dichlorvos (DDVP)

17

17

NA

NA

rats

NA

oral

NA

NA

unknown primary reference

NA

Japan Pesticide Information. (Japan Plant Protection Assoc., 1-43-11, Komagome, Toshima-ku,
Tokyo 170, Japan) No.1-61, 1969-92. 1972. (RTECS REFERENCE)

Dichlorvos (DDVP)

17

50

NA

Litchfield andWilcoxon method(1949)

CFY strain rats; 120+ g;
adult

female

oral

NA

NA

NA

93% pure; CibaGeigy, Switzerland

Desi I. 1983. Neurotoxicological investigaton of pesticides in animal experiments. Neurobehav
Toxicol 5:503-515. National Institute of Hygiene, Hungary

Dichlorvos (DDVP)

1754(calculated fromnegative log in
mol/kg [3.61])

24 - 111(CL)

Litchfield andWilcoxon method(1949)

Wistar rats; 150 g

femaleintragastric-ally(metal tube)

ethanol: water 1:4 solution used
observed for 72 hours; decreased body weight
as solvent; 2 mL/kg dosage;

30 rats tested (5 groups of 6 rats)

95% pure

Gajewski D, Katkiewicz M. 1981. Activity of certain enzymes and histomorphological changes insubacute intoxication of rats with selected organophosphates. Acta Physiol Pol 32(5):507-520.
Agicultural Academy (and others), Warsaw, Poland

Dichlorvos (DDVP)

1756

48 - 65(95% CL)

Litchfield andWilcoxon method(1949)

Sherman strain rats; min.wt.:
female = 200 g; min.age of female
90 daysoral; stomach tube

chemical in peanut oil; 0.005
mL/g of bw

80 rats tested; LD50 value from
Durham et al. 1957

technical grade

Dichlorvos (DDVP)

1756

48 - 65(95% CL)

Litchfield andWilcoxon method(1949)

Sherman albino rats

femaledissolved in peanut oil; dosage bulging eyes, excessive lacrimation, sialorrhea, generalized muscle
oral; stomach tube rate of 5ul/g; DDVP
fasiculations, tremors; killed rats dead within 1 hour; all survivors
concentration variedcompletely recovered within 24 hours

NA

technical grade,
90%DDVP

Durham WF, Gaines TB, McCauley RH, Sedlak VA, Mattson MA, Hayes WJ. 1957. Studies on the
toxicity of 0,0-dimethyl-2,2-dichlorovinyl phosphate (DDVP). AMA Arch Ind Health 15:340-349.
U.S. Dept. of Health, Education and Welfare, Savannah, GA

Dichlorvos (DDVP)

17

68

59 - 79(95% CL)

Litchfield andWilcoxon method(1949)

Sherman albino rats

femaledissolved in peanut oil; dosage bulging eyes, excessive lacrimation, sialorrhea, generalized muscle
oral; stomach tube rate of 5uL/g; DDVP
fasiculations, tremors; killed rats dead within I hour; all survivors
concentration variedcompletely recovered within 24 hours

NA

99% pure DDVP

Durham WF, Gaines TB, McCauley RH, Sedlak VA, Mattson MA, Hayes WJ. 1957. Studies on the
toxicity of 0,0-dimethyl-2,2-dichlorovinyl phosphate (DDVP). AMA Arch Ind Health 15:340-349.
U.S. Dept. of Health, Education and Welfare, Savannah, GA

Litchfield andWilcoxon method(1949)

Sherman strain rats; min.
wt.: male = 175 g; min. ageof 90 days

malechemical in peanut oil; 0.005
oral; stomach tube
mL/g of bw

observed hourly on first day of dosage and twice a day thereafter until
time of death; max survival = died within 1 hour

59 rats tested; LD50 value fromtechnical grade
reseach paper of Durham et al. 1957

Litchfield andWilcoxon method(1949)

CFY strain rats; 120+ g;
adult

male

oral

NA

NA

Traub R, DeWitt JB, Welch JF, Newman DJ. 1950. Toxicity and repellency to rats of actidione. J Am
Pharm Assoc (Sci. Ed.) 39(10):552 - 555.
Army Medical Department Research and Graduate School, Washington, D.C.

Gaines TB. 1960. The acute toxicity of pesticides to rats. Toxicol Appl Pharmacol 2:88-99.
U.S. Dept. of Health, Education, and Welfare, Savannah, GA
observed hourly on first day of dosage and twice a day thereafter until
time of death; max survival = died within 1 hour

Mattson AM, Spillane JT, Pearce GW. 1955. Dimethyl 2,2-dichlorvinyl phosphate (DDVP), an
organic phosphorous compound highly toxic to insects. J Agr Food Chem 3:319-321.
Communicable Disease Center, Savannah, GA

Gaines TB. 1960. The acute toxicity of pesticides to rats. Toxicol Appl Pharmacol 2:88-99.
U.S. Dept. of Health, Education, and Welfare, Savannah, GA
Dichlorvos (DDVP)

1780

62 - 104(95% CL)

Dichlorvos (DDVP)

17

80

NA

NA

H-17

93% pure; CibaGeigy, Switzerland

Mattson AM, Spillane JT, Pearce GW. 1955. Dimethyl 2,2-dichlorvinyl phosphate (DDVP), an
organic phosphorous compound highly toxic to insects. J Agr Food Chem 3:319-321.
Communicable Disease Center, Savannah, GA

Desi I. 1983. Neurotoxicological investigaton of pesticides in animal experiments. Neurobehav
Toxicol 5:503-515. National Institute of Hygiene, Hungary

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

Dichlorvos (DDVP)

1780

62 - 104(95% CL)

Litchfield andWilcoxon method(1949)

Sherman albino ratsmale

dissolved in peanut oil; dosage bulging eyes, excessive lacrimation, sialorrhea, generalized muscle
oral; stomach tube rate of 5 ul/g; DDVP
fasiculations, tremors; killed rats dead within 1 hour; all survivors
concentration variedcompletely recovered within 24 hours

NA

technical grade,
90%DDVP

Durham WF, Gaines TB, McCauley RH, Sedlak VA, Mattson MA, Hayes WJ. 1957. Studies on the
toxicity of 0,0-dimethyl-2,2-dichlorovinyl phosphate (DDVP). AMA Arch Ind Health 15:340-349.
U.S. Dept. of Health, Education and Welfare, Savannah, GA

Dichlorvos (DDVP)

1780

71 - 90(95% CL)

Litchfield andWilcoxon method(1949)

Sherman albino rats

femaledissolved in peanut oil; dosage bulging eyes, excessive lacrimation, sialorrhea, generalized muscle
oral; stomach tube rate of 5 ul/g; DDVP
fasiculations, tremors; killed rats dead within 1 hour; all survivors
concentration variedcompletely recovered within 24 hours

NA

technical grade,
90%DDVP

Durham WF, Gaines TB, McCauley RH, Sedlak VA, Mattson MA, Hayes WJ. 1957. Studies on the
toxicity of 0,0-dimethyl-2,2-dichlorovinyl phosphate (DDVP). AMA Arch Ind Health 15:340-349.
U.S. Dept. of Health, Education and Welfare, Savannah, GA

Dichlorvos (DDVP)

17

97.5

Fischer 344 rats; 7 weeks

maleoral gavage

Ikeda T, Kojima T, Yoshida M, Takahashi H, Tsuda S, Shirasu Y. 1990. Pretreatment of rats with an
24 hour observation; anti-cholinesterase signs of salivation,
98.7% pure; Nippon
dissolved in olive oil; 5 mL/kg
aclimated for 1 week before dosing;organophosphorous insecticide, chlorfenvinphos, protects against subsequent challenge with the same
fasiculation, lacrimation, tremors, irregular respiration, prostration; all
Chemical Industrial
dosing solution; 4 -5 dosages
5 - 10 animals per each dosage
compound. Fundam Appl Toxicol 14(3):560-567.
deaths observed between 2 -24 hours
Company, Ltd.
Mitsukaido Laboratories, Institute of Environmental Toxicology, Japan

Diethyl phthalate

8600> 5590(reported as >5.0 mL/kg;
specific density= 1.118)

95% CL (where
possible);

Diethyl phthalate

8600

8600

Diethyl phthalate

8600

Digoxin28.3

Dimethylformamide

2800

Dimethylformamide2800> 2000NA

acceptable methods(e.g., Bliss, Litchfield
Sprague-Dawley ratsand Wilcoxon, Weil,
Thompson, etc.)

Dimethylformamide

2800

2800

NA

NA

Dimethylformamide

2800

Dimethylformamide

2800

Dimethylformamide

2800

88.6 - 107
Litchfield and(95% CL; slope = Wilcoxon method
1.24 [1.15 - 1.34]) (1949)

Litchfield andWilcoxon method(1949)

Wistar albino rats; 139-164 male andgfemaleoral; gavage

0.5, 1, 2, 5 mL/kg; single dose

observed at 1, 3, 6, and 24 hours after dosing; then observed daily for
14 days; 2 rats dead8 groups of 10 rats (5M, 5F); 80 rats
NAused; fasted overnight

data from EPA TSCATS database; ORAL LD50 TEST IN RATS OF DIETHYL PHTHALATEWITH COVER LETTER DATED 05/09/94 (SANITIZED) (1978) EPA Document No. 86940000887S Fiche No. OTS0557297; Consumer Product Testing, Fairfield, NJ

7840 - 9890

NA

rats

NA

oral

NA

NA

NA

10100

8920 - 11280

NArats; 220 +/- 40 gNAoral; intragastricNANA(source of information not provided) NA

28.27

24.85 - 32.17(limits of error[P=0.95])

Probit method

rats; 250-310 g

male andfemale (equal oral
numbers)NA

mortality rate computed 7 days after administration

3 or 4 groups of 10; 30 - 40 rats used;NA
fasted overnightoral

solvent used in undiluted form

animals observed for a week after medication

nonfasted rats; 6-12 rats of both
sexes used for studies; solvent usedin undiluted form

analytical grade
meeting A.C.S.
specifications

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, IL

male andfemaleoral gavage

single dose

14 day observation; toxicity symptoms: Ptosis, posture, respiratory
effects, lethargy, abnormal gait, tremors, convulsions, prostrate coma;time to onset of signs --; duration of signs -- no signs reported; 0 rats
dead (average per test)

3 dose levels (5 male and 5 femaleeach); 30 rats used; OECD TG401(1981) followed for experimental
procedures

NA

Vandenheuvel MJ, Clark DG, Fielder RJ, Koundakjian PP, Oliver GJA, Pelling D, Tomlinson NJ,
Walker AP. 1990. Jul. The International Validation Of A Fixed-Dose Procedure As An Alternative To
The Classical LD50 Test Food And Chemical Toxicology 28(7):469-482.

NA

oral

NA

NA

NA

NA

Druckery H, Preussmann R, Ivankovic S, Schmahl D. 1966. Organotrope carcinogene Wirkungen bei
65 verschiedenen N-Nitroso-Verbindungen an BD-Ratten. Zeitschrift fur Krebsforschung 69:103201. (RTECS REFERENCE)

3990
2565 - 6270
(4.2 mL/kg;(95% CL; 2.7 - 6.6 Litchfield andSprague-Dawley rats; 80-160
converted to mL/kg; converted to Wilcoxon method andmaleg; young adultmg/kg using
mg/kg using density probit analysis
density = 0.950)= 0.950)oral

solvent used in undiluted form

animals observed for a week after medication

nonfasted rats; groups of 6 rats used analytical grade
for the studies; solvent used inmeeting A.C.S.
undiluted form
specifications

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, ILNAoral; intragastricNANA(source of information not provided) NA

Izmerov NF, Sanotsky IV, Sidorov KK. 1982. Toxicometric Parameters of Industrial Toxic Chemicals
under Single Exposure. International Register of Potentially Toxic Chemicals (IRPTC). United
Nations Environment Programme (UNEP). Centre of International Projects, GKNT. Moscow, Russia.

male

oral

solvent used in undiluted form

animals observed for a week after medication

nonfasted rats; groups of 6 rats used analytical grade
for the studies; solvent used inmeeting A.C.S.
undiluted form
specifications

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, IL

Gigiena Truda i Professional'nye Zabolevaniya. Labor Hygiene and Occupational Diseases. (V/O
Mezhdunarodnaya Kniga, 113095 Moscow, USSR) V.1-36, 1957-1992. 1980.

1425
855 - 2565
(1.5 mL/kg;(95% CL; 0.9 - 2.7 Litchfield andSprague-Dawley rats; 16-50 male andconverted to mL/kg; converted to Wilcoxon method andg; 14 days
femalemg/kg using
mg/kg using density probit analysis
density = 0.950)= 0.950)5800+/- 1200

6840

5700 - 8170

NA

rats

rats; 220 +/- 40 g

(7.2 mL/kg; sp. (95% CL; 6.0 - 8.6 Litchfield andSprague-Dawley rats; 300density = 0.950; mL/kg; sp. density is Wilcoxon method and
470 g; older adultconvert LD50 to 0.950; convert LD50 probit analysis
mg/kg)
to mg/kg)

NA

Timofeevshaia LA, Ivanova NI, Balinina ES. 1980. Toxicology of O-phthalate acid esters andhygiene reglamentation. Gigiena Truda i Professional'nye Zabolevaniya 24(3):25-27.(RTECSREFERENCE)

Izmerov NF, Sanotsky IV, Sidorov KK. 1982. Toxicometric Parameters of Industrial Toxic Chemicals
under Single Exposure. International Register of Potentially Toxic Chemicals (IRPTC). United
Nations Environment Programme (UNEP). Centre of International Projects, GKNT. Moscow, Russia.

Archives Internationales de Pharmacodynamie et de Therapie. (Heymans Institute of Pharmacology,
DePintelaan 185, B-9000 Ghent, Belgium) V.4- 1898, 1966.

H-18

Georges A, Page J, Duvernay G. 1966. Cardiotonic properties of formiloxin: a semi-synthetic cardiac
glycoside. Arch Int Pharmacodyn 164(1):47-55. Research Dept., A. Christianens, S.A., Brussels,
Belgium (RTECS REFERENCE)In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference SubstanceDimethylformamide

LD501
mg/kg

2800

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

7000

NA

Animal Information(stock, weight, age)

2800

7182
(7.6 mL/kg; sp.
density listed as
0.945; convert
LD50 to mg/kg)

Diquat dibromide

231

231

NA

Diquat dibromide

231

121

Diquat dibromide

231

Diquat dibromide

Route and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

oral gastricintubation

in aqueous solution; doses (ing/kg) differ by 1 log to bracket
LD50, then refine LD50 with
LD50 based on mortalities during a 14 day period
doses in a series of antilog 1.1,
1.3, 1.5, etc

6 rats/dose at doses that differ by 1log to bracket LD50 (given 1 week
apart); then refined LD50 with 10
rats/dose in a dose series of antilog
reagent grade
1.1, 1.3, 1.5, etc.; assumed to usematerials/methods of Smyth &Carpenter (1944) except for reportedchangesmale andfemaleoral; stomach tube

diluted in 0.9% saline; 20 - 30
mL/kg dose

observed up to 7 days after administration; all deaths occurred within
24 hour

10 animals per dose (5 male, 5female)pure DMF

Bartsch W, Sponer G, Dietmann K, Fuchs G. 1976. Acute toxicity of various solvents in the mouseand rat. LD50 of ethanol, diethylacetamide, dimethylformamide, dimethylsulfoxide, glycerine, Nmethylpyrrolidone, polyethylene glycol 400, 1,2- propanediol and Tween 20. Arzneimittelforschung26(8):1581-1583.

assumed to be the value from Clark& Hurst 1970

NA

Pesticide Manual. (The British Crop Protection Council, 20 Bridport Rd., Thornton Heath CR4 7QG,UK) V.1- 1968. 1991. (RTECS REFERENCE)

based on assumptionthat probit mortality
Sherman rats; 90-120 g; 4-5
vs log dose has same
male
weeks
slope as similar
chemical

6804 - 7655
(95% CL; 7.2 - 8.1
mL/kg; sp. density Finney (1962) Probit Sprague-Dawley SPF rats;
listed as 0.945;
Analysis170-230 gconvert LD50 to
mg/kg; slope=1.11)

Dimethylformamide

GenderSmyth HF Jr, Carpenter CP. 1948. Further experience with the range finding test in the industrial
toxicology laboratory. J Ind Hyg Toxicol 30: 63-68. (LD50 value)
Smyth HF Jr, Carpenter CP. 1944. The place of the range-finding test in the industrial toxicology
laborotory. J Ind Hyg Toxicol 26:269-273. (most materials/methods)

NA

rats

NA

oral

NA

NA

108 - 136
(95% CL; slope =12.2)

Litchfield andWilcoxon method(1949)

Sherman strain rats (SPF);
min. wt. = 200 g; min. ageof 90 days

femaleoral; stomach tube

chemical in peanut oil; 0.005
mL/g of bw

observed for at least 14 days after dosing or until recovered from signs 40 rats used; min. of 10 animals perof toxicity
group tested

technical grade

Gaines TB, Linder RE. 1986. Acute toxicity of pesticides in adult and weanling rats. Fundam Appl
Toxicol 7(2):299-308. Health Effects Research Laboratory, U.S. EPA, Research Triangle Park,NC

147

138 - 155
(95% CL; slope =22.5)

Litchfield andWilcoxon method(1949)

Sherman strain rats (SPF);
min. wt. = 175 g; min. age of male
90 daysoral; stomach tube

chemical in peanut oil; 0.005
mL/g of bw

observed for at least 14 days after dosing or until recovered from signs 40 rats used; min. of 10 animals perof toxicity
group tested

technical grade

Gaines TB, Linder RE. 1986. Acute toxicity of pesticides in adult and weanling rats. Fundam Appl
Toxicol 7(2):299-308. Health Effects Research Laboratory, U.S. EPA, Research Triangle Park,
NC

231

231
(diquat ion per
kg bw)

194 - 274
(95% CL)

Thompson (1947);
Alderly Park albino rats
moving average(SPF); 180-200 g; young,interpolation method mature

femaleoral; stomach tube

chemical dissolved in water orphysiological saline

observed for 14 days; lethargy, weight loss, respiratory difficultyNA

99% pure diquat
Clark DG, Hurst EW. 1970. The toxicity of diquat. Br J Ind Med Jan;27(1):51-55.
dichloride or diquat
Imperial Chemical Industries Limited, Cheshire, UKdibromide

Disulfoton

2.6

2.3

1.7 - 3.1
(95% CL)

Litchfield andWilcoxon method(1949)

Sherman strain rats; min. wt.
female= 200 g; min. age of 90 days

oral; stomach tube

chemical in peanut oil;
0.005mL/g of bw

observed hourly on first day of dosage and twice a day thereafter until
time of death; max survival 3 days

50 rats tested

technical grade

Gaines TB. 1969. Acute toxicity of pesticides. Toxicol Appl Pharmacol 14(3): 515-34.
U.S. Dept. of Health, Education, and Welfare, Atlanta, GA

Disulfoton

2.6

2.6

NA

estimated by theSprague-Dawley rats; 175 logarithm-probability
225 gmethodfemaleNAanimals observed for 10 days; death or complete recovery occurred
dissolved in 10% ETOH, 90%
within this time; acute toxic dose symptoms typical of those produced
propylene glycol; strength ofby cholinergic organic phosphates; single doses produced effects
solutions adjusted so that less25 rats usedresembling those resulting from excessive stimulation of the central
than 0.3% bw was administerednervous system, the patasympathetic nervous system and somatic motor
to the rats
nerves; after lethal doses death usually occurred within 48 hour

Chemagro Corp.,
New York

Bombinski TJ, Dubois KP. 1958. Toxicity and mechanism of action of Di-syston. AMA Arch Ind
Health 17:192-199.

Disulfoton

2.6

2.6

NA

NA

female

oral

NA

NA

Yakkyoku. Pharmacy. (Nanzando, 4-1-11, Yushima, Bunkyo-ku, Tokyo, Japan) V.1- 1950. 1986.
(see Bombinski and Dubois [1958]) (RTECS REFERENCE)

Pawar SS, Fawade MM. 1978. Alterations in the toxicity of thiodemeton due to the pretreatment of
inducers, substrate, and inhibitors of mixed function oxidase system. Bull Environ Contam Toxicol
20:805-810. Marathwada University, India

rats

reference is a review article inJapanese; this LD50 value isassumed to be from Bombinski andDubois 1958

NA

Disulfoton

2.6

3.2

3.0 - 3.3
(95% CL)NA

Hindustan Antibiotics strain
femalerats; adultoral

overnight fasted; rats pretreated with
one of the following: saline, oil,phenobarbital, 3-methylacute 24 hour LD50 determination; percent mortality given for different cholanthourene, nickel chloride,1 - 10 mg/kg doses; 6 different
timepoints within the 24 hour period; pretreatment of rats reduced
cobalt chloride, cycloheximide orNAdose levels
mortality in some cases
ethylmorphine; reference doesn't
adequately define which rats received
what and if all data were used in
LD50 determinations

Disulfoton

2.6

6.8

5.9 - 7.8
(95% CL)

Litchfield andWilcoxon method(1949)

Sherman strain rats; min. wt.
male= 175 g; min age of 90 days

oral; stomach tube

chemical in peanut oil;
0.005mL/g of bw

oral

overnight fasted; rats pretreated with
one of the following: saline, oil,phenobarbital, 3-methylacute 24 hour LD50 determination; percent mortality given for different cholanthourene, nickel chloride,1 - 10 mg/kg doses; 6 different
timepoints within the 24 hour period; pretreatment of rats reduced
cobalt chloride, cycloheximide orNAdose levels
mortality in some cases
ethylmorphine; reference doesn't
define which rats received what andif all data were used in LD50
determinations

Disulfoton

2.6

7.2

7.0 - 7.3
(95% CL)NA

Hindustan Antibiotics strain
malerats; adultobserved hourly on first day of dosage and twice a day thereafter until
time of death; max survival 2 days

H-19

69 rats tested

technical grade

Gaines TB. 1969. Acute toxicity of pesticides. Toxicol. Appl. Pharmacol. 14(3):515-34.
U.S. Dept. of Health, Education, and Welfare, Atlanta, GA

Pawar SS, Fawade MM. 1978. Alterations in the toxicity of thiodemeton due to the pretreatment of
inducers, substrate, and inhibitors of mixed function oxidase system. Bull Environ Contam Toxicol
20:805-810. Marathwada University, India

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Disulfoton

2.6

12.6

NA

estimated by theSprague-Dawley rats; 175logarithm-probability
225 gmethodmale

NAanimals observed for 10 days; death or complete recovery occurred
dissolved in 10% ETOH, 90%
within this time; acute toxic dose symptoms typical of those produced
propylene glycol; strength ofby cholinergic organic phosphates; single doses produced effects
solutions adjusted so that less
39 rats usedresembling those resulting from excessive stimulation of the central
than 0.3% bw was administerednervous system, the patasympathetic nervous system and somatic motor
to the rats
nerves; after lethal doses death usually occurred within 48 hour

Endosulfan

18

18

NA

NA

NA

NA

NA

NA

NA

Endosulfan

18

18

15 - 21(95% CL)

Litchfield andWilcoxon method(1949)

Sherman strain rats; min wt.
female= 200 g; min age of 90 days

oral; stomach tube

chemical in peanut oil; 0.005
mL/g of bw

Endosulfan

18

43

41 - 46(95% CL)

Litchfield andWilcoxon method(1949)

Sherman strain rats; min wt.
male= 175 g; min age of 90 days

oral; stomach tube

4
(mouse oral)

NA

+/- 1

NA

NA

Epinephrine bitartrate

Reference
Substance Source

Primary Reference

Chemagro Corp.,
New York

Bombinski TJ, Dubois KP. 1958. Toxicity and mechanism of action of Di-syston. AMA Arch Ind
Health 17:192-199.

assumed to be the values from
Gaines 1969

NA

Agricultural Research Service, USDA Information Memorandum. (Beltsville, MD 20705): 20,9,1966.
(see Gaines 1969) (RTECS REFERENCE)

observed hourly on first day of dosage and twice a day thereafter until
time of death; max survival 2 days

60 rats tested

technical grade

Gaines TB. 1969. Acute toxicity of pesticides. Toxicol Appl Pharmacol 14(3):515-34.
U.S. Dept. of Health, Education, and Welfare, Atlanta, GA

chemical in peanut oil; 0.005
mL/g of bw

observed hourly on first day of dosage and twice a day thereafter until
time of death; max survival 5 days

70 rats tested

technical grade

Gaines TB. 1969. Acute toxicity of pesticides. Toxicol Appl Pharmacol 14(3):515-34.
U.S. Dept. of Health, Education, and Welfare, Atlanta, GA

NA

NA

observed for 5 days

NA

NA

Acta Pharmacologica et Toxicologica. (Copenhagen, Denmark) V.1-59, 1945-86. 1972. (RTECSREFERENCE)

6162(7.8 mL/kg;
4977 - 7663
Litchfield andSprague-Dawley rats; (16-50 male andconverted to(95% CL; 6.3 - 9.7 Wilcoxon method andg); 14 days
femalemg/kg using
mL/kg)
probit analysis
density of 0.790)

oral

solvent used in undiluted form

animals observed for a week after medication

nonfasted rats; 6-12 rats of both
sexes used for studies; solvent usedin undiluted form

analytical grade
meeting A.C.S.
specifications

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, IL

moving average ofWeil (1952) or
Litchfield andWilcoxon method(1949)

Wistar albino rats; old adult;
male
11-12 months

oral

dose interval 1.1; ethanol
concentration of 40% w/v

acute (24 hour) toxicity; respiratory failure

fasted overnight; 6 - 8 grouped of 10
NArats eachNA

malerats; 150-250 g; 70- 100 days (predominate orally)NAobserved for 6 days

18 hour fasting before dosing

NA

Ethanol

7060

Ethanol

7060

7060

6670 - 7460
(95% CL)

Ethanol

7060

7400

NA

Ethanol

7060

10600

10000 - 11200(95% CL)

Litchfield andWilcoxon method(1949) or moving
average of Weil
(1952)

Wistar albino rats; young
adult; 100 days

male

oral

dose interval 1.1; ethanol
concentration of 40% w/v

acute (24 hour) toxicity; respiratory failure

fasted overnight; 6 - 8 grouped of 10
NArats each

Wiberg GS, Trenholm HL, Coldwell BB. 1970. Increased ethanol toxicity in old rats: changes in
LD50, in vivo and in vitro metabolism, and liver alcohol dehydrogenase activity. Toxicol. Appl.
Pharmacol. May. 16(3):718-727. Dept. of National Health and Welfare, Ottawa, Canada

Ethanol

7060

11290 - A
11204 - B
(A = 14.31
mL/kg; B =14.20 mL/kg;
used density of
0.789 to convert
to mg/kg)

NA

A: Behrens (1929)
B: Bliss (1938)

rats

NA

oral

NA

NA

40 - 90 animals used; NICEATM
used value B since authors stated it
was more accurate

Deichmann WB, Mergard EG. 1948. Comparative evaluation of methods employed to express thedegree of toxicity of a compound. J Ind Hyg Toxicol 30:373-378.
Albany Medical College, Albany, NY; University of Cincinnati, Cincinnati, OH

Ethanol

7060

11534
(14.6 mL/kg;
10112 - 13193
Litchfield andSprague-Dawley rats; 300used density of (95% CL; 12.8 - 16.7 Wilcoxon method and
470 g; older adult
0.790 to convert
mL/kg)
probit analysis
to mg/kg)

male

oral

solvent used in undiluted form

animals observed for a week after medication

nonfasted rats; groups of 6 rats used analytical grade
for the studies; solvent used inmeeting A.C.S.
undiluted form
specifications

Ethanol

7060

13660

7060

15543
(19.7 mL/kg;
used density of
0.789 to convert
to mg/kg)

11170 - 16710(95% probability; +/probits (Bliss)
1.96 S.D.; slope =4.57)

Wistar albino rats; 90-120 g maleoral; stomach tube; 50% concentration in water;most deaths occurred in 2 days; all deaths occurred in 14 days
single doses
largest dose given was 50 g/kg

NA

NA

Wiberg GS, Trenholm HL, Coldwell BB. 1970. Increased ethanol toxicity in old rats: changes in
LD50, in vivo and in vitro metabolism, and liver alcohol dehydrogenase activity. Toxicol Appl
Pharmacol May 16(3):718-727.
Dept. of National Health and Welfare, Ottawa, Canada (RTECS REFERENCE)
Welch H, Slocum GG. 1943. Relation of length of carbon chain to the primary and functional
toxicities of alcohols. J Lab Chem Med 28:1440-1445. U.S. FDA, Washington, D.C.

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, IL

Smyth HF Jr, Seaton J, Fischer, L. 1941. The single dose toxicity of some glycols and derivatives. J
groups of 10 animals; 10 animals per purified commercial
Ind Hyg Toxicol 23:259-268. Mellon Institute, Pittsburgh, PA
dose
grade
(This was the value used by the RC [from 1977 RTECS]).
Smyth HF, Weil CS, West JS, Carpenter CP. 1970. An exploration of joint toxic action:II. Equitoxic
versus equivolume mixtures. Toxicol Appl Pharmacol 17:498-503. (LD50 value)

Ethanol

Thompson method;
Weil tables

Carworth-Wistar rats; 90120 g; 4-5 weeks

maleoral gastricintubation

in aqueous solution;
concentration intubated = 10
mg/mL; dosages arranged in a LD50 based on mortalities during a 14 day period
logarithmic series differing by afactor of 2

H-20

non-fasted; groups of 5 rats; single
oral dose toxicity

reagent grade

Smyth HF Jr., Carpenter CP., Weil CS., Pozzani, UC., Striegel, JA. And Nycum, JS. 1969. Rangefinding toxicity data: List VII. Am Ind Hyg Assoc J 30:470-476.
Carnegie-Mellon University, Pittsburgh, PA
Smyth HF Jr., Carpenter CP., Weil CS., Pozzani, UC., and Striegel, JA. 1962. Range-finding toxicity
data: List VI. Am Ind Hyg Assoc J 23:95-107.
Mellon Institute of Industrial Research, Pittsburg, PA (experimental parameters)

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

Gender17775(22.5 mL/kg;
14852 - 21330
Litchfield andSprague-Dawley rats; 80-160
used density of (95% CL; 18.8 - 27.0 Wilcoxon method andmaleg); young adult
0.790 to convert
mL/kg)
probit analysis
to mg/kg)

Route and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

oral

solvent used in undiluted form

animals observed for a week after medication

nonfasted rats; groups of 6 rats used analytical grade
for the studies; solvent used inmeeting A.C.S.
undiluted form
specifications

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, IL

Fischer 344 (COB CD F/CrlBR) rats; 150-200 g; 12-14 female
weeksoral intubation

0.1 log dosages with 5 rats per
levelanimals observed for mortality daily for 14 days

fasted overnight; no dosage exceeded
Aldrich Chemical
24 g/kg bw; LD50 and 95%
Co.; high purity;
confidence limits calculated at 24> 99% ethylene
hour post-treatment; no deaths
glycolbeyond 72 hour post-treatment

Clark CR, Marshall TC, Merickel BS, et al. 1979. Toxicological assessment of heat transfer fluids
proposed for use in solar energy applications. Toxicol Appl Pharmacol 5(1):529-535.
Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental researchInstitute, Alburquerque, NM

rats

NA

oral

NA

NA

reference in intranslated Russian;same reference was cited in 1983/84
RTECs, but this is not the LD50 used NA
by RC (ZEBET did not provide thereference)

Filatova VS, Smirkova ES. 1982. Derivation of the maximum permissable concentration of ethylen
glycol in the air of worksites. Gigiena Truda i Professional'nye Zabolevaniya. 26(6):28-30. (RTECSREFERENCE)

NA

Holzman Sprague-Dawley
rats

male

oral gavage

50 mg/kg, 500 mg/kg, and 5000 clinical observations included depression, labored breathing,mg/kg in corn oil
emaciation, and alopecia

3 groups of 10 males; no mortalities
NA
were observed

from EPA TSCATS database; Acute Toxicity Study in Rats Administered 10 Materials (final report)with Cover Letter dated 062669, (1969), EPA Doc. No. 40-6942188, Fiche No. OTS0519234;
FMC Corporation

5053 - 7106
(95% probability;
4.53 - 6.37 cc/kg)

probits (Bliss)rats from the same strain;
275 +/- 25 g; 3 months +/- 9 NAdays

5 doses for 11 animals each dose; 55
NArats used

Laug EP, Calvery HO, Morris HJ, Woodard G. 1939. The toxicology of some glycols and derivatives.
J Ind Hyg Toxicol 21:173-201. Division of Pharmacology, Food and Drug Administration, U.S.
Dept. of Agriculture, Washington, D.C.

6135
(5.50 cc/kg;
converted to
mg/kg using
density of
1.1155)5578 - 6749
(95% probability;
5.00 - 6.05 cc/kg)

probits (Bliss)rats from different sources;
175-325 g

Ethanol

7060

Ethylene glycol

4700

4000

3100 - 5200
(95% CI; slope =258)

Ethylene glycol

4700

4700

NA

Ethylene glycol

4700

>5000

NA

Ethylene glycol

4700

5890
(5.28 cc/kg;
converted to
mg/kg using
density of
1.1155)Ethylene glycol

4700

Litchfield andWilcoxon methodmale andfemale (~equal)

single doses; 3904 mg/kg--7028
most deaths occurred in 1 - 5 days; weakness and lack of muscular
oral; stomach tube; mg/kg; log doses 0.544, 0.608,
coordination; no deaths per dose: 3904 mg/kg -- 2/11; 4440 mg/kg -single doses
0.672, 0.735, 0.799; diluted 1 +3/11; 5243 mg/kg -- 3/11; 6057 mg/kg -- 5/11; 7028 mg/kg -- 8/11
3oral; stomach tube; single doses; 3904 mg/kg -single doses
8366 mg/kg

most deaths occurred in 1 - 5 days; weakness and lack of muscular
coordination; no deaths per dose: 3904 mg/kg - 0/7; 4462 mg/kg - 4/20;
rats fasted for about 18 hours; 147
5020 mg/kg - 3/10; 5578 mg/kg - 11/20; 6135 mg/kg - 15/20; 6693
rats used; 76 diedmg/kg - 4/10; 6972 mg/kg - 7/10; 7251 mg/kg - 2/10; 7809 mg/kg -NA

Laug EP, Calvery HO, Morris HJ, Woodard G. 1939. The toxicology of some glycols and derivatives.
J Ind Hyg Toxicol 21:173-201. Division of Pharmacology, Food and Drug Administration, U.S.
Dept. of Agriculture, Washington, D.C.

NA

Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388.
Mellon Institute, Pittsburgh, PA and The Dow Chemical Company, Midland, MI

13/20; 8366 mg/kg - 17/20

Thompson (1947) andManor farms Wistar rats;
Weil (1952); moving
150-200 g
average tables

single dose; geometric factor
between dosage levels=2;
undiluted

14 day observation

5 rats per dosage level; fasted
overnight

most deaths occurred in 1 - 5 days; weakness and lack of muscular
coordination; no deaths per dose: 3904 mg/kg -- 2/11; 4440 mg/kg -2/11; 5243 mg/kg -- 4/11; 6057 mg/kg -- 5/11; 7028 mg/kg -- 6/11

5 doses for 11 animals each dose; 55
NArats used

Laug EP, Calvery HO, Morris HJ, Woodard G. 1939. The toxicology of some glycols and derivatives.
J Ind Hyg Toxicol 21:173-201. Division of Pharmacology, Food and Drug Administration, U.s.
Dept. of Agriculture, Washington, D.C.

single dose; geometric factor
between dosage levels = 2;
undiluted

14 day observation

5 rats per dosage level; fasted
overnight

NA

Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388.
Mellon Institute, Pittsburgh, PA and The Dow Chemical Company, Midland, MI

oral; stomach
intubation

single dose; geometric factor
between dosage levels = 2;
undiluted

14 day observation

5 rats per dosage level; fasted
overnight

NA

Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388.
Mellon Institute, Pittsburgh, PA and The Dow Chemical Company, Midland, MIoral gastricintubation

in aqueous solution;
concentration intubated = 10
mg/mL; dosages arranged in a LD50 based on mortalities during a 14 day period
logarithmic series differing by afactor of 2

non-fasted; groups of 5 rats; single
oral dose toxicity

single dose; geometric factor
between dosage levels = 2;
undiluted

5 rats per dosage level; fasted
overnight

4700

6500

NA

Ethylene glycol

4700

6537
(5.86 cc/kg;
converted to
mg/kg using
density of
1.1155)5064 - 8455
(95% probability;
4.54 - 7.58 cc/kg)

Ethylene glycol

4700

6860

NA

Thompson (1947) andManor farms Wistar rats;
Weil (1952); moving
150-200 g
average tables

male

oral; stomach
intubation

Ethylene glycol

4700

7460

NA

Thompson (1947) andManor farms Wistar rats;
Weil (1952); moving
150-200 g
average tables

male

4700

7887
(7.07 mL/kg;
converted to
mg/kg using
density of
1.1155)

NA

8000

NA

probits (Bliss)maleoral; stomach
intubation;

Ethylene glycol

single doses; 3904 mg/kg -oral; stomach tube; 7028 mg/kg; log doses 0.544,
single doses
0.608, 0.672, 0.735, 0.799;
undiluted

rats from the same strain;
275 +/- 25 g; 3 months +/- 9
days

Smyth HF, Weil CS, West JS, Carpenter CP. 1970. An exploration of joint toxic action:II. Equitoxic
versus equivolume mixtures. Toxicol Appl Pharmacol 17:498-503. (LD50 value)

Ethylene glycol

Ethylene glycol

4700

Thompson method;
Weil tables

Carworth-Wistar rats; 90120 g; 4-5 weeks

Thompson (1947) andManor farms Wistar rats;
Weil (1952); moving
150-200 g
average tables

male

male

oral; stomach
intubation

14 day observation

H-21

reagent grade

Smyth HF Jr., Carpenter CP., Weil CS., Pozzani, UC., Striegel, JA. And Nycum, JS. 1969. Rangefinding toxicity data: List VII. Am Ind Hyg Assoc J 30:470-476.
Carnegie-Mellon University, Pittsburgh, PA
Smyth HF Jr., Carpenter CP., Weil CS., Pozzani, UC., and Striegel, JA. 1962. Range-finding toxicity
data: List VI. Am Ind Hyg Assoc J 23:95-107.
Mellon Institute of Industrial Research, Pittsburg, PA (experimental parameters)

NA

Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388. Mellon Institute, Pittsburgh, PA and The Dow
Chemical Company, Midland, MI

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

Ethylene glycol

4700

8120

NA

Thompson (1947) andManor farms Wistar rats;
Weil (1952); moving
150-200 g
average tables

male

oral; stomach
intubation

single dose; geometric factor
between dosage levels = 2;
undiluted

14 day observation

5 rats per dosage level; fasted
overnight

NA

Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388. Mellon Institute, Pittsburgh, PA and The Dow
Chemical Company, Midland, MI

Ethylene glycol

4700

8480

NA

Thompson (1947) andManor farms Wistar rats;
Weil (1952); moving
150-200 g
average tables

male

oral; stomach
intubation

single dose; geometric factor
between dosage levels = 2;
undiluted

14 day observation

5 rats per dosage level; fasted
overnight

NA

Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388. Mellon Institute, Pittsburgh, PA and The Dow
Chemical Company, Midland, MI

Ethylene glycol

4700

8540

4700

9058
(8.12 mL/kg;
converted to
mg/kg using
density of
1.1155)NA7310 - 9990
(95% probability; +/probits (Bliss)
1.96 S.D.; slope =5.71)

Wistar albino rats; 90-120 g maleoral; stomach tube; 50% concentration in water;most deaths occurred in 2 days; all deaths occurred in 14 days
single doses
largest dose given was 50 g/kg

groups of 10 animals; 10 animals percommercial grade
dose

Smyth HF Jr, Seaton J, Fischer L. 1941. The single dose toxicity of some glycols and derivatives. J
Ind Hyg Toxicol 23:259-268. Mellon Institute, Pittsburgh, PA.
(This is the value used by the RC [from 1981/82 RTECS]).
Smyth HF, Weil CS, West JS, Carpenter CP. 1970. An exploration of joint toxic action:II. Equitoxic
versus equivolume mixtures. Toxicol Appl Pharmacol 17:498-503. (LD50 value)

Ethylene glycol

Thompson method;
Weil tables

Carworth-Wistar rats; 90120 g; 4-5 weeks

maleoral gastricintubation

in aqueous solution;
concentration intubated = 10
mg/mL; dosages arranged in a LD50 based on mortalities during a 14 day period
logarithmic series differing by afactor of 2

non-fasted; groups of 5 rats; single
oral dose toxicity

reagent grade

Smyth HF Jr., Carpenter CP., Weil CS., Pozzani, UC., Striegel, JA. And Nycum, JS. 1969. Rangefinding toxicity data: List VII. Am Ind Hyg Assoc J 30:470-476.
Carnegie-Mellon University, Pittsburgh, PA
Smyth HF Jr., Carpenter CP., Weil CS., Pozzani, UC., and Striegel, JA. 1962. Range-finding toxicity
data: List VI. Am Ind Hyg Assoc J 23:95-107.
Mellon Institute of Industrial Research, Pittsburg, PA (experimental parameters)

Ethylene glycol

4700

9850

NA

Thompson (1947) andManor farms Wistar rats;
Weil (1952); moving
150-200 g
average tables

male

oral; stomach
intubation

single dose; geometric factor
between dosage levels = 2;
undiluted

14 day observation

5 rats per dosage level; fasted
overnight

NA

Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388. Mellon Institute, Pittsburgh, PA and The Dow
Chemical Company, Midland, MI

Ethylene glycol

4700

9900

NA

Thompson (1947) andManor farms Wistar rats
Weil (1952); moving(SPF); 150-200 g
average tables

male

oral; stomach
intubation

single dose; geometric factor
between dosage levels = 2;
undiluted

14 day observation

5 rats per dosage level; fasted
overnight

NA

Weil CS, Wright GJ. 1967. Intra- and Interlaboratory Comparative Evaluation of Single Oral Test.
Toxicology and Applied Pharmacology 11:378-388.
Mellon Institute, Pittsburgh, PA and The Dow Chemical Company, Midland, MI

Ethylene glycol

4700> 10000

NA

NA

Sprague-Dawley rats

femaleoral; gavage

single dose; 1250, 2500, 5000,
10000 mg/kg doses

14 day observation; no rats diedethylene glycol engine coolant; test
material is 50/50 (vol.) ethylene
NAglycol and water mix with 1.5 oz./galof DCA inhibitor

from EPA TSCATS database; Initial Submission: Acute Toxicological Properties & HandlingHazards With Ethylene Glycol Tested In Rats (Final Report) With Cover Letter Dated 051492; EPA
Doc. No. 88-920003189 Fiche No.OTS0539777. The Dow Chemical Co.

Ethylene glycol

4700

17800

NA

Litchfield andWilcoxon method

Holzman Sprague-Dawley
rats; 243- 274 gmaleoral intubation

316 mg/kg, 1000 mg/kg, 3160
mg/kg, 10000 mg/kg, 31600
mg/kg in corn oil

clinical observations included depression, rapid respiration andhunching; 2 rats dead at highest dose

5 groups of 2 males; only mortalities
were both rats at the 31600 mg/kg
NAdose; fasted overnightfrom EPA TSCATS database; Acute Toxicity Study in Rats Administered One of 10 Materials (final
report) with Cover Letter dated 090869, (1969), EPA Doc. No. 40-6942189, Fiche No. OTS0519235.
FMC Corporation

Fenpropathrin

18

18 - 24NA

NA

Charles River (?) rats

femaleoral

5% solution in DMSO

mortalities recorded 10 days after dosing

15 male, 15 female rats used; 30 total
rats; rats injected with 0.9% salineNAi.p. (1 mL/kg) 2 hour before dosing

Crawford MJ, Hutson DH. 1977. The metabolism of the pyrthroid insecticide (+/-)-a-cyano-3phenoxybenzyl 2,2,3,3-tetramethyl-cyclopropanecarboxylate, WL 41706, in the rat. Pestic Sci 8:579599. Shell Research Limited, Kent, UK (RTECS REFERENCE)

Fenpropathrin

18

24 - 36NA

NACharles River (?) rats

male

oral

5% solution in DMSO

mortalities recorded 10 days after dosing

15 male, 15 female rats used; 30 total
rats; rats injected with 0.9% salineNAi.p. (1 mL/kg) 2 hour before dosing

Crawford MJ, Hutson DH. 1977. The metabolism of the pyrthroid insecticide (+/-)-a-cyano-3phenoxybenzyl 2,2,3,3-tetramethyl-cyclopropanecarboxylate, WL 41706, in the rat. Pestic Sci 8:579599. Shell Research Limited, Kent, UK

Fenpropathrin

18

24 - 36NA

NACharles River (?) rats

femaleoral

5% solution in DMSO

mortalities recorded 10 days after dosing

12 male, 12 female rats used; 24 total
rats; rats pretreated with corn oil 18 NAhour before dosing

Crawford MJ, Hutson DH. 1977. The metabolism of the pyrthroid insecticide (+/-)-a-cyano-3phenoxybenzyl 2,2,3,3-tetramethyl-cyclopropanecarboxylate, WL 41706, in the rat. Pestic Sci 8:579599. Shell Research Limited, Kent, UK

Fenpropathrin

18

24 - 36NA

NACharles River (?) rats

male

oral

5% solution in DMSO

mortalities recorded 10 days after dosing

12 male, 12 female rats used; 24 total
rats; rats pretreated with corn oil 18 NAhour before dosing

Crawford MJ, Hutson DH. 1977. The metabolism of the pyrthroid insecticide (+/-)-a-cyano-3phenoxybenzyl 2,2,3,3-tetramethyl-cyclopropanecarboxylate, WL 41706, in the rat. Pestic Sci 8:579599. Shell Research Limited, Kent, UK

Fenpropathrin

18

48.5

37.6 - 62.6
(CL)

NA

rats

femaleoral gavage

single doses (mg/kg): 15, 20,
30, 50, 59, 77, 100, 120, 169;
doses in corn oil

observed for 14 days; decrease of spontaneous motor activity,
hypersensitivity, fibrillation, tremor, clonic convulsion, salivation,
lacrimation, incontinence, hind limb ataxia; deaths resulted within 24
hour and signs of intoxication dissapeared in 24 - 48 hour; min. toxic
dose was 20 mg/kg

8 groups of 10 rats; 80 rats used

Fenpropathrin 97%(S-3206 lot. No.
022018)

Sumitomo Chemical Co., Japan; FT-50-0018; Jan. 1, 1979; U.S. EPA, Office of Pesticide Programs;
Health Effects Division; Tox Oneliners; MRID No. 00127343; EPA Chem. Code: 127901; Core
Grade/Tox Record No. minimum 004567; EPA Accession No. 249937

Fenpropathrin

18

49

NA

NA

rats

female

oral

NA

NA

assumed to be same LD50 value as
Sumitomo 1979

NA

Fujita Y. 1981. Meothrin (Fenpropathrin). Japan Plant Protection Assoc. Japan Pesticide Information
38:21 -25.

Fenpropathrin

185443.5 - 67.0
(CL)

NA

rats

male

oral gavage

single doses (mg/kg): 15, 20,
30, 50, 59, 77, 100, 120, 169;
doses in corn oil

observed for 14 days; decrease of spontaneous motor activity,
hypersensitivity, fibrillation, tremor, clonic convulsion, salivation,
lacrimation, incontinence, hind limb ataxia; deaths resulted within 24
hour and signs of intoxication dissapeared in 24 - 48 hour; min. toxic
dose was 20 mg/kg

9 groups of 10 rats; 90 rats used

Fenpropathrin 97%(S-3206 lot. No.
022018)

Sumitomo Chemical Co., Japan; FT-50-0018; Jan. 1, 1979; U.S. EPA, Office of Pesticide Programs;
Health Effects Division; Tox Oneliners; MRID No. 00127343; EPA Chem. Code: 127901; Core
Grade/Tox Record No. minimum 004567; EPA Accession No. 249937

Fenpropathrin

18

54

NA

NA

rats

male

oral

NA

NA

assumed to be same LD50 value as
Sumitomo 1979

NA

Fujita Y. 1981. Meothrin (Fenpropathrin). Japan Plant Protection Assoc. Japan Pesticide Information
38:21-25.

H-22

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

Fenpropathrin

18

66.7

50.6 - 87.9
(CL)

NA

Sprague Dawley rats

femaleoral gavage

observed for 14 days; signs of intoxication with doses 25 mg/kg andabove; muscular fibrillation, soft feces, diarrhea, tremor, decreased
single doses (mg/kg): 0, 10, 25,
spontaneous activity, ataxia, limb paralysis, irregular respiration, slight rats fasted 20 hour before dosing; 950, 60, 72, 86, 104, 125; doses
salivation, urinary incontinence; signs developed an hour after dosing groups of 10 rats; 90 rats usedin corn oil
but rats recovered after 3 days; deaths resulted on day of dosing or day
after dosing

Fenpropathrin 91.8%
Sumitomo Chemical Co., Japan; FT-30-0081; Jan. 17, 1983; U.S. EPA, Office of Pesticide Programs;(S-3206 technical
Health Effects Division; Tox Oneliners; MRID No. 00127342; EPA Chem. Code: 127901; Core
grade, lot. No.
Grade/Tox Record No. guideline 004567; EPA Accession No. 249937
2TC019)Fenpropathrin

18

70.6

53.7 - 92.7
(CL)

NA

Sprague Dawley ratsmaleoral gavage

observed for 14 days; signs of intoxication with doses 25 mg/kg andabove; muscular fibrillation, soft feces, diarrhea, tremor, decreased
single doses (mg/kg): 0, 10, 25,
spontaneous activity, ataxia, limb paralysis, irregular respiration, slight rats fasted 20 hour before dosing; 950, 60, 72, 86, 104, 125; doses
salivation, urinary incontinence; signs developed an hour after dosing groups of 10 rats; 90 rats usedin corn oil
but rats recovered after 3 days; deaths resulted on day of dosing or day
after dosing

Fenpropathrin 91.8%
Sumitomo Chemical Co., Japan; FT-30-0081; Jan. 17, 1983; U.S. EPA, Office of Pesticide Programs;(S-3206 technical
Health Effects Division; Tox Oneliners; MRID No. 00127342; EPA Chem. Code: 127901; Core
grade, lot. No.
Grade/Tox Record No. guideline 004567; EPA Accession No. 249937
2TC019)Fenpropathrin

18

71.6

56.1 - 92.0

NA

rats

female

oral

NA

NA

NA

International Reseach & Development Corp.; 491-003; FT-11-0052; Oct. 26, 1981; U.S. EPA, Office
Danitol S-3206 (2.4of Pesticide Programs; Health Effects Division; Tox Oneliners; MRID No. 00128341; EPA Chem.
lb/GEC)
Code: 127901; Core Grade/Tox Record No. guideline 003814

Fenpropathrin

18

72.1

53.0 - 82.5

NA

rats

male andfemale

oral

NA

NA

NA

International Reseach & Development Corp.; 491-003; FT-11-0052; Oct. 26, 1981; U.S. EPA, Office
Danitol S-3206 (2.4of Pesticide Programs; Health Effects Division; Tox Oneliners; MRID No. 00128341; EPA Chem.
lb/GEC)
Code: 127901; Core Grade/Tox Record No. guideline 003814

Fenpropathrin

18

72.4

62.1 - 84.3

NA

rats

male

oral

NA

NA

NA

International Reseach & Development Corp.; 491-003; FT-11-0052; Oct. 26, 1981; U.S. EPA, Office
Danitol S-3206 (2.4of Pesticide Programs; Health Effects Division; Tox Oneliners; MRID No. 00128341; EPA Chem.
lb/GEC)
Code: 127901; Core Grade/Tox Record No. guideline 003814

Fenpropathrin

18

107

69.8 - 164
(CL)

NA

Sprague Dawley rats

femaleoral gavage

observed for 14 days; toxic signs noted at 50 mg/kg and above;
single doses (mg/kg): 0, 25, 50,
muscular fibrillation, tremor, ataxia, limb paralysis, irregular90, 120, 160, 220, 300
respiration, lacrimation, salivation, urinary incontinence, diarrhea

8 groups of 10 rats; 80 rats used

Fenpropathrin 97.3% Sumitomo Chemical Co., Japan; FT-20-0076; Sept. 12, 1982; U.S. EPA, Office of Pesticide(S-3206 lot. No. T- Programs; Health Effects Division; Tox Oneliners; MRID No. 00127344; EPA Chem. Code: 127901;
1)Core Grade/Tox Record No. guideline 004567; EPA Accession No. 249937

Fenpropathrin

18

164

115 - 234
(CL)NA

Sprague Dawley ratsmaleoral gavage

observed for 14 days; toxic signs noted at 50 mg/kg and above;
single doses (mg/kg): 0, 25, 50,
muscular fibrillation, tremor, ataxia, limb paralysis, irregular90, 120, 160, 220, 300
respiration, lacrimation, salivation, urinary incontinence, diarrhea

8 groups of 10 rats; 80 rats used

Fenpropathrin 97.3% Sumitomo Chemical Co., Japan; FT-20-0076; Sept. 12, 1982; U.S. EPA, Office of Pesticide(S-3206 lot. No. T- Programs; Health Effects Division; Tox Oneliners; MRID No. 00127344; EPA Chem. Code: 127901;
1)Core Grade/Tox Record No. guideline 004567; EPA Accession No. 249937

Gibberellic acid

6300

> 5000

NA

NA

rats

male andfemale

oral

NA

NA

NA

Gibberellins Tech.
GA47A, 90%Hazleton Laboratories, Inc.; HLA 80602323; Aug. 29, 1988; U.S. EPA, Office of Pesticide Programs;
Health Effects Division; Tox Oneliners; MRID No. 40873201; EPA Chem. Code: 043801; Core
Grade/Tox Record No. Guideline 007756; FEB. 9, 1990

Gibberellic acid

6300

> 5000

NA

NA

rats

female

oral

NA

NA

NA

Pro Gibb 4%(gibberellic acid);
Lot 28-T80-CF

Abbott Research Center; TA89-363; Feb. 20, 1990; U.S. EPA, Office of Pesticide Programs; Health
Effects Division; Tox Oneliners; MRID No.41558201; EPA Chem. Code: 043801; Core Grade/Tox
Record No. Guideline 008645; Oct. 8, 1991

Gibberellic acid

6300

> 5000

NA

NA

rats

NA

oral

5000 mg/mL

NA

NA

cytokinin (as kinetin) University of Utah Reearch Institute 03-80; TR 05-485-002A; Jan. 20, 1984; U.S. EPA, Office of
0.012%; Gibberellic Pesticide Programs; Health Effects Division; Tox Oneliners; MRID No. 00142864; EPA Chem.
acid 0.0007%
Code: 043801; Core Grade/Tox Record No. Guideline 006198

Gibberellic acid

6300

> 5000

NA

NA

rats

NA

oral

NA

NA

NA

Ricerca, Inc.; 90-0138; May 31, 1990; U.S. EPA, Office of Pesticide Programs; Health Effects
Pro Gibb (gibberellic
Division; Tox Oneliners; MRID No. 41560401; EPA Chem. Code: 043801; Core Grade/Tox Record
acid 10%);
No. supplementary 008876; Dec. 5, 1991

Gibberellic acid

6300

> 5000

NA

NA

rats

male andfemale

oral

NA

NA

NA

Gibberellic acid
7.5% a.l.

Ricerca, Inc.; 90-0138; May 31, 1990; U.S. EPA, Office of Pesticide Programs; Health Effects
Division; Tox Oneliners; MRID No. 41591103; EPA Chem. Code: 043801; Core Grade/Tox Record
No. Guideline 008571; Sept. 11, 1991

Gibberellic acid

6300

> 5000NA

NACharles River Crl CD; 271293 g; young adult

male

oral

5000 mg/mL in corn oil; 10
mL/kg dose;

14 day observation; 0/5 animals dead; dyspnea

5 animals used; tan to white powder

Gibberellins Tech.,
88.0%

Hazleton Laboratories, Inc.; HLA 90305639; June 22, 1989; U.S. EPA, Office of Pesticide Programs;
Health Effects Division; Tox Oneliners; MRID No. 41605801; EPA Chem. Code: 043801; Core
Grade/Tox Record No. Guideline 008916; Dec. 17, 1991

Gibberellic acid

6300

> 5000NA

NACharles River Crl CD; 245271 g; young adultfemaleoral

5000 mg/mL in corn oil

14 day observation; 0/5 animals dead; dyspnea

5 animals used; tan to white powder

Gibberellins Tech.,
88.0%

Hazleton Laboratories, Inc.; HLA 90305639; June 22, 1989; U.S. EPA, Office of Pesticide Programs;
Health Effects Division; Tox Oneliners; MRID No. 41605801; EPA Chem. Code: 043801; Core
Grade/Tox Record No. Guideline 008916; Dec. 17, 1991

Gibberellic acid

6300

5780

NA

NA

rats

male

oral

NA

NA

NA

Pro Gibb 4%(gibberellic acid);
Lot 28-T80-CF

Abbott Research Center; TA89-363; Feb. 20, 1990; U.S. EPA, Office of Pesticide Programs; Health
Effects Division; Tox Oneliners; MRID No.41558201; EPA Chem. Code: 043801; Core Grade/Tox
Record No. Guideline 008645; Oct. 8, 1991

Gibberellic acid

6300

6300

NA

NA

rats

NA

oral

NA

NA

NA

NA

Agricultural Chemicals. Thomson, W.T., 4 vols., Fresno, CA, Thomson Publications, 1976/77
revision (RTECS REFERENCE)

Glutethimide

600

600

NA

NA

rats

NA

oral

NA

NA

NA

NA

Psychotropic Drugs and Related Compounds," 2nd ed., Usdin, E., and D.H. Efron, Dept. of Health,
Education and Welfare, Washington, DC, 1972. (RTECS REFERENCE)

Glycerol

12600

12600

NA

NA

rats

NA

oral

NA

NA

reference in Russian

NA

Farmatsevtichnii Zhurnal (Kiev). (V/O Mezhdunarodnaya Kniga, 113095 Moscow, USSR) V.31930. 1977. (RTECS REFERENCE)

H-23

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Glycerol

12600

15890
(12.6 cc/kg; used
density of 1.261
for conversion)

Glycerol

12600

27500

Glycerol

12600

26730 - A
27650 - B
(A = 21.2
mL/kg; B =21.93 mL/kg;
used density of
1.261 to convert
to mg)NA

Haloperidol

128

128

Haloperidol

128

Haloperidol

NANA

23950 - 31610(95% probability; +/probits (Bliss)
1.96 S.D.; slope =8.90)

Animal Information(stock, weight, age)rats

GenderNA

Route and/or
Method ofExposure

oral

Dose

NA

Observations

Notes

Reference
Substance Source

Reference provided by ZEBET assource of RC value (i.e., from
1983/84 RTECS), but mg/kg value
calculated from cc/kg value isNAdifferent from RC value (12691 vs
15890 mg/kg). Maybe ZEBET didn't
use density? This is not a primaryreference.

NA

Primary Reference

Woodard G, Johnson VD, Nelson AA. 1945. Acute toxicity of 2-methyl, 2-4 pentanediol. Fed Proc
4:142-143. (Supposed 1983/84 RTECS reference)

Wistar albino rats; 90-120 g maleoral; stomach tube; 50% concentration in water;most deaths occurred in 2 days; all deaths occurred in 14 days
single doses
largest dose given was 50 g/kg

groups of 10 animals; 10 animals per purified commercial Smyth HF Jr, Seaton J, Fischer L. 1941. The single dose toxicity of some glycols and derivatives. J
dose
grade
Ind Hyg Toxicol 23:259-268. Mellon Institute, Pittsburgh, PA

A: Behrens (1929)
B: Bliss (1938)

rats

NA

oral

NA

NA

40 - 90 animals used; NICEATM
used value B since authors stated it
was more accurate

NA

Deichmann WB, Mergard EG. 1948. Comparative evaluation of methods employed to express thedegree of toxicity of a compound. J Ind Hyg Toxicol 30:373-378.
Albany Medical College, Albany, NY; University of Cincinnati, Cincinnati, OH

77 - 212

NA

rat

NA

oral

NA

NA

unknown primary source of
information

NA

Niemegeers CJC, Janssen PAJ. 1974. Bromoperidol, a new potent neuroleptic of the butyrophenone
series. Arzneimittel-Forschung Drug Research 24 (1):45-52.
Janssen Pharmaceutica, Belgium (RTECS REFERENCE)

165

NA

NA

CFN; newborn

NA

oral

NA

NA

information from: drug applications
from pharmaceutical manufacturers, NA
the literature, and FDA labs

Goldenthal EI. 1971. A compilation of LD50 values in newborn and adult animals. Toxicology andApplied Pharamacology 18:185-207. Bureau of Drugs, Food and Drug Administration, Dept. of
Health, Education, and Welfare, Rockville, MD.

128

850

617 - 1173

NA

Holtzman; adult

male

oral

NA

NA

information from: drug applications
from pharmaceutical manufacturers, NA
the literature, and FDA labs

Goldenthal EI. 1971. A compilation of LD50 values in newborn and adult animals. Toxicology andApplied Pharamacology 18:185-207. Bureau of Drugs, Food and Drug Administration, Dept. of
Health, Education, and Welfare, Rockville, MD.

Hexachlorophene

56

9

2
(S.E.)

Miller and Tainter(1944)

Sprague-Dawley rats; 10 day

male andfemaleoral; stomach tube 1% carboxymethylcellulose

observed for 10 day

approximately equal numbers ofmales and females; 28 rats

NA

Nieminen L, Bjondahn K, Mottonen M. 1973. Effect of hexachlorophene on the rat brain during
ontogenesis. Fd Cosmet Toxicol 11:635-639.

Hexachlorophene

56

42

5
(S.E.)

Miller and Tainter(1944)

Sprague-Dawley rats; 20 day

male andfemaleoral; stomach tube 1% carboxymethylcellulose

observed for 10 day

approximately equal numbers ofmales and females; 22 rats; values
from graph

NA

Nieminen L, Bjondahn K, Mottonen M. 1973. Effect of hexachlorophene on the rat brain during
ontogenesis. Fd Cosmet Toxicol 11:635-639.

Hexachlorophene

56

56

8
(S.E.)

Miller and Tainter(1944)

Sprague-Dawley rats; 300
daymale andfemaleoral; stomach tube 1% carboxymethylcellulose

observed for 10 day

approximately equal numbers ofmales and females; 14 rats; values
from graph

NA

Nieminen L, Bjondahn K, Mottonen M. 1973. Effect of hexachlorophene on the rat brain during
ontogenesis. Fd Cosmet Toxicol 11:635-639.

Hexachlorophene

56

56

51 - 62(95% CI)

Litchfield andWilcoxon method(1949)

Sherman strain rats (SPF);
adult;

femaleoral; stomach tube peanut oil solution

died within 3 days; severe depression and diarrhea

5 or more groups of 10 rats each

USP

Gaines TB, Kimbrough RD, Linder RE. 1973. The oral and dermal toxicity of hexachlorophene.
Toxicology and Applied Pharmacology 25:332-343. (RTECS REFERENCE)

Hexachlorophene

56

57

52 - 61(95% CL; slope =13.5)

Finney's maximum
likelihood probit

Sherman strain rats (SPF);
min wt. = 200 g; min age of female
90 daysoral; stomach tube

chemical in peanut oil;
0.005mL/g of bw

At least 40 rats used; min. of 10
observed for at least 14 days after dosing or until recovered from signs animals per group tested; min. of 4
of toxicity
doses; animals used are the same as
Gaines 1973

technical grade

Gaines TB, Linder RE. 1986. Acute toxicity of pesticides in adult and weanling rats. Fundam Appl
Toxicol 7(2):299-308. Health Effects Research Laboratory, U.S. EPA, Research Triangle Park,NC

Hexachlorophene

56

57.6

50.8 - 65.5
(95% CI)

Weil (1952) method

Wistar albino rats; 400 g; 17
male
weeks

oral

corn oil solution; geometricdose factor of 1.2

preliminary observations over a 1 - 2 week period after dosing; nosignificant mortalities occurred after 5 days; toxicity signs: lethargy,
posterior paralysis, increased rate of respiration, hyperthermia, anddiarrhea

16 rats at 4 dosage levels; fasted
overnight

U.S.P. grade;
Givaudan Corp.,
Clifton, NJ

Nakaue HS, Dost FN, Buhler DR. 1973. Studies On The Toxicity Of Hexachlorophene In Rats.
Toxicol Appl Pharmacol 24:239-49.A19 Oregon State University, Corvallis, OR

Hexachlorophene

56

60

4
(S.E.)

Miller and Tainter(1944)

Sprague-Dawley; 70 day

observed for 10 day

approximately equal numbers ofmales and females; 84 rats; values
from graph

NA

Nieminen L, Bjondahn K, Mottonen M. 1973. Effect of hexachlorophene on the rat brain during
ontogenesis. Fd Cosmet Toxicol 11:635-639.

Hexachlorophene

56

60.3

55.0 - 66.0
(95% CI)

Weil (1952) method

Wistar albino rats; 100 g; 45
male
weeks

oral

corn oil solution; geometricdose factor of 1.2

preliminary observations over a 1 - 2 week period after dosing; nosignificant mortalities occurred after 5 days; toxicity signs: lethargy,
posterior paralysis, increased rate of respiration, hyperthermia, anddiarrhea

16 rats at 4 dosage levels; fasted
overnight

U.S.P. grade;
Givaudan Corp.,
Clifton, NJ

Nakaue HS, Dost FN, Buhler DR. 1973. Studies On The Toxicity Of Hexachlorophene In Rats.
Toxicol Appl Pharmacol 24:239-49.A19 Oregon State University, Corvallis, OR

Hexachlorophene

566355.5 - 71.8
(95% CI)

Weil (1952) method

Wistar albino rats; 300 g; 10
male
weeks

oral

corn oil solution; geometricdose factor of 1.2

preliminary observations over a 1 - 2 week period after dosing; nosignificant mortalities occurred after 5 days; toxicity signs: lethargy,
posterior paralysis, increased rate of respiration, hyperthermia, anddiarrhea

16 rats at 4 dosage levels; fasted
overnight

U.S.P. grade;
Givaudan Corp.,
Clifton, NJ

Nakaue HS, Dost FN, Buhler DR. 1973. Studies On The Toxicity Of Hexachlorophene In Rats.
Toxicol Appl Pharmacol 24:239-49.A19 Oregon State University, Corvallis, OR

Hexachlorophene

566345.9 - 87.2
(95% CI)

Weil (1952) method

Wistar albino rats; 200 g; 9
weeks

oral

corn oil solution; geometricdose factor of 1.2

preliminary observations over a 1 - 2 week period after dosing; nosignificant mortalities occurred after 5 days; toxicity signs: lethargy,
posterior paralysis, increased rate of respiration, hyperthermia, anddiarrhea

16 rats at 4 dosage levels; fasted
overnight

U.S.P. grade;
Givaudan Corp.,
Clifton, NJ

Nakaue HS, Dost FN, Buhler DR. 1973. Studies On The Toxicity Of Hexachlorophene In Rats.
Toxicol Appl Pharmacol 24:239-49.A19 Oregon State University, Corvallis, OR

male andfemalefemaleoral; stomach tube 1% carboxymethylcellulose

H-24

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference59 - 75
Finney's maximum
95% CL; slope 10.6 likelihood probit

Route and/or
Method ofExposure

Dose

Sherman strain rats (SPF);
min wt. = 175 g; min age of male
90 daysoral; stomach tube

chemical in peanut oil; 0.005
mL/g of bw

Animal Information(stock, weight, age)

Gender

Notes

Reference
Substance Source

Primary Reference

At least 40 rats used; min. of 10
observed for at least 14 days after dosing or until recovered from signs animals per group tested; min. of 4
of toxicity
doses; animals used are the same as
Gaines 1973

technical grade

Gaines TB, Linder RE. 1986. Acute toxicity of pesticides in adult and weanling rats. Fundam Appl
Toxicol 7(2):299-308. Health Effects Research Laboratory, U.S. EPA, Research Triangle Park,NC

died within 12 days; severe depression and diarrhea

5 or more groups of 10 rats each;NA

Gaines TB, Kimbrough RD, Linder RE. 1973. The oral and dermal toxicity of hexachlorophene.
Toxicology and Applied Pharmacology 25:332 -343. Environmental Protection Agency, Chamblee,GAcorn oil solution; geometricdose factor of 1.2

preliminary observations over a 1 - 2 week period after dosing; nosignificant mortalities occurred after 5 days; toxicity signs: lethargy,
posterior paralysis, increased rate of respiration, hyperthermia, anddiarrhea

16 rats at 4 dosage levels; fasted
overnight

U.S.P. grade;
Givaudan Corp.,
Clifton, NJ

Nakaue HS, Dost FN, Buhler DR. 1973. Studies On The Toxicity Of Hexachlorophene In Rats.
Toxicol Appl Pharmacol 24:239-49.A19 Oregon State University, Corvallis, OR

corn oil solution; geometricdose factor of 1.2

preliminary observations over a 1 - 2 week period after dosing; nosignificant mortalities occurred after 5 days; toxicity signs: lethargy,
posterior paralysis, increased rate of respiration, hyperthermia, anddiarrhea

16 rats at 4 dosage levels; fasted
overnight

U.S.P. grade;
Givaudan Corp.,
Clifton, NJ

Nakaue HS, Dost FN, Buhler DR. 1973. Studies On The Toxicity Of Hexachlorophene In Rats.
Toxicol Appl Pharmacol 24:239-49.A19 Oregon State University, Corvallis, OR

oral; stomach tube 1% carboxymethylcellulose

observed for 10 day

approximately equal numbers ofmales and females; 12 rats; values
from graph

NA

Nieminen L, Bjondahn K, Mottonen M. 1973. Effect of hexachlorophene on the rat brain during
ontogenesis. Fd Cosmet Toxicol 11:635-639.

male andfemaleoral; stomach tube 1% carboxymethylcellulose

observed for 10 day

approximately equal numbers ofmales and females; 16 rats; values
from graph

NA

Nieminen L, Bjondahn K, Mottonen M. 1973. Effect of hexachlorophene on the rat brain during
ontogenesis. Fd Cosmet Toxicol 11:635-639.

Wistar albino rats; 67 g; 4
weeks

male

oral

corn oil solution; geometricdose factor of 12

preliminary observations over a 1 - 2 week period after dosing; nosignificant mortalities occurred after 5 days; toxicity signs: lethargy,
posterior paralysis, increased rate of respiration, hyperthermia, anddiarrhea

16 rats at 4 dosage levels; fasted
overnight

U.S.P. grade;
Givaudan Corp.,
Clifton, NJ

Nakaue HS, Dost FN, Buhler DR. 1973. Studies On The Toxicity Of Hexachlorophene In Rats.
Toxicol Appl Pharmacol 24:239-49.A19 Oregon State University, Corvallis, OR

Wistar albino rats; 68 g; 4
weeks

femaleoral

corn oil solution; geometricdose factor of 12

preliminary observations over a 1 - 2 week period after dosing; nosignificant mortalities occurred after 5 days; toxicity signs: lethargy,
posterior paralysis, increased rate of respiration, hyperthermia, anddiarrhea

16 rats at 4 dosage levels; fasted
overnight

U.S.P. grade;
Givaudan Corp.,
Clifton, NJ

Nakaue HS, Dost FN, Buhler DR. 1973. Studies On The Toxicity Of Hexachlorophene In Rats.
Toxicol Appl Pharmacol 24:239-49.A19 Oregon State University, Corvallis, ORNA

40, 80, 120, 160, 200 mg/kg

25 rats used; 12 dead within 40 hours

5 groups of 5 rats eachNA

Chung HL., 1963. Hexachlorophene (G-11) as a new specific drug against Clonorchiasis Sinensis.
Chinese Medical Journal. 82. No. 11. November.
Peking Sino-Soviet Friendship Hospital, Peking, China

oral; stomach tube 1% carboxymethylcellulose

observed for 10 days

approximately equal numbers ofmales and females; 66 rats

NA

Nieminen L, Bjondahn K, Mottonen M. 1973. Effect of hexachlorophene on the rat brain during
ontogenesis. Fd Cosmet Toxicol 11:635-639.

oral; stomach tube peanut oil solution

died within 5 days; depression and posterior paralysis

5 or more groups of 10 rats eachNA

Gaines TB, Kimbrough RD, Linder RE. 1973. The oral and dermal toxicity of hexachlorophene.
Toxicology and Applied Pharmacology 25:332 -343. Environmental Protection Agency, Chamblee,
GA

Hexachlorophene

56

66

Hexachlorophene

56

66

57 - 75(95% CI)

Litchfield andWilcoxon method(1949)

Sherman strain rats (SPF);
adult

male

oral; stomach tube peanut oil solution

Hexachlorophene

56

69.1

64.6 - 94.2
(95% CI)

Weil (1952) method

Wistar albino rats; 100 g; 5
weeks

femaleoral

Hexachlorophene

56

69.2

55.5 - 86.2
(95% CI)

Weil (1952) method

Wistar albino rats; 200 g; 7
weeks

male

oral

Hexachlorophene

56

83

6
(S.E.)

Miller and Tainter(1944)

Sprague-Dawley rats; 25 day

male andfemaleHexachlorophene

56

84

8
(S.E.)

Miller and Tainter(1944)

Sprague-Dawley rats; 50 day

Hexachlorophene

568779.2 - 95.5
(95% CI)

Weil (1952) method

Hexachlorophene

568779.5 - 95.0
(95% CI)

Weil (1952) method

Hexachlorophene56104.03

Hexachlorophene

56

111

12
(S.E.)

Miller and Tainter(1944)

Sprague-Dawley rats; 32 day

male andfemaleHexachlorophene

56

120

110 - 131
(95% CI)

Litchfield andWilcoxin 1949

Sherman strain rats (SPF);
weanling

femaleHexachlorophene

56

121

Hexachlorophene

56

165

149 - 179
(95% CI)

Hexachlorophene

56

215

191 - 237
(95% CI)

Lactic acid

3543

3543

NA

Lactic acid

3543

3730

Lindane7676 - 200

84.45 - 128.20
Bliss method(95% fiducial limit)

Observations

normal white rats; 150-250 g NA

Sherman strain rats (SPF); 4female
6 weeks

oral; stomach tube

chemical in peanut oil; 0.005
mL/g of bw

At least 40 rats used; min. of 10
observed for at least 14 days after dosing or until recovered from signs animals per group tested; min. of 4
of toxicity
doses; animals used are the same as
Gaines 1973

technical grade

Gaines TB, Linder RE. 1986. Acute toxicity of pesticides in adult and weanling rats. Fundam Appl
Toxicol 7(2):299-308. Health Effects Research Laboratory, U.S. EPA, Research Triangle Park,NC

Probit analysis

Crl-CD rats from Charles
River Breeding lab; 220 -280 maleg; 60 days

oral; intragastricintubation

0.5 - 3.9% suspens; dissolved orsuspended in corn oil; single
observed daily for 14 days; death within 6 days; toxic symptoms:
non fasted; 4 groups of 10; 40 rats
dose; 100, 140, 175, 200 mg/kg staining of the face and perineal area, weakness, diarrhea, weight loss used; 17 rats dieddoses

99+% pure;Givaudan Corp.,
Clifton, NJ

Dashiell OL, Kennedy GL Jr. 1984. The effects of fasting on the acute oral toxicity of nine chemicals
in the rat. J Appl Toxicol 4(6): 320-325. E.I. Du Pont de Nemours & Co., Newark, DE

Probit analysis

Crl-CD rats from Charles
River Breeding lab; 220 -280 maleg; 60 days

oral; intragastricintubation

0.26 - 1.4% suspens dissolved
fasted 24 hours before dosing; 5or suspended in corn oil; single observed daily for 14 days; death within 6 days; toxic symptoms:
groups of 10; 50 rats used; 16 rats
dose; 50, 100, 170, 225, 275
staining of the face and perineal area, weakness, diarrhea, weight loss
died
mg/kg doses

99+% pure;Givaudan Corp.,
Clifton, NJ

Dashiell OL, Kennedy GL Jr. 1984. The effects of fasting on the acute oral toxicity of nine chemicals
in the rat. J Appl Toxicol 4(6): 320-325. E.I. Du Pont de Nemours & Co., Newark, DE

NA

NA

NA

NA

NA

Farm Chemicals Handbook. (Meister Pub., 37841 Euclid Ave., Willoughy, OH 44094). 1991.
(RTECS REFERENCE)

112 - 133
Finney's maximum
95% CL; slope 14.8 likelihood probit

3020 - 4610
(95% probability; +/probits (Bliss)
1.96 S.D. slope =
4.04)

NA

NA

NA

Wistar albino rats; 90-120 g maleoral; stomach tube; concentration in water; largest
single doses
dose given was 50 g/kg

most deaths occurred in 2 days; all deaths occurred in 14 days

groups of 10 animals; 10 animals per purified commercial Smyth HF Jr, Seaton J, Fischer L. 1941. The single dose toxicity of some glycols and derivatives. J
dose
grade
Ind Hyg Toxicol 23:259-268. Mellon Institute, Pittsburgh, PA

rats

oral

NA

secondary source; unknown primaryNAsourceSpecial Publication of the Entomological Society of America. (4603 Calvert Rd., College Park, MD
20740). 1978.
NA

NA

NA

NA

H-25

Kenaga EE, Morgan RW. 1978. Commercial and Experimental Organic Insecticides. 1978 Revision.
Special Publication 78-1:1-76. The Dow Chemical Company, Midland, MI (RTECSREFERENCE)In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

Lindane7688

76 - 101(95% CL)

Litchfield andWilcoxon method(1949)

Sherman strain rats; min. wt.
male= 175 g; min. age of 90 days

oral; stomach tube

chemical in peanut oil; 0.005
mL/g of bw

observed hourly on first day of dosage and twice a day thereafter until
time of death; max survival 8 days; 14 days observation

89 rats tested; not fasted

technical grade

Gaines TB. 1960. The acute toxicity of pesticides to rats. Toxicol Appl Pharmacol 2:88-99
U.S. Dept. of Health, Education, and Welfare, Savannah, GA

Lindane76

91

83 - 100(95% CL)

Litchfield andWilcoxon method(1949)

Sherman strain rats; min. wt.
female= 200 g; min. age of 90 days

oral; stomach tube

chemical in peanut oil;
0.005mL/g of bw

observed hourly on first day of dosage and twice a day thereafter until
time of death; max survival 7 days; 14 days observation

69 rats tested; not fasted

technical grade

Gaines TB. 1960. The acute toxicity of pesticides to rats. Toxicol Appl Pharmacol 2:88-99
U.S. Dept. of Health, Education, and Welfare, Savannah, GA

Lindane

76

100

NA

Litchfield andWilcoxon method(1949)

CFY strain rats; 120+ g;
adult

female

oral

NA

NA

NA

99.5% pure;Budapest Chemical
Works

Lindane

76

125

NA

NA

rats

NA

oral; stomach tube NA

hypersensitivity and convulsions

information from the laboratories ofDivision of Pharmacology, U.S.
NA
FDA.; fasted animals

Litchfield andWilcoxon method

Wistar rats; 180 g (ave)femaleoral

7 days observation; deaths/dose (mg/kg): 347-0/10, 417-1/10, 500-3/10,
in solution; 347, 417, 500, 600,
Used 10 rats/dose; RTECS reference;
600-5/10, 720-8/10, 864-10/10; 14 deaths on day 1, 12 deaths on day 2
reagent grade
720, 864 mg/kg
in Japanese, 1 death on day 3; all rats at highest dose dead by day 2

NA

rats

NA

oral

NA

male

oral

7 d observation; deaths/dose (mg/kg): 347- 0/10, 417- 2/10, 500- 3/10,
in solution; 347, 417, 500, 600,
Used 10 rats/dose; RTECS reference,
600- 5/10, 720 - 8/10, 864- 10/10; most deaths on day 2; 3 deaths onreagent grade
720, 864 mg/kg
in Japaneseday 1 at highest dose; 3 deaths at lower doses on day 3

oral gastricintubation

in aqueous solution;
concentration intubated = 200
mg/mL; dosages arranged in a LD50 based on mortalities during a 14 day period;
logarithmic series differing by afactor of 2

non-fasted; groups of 5 rats; single
oral dose toxicity

reagent grade

Lithium I carbonate

525

553

525

460-598(95% CI)

Lithium I carbonate

525

553

553

NA

Lithium I carbonate

525

553

590

505-691(95% CI)

Litchfield andWilcoxon method

Wistar rats; 220 g (ave)NA

Thompson method;
Weil tables

Carworth-Wistar rats; 90120 g; 4-5 weeks

male

Lithium I carbonate

525

553

710

RTECS reference that provides
summary data only. LD50 value isunreferenced and unsupportedNA

reagent grade

Desi I. 1983. Neurotoxicological investigaton of pesticides in animal experiments. Neurobehav
Toxicol 5:503-515. National Institute of Hygiene, Hungary
Lehman AJ. 1951. Chemicals in Foods: a report to the association of food and drug officials on
current developments. Part II. Pesticides. Quarterly Bulletine (Association of Food and Drug
Officials of the United States). Vol. 15:122-133. U.S. FDA

Nakasawa M, et al. 1973. Lithium carbonate toxicity tests, rat and mouse acute toxicity. Kiso to
Rinsho Clinical Report 7:1273-1277. (RTECS REFERENCE)

Filov VA, Ivin BA, Bandman AL (eds).1993. Harmful Chemical substances. Volume 1: Elements inGroups I-IV of the Periodic Table and their Inorganic Compounds. Ellis Horwood Limited(publisher). First published in Russian asVrednye khimichesklye vechestra. Neorganicheskiye
soyedineniga elementor I-IV grup. VA Filov, ed. Khimiya, St. Petersburg. 1988.

Nakasawa M, et al. 1973. Lithium carbonate toxicity tests, rat and mouse acute toxicity. Kiso to
Rinsho Clinical Report 7:1273-1277.
Smyth HF Jr, Carpenter CP, Weil CS, Pozzani UC, Striegel JA, Nycum JS. 1969. Range-finding
toxicity data: List VII. Am Ind Hyg Assoc J 30:470-476.
Carnegie-Mellon University, Pittsburgh, PA (LD50 value).
Smyth HF Jr, Carpenter CP, Weil CS, Pozzani UC, Striegel JA. 1962. Range-finding toxicity data:
List VI. Am Ind Hyg Assoc J 23:95-107.
Mellon Institute of Industrial Research, Pittsburg, PA (experimental parameters)

Meprobamate

794

486

+/- 24(S.E.)

Miller and Tainter(1944)

FDRL-strain rats; 21 days

femaleoral

NAobserved for 7 days post-treatment

NA

NA

Weinberg MS, Goldhamer RE, Carson S. 1966. Acute oral toxicity of various drugs in newborn rats
after treatment of the dam during gestation. Toxic Appl Pharmac 9:234-239.
Food and Drug Research Laboratories, Inc., Maspeth, NY

Meprobamate

794

794
(outlier)

584 - 1080
(95% CL)

Litchfield andWilcoxon method(1949)

rats; 117-180 g; adultfemaleoral

suspension; 2.3 - 23.2 mg/kg
dose levels

hypothermia, prostration, bradypnea, ptosis, sluggish corneal reflex

5 rats per dose level; 20 rats used

NA

Franko BV, Ward JW, Gilbert DL, Woodard G. 1971. Toxicologic studies of glycopyrralate incombination with other drugs. Toxicology and Appled Pharmacology 19:93-102.
Woodard Research Corporation, Herndon, VA (RTECS REFERENCE)

Meprobamate

794

1286

+/- 81(S.E.)

Miller and Tainter(1944)

FDRL-strain rats; 100 days

male

oral

NA

observed for 7 days post-treatment

NA

NA

Weinberg MS, Goldhamer RE, Carson S. 1966. Acute oral toxicity of various drugs in newborn rats
after treatment of the dam during gestation. Toxic Appl Pharmac 9:234-239.
Food and Drug Research Laboratories, Inc., Maspeth, NY

Meprobamate

794

1290

+/- 104(S.E.)

Miller and Tainter(1944)

FDRL-strain rats; 63 days

male

oral

NA

observed for 7 days post-treatment

NA

NA

Weinberg MS, Goldhamer RE, Carson S. 1966. Acute oral toxicity of various drugs in newborn rats
after treatment of the dam during gestation. Toxic Appl Pharmac 9:234-239.
Food and Drug Research Laboratories, Inc., Maspeth, NY

Meprobamate

794

1346

+/- 82(S.E.)

Miller and Tainter(1944)

FDRL-strain rats; 21 days

male

oral

NA

observed for 7 days post-treatment

NA

NA

Weinberg MS, Goldhamer RE, Carson S. 1966. Acute oral toxicity of various drugs in newborn rats
after treatment of the dam during gestation. Toxic Appl Pharmac 9:234-239.
Food and Drug Research Laboratories, Inc., Maspeth, NY

Meprobamate

794

1361

+/- 76(S.E.)

Miller and Tainter(1944)

FDRL-strain rats; 100 days

femaleoral

NAobserved for 7 days post-treatment

NA

NA

Weinberg MS, Goldhamer RE, Carson S. 1966. Acute oral toxicity of various drugs in newborn rats
after treatment of the dam during gestation. Toxic Appl Pharmac 9:234-239.
Food and Drug Research Laboratories, Inc., Maspeth, NY

Meprobamate

794

1410

+/- 83(S.E.)

Miller and Tainter(1944)

FDRL-strain rats; 63 days

femaleoral

NAobserved for 7 days post-treatment

NA

NA

Weinberg MS, Goldhamer RE, Carson S. 1966. Acute oral toxicity of various drugs in newborn rats
after treatment of the dam during gestation. Toxic Appl Pharmac 9:234-239.
Food and Drug Research Laboratories, Inc., Maspeth, NY

Meprobamate

794

1470

Litchfield andWilcoxon method(1949)

rats; 117-180 g; adult

male

oral

suspension; 2.3 - 23.2 mg/kg
dose levels

hypothermia, prostration, bradypnea, ptosis, sluggish corneal reflex

5 rats per dose level; 20 rats used

NA

Franko BV, Ward JW, Gilbert DL, Woodard G. 1971. Toxicologic studies of glycopyrralate incombination with other drugs. Toxicology and Appled Pharmacology 19:93-102.
Woodard Research Corporation, Herndon, VA

Meprobamate

794

1522

+/- 16(S.E.)

Miller and Tainter(1944)Charles River CD andSprague-Dawley strains; >100 g; adultNA

oral intubation

up to 50 mL/kg

rats observed for 7 days; observed up to 14 days when heavy metals or
fasted overnight
other compounds that produce latent death were investigatedNA

Yeary RA, Benish RA, Finkelstein M. 1966. Acute Toxicity of Drugs in Newborn Animals. Journalof Pediatrics 69 (4):663-667.
Dept. of Veterinary Preventive Medicine, Ohio State University, Columbus, OH

Mercury II chloride

1

1-5

NA

NA

rats

NA

oral

NA

NA

NA

Pesticide Manual. (The British Crop Protection Council, 20 Bridport Rd., Thornton Heath CR4 7QG,UK) V.1- 1968. 1991. (RTECS REFERENCE)

Mercury II chloride1

12

9 - 17(95% CL)

acceptable methods(e.g., Bliss, Litchfield Sprague-Dawley rats; 190and Wilcoxon, Weil, 300 g
Thompson, etc.)

femaleoral gavage

single dose

14 day observation; toxicity symptoms: motor activity decrease,respiratory effects, tremors, blanching, piloerection, diarrhea,
UDP Test
chouromodacryorrhoea; time to onset of signs < 1day; duration of signs
11 days; animals fasted 16 -20 hours before administrationNA

Yam J, Reer PJ, Bruce RD. 1991. Comparison of the up-and-down method and the fixed-doseprocedure for acute oral toxicity testing. Food Chem Toxicol 29(4):259-264.
The Procter and Gamble Co., Cincinnati, OH

lists LD50 range as 1 - 5 mg/kg

H-26

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

Mercury II chloride

Mercury II chloride

LD501
mg/kg

1

1

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

24

32

Animal Information(stock, weight, age)

Sprague-Dawley rats; 5
weeks

Gender

17.9 - 32.2

Bliss-Probit method

NA

Rosiello (1979) andBliss (1938)

rats

male

rats

male

male

Route and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

dissolved in saline; range(mg/kg) of doses 10.6, 13.8,
17.9, 23.3, 30.3, 39.7

observed at 6 hours after dosing and a once a day for 1-2 weeks; most animals acclimated to environment
dead within 3 days; 25/60 died; toxic symptoms: piloerection, drooling, for 1 week before testing; 6 groups
Kishida Chemical
hypothermia, abdominal posture, tremor, and diarrhea; dose (mg/kg), of10 rats each; fasted 16 hours before
Co., Ltd.
dead rats per dose: 10.6-0/10; 13.8-1/10; 17.9-1/10; 23.3-4/10; 30.3dosing; 100% lethal dose = 39.7
9/10; 39.7-10/10
mg/kg; 0% lethal dose = 10.6 mg/kg

Kitagawa H, Saito H, Sugimoto T, Yanaura S, Kitagawa H, Hosokawa T, Sakamoto K. 1982. Effects
of diiospropyl-1,3-dithiol-2-ylidene malonate (NKK-105) on acute toxicity of various drugs andheavy metals. J Toxicol Sci 7(2):123-34.
Chiba University; Hoshi College of Pharmacy; Showa University -- Japan

10, 15, 25, 40, 60, 100 mg/kg

acclimated for 5 days; observed for
15 mg/kg: 0/3 dead; 25mg/kg: 0/3 dead; 40 mg/kg: 3/3 dead; 60 mg/kg: 14 days; 4 groups used for each dose
3/3 dead; 6/12 rats dead; LD50 from 12 rats used; LD50 recalculated (1, 2, 3, 5 animals per group; total ofusing US EPA Benchmark Dose soft-ware; Lorke used data from 10
11 rats per dose; range finder: 10
NAand 100 mg/kg in range finder for all animal groups; omitted this data mg/kg - 0/3 dead; 100 mg/kg - 3/3
in recalculation; orginial LD50 from Lorke = 32 mg/kg
dead; 1000 mg/kg - 3/3 dead; 9 rats
in range finder

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

oral

10, 15, 25, 40, 60, 100 mg/kg

acclimated for five days; observed
15 mg/kg: 1/11 dead; 25mg/kg: 1/11 dead; 40 mg/kg: 7/11 dead; 60
for 14 days; 4 groups used for each
mg/kg: 10/11 dead; 19/44 rats dead; LD50 from 44 rats used; LD50
dose (1, 2, 3, 5 animals per group;
recalculated using US EPA Benchmark Dose software; Lorke used data
total of 11 rats per dose; range finder NAfrom 10 and 100 mg/kg in range finder for all animal groups; omitted
showed: 10 mg/kg - 0/3 dead; 100
this data in recalculation; Orginial LD50 from Lorke = 37 mg/kg; this
mg/kg - 3/3 dead; 1000 mg/kg - 3/3
value based on accumulated data from 4 different test groups
dead; 9 rats in range finder

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

oral gavage

oral

Mercury II chloride

1

39

NA

Rosiello (1979) andBliss (1938)

Mercury II chloride

1

40

NA

Rosiello (1979) andBliss (1938)

rats

male

oral

10, 15, 25, 40, 60, 100 mg/kg

acclimated for five days; observed
15 mg/kg: 1/5 dead; 25mg/kg: 1/5 dead; 40 mg/kg: 3/5 dead; 60 mg/kg: for 14 days; 4 groups used for each
5/5 dead; 10/20 rats dead; LD50 based on 20 rats used; LD50
dose (1, 2, 3, 5 animals per group;
recalculated using US EPA Benchmark Dose software; Lorke used data total of 11 rats per dose; range finder NAfrom 10 and 100 mg/kg in range finder for all animal groups; omitted showed: 10 mg/kg - 0/3 dead; 100
this data in recalculation; orginial LD50 from Lorke = 32 mg/kg
mg/kg - 3/3 dead; 1000 mg/kg - 3/3
dead; 9 rats in range finder

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

Mercury II chloride

1

49

NA

Rosiello (1979) andBliss (1938)

rats

male

oral

10, 15, 25, 40, 60, 100 mg/kg

acclimated for five days; observedfor 14 days; 4 groups used for eachdose (1, 2, 3, 5 animals per group;
15 mg/kg: 0/1 dead; 25mg/kg: 0/1 dead; 40 mg/kg: 0/1 dead; 60 mg/kg:
total of 11 rats per dose; range finder NA
1/1 dead; 1/4 rats dead; LD50 from 4 rats used; T306
showed: 10 mg/kg - 0/3 dead; 100
mg/kg - 3/3 dead; 1000 mg/kg - 3/3
dead; 9 rats in range finder

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

Mercury II chloride

1

50

40 - 63

Thompson and Weil;
1952; method ofalbino rats; 18 weeks
moving averages

femaleoral; stomach tube 1 mL/200 g bw

observed after 8 days after single oral administration

6 dose levels per group, 6 rats pergroup; 36 rats used

NA

Kostial K, Kello D, Jugo S, Rabar I, Maljkovic, T. 1978. Influence of age on metal metabolism andtoxicity. Environ Health Perspect 25:81-8.
Yugoslav Academy of Sciences and Art, Zagreb, Yugoslavia

Mercury II chloride

1

50

43 - 59

Thompson and Weil;
1952; method ofalbino rats; 54 weeks
moving averages

femaleoral; stomach tube 1 mL/200 g bw

observed after 8 days after single oral administration

6 dose levels per group, 6 rats pergroup; 36 rats used

NA

Kostial K, Kello D, Jugo S, Rabar I, Maljkovic, T. 1978. Influence of age on metal metabolism andtoxicity. Environ Health Perspect 25:81-86.
Yugoslav Academy of Sciences and Art, Zagreb, Yugoslavia

Mercury II chloride

1

51

39 - 66(95% CL)

acceptable methods(e.g., Bliss, Litchfield
Sprague-Dawley ratsand Wilcoxon, Weil,
Thompson, etc.)

maleoral gavage

single dose

14 day observation; toxicity symptoms: posture, respiratory effects,
lethargy, abnormal gait, prostrate coma, salivation; time to onset ofsigns < 1 day; duration of signs 5 days

3 dose levels (5 male each); 15 rats
used; OECD TG401 (1981) followedNAfor experimental procedures; 8 rats
dead (average per test)

Mercury II chloride

1

52

NA

Rosiello (1979) andBliss (1938)

male

oral

10, 15, 25, 40, 60, 100 mg/kg

acclimated for five days; observed
15 mg/kg: 0/2 dead; 25mg/kg: 0/2 dead; 40 mg/kg: 1/2 dead; 60 mg/kg: for 14 days; 4 groups used for each
1/2 dead; 2/8 rats dead; LD50 based on 8 rats used; LD50 recalculated dose (1, 2, 3, 5 animals per group;
using US EPA Benchmark Dose software; Lorke used data from 10 and total of 11 rats per dose; range finder NA
100 mg/kg in range finder for all animal groups; omitted this data inshowed: 10 mg/kg - 0/3 dead; 100
recalculation; orginial LD50 from Lorke = 50 mg/kg
mg/kg - 3/3 dead; 1000 mg/kg - 3/3
dead; 9 rats in range finder

Mercury II chloride

1

92

77 - 108

Thompson and Weil;
1952; method ofalbino rats; 6 weeks
moving averages

femaleoral; stomach tube

1 mL/200 g bw; 6 dose levels in
observed after 8 days after single oral administrationeach group

Mercury II chloride

1

160(outlier)

119 - 235
(95% CL)

acceptable methods(e.g., Bliss, Litchfield
Sprague-Dawley ratsand Wilcoxon, Weil,
Thompson, etc.)

femaleoral gavage

single dose

14 day observation; toxicity symptoms: posture, respiratory effects,
lethargy, abnormal gait, prostrate coma, salivation; time to onset ofsigns < 1 day; duration of signs 5 days

3 dose levels (5 female each); 15 rats
used; OECD TG401 (1981) followedNAfor experimental procedures; 8 rats
dead (average per test)

Vandenheuvel MJ, Clark DG, Fielder RJ, Koundakjian PP, Oliver GJA, Pelling D, Tomlinson NJ,
Walker AP. 1990. Jul. The International Validation Of A Fixed-Dose Procedure As An Alternative To
The Classical LD50 Test Food And Chemical Toxicology 28(7):469-482.

5628

5628

4613 - 6866

NA

NA

oral

NA

NA

reference in Russian; was also citedin 1983/84 RTECS but value wasdifferent from that used by RC andreference was not provided by
ZEBET

Lazinov AG, Broitman AT. 1975. On the combined action of 2, 6-dimethylphenol and methanol.
Gigiena Truda i Professional'nye Zabolevaniya 19(11):27-30. (RTECS REFERENCE)

Methanol

rats

rats

H-27

6 dose levels per group, 6 rats pergroup; 36 rats used

NA

NA

Vandenheuvel MJ, Clark DG, Fielder RJ, Koundakjian PP, Oliver GJA, Pelling D, Tomlinson NJ,
Walker AP. 1990. Jul. The International Validation Of A Fixed-Dose Procedure As An Alternative To
The Classical LD50 Test Food And Chemical Toxicology 28(7):469-482.

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

Kostial K, Kello D, Jugo S, Rabar I, Maljkovic, T. 1978. Influence of age on metal metabolism andtoxicity. Environ Health Perspect 25:81-86.
Yugoslav Academy of Sciences and Art, Zagreb, Yugoslavia

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

Methanol

5628

5890
(7.4 mL/kg; used
4776 - 7244
Litchfield andSprague-Dawley rats; 16-50 male anddensity of 0.796 (95% CL; 6.0 - 9.1 Wilcoxon method andg; 14 days
femaleto convert tomL/kg)
probit analysis
mg/kg)

oral

solvent used in undiluted form

animals observed for a week after medication

nonfasted rats; 6-12 rats of both
sexes used for studies; solvent usedin undiluted form

analytical grade
meeting A.C.S.
specifications

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, IL

Methanol

5628

7005
(8.8 mL/kg; used5731 - 8597
Litchfield andSprague-Dawley rats; 300density of 0.796 (95% CL; 7.2 - 10.8 Wilcoxon method and
470 g; older adultto convert tomL/kg)
probit analysis
mg/kg)

oral

solvent used in undiluted form

animals observed for a week after medication

nonfasted rats; groups of 6 rats used analytical grade
for the studies; solvent used inmeeting A.C.S.
undiluted form
specifications

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, IL

Methanol

5628

NA

observed for 6 days

18 hour fasting before dosing

Methanol

5628

10348
(13.0 mL/kg;
9472 - 11303
Litchfield andSprague-Dawley rats; 80-160
used density of (95% CL; 11.9 - 14.2 Wilcoxon method andmaleg; young adult
0.796 to convert
mL/kg)
probit analysis
to mg/kg)

oral

solvent used in undiluted form

animals observed for a week after medication

nonfasted rats; groups of 6 rats used analytical grade
for the studies; solvent used inmeeting A.C.S.
undiluted form
specifications

Kimura ET, Ebert DM, Dodge PW. 1971. Acute toxicity and limits of solvent residue for sixteenorganic solvents. Toxicol Appl Pharmacol 19:699-704. Abbott Laboratories, Chicago, IL

Methanol

5628

12086 - A
11303 - B
(A = 15.28
mL/kg; B =14.29 mL/kg;
used density of
0.791 forconversion to
mg/kg)

oral

NA

NA

40 - 90 animals used; NICEATM
used value B since authors stated it
was more accurate

Deichmann WB, Mergard EG. 1948. Comparative evaluation of methods employed to express thedegree of toxicity of a compound. J Ind Hyg Toxicol 30:373-378
Albany Medical College, Albany, NY; University of Cincinnati, Cincinnati, OH

Methanol

5628

12880

Nicotine

50

50 - 60

NA

Nicotine

50

50 - 60

NA

Nicotine

50

68

Nicotine

50

70

49 - 109(95% CL)

Nicotine

50

70

Nicotine

50

Paraquat

57

7400

NA

NA

NA

A= Behrens (1929)
B = Bliss (1938)

11440 - 14460(95% probability; +/probits (Bliss)
1.96 S.D. slope =
8.53)

male

male
rats; 150-250 g; 70- 100 days (predominate oral
ly)

rats

NA

NA

NA

Welch, H, Slocum GG. 1943. Relation of length of carbon chain to the primary and functional
toxicities of alcohols. J Lab Chem Med 28:1440-1445. U.S. FDA, Washington, D.C.

Wistar albino rats; 90-120 g maleoral; stomach tube; 50% concentration in water;most deaths occurred in 2 days; all deaths occurred in 14 days
single doses
largest dose given was 50 g/kg

Smyth HF Jr, Seaton J, Fischer, L. 1941. The single dose toxicity of some glycols and derivatives. J
groups of 10 animals; 10 animals per purified commercial
Ind Hyg Toxicol 23:259-268. Mellon Institute, Pittsburgh, PA (
dose
grade
This was the value used by the RC [from 1977 RTECS]).

NA

rats

NA

oral

NA

reference is secondary; assumed to beNAvalues from Lehman (1951)

Farm Chemicals Handbook. (Meister Pub., 37841 Euclid Ave., Willoughy, OH 44094). 1991.
(RTECS REFERENCE)

NA

rats

NA

oral; stomach tube NA

clonic convulsions; onset within minutes; paralysis of respiratory
muscles and death

information from the laboratories ofDivision of Pharmacology, U.S.
NA
FDA.; fasted animals

Lehman AJ. 1951. Chemicals in Foods: a report to the association of food and drug officials on
current developments. Part II. Pesticides. Quarterly Bulletine (Association of Food and Drug Officialsof the United States). Vol.15:122-133. U.S. FDA

maleoral gavage

single dose

14 day observation; toxicity symptoms: Ptosis, posture, respiratory
effects, lethargy, abnormal gait, tremors, convulsions, prostrate coma;time to onset of signs < 1day; duration of signs 3 days; 13 rats dead(average per test)

3 dose levels (5 male each); 15 rats
used; OECD TG401 (1981) followed NAfor experimental procedures

Vandenheuvel MJ, Clark DG, Fielder RJ, Koundakjian PP, Oliver GJA, Pelling D, Tomlinson NJ,
Walker AP. 1990. Jul. The International Validation Of A Fixed-Dose Procedure As An Alternative To
The Classical LD50 Test Food And Chemical Toxicology 28(7):469-482.

acceptable methods(e.g., Bliss, Litchfield
Sprague-Dawley ratsand Wilcoxon, Weil,
Thompson, etc.)

male andfemaleoral gavage

single dose

14 day observation; toxicity symptoms: Ptosis, posture, respiratory
effects, lethargy, abnormal gait, tremors, convulsions, prostrate coma;time to onset of signs < 1day; duration of signs 3 days; 13 rats dead(average per test)

3 dose levels (5 male each and 5
female); 30 rats used; OECD TG401
NA(1981) followed for experimental
procedures

Vandenheuvel MJ, Clark DG, Fielder RJ, Koundakjian PP, Oliver GJA, Pelling D, Tomlinson NJ,
Walker AP. 1990. Jul. The International Validation Of A Fixed-Dose Procedure As An Alternative To
The Classical LD50 Test Food And Chemical Toxicology 28(7):469-482.

51 - 96(95% CL)

acceptable methods(e.g., Bliss, Litchfield Sprague-Dawley rats; 190and Wilcoxon, Weil, 300 g
Thompson, etc.)

femaleoral gavage

single dose

14 day observation; toxicity symptoms: motor activity decrease,respiratory effects, tremors, blanching, piloerection, ataxia,
convulsions, extension of the limbs; time to onset of signs < 1day;
duration of signs 5 days; animals fasted 16 -20 hours before
administration

UDP Test

Yam J, Reer PJ, Bruce RD. 1991. Comparison of the up-and-down method and the fixed-doseprocedure for acute oral toxicity testing. Food Chem Toxicol 29(4):259-264.
The Procter and Gamble Co., Cincinnati, OH

71

42 - 128(95% CL)

acceptable methods(e.g., Bliss, Litchfield
Sprague-Dawley ratsand Wilcoxon, Weil,
Thompson, etc.)

femaleoral gavage

single dose

14 day observation; toxicity symptoms: Ptosis, posture, respiratory
effects, lethargy, abnormal gait, tremors, convulsions, prostrate coma;time to onset of signs < 1day; duration of signs 3 days; 13 rats dead(average per test)

3 dose levels (5 female each); 15 rats
used; OECD TG401 (1981) followed NAfor experimental procedures

57

NA

NA

NA

oral

NA

NA

NA

acceptable methods
41 -129(e.g., Bliss, Litchfield(95% CL; slope = 3.0
Sprague-Dawley ratsand Wilcoxon, Weil,[S.E. 0.8])
Thompson, etc.)

NA

NA

Vandenheuvel MJ, Clark DG, Fielder RJ, Koundakjian PP, Oliver GJA, Pelling D, Tomlinson NJ,
Walker AP. 1990. Jul. The International Validation Of A Fixed-Dose Procedure As An Alternative To
The Classical LD50 Test Food And Chemical Toxicology 28(7):469-482.

Residue Reviews. (Springer-Verlag New York, Inc., Service Center, 44 Hartz Way, Secaucus, NJ
07094) V.1- 1962. 1965.
rats

H-28

NA

Bailey GW, White JL. 1965. Herbicides: a compilation of their physical, chemical, and biologicalproperties. Journal paper no. 2413. Purdue University Agricultural Experiment Station. Residue
Reviews 10:7-122. (RTECS REFERENCE)In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

oral intubation

single dose

observe several times daily and at least once on weekends for 30 days;
most of the rats that died did so within 5 days of administration; weightused 29 paraquat-dichloride
loss, diarrhea, piloerection and red drainage around mouth, eyes, andnose

Sharp CW, Ottolenghi A, Posner HS. 1972. Correlation of paraquat toxicity woth tissue
Ortho Chemical Co. concentrations and weight loss of the rat. Toxicology and Appied Pharmacology 22:241-251.
NIEHS, RTP, NC USA

Sherman strain rats; min. wt.
male= 175 g; min. age of 90 days

oral; stomach tube

chemical in peanut oil; 0.005
mL/g of bw

observed hourly on first day of dosage and twice a day thereafter until
time of death; max survival 14 days

50 rats tested

technical grade

Gaines TB. 1969. Acute toxicity of pesticides. Toxicol Appl Pharmacol 14(3):515-34.
U.S. Dept. of Health, Education, and Welfare, Atlanta, GA

Sherman strain rats; min. wt.
female= 200 g; min. age of 90 days

oral; stomach tube

chemical in peanut oil; 0.005
mL/g of bw

observed hourly on first day of dosage and twice a day thereafter until
time of death; max survival 13 days

50 rats tested

technical grade

Gaines TB. 1969. Acute toxicity of pesticides. Toxicol Appl Pharmacol 14(3):515-34.
U.S. Dept. of Health, Education, and Welfare, Atlanta, GA

oral; in foodsingle dose; mixed salt of
paraquat in food with 20% malt fasted overnight; observed up to 12 days
extract and fed to rats

6 rats per group

99.9% pure paraquat Clark DG, McElligott TF, Hurst EW. 1966. The toxicity of paraquat. Br J Ind Med 23:126-132.
dichloride
Imperial Chemical Industries Limited, Cheshire, UK

oral intubation

single dosemale andfemaleoral; in foodsingle dose; mixed salt of
paraquat in food with 20% malt fasted overnight; observed up to 12 days
extract and fed to rats

6 rats per group

99.9% pure paraquat Clark DG, McElligott TF, Hurst EW. 1966. The toxicity of paraquat. Br J Ind Med 23:126-132.
dimetho-sulfate
Imperial Chemical Industries Limited, Cheshire, UK

male andfemaleoral; in foodsingle dose; mixed salt of
paraquat in food with 20% malt fasted overnight; observed up to 12 days
extract and fed to rats

10 rats per group

99.9% pure paraquat Clark DG, McElligott TF, Hurst EW. 1966. The toxicity of paraquat. Br J Ind Med 23:126-132.
dichloride
Imperial Chemical Industries Limited, Cheshire, UK

Osborne-Mendel (?) rats

femaleoral

5 dose levels; constant vol. doseof solvent of 5 mL/kg; single
observed for 24 hours; deaths infrequent after 24 hour; onset ofdose; aqueous solution (sodium
anticholinesterase poisoning syptoms slower with corn oil than DMSO fasted for 20 hours
carboxymethyl-cellulose, 0.5%;
or aqueous
NaCl, 0.9%; benzyl alcohol,
0.2% v/v; Tween 80, 0.4%)

NA

Weis LR, Orzel RA. 1967. Some comparative toxicologic and pharmacologic effects of dimethyl
sulfoxide as a pesticide solvent. Toxicology and Applied Pharmacology 11:546-557.
U.S. FDA, Washington, D.C. (RTECS REFERENCE)

Litchfield andWilcoxon method(1949)

Osborne-Mendel (?) rats

femaleoral

5 dose levels; constant vol. dose
observed for 24 hours; deaths infrequent after 24 hour; onset ofof solvent of 5 mL/kg; single
anticholinesterase poisoning syptoms slower with corn oil than DMSO fasted for 20 hours
dose; cmpd dissolved in DMSO
or aqueous(industrial grade, 99% pure)NA

Weis LR, Orzel RA. 1967. Some comparative toxicologic and pharmacologic effects of dimethyl
sulfoxide as a pesticide solvent. Toxicology and Applied Pharmacology 11:546-557.
U.S. FDA, Washington, D.C.

NA

rats

NA

oral; stomach tube NA

generalized fibrillary tremors, salivation, lacrimation, diarrhea, andconvulsions; onset within 1 hour

information from the laboratories ofDivision of Pharmacology, U.S.
NA
FDA.; fasted animals; LD50 value isfrom research by Frawley et al. 1952

oral; stomach tube cmpd in corn oil

toxicity symptoms: muscle fibrillation, red colored lacrimation,
diarrhea, dyspnea, convulsions; respiratory paralysis, anoxia, terminal
convulsion

rats fasted for 24 hours; LD50 valueNA
was used in Lehman 1951

Paraquat

57

95

79-114(95 % CL)

Litchfield andWilcoxon method(1949)

Wistar rats; 292 +/- 13 g

Paraquat

57

100

85 - 117(95% CL)

Litchfield andWilcoxon method(1949)

Paraquat

57

110

90 - 134(95% CL)

Litchfield andWilcoxon method(1949)

Paraquat

57

112(paraquat ion)

104-122(95% CL)

Thompson (1947);
moving averagerats; 130-160 g
interpolation method

Paraquat

57

115

90-150(95% CL)

Litchfield andWilcoxin method(1949)

Paraquat

57

141(paraquat ion)

140-142(95% CL)

Thompson (1947);
moving averagerats; 130-160 g
interpolation method

Paraquat

57

150(paraquat ion)139-162(95% CL)

Thompson (1947);
moving averagerats; 150-205 g
interpolation method

Parathion2

1.8(actual value)

Parathion2

2.1

1.72 - 2.56
(95% CL; slope =1.25 [1.01 - 1.55
95% CL])

Parathion

2

3

NA

Parathion2

3+/- 0.25(S.E.)

Litchfield and Fertig Osborne-Mendel strain rats;
female(1941)
180-200 g

Sherman strain rats; min. wt.
female= 200 g; min. age of 90 days

oral; stomach tube

1.26 - 2.57
Litchfield and(95% CL; slope = 1.5
Wilcoxin method[1.0 - 2.25 95%(1949)
CL])male

male andfemaleSprague Dawley rat; 290 +/male
37 gobserve several times daily and at least once on weekends for 30 days;
most of the rats that died did so within 5 days of administration; weightused 29 paraquat-dichloride
loss, diarrhea, piloerection and red drainage around mouth, eyes, andnose

Sharp CW, Ottolenghi A, Posner HS. 1972. Correlation of paraquat toxicity woth tissue
Ortho Chemical Co. concentrations and weight loss of the rat. Toxicology and Appied Pharmacology 22:241-251.
NIEHS, RTP, NC USA

Lehman AJ. 1951. Chemicals in Foods: a report to the association of food and drug officials on
current developments. Part II. Pesticides. Quarterly Bulletine (Association of Food and Drug Officialsof the United States). Vol.15:122-133. U.S. FDA

Frawley JP, Hagan EC, Fitzhugh OG. 1952. A comparative pharmacological and toxicological studyof organic phosphate-anticholinesterase compounnds. J Pharmacol Exp Ther 152:156-165.
U.S. FDA, Washington, D.C.
Gaines TB. 1960. The acute toxicity of pesticides to rats. Toxicol Appl Pharmacol 2:88-99.
U.S. Dept. of Health, Education, and Welfare, Savannah, GA

Parathion2

3.6

3.2 - 4.0
(95% CL)

Litchfield andWilcoxin method(1949)

Parathion2

3.6

3.2 - 4.0
(95% CL)

Litchfield andWilcoxin method(1949)

Sherman albino rats

femaleoral; stomach tube NA

Parathion2

4.7

3.98 - 5.55
(95% CL; slope =1.21 [0.98 - 1.50
95% CL])

Litchfield andWilcoxin method(1949)

Osborne-Mendel (?) rats

femaleoral

chemical in peanut oil; 0.005
mL/g of bw

observed hourly on first day of dosage and twice a day thereafter until
time of death; max survival 3 days

70 rats tested

NA

LD50 value from research in Gaines
NA1960

5 dose levels; constant vol. doseof solvent of 5 mL/kg; single
observed for 24 hours; deaths infrequent after 24 hour; onset ofdose; cmpd dissolved in corn oil anticholinesterase poisoning syptoms slower with corn oil than DMSO fasted for 20 hours
mixture (90% corn oil, 10% N, or aqueous
N-dimethyl formamide)H-29

technical grade

NA

Mattson AM, Spillane JT, Pearce GW. 1955. Dimethyl 2,2-dichlorvinyl phosphate (DDVP), an
organic phosphorous compound highly toxic to insects. J Agr Food Chem 3:319-321.
Communicable Disease Center, Savannah, GA

Durham WF, Gaines TB, McCauley RH, Sedlak VA, Mattson MA, Hayes WJ. 1957. Studies on the
toxicity of 0,0-dimethyl-2,2-dichlorovinyl phosphate (DDVP). AMA Arch Ind Health 15:340-349.
U.S. Dept. of Health, Education and Welfare, Savannah, GA

Weis LR, Orzel RA. 1967. Some comparative toxicologic and pharmacologic effects of dimethyl
sulfoxide as a pesticide solvent. Toxicology and Applied Pharmacology 11:546-557.
U.S. FDA, Washington, D.C.

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

Parathion

26

4.6 - 7.8
(95% CL)

Litchfield andWilcoxin method(1949)CD (COBS) rats Charles
River, France; 120-200 g

femaleoral gavage

cmpd dissolved in 1 mL
methylene chloride; emulsified
LD50 determined after 10 days of observationin 10% arabic gum solution
with Tween 80; dose 5 mL/kg

5 dose levels; 10 female per dose; 5095+% purerats used

Pasquet J, Mazuret A, et al. 1976. Acute oral and percutaneous toxicity of phosalone in the rat, incomparison with azinphosmethyl and parathion. Toxicol Appl Pharmacol 37(1):85-92.
Rhone-Poulenc, France

Parathion2

10

8 - 13(95% CL)

Litchfield andWilcoxin method(1949)CD (COBS) rats Charles
River, France; 120-200 g

male andfemaleoral gavage

cmpds dissolved in 1 mL
methylene chloride andemulsified in 10% arabic gum
solution with Tween 80; dose
5mL/kg5 dose levels; 10 male and 10 female
95+% pure
per dose; 100 rats used

Pasquet J, Mazuret A, et al. 1976. Acute oral and percutaneous toxicity of phosalone in the rat, incomparison with azinphosmethyl and parathion. Toxicol Appl Pharmacol 37(1):85-92.
Rhone-Poulenc, France

Litchfield andWilcoxin method(1949)

Sherman strain rats; min. wt.
male= 175 g; min. age of 90 days

chemical in peanut oil; 0.005
oral; stomach tube
mL/g of bw

observed hourly on first day of dosage and twice a day thereafter until
time of death; max survival 3 days

50 rats tested

NA

LD50 value from research in Gaines
NA
1960

Durham WF, Gaines TB, McCauley RH, Sedlak VA, Mattson MA, Hayes WJ. 1957. Studies on the
toxicity of 0,0-dimethyl-2,2-dichlorovinyl phosphate (DDVP). AMA Arch Ind Health 15:340-349.
U.S. Dept. of Health, Education and Welfare, Savannah, GA

5 dose levels; 10 male per dose; 50
rats used

95+% pure

Pasquet J, Mazuret A, et al. 1976. Acute oral and percutaneous toxicity of phosalone in the rat, incomparison with azinphosmethyl and parathion. Toxicol Appl Pharmacol 37(1):85-92.
Rhone-Poulenc, France

NA

LD50 determined after 10 days of observation

Gaines TB. 1960. The acute toxicity of pesticides to rats. Toxicol Appl Pharmacol 2:88-99.
U.S. Dept. of Health, Education, and Welfare, Savannah, GA
Parathion2

13

10 - 17(95% CL)

Parathion2

15

10.2 - 16.5
(95% CL)

Litchfield andWilcoxin method(1949)

Sherman albino ratsmaleoral; stomach tube NA

Parathion2

16

13 - 20(95% CL)

Litchfield andWilcoxin method(1949)CD (COBS) rats Charles
River, France; 120-200 gmaleoral gavage

Parathion230+/- 3.6(S.E.)

Litchfield and Fertig Osborne-Mendel strain rats;male(1941)
180 - 200 g

oral; stomach tube cmpd in corn oil

toxicity symptoms: muscle fibrillation, red colored lacrimation,
diarrhea, dyspnea, convulsions; respiratory paralysis, anoxia, terminal
convulsion

rats fasted for 24 hours;

Phenobarbital

162

162

+/- 14NA

Wistar rats; adult

NA

oral

NA

NA

information from: drug applications
from pharmaceutical manufacturers, NA
the literature, and FDA labs

Goldenthal EI. 1971. A compilation of LD50 values in newborn and adult animals. Toxicology andApplied Pharamacology 18:185-207. Bureau of Drugs, Food and Drug Administration, Dept. of
Health, Education, and Welfare, Rockville, MD. (RTECS REFERENCE)

Phenobarbital

162

220

NA

NA

MJ rats; 80 - 100 days

NA

oral

NA

NA

information from: drug applications
from pharmaceutical manufacturers, NA
the literature, and FDA labs

Goldenthal EI. 1971. A compilation of LD50 values in newborn and adult animals. Toxicology andApplied Pharamacology 18:185-207. Bureau of Drugs, Food and Drug Administration, Dept. of
Health, Education, and Welfare, Rockville, MD.

Phenobarbital

162

318

+/- 23(S.E.)

Miller and Tainter(1944)Charles River CD andSprague-Dawley rat strains; NA> 100 g; adultoral intubation; up
NAto 50 mL/kg

Phenol317

317(0.30 cc/kg of
drug lethal to
50% of rats;
density = 1.055)

NAgraphicallywhite rats

NAoral; stomach tube

5% ethylene glycol added tomost rats died within 2 - 6 hour; practically all dead within 8 - 12 hour;phenol to liquify it so that it
convulsions began several minutes after dosing and continued forNAwould pass through the stomach
several hours
tube

Phenol

317

340

NA

NA

Wistar rats; 100- 200 g

male andfemaleoral

20% aqueous emulsion 0.3, 0.4, 45 rats used; 30 dead; death within 1 hour; twitching, weak pulse and
0.5 g/kg doses
respiration, salivation, dyspnea

45 rats used (equal numbers of male Merck reagent
and female used)
quality

Phenol317

400

297 - 539
(95% CL)

Dixon (1965) and
Bruce (1985)

Fischer 344 rats; 77 days old
femaleat testoral gavage

in deionized water; maximum
volume dose 10mL/kg; 5 doselevels: 0, 12, 40, 120, 224
mg/kg; single dose

fasted overnight; initial dose levels
were 100, 1000, and 5000 mg/kg;
analytical grad_;Berman E, Schlicht M, Moser VC, MacPhail RC. 1995. A multidisciplinary approach to toxicologicalsubsequent doses selected by up-and99+% pure; Aldrich screening: I. Systemic toxicity. J Toxicol Environ Health 45(2): 127-43.
down method (Bruce, 1985, 1987); 5
Chemical Co.
Health Effects Res. Lab., U.S.EPA, Research Triangle Park, NC
groups of 8 rats each; 40 rats used; 7 15 rats used in first LD50 estimate

Phenol

317

445

NA

Probit method

Sprague-Dawley rats; 190200 g

femaleoral

geometric progression of 14 for
9 dead; observed for 14 days
dosing; in water or neat

non-fasted; 4 groups of 5 female; 20 Polysciences, Inc.
rats used
Warrington, PA

Thompson ED, Gibson DP. 1984. A method for determining the maximum tolerated dose for acute invivo cytogenetic studies. Food Chem Toxicol 22(8):665-76.
The Procter and Gamble Co., Cincinnati, OH

Phenol317

512

455 - 568

NArats; 220 +/- 40 gNAoral; intragastricNA(source of information not provided) NA

Izmerov NF, Sanotsky IV, Sidorov KK. 1982. Toxicometric Parameters of Industrial Toxic Chemicals
under Single Exposure. International Register of Potentially Toxic Chemicals (IRPTC). United
Nations Environment Programme (UNEP). Centre of International Projects, GKNT. Moscow, Russia.

Phenol

317

520

NA

Probit method

Sprague-Dawley rats; 190200 g

male

oral

geometric progression of 14 for
10 dead; observed for 14 days
dosing; in water or neat

non-fasted; 3 groups of 5 male; 1
group of 10 male; 25 rats used

Thompson ED, Gibson DP. 1984. A method for determining the maximum tolerated dose for acute invivo cytogenetic studies. Food Chem Toxicol 22(8):665-76.
The Procter and Gamble Co., Cincinnati, OH

cmpds dissolved in 1 mL
methylene chloride andemulsified in 10% arabic gum LD50 determined after 10 days of observation
solution with Tween 80; dose 5
mL/kg

rats observed for 7 days; observed up to 14 days when heavy metals or
fasted overnight
other cmpds that produce latent death were investigated

7 day survival time

NA

H-30

technical grade

NA

NA

Polysciences, Inc.
Warrington, PA

Mattson AM, Spillane JT, Pearce GW. 1955. Dimethyl 2,2-dichlorvinyl phosphate (DDVP), an
organic phosphorous compound highly toxic to insects. J Agr Food Chem 3:319-321.
Communicable Disease Center, Savannah, GA

Frawley JP, Hagan EC, Fitzhugh OG. 1952. A comparative pharmacological and toxicological studyof organic phosphate-anticholinesterase compounnds. J Pharmacol Exp Ther 152:156-165.
U.S. FDA, Washington, D.C.

Yeary RA, Benish RA, Finkelstein M. 1966. Acute Toxicity of Drugs in Newborn Animals. Journalof Pediatrics 69 (4):663-667.
Dept. of Veterinary Preventive Medicine, Ohio State University, Columbus, OH

Gigiena i Sanitariya. (V/O Mezhdunarodnaya Kniga, 113095 Moscow, USSR) V.1- 1936. 1976.---Brown HW, Lamson PD. 1935. Oral Toxicity of Ortho-n-alkylphenols to White Rats. Proc Soc Exp
Biol Med 32:592-594. (RTECS REFERENCE)

Deichmann WB, Witherup S. 1944. Phenol Studies VI: the acute and comparative toxicity of phenol
and o-, m-, and p-cresols for experimental animals. J of Pharmacol and Exp Therapeutics 80:233-240.
College of Medicine, University of Cincinnati, Cincinnati, OH.

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

Phenol

317

530

NA

NA

Wistar rats; 100- 200 g

male andfemaleoral

2% aqueous solution; 0.4, 0.5,
0.6, 0.7, 0.8 g/kg doses

45 rats used; 32 dead; death within 3 hours; twitching, weak pulse and 45 rats used (equal numbers of male Merck reagent
respiration, salivation, dyspnea
and female used)
quality

Deichmann WB, Witherup S. 1944. Phenol Studies VI: the acute and comparative toxicity of phenol
and o-, m-, and p-cresols for experimental animals. J of Pharmacol and Exp Therapeutics 80:233-240.
College of Medicine, University of Cincinnati, Cincinnati, OH.

Phenol

317

530

NA

NA

Wistar rats; 100- 200 g

male andfemaleoral

5% aqueous solution; 0.4, 0.5,
0.6, 0.7 g/kg doses

45 rats used; 27 dead; death within 80 minutes twitching, weak pulse
and respiration, salivation, dyspnea

45 rats used (equal numbers of male Merck reagent
and female used)
quality

Deichmann WB, Witherup S. 1944. Phenol Studies VI: the acute and comparative toxicity of phenol
and o-, m-, and p-cresols for experimental animals. J of Pharmacol and Exp Therapeutics 80:233-240.
College of Medicine, University of Cincinnati, Cincinnati, OH.

Phenol

317

540

NA

NA

Wistar rats; 100- 200 g

male andfemaleoral

10% aqueous emulsion 0.5, 0.6, 40 rats used; 28 dead; death within 120 minutes; twitching, weak pulse 40 rats used (equal numbers of male Merck reagent
0.7, 0.8 g/kg doses
and respiration, salivation, dyspnea
and female used)
quality

Deichmann WB, Witherup S. 1944. Phenol Studies VI: the acute and comparative toxicity of phenol
and o-, m-, and p-cresols for experimental animals. J of Pharmacol and Exp Therapeutics 80:233-240.
College of Medicine, University of Cincinnati, Cincinnati, OH.

Phenol317

550 - A
530 - BNA

A= Behrens (1929)
B = Bliss (1938)

rats

NA

oral

2% aqueous solutionNA

41 - 90 animals used; NICEATM
used value B since authors stated it
was more accurate

NA

Deichmann WB, Mergard EG. 1948. Comparative evaluation of methods employed to express thedegree of toxicity of a cmpd. J Ind Hyg Toxicol 30:373-378.
Albany Medical College, Albany, NY; University of Cincinnati, Cincinnati, OH

Phenol317

580 - A
540 - BNA

A= Behrens (1929)
B = Bliss (1938)

rats

NA

oral

10% aqueous solutionNA

42 - 90 animals used; NICEATM
used value B since authors stated it
was more accurate

NA

Deichmann WB, Mergard EG. 1948. Comparative evaluation of methods employed to express thedegree of toxicity of a compound. J Ind Hyg Toxicol 30:373-378.
Albany Medical College, Albany, NY; University of Cincinnati, Cincinnati, OH

Phenol317

550 - 650

NA

NA

Normal albino rats

male andfemaleoral

dose (mg/kg), percent mortality, minutes till death: 400, 10%, 20; 450,
single doses in mg/kg: 400, 450,
rats divided into 5 test groups and 1
20%, 10 to 80; 500, 30%, 10 to 30; 500, 30%, 10 to 30; 550, 50%, 5 to500, 550, 600, 650, 700; phenol
control; 10 rats per group; 80 rats
90; 600, 60%, 3 to 8; 650, 60%, 4 to 60; 700, 90%, 4 to 50; 500 mg/kg
as 5% aqueous solutionusedrepeated in reference paper

NA

Deichmann W, Oesper P. 1940. Ingestion of phenol: effects on the albino rat. Industr Med 9:296-298.

NAalbino ratsmaleoral; stomach
intubation

4 doses: 200, 398, 795, 1580
mg/kg; single dose

4 groups of 5 rats; 20 rats used; test
procedures outlined in the Federal
Hazardous Substances Act (FSHA)
observed for 14 days; 9 of 20 rats dead; dose (mg/kg), rats dead: 200 Flickinger CW. 1976. The benzenediols: catechol, resorcinol and hydroquinone -- a review of thein the Federeal Register 8/12/61,
0/5; 398 - 0/5; 795 - 4/5 (dead within 1 day after dosing); 1580 - 5/5
Fisher Scientific Co. industrial toxicoloogy and current industrial exposure limits. Am Ind Hyg Assoc J 37:596-606.
pages 7333-7341, entitled "Part 191 (dead < 2 hour after dosing)
Koppers Company, Inc., Monroeville, PA
Hazardous Sub-stances: Definitions
and Procedural and Interpretive
Regulations, Final Order"NA

albino rats; 90-120 g

maleoral; stomach tube

5% phenol solution in water;
single dose

observed for 14 days; 10 rats deadnon-fasted; 4 groups of 10 rats

rwagent gradefrom EPA TSCATS database; Acute Toxicity of Phenol (1949), EPA Document No. 86-870001405
Fiche No. OTS0515567 Mellon Institute of Industrial research, Univ. of Pittsburgh, Pittsburgh,
PA

NA

oral

10% solution in olive oilNA40 - 90 animals used; NICEATM
used value B since authors stated it
was more accurate

NA

Deichmann WB, Mergard EG. 1948. Comparative evaluation of methods employed to express thedegree of toxicity of a cmpd. J Ind Hyg Toxicol 30:373-378.
Albany Medical College, Albany, NY; University of Cincinnati, Cincinnati, OH

oral

NA

NA

value cited from unknown reference NA

Phenol317

650

490 - 860
(95% CL)

Phenol317

1030

940 - 1120

Phenol317

1460 - A
1500 - B

NA

A= Behrens (1929)
B = Bliss (1938)

rats

Phenylthiourea

3

3.1

NA

NA

rats

Phenylthiourea

3< 21.5NA

NA

Fischer rats; 6 weeks

male andfemaleoral intubationNA

observed up to 14 days

NA

NA

4.5

4.5

NA

NA

rat

NA

oral

NA

NA

NA

NA

Potassium I chloride

2600

2600

2330 - 2900

Bliss method

Wistar rats; 110- 140 gmaleoral gavage

approximately 5 doses; in water
14 day observation period;or oil solution

Potassium I chloride

2600

3020

+/- 140(S.E.)

Croxton (1953) Leastsquares linear
Wistar albino rats; adultregression.

femalein distilled water: 0, 2.1, 2.4,
respiratory failure, convulsions, gastroenteritis, anorexia, polydipsia,
oral; stomach tube 2.7, 3.3, 3.6, and 3.9 g/kg bw
polyurea, fever; 14 day observation; death occurred in about half thedoses; volume of 20 mL/kg bw rats

Potassium cyanide

5

5

NA

Rosiello (1979) andBliss (1938)

male

oral

Scheline RR, Smith RL, Williams RT. 1961. The metabolism of arylthioureas -- II. The metabolism

Physostigmine(Eserine)rats

2, 4, 9, 14 mg/kg

2 mg/kg: 0/11 dead; 4 mg/kg:2/11 dead; 9 mg/kg:10/11 dead; 14
mg/kg:11/11 dead; 23 of 44 rats dead; LD50 based on groups
containing 3 and 5 rats

H-31

of 14C- and 35S-labelled 1-phenyl-2-thiourea and its derivatives. Journal of Medicinal andPharmaceutical Chemistry 4(1):109-134. University of London, UK (RTECS REFERENCE)

Carcinogenesis bioassay of environmental chemicals annual progress report NIH-NCI-E-C-72-3252.
5/13/71 -- 8/6/73 and Final report NIH-NCI-E-71-2146. Submitted to The National Cancer Institute,
National Institutes of Health, Bethesda, MD. 8/15/73 (revised 8/10/73).
Litton Bionetics, Inc. Bethesda, MD.
Alisi MA, Brufani M, Cesta MC, Filocamo L, Gostoli G, Lappa S, et al. 1994. U.S. Patent 5,302,593.
Aminoalkylcarbamic esters of eseroline suitable for use as cholinesterase activity inhibitors (April 12,1994). (RTECS REFERENCE)

reference in Czechoslovakian; intro
to reference in English; generally 10 NAanimals per dose; up to 50 rats used

Sbornik Vysledku Toxixologickeho Vysetreni Latek A Pripravku. Marhold, J.V., Institut Pro
Vychovu Vedoucicn Pracovniku Chemickeho Prumyclu Praha, Czechoslovakia, 1972. (RTECSREFERENCE)

109 female rats used; fasted for 16
hours

Boyd EM, Shanas MN. 1961. The Acute Oral Toxicity of Potassium Chloride. Arch Int Pharmacodyn
133:275. Queen's University, Kingston, Ontario, CanadaNAacclimated for 5 days; observed for
14 days; 4 groups used for each dose
(1, 2, 3, 5 animals per group; total of NA
11 rats per dose); 9 rats used for
initial range finding

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany (RTECS REFERENCE)In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

Potassium cyanide

5

5

NA

Rosiello (1979) andBliss (1938)

rats

male

oral

2, 4, 9, 14 mg/kg

acclimated for 5 days; observed for
2 mg/kg: 0/3 dead; 4 mg/kg: 1/3 dead; 9 mg/kg: 3/3 dead; 14 mg/kg:
14 days; 4 groups used for each dose
3/3 dead; 7 of 12 rats dead; LD50 based on 12 rats used; used same rats (1, 2, 3, 5 animals per group; total of NA
as experiments using 44 or 20 rats
11 rats per dose); 9 rats used for
initial range finding

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

Potassium cyanide

5

5

NA

Rosiello (1979) andBliss (1938)

rats

male

oral

2, 4, 9, 14 mg/kg

2 mg/kg: 0/5 dead; 4 mg/kg: 1/5 dead; 9 mg/kg: 5/5 dead; 14 mg/kg:
5/5 dead; 11 of 20 rats dead; LD50 based on 20 rats usedacclimated for 5 days; observed for
14 days; 4 groups used for each dose
(1, 2, 3, 5 animals per group; total of NA
11 rats per dose); 9 rats used for
initial range finding

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

Potassium cyanide

5

6

NA

Rosiello (1979) andBliss (1938)

rats

male

oral

2, 4, 9, 14 mg/kg

acclimated for 5 days; observed for
2 mg/kg: 0/1 dead; 4 mg/kg: 0/1 dead; 9 mg/kg:1/1 dead; 14 mg/kg:1/1 14 days; 4 groups used for each dose
dead; 2 of 4 rats dead; LD50 based on 4 rats used; used same rats as(1, 2, 3, 5 animals per group; total of NA
experiments using 44 rats
11 rats per dose); 9 rats used for
initial range finding

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

Potassium cyanide

5

6

NA

Rosiello (1979) andBliss (1938)

rats

male

oral

2, 4, 9, 14 mg/kg

acclimated for 5 days; observed for
2 mg/kg: 0/11 dead; 4 mg/kg:2/11 dead; 9 mg/kg:10/11 dead; 14
14 days; 4 groups used for each dose
mg/kg:11/11 dead; 23 of 44 rats dead; LD50 based on all rats used (44);(1, 2, 3, 5 animals per group; total of NAsummary data from four tests; Test 1 = 4 rats; test 2 = 8 rats; test 3 = 12
11 rats per dose); 9 rats used for
rats; test 4 = 20 rats
initial range finding

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

Potassium cyanide

5

7.26

6.50 - 8.09

Bliss-Probit method

Sprague-Dawley rats; 5
weeks

male

oral gavage

dissolved in saline; range(mg/kg) of doses 4.9, 5.8, 7.0,
8.4, 10.1, 12.1

rats observed at 6 hours after dosing and a once a day for 1-2 weeks;most dead within 3 days; 33/60 rats died; toxic symptoms: decrease inspontaneous movement, abdominal posture, apsychia andhyperventilation within seconds or minutes of all rats dosed with 84
mg/kg or greater; in all dead rats, convulsion due to asphyxia; dose
(mg/kg), dead rats per dose: 49-0/10; 58-3/10; 70-5/10; 84-7/10; 1018/10; 121-10/10

animals acclimated to environmentfor 1 week before testing; 6 groups
Wako Pureof 10 rats each; fasted 16 hours
Chemicals Co.
before dosing; 100% mortality = 12.1
mg/kg; 0% mortality = 4.9 mg/kg

Kitagawa H, Saito H, Sugimoto T, Yanaura S, Kitagawa H, Hosokawa T, Sakamoto K. 1982. Effects
of diiospropyl-1,3-dithiol-2-ylidene malonate (NKK-105) on acute toxicity of various drugs andheavy metals. J Toxicol Sci 7(2):123-34.
Chiba University; Hoshi College of Pharmacy; Showa University -- Japan

Potassium cyanide

5

9

NA

Rosiello (1979) andBliss (1938)

rats

male

oral

2, 4, 9, 14 mg/kg

2 mg/kg: 0/2 dead; 4 mg/kg: 0/2 dead; 9 mg/kg: 1/2 dead; 14 mg/kg:
2/2 dead; 3 of 8 rats dead; LD50 based on 8 rats usedacclimated for 5 days; observed for
14 days; 4 groups used for each dose
(1, 2, 3, 5 animals per group; total of NA
11 rats per dose); 9 rats used for
initial range finding

Lorke D. 1983. A new approach to practical acute toxicity testing. Arch Toxicol 54(4):275-288.
Institut fur Toxikologie, Wuppertal, Federal Republic of Germany

Potassium cyanide

510

8.7 - 11.5
(95% CL)

Litchfield andWilcoxin method(1949)

Sherman strain rats; min. wt.
male= 175 g; min. age of 90 days

oral; stomach tube

chemical in peanut oil; 0.005
mL/g of bw

observed hourly on first day of dosage and twice a day thereafter until
time of death; max survival = died within 1 hour

50 rats tested

technical grade

Potassium cyanide

510

9 - 12(95% CL; slope =14.5)

Finney (1971)

Crl: CD rats; ave bw = 243male
251 g; young adultoral; intragastricintubation

single dose as suspension in
observed for 14 days; 16 rats dead; all deaths occurred within 1 hour;corn oil (0.1% susp.); 5, 8, 10,
convulsions, tremors, fascilations, gasping, lethargy, weakness,
15 mg/kg dose; dose = 126-377
hyperemia, weight lossmL

4 groups of 10 rats

NAfrom EPA TSCATS database; INITIAL SUBMISSION: ORAL LD50 TEST OF POTASSIUMCYANIDE IN RATS WITH COVER LETTER DATED 08/10/92; EPA Document No. 88920009041 Fiche No. OTS0555358; E.I Dupont DeNemours & Co., Inc./Haskell Labs

Procainamide

1950

1950

NA

NA

rats

NA

oral

NA

NA

no source given for LD50 value

NA

Protiva M, Valenta V, Trcka V, Hladovec J, Nemec J. 1977. Basic amided of 3,4,5trimethoxyphenoxyacetic acid; synthesis and phaarmacology of trimethoxamide and analogues.
Collection of Czechoslovak Chemical Communications 42:3628-3642.
Research Institute for Pharmacy and Biochemistry, Prague, Czechoslovakia (RTECSREFERENCE)

Procainamide1950> 2000NA

Litchfield andWilcoxon method or Wistar rats
Thompson method

male

oral

single doseNA

20 rats used

NA

Turba C, Sanna GP, Bianchi C. 1968. 1: Acute toxicity and general pharmacologic properties of 1,5dimorpholino-3-(1-naphthyl)-pentane: DA 1686. Arzneimittelforschung Sep. 18(9):1127-1132.
LABORATORI RICERCHE ISTITUTO DE ANGLELI, MILANO, ITALY

Propranolol HCl

466

466

NA

Litchfield andWilcoxon method

Sprague-Dawley rats; 2monthsmale

gastric intubation;
single high oral
NA
doses

determined at 10 days by administering po to groups of 5 animals foreach dose a series of doses increasing serially by a factor of 2

fasted 12 hour before dosing

pharmaceu-tical
grade

Maura A, Carlo P, et al. 1985. Absence of DNA damage in mice and rats given high doses of five betaadrenergic blocking agents. Arzneimittelforschung 35(8):1236-1238.
University of Genova, Italy (RTECS REFERENCE)

Propylparaben

6332(mouse oral)

6332(mouse)5740 - 6984
(S.E.)

NAdd strain mice

NA

oral

NA

NA

NA

NA

Sado I. 1973. Synergistic toxicity of officially permitted food preservatives. Nippon Eiseigaku Zasshi
28(5):463-476. (RTECS REFERENCE)

Propylparaben

6332(mouse oral)> 8000(mouse)NA

Miller and Tainter(1944)

uniform strain of albino miceNAfrom a single sourceoralsuspended in 3% starch,
proplene glycol, or olive oil

rapid onset of ataxia, deep depression resembling anesthesia; deaths
usually occurred within 1 hour; recovery from nonfatal doses seldom
lasted > 30 minutes

fasted 12 hour prior to dosing

NA

Matthews C, Davidson J, Bauer E, Morrison JL, Richardson AP. 1956. p- Hydroxybenzoic acide
esters as preservatives II. Acute and chronic toxicity in dogs, rats, and mice. J Am Pharmaceut Assoc
45:260-267.

Sodium arsenite

41

36

acceptable methods
27 - 52(e.g., Bliss, Litchfield(95% CL; slope =Sprague-Dawley ratsand Wilcoxon, Weil,
7.6 [S.E. 2.7])
Thompson, etc.)

maleoral gavage

single dose

14 day observation; toxicity symptoms: diarrhea, diuresis, posture,
respiratory effects, lethargy, abnormal gait; time to onset of signs <1day; duration of signs 3 days; 9 rats dead (average per test)

3 dose levels (5 male each); 15 rats
used; OECD TG401 (1981) followed NAfor experimental procedures

Vandenheuvel MJ, Clark DG, Fielder RJ, Koundakjian PP, Oliver GJA, Pelling D, Tomlinson NJ,
Walker AP. 1990. Jul. The International Validation Of A Fixed-Dose Procedure As An Alternative To
The Classical LD50 Test Food And Chemical Toxicology 28(7):469-482.

Sodium arsenite

41

41

31 - 53
Thompson method;(these limits are +/Weil tables
1.96 S.D.)

maleoral gastricintubation

in aqueous solution;
concentration intubated = 10
mg/mL; dosages arranged in a LD50 based on mortalities during a 14 day period
logarithmic series differing by afactor of 2

Carworth-Wistar rats; 90120 g; 4-5 weeks

H-32

non-fasted; groups of 5 rats; single
oral dose toxicity

Gaines TB. 1969. Acute toxicity of pesticides. Toxicol Appl Pharmacol 14(3):515-34.
U.S. Dept. of Health, Education, and Welfare, Atlanta, GA

Smyth HF Jr, Carpenter CP, Weil CS, Pozzani UC, Striegel JA, Nycum, JS. 1969. Range-finding
toxicity data: List VII. Am Ind Hyg Assoc J 30: 470-476.
Carnegie-Mellon University, Pittsburgh, PA (LD50 value) (RTECS REFERENCE)
reagent grade
Smyth HF Jr, Carpenter CP, Weil CS, Pozzani UC, Striegel JA. 1962. Range-finding toxicity data:
List VI. Am Ind Hyg Assoc J 23:95-107.
Mellon Institute of Industrial Research, Pittsburg, PA (experimental parameters)

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

Route and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

male andfemaleoral, gelatin
capsules20, 50, 100, 200 (all in mg/kg)

death occurred within 4 days

approximately 40 rats used; 24 hour Baker Analyzed
Done AK, Peart AJ. 1971. Acute Toxicities of Arsenical Herbicides. Cinical Toxicology, 4(3):343fasting before dosing; rats dosed
Reagent with 0.02%
355. University of Utah, Salt Lake City, UT
under light anesthesia
impurities

acceptable methods(e.g., Bliss, Litchfield
Sprague-Dawley ratsand Wilcoxon, Weil,
Thompson, etc.)

male andfemaleoral gavage

single dose

14 day observation; toxicity symptoms: diarrhea, diuresis, posture,
respiratory effects, lethargy, abnormal gait; time to onset of signs < 1day; duration of signs 3 days; 9 rats dead (average per test)

3 dose levels (5 male each and 5
female); 30 rats used; OECD TG401
NA(1981) followed for experimental
procedures

Vandenheuvel MJ, Clark DG, Fielder RJ, Koundakjian PP, Oliver GJA, Pelling D, Tomlinson NJ,
Walker AP. 1990. Jul. The International Validation Of A Fixed-Dose Procedure As An Alternative To
The Classical LD50 Test Food And Chemical Toxicology 28(7):469-482.

37 - 76(95% CL)

acceptable methods(e.g., Bliss, Litchfield
Sprague-Dawley ratsand Wilcoxon, Weil,
Thompson, etc.)

femaleoral gavage

single dose

14 day observation; toxicity symptoms: diarrhea, diuresis, posture,
respiratory effects, lethargy, abnormal gait; time to onset of signs < 1day; duration of signs 3 days; 9 rats dead (average per test)

3 dose levels (5 female each); 15 rats
used; OECD TG401 (1981) followed NAfor experimental procedures

Vandenheuvel MJ, Clark DG, Fielder RJ, Koundakjian PP, Oliver GJA, Pelling D, Tomlinson NJ,
Walker AP. 1990. Jul. The International Validation Of A Fixed-Dose Procedure As An Alternative To
The Classical LD50 Test Food And Chemical Toxicology 28(7):469-482.

53

39 - 74(95% CL)

acceptable methods(e.g., Bliss, Litchfield Sprague-Dawley rats; 190and Wilcoxon, Weil, 300 g
Thompson, etc.)

femaleoral gavage

single dose

14 day observation; toxicity symptoms: motor activity decrease,respiratory effects, blanching, piloerection, salivation, diarrhea; time to
UDP Test
onset of signs < 1 day; duration of signs 3 days; animals fasted 16 -20
hours before administrationNA

Yam J, Reer PJ, Bruce RD. 1991. Comparison of the up-and-down method and the fixed-doseprocedure for acute oral toxicity testing. Food Chem Toxicol 29(4):259-264.
The Procter and Gamble Co., Cincinnati, OH

3000

3000

NA

NA

rats

NA

oral

NA

NA

No information/reference provided.

NA

Tucker RK, Haegel MA. 1971. Compararive acute oral toxicity of pesticides to six species of birds.
Toxicology and Applied Pharmacology 20:57-65. (RTECS REFERENCE)

3000

3620

+/-300(S.E.)

Croxton (1953) and
Waugh (1952)

Wistar albino rats; female:
167+/-27 g; young adultfemaleoral; intragastrictube

doses = 0, 0.8, 3, 3.2, 3.5, 3.8,
4, 5, 10, 16 g/kg in water; 20
mL/kg dose; 2 largest doses inlarger volumes

convulsive movements, diarrhea, muscular rigidity, prostration,
respiratory failure; death within 14 hours

fasted for 16 hours; 84 rats used; 12 NA
44 rats per dose

Boyd EM, Shanas MN. 1963. The acute oral toxicity of sodium chloride. Arch Internat Pharmacodyn
144:86-96. Quebecs' University, Kingston, Ontario, Canada

male andfemale(equalnumbers)

oral; intragastrictube

doses = 0, 0.8, 3, 3.2, 3.5, 3.8,
4, 5, 10, 16 g/kg in water; 20
mL/kg dose; 2 largest doses inlarger volumes

convulsive movements, diarrhea, muscular rigidity, prostration,
respiratory failure; death within 14 hours

fasted for 16 hours; 168 rats used;equal numbers of male and female;
12-44 rats per dose; this LD50 isdetermined from the data used toNAdetermine LD50 of 3620 mg/kg(female) and 3890 mg/kg (male) also
reported in this reference [Boyd andShanas 1963]

Boyd EM, Shanas MN. 1963. The acute oral toxicity of sodium chloride. Arch Internat Pharmacodyn
144:86-96. Quebecs' University, Kingston, Ontario, Canada

Sodium arsenite

41

42

35 - 50(95% CL)

Litchfield andWilcoxon method

Sodium arsenite

41

42

35 - 58(95% CL)

Sodium arsenite

41

48

Sodium arsenite

41

Sodium chloride

Sodium chloride

Holtsman rats; 300- 500 g;
100-300 days (13 - 41
weeks)

GenderSodium chloride

3000

3750

+/-430(S.E.)

Croxton (1953) and
Waugh (1952)

Wistar albino rats; male:202+/-42 g; female: 167+/27 g; young adult

Sodium chloride

3000

3890

+/-300(S.E.)

Croxton (1953) and
Waugh (1952)

Wistar albino rats; male:202+/-42 g; young adultmaleoral; intragastrictube

doses = 0, 0.8, 3, 3.2, 3.5, 3.8,
4, 5, 10, 16 g/kg in water; 20
mL/kg dose; 2 largest doses inlarger volumes

convulsive movements, diarrhea, muscular rigidity, prostration,
respiratory failure; death within 14 hours

fasted for 16 hours; 84 rats used; 12 NA
44 rats per dose

Boyd EM, Shanas MN. 1963. The acute oral toxicity of sodium chloride. Arch Internat Pharmacodyn
144:86-96. Quebecs' University, Kingston, Ontario, Canada

Sodium chloride

3000

4200

3980 - 4430
(95% CL)

Litchfield andWilcoxon method(1949)

rats

NA

oral

NA

NA

reference in Italian

Scognamiglio WP, Amorico L, Gatti GL. 1972. Esperienze di tossicita e di tolleranza almonosioglutammato con un saggio di condizionamento di salvaguardia. Il Farmaco Edizone Pratica
27:19-27.

Sodium chloride

3000

6140

+/-310(S.E.)NA

CBL Wistar albino rats; 150male
200 goral; intragastrictube

single dose; 5000 - 7500 mg/kg
dose range; cmpd dissolved inobserved for 5 days; premortal diarrhea; convulsive movements
distilled water; 20 mL/kg
dosage5 rats per dose; 30 rats used; rats not
Merck Reagent
fastedBoyd EM, Abel MM, Knight LM. 1966. The chronic oral toxicity of sodium chloride at the range of
the LD50 (0.1L). Canad J Physiol Pharmacol 44:157-172.
Queen's University, Ontario, Canada

Sodium dichromate (Sodium
bichromate VI)

50

34.17

+/- 20.95(S.D.)

Gad and Weil (1982) Fischer 344 rats (Harlen
Probit analysis
Sprague Dawley)femaleoral gavage

single dose: 40,60,80 mg/kg;
dosing solution: 10,5,1,0.5%(w/v); dosing vol: 0.4-8.0
mL/kg (40 mg/kg); 0.6-12
mL/kg (60 mg/kl); 0.8-16
mL/kg (80 mg/kg); doses indistilled water; 10% dose

observed first 6 hours then day 1, 7 and 14; hypoactivity, lacrimation,
mydriasis, diarrhea, change in body weight; LD50 increased as theconcentration of the dosing solution increased

animals acclimated for 2 weeks
before dosing; animals fasted
overnight; 5 animals usedmember companiesof the Industrial
Health Foundation

Gad SC, Powers WJ, Dunn BJ, Hoffman GM, Siino KM, Walsh RD. 1986. Acute toxicity of fourchromate salts. Proceedings of the Chromium Symposium, pp. 43-58.
G.D. Searle and Co., Skokie, IL

Sodium dichromate (Sodium
bichromate VI)

50

38.55

+/- 7.79(S.D.)

Gad and Weil (1982) Fischer 344 rats (Harlen
Probit analysis
Sprague Dawley)femaleoral gavage

single dose: 40,60,80 mg/kg;
dosing solution: 10,5,1,0.5%(w/v); dosing vol: 0.4-8.0
mL/kg (40 mg/kg); 0.6-12
mL/kg (60 mg/kl); 0.8-16
mL/kg (80 mg/kg); doses indistilled water; 5% dose

observed first 6 hours then day 1, 7 and 14; hypoactivity, lacrimation,
mydriasis, diarrhea, change in body weight; LD50 increased as theconcentration of the dosing solution increased

animals acclimated for 2 weeks
before dosing; animals fasted
overnight; 5 animals/dose

member companiesof the Industrial
Health Foundation

Gad SC, Powers WJ, Dunn BJ, Hoffman GM, Siino KM, Walsh RD. 1986. Acute toxicity of fourchromate salts. Proceedings of the Chromium Symposium, pp. 43-58.
G.D. Searle and Co., Skokie, IL

Sodium dichromate (Sodium
bichromate VI)

50

39.02

+/- 13.54(S.D.)

Gad and Weil (1982) Fischer 344 rats (Harlen
Probit analysis
Sprague Dawley)femaleoral gavage

single dose: 40, 50, 60, 80, 100
observed first 6 hours then day 1, 7 and 14; hypoactivity, lacrimation,
mg/kg; dosing solution 50%
mydriasis, diarrhea, change in body weight; LD50 increased as the(w/v); 0.8-2.0 mL/kg dosing
concentration of the dosing solution increased
volume; doses in distilled water

animals acclimated for 2 weeks
before dosing; animals fasted
overnight; 5 male and 5 female rats
per dose; 10 rats/dose; 5 female
rats/dose for this value

member companiesof the Industrial
Health Foundation

Gad SC, Powers WJ, Dunn BJ, Hoffman GM, Siino KM, Walsh RD. 1986. Acute toxicity of fourchromate salts. Proceedings of the Chromium Symposium, pp. 43-58.
G.D. Searle and Co., Skokie, IL

H-33

NA

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

Sodium dichromate (Sodium
bichromate VI)

50

48.98

+/- 10.50(S.D.)

Sodium dichromate (Sodium
bichromate VI)

50

50

NA

Gad and Weil (1982) Fischer 344 rats (Harlen
Probit analysis
Sprague Dawley)

GenderDose

Observations

Notes

Reference
Substance Source

Primary Reference

oral gavage

single dose: 40,60,80 mg/kg;
dosing solution: 10,5,1,0.5%(w/v); dosing vol: 0.4-8.0
mL/kg (40 mg/kg); 0.6-12
mL/kg (60 mg/kl); 0.8-16
mL/kg (80 mg/kg); doses indistilled water; 10% dose

observed first 6 hours then day 1, 7 and 14; hypoactivity, lacrimation,
mydriasis, diarrhea, change in body weight; LD50 increased as theconcentration of the dosing solution increased

animals acclimated for 2 weeks
before dosing; animals fasted
overnight; 5 animals/dose

member companiesof the Industrial
Health Foundation

Gad SC, Powers WJ, Dunn BJ, Hoffman GM, Siino KM, Walsh RD. 1986. Acute toxicity of fourchromate salts. Proceedings of the Chromium Symposium, pp. 43-58.
G.D. Searle and Co., Skokie, IL

NA

NA

NA

reference in Russian

NA

Gigiena Truda i Professional'nye Zabolevaniya. Labor Hygiene and Occupational Diseases. (V/O
Mezhdunarodnaya Kniga, 113095 Moscow, USSR) V.1-36, 1957-1992. 1978.
(RTECS REFERENCE)

male andfemaleoral gavage

single dose: 40, 50, 60, 80, 100
observed first 6 hours then day 1, 7 and 14; hypoactivity, lacrimation,
mg/kg; dosing solution 50%
mydriasis, diarrhea, change in body weight; LD50 increased as the(w/v); 0.8-20 mL/kg dosing
concentration of the dosing solution increased
volume; doses in distilled water

animals acclimated for 2 weeks
before dosing; animals fasted
overnight; 5 male and 5 female rats
member companies
per dose; 10 rats/dose; this LD50 isof the Industrial
determined from the data used to
Health Foundation
determine LD50 of 39.02 mg/kg(female) and 58.84 mg/kg (male) also
reported in this reference

Gad SC, Powers WJ, Dunn BJ, Hoffman GM, Siino KM, Walsh RD. 1986. Acute toxicity of fourchromate salts. Proceedings of the Chromium Symposium, pp. 43-58.
G.D. Searle and Co., Skokie, IL

maleoral gavage

single dose: 40,60,80 mg/kg;
dosing solution: 10,5,1,0.5%(w/v); dosing vol: 0.4-8.0
mL/kg (40 mg/kg); 0.6-12
mL/kg (60 mg/kl); 0.8-16
mL/kg (80 mg/kg); doses indistilled water; 5% dose

observed first 6 hours then day 1, 7 and 14; hypoactivity, lacrimation,
mydriasis, diarrhea, change in body weight; LD50 increased as theconcentration of the dosing solution increased

animals acclimated for 2 weeks
before dosing; animals fasted
overnight; 5 animals/dose

member companiesof the Industrial
Health Foundation

Gad SC, Powers WJ, Dunn BJ, Hoffman GM, Siino KM, Walsh RD. 1986. Acute toxicity of fourchromate salts. Proceedings of the Chromium Symposium, pp. 43-58.
G.D. Searle and Co., Skokie, IL

observed first 6 hours then day 1, 7 and 14; hypoactivity, lacrimation,
mydriasis, diarrhea, change in body weight; LD50 increased as theconcentration of the dosing solution increased

animals acclimated for 2 weeks
before dosing; animals fasted
overnight; 5 animals/dose

member companiesof the Industrial
Health Foundation

Gad SC, Powers WJ, Dunn BJ, Hoffman GM, Siino KM, Walsh RD. 1986. Acute toxicity of fourchromate salts. Proceedings of the Chromium Symposium, pp. 43-58.
G.D. Searle and Co., Skokie, IL

Gad and Weil (1982) Fischer 344 rats (Harlen
Probit analysis
Sprague Dawley)

male

NA

NA

rats

Route and/or
Method ofExposure

Sodium dichromate (Sodium
bichromate VI)

50

51.1

+/- 5.93(S.D.)

Sodium dichromate (Sodium
bichromate VI)

50

55.75

+/- 15.98(S.D.)

Gad and Weil (1982) Fischer 344 rats (Harlen
Probit analysis
Sprague Dawley)

Gad and Weil (1982) Fischer 344 rats (Harlen
Probit analysis
Sprague Dawley)femaleoral gavage

single dose: 40,60,80 mg/kg;
dosing solution: 10,5,1,0.5%(w/v); dosing vol: 0.4-8.0
mL/kg (40 mg/kg); 0.6-12
mL/kg (60 mg/kl); 0.8-16
mL/kg (80 mg/kg); doses indistilled water; 0.5% dose

Sodium dichromate (Sodium
bichromate VI)

50

57.13

+/- 8.81(S.D.)

Sodium dichromate (Sodium
bichromate VI)

50

58.84

+/- 5.78(S.D.)

Gad and Weil (1982) Fischer 344 rats (Harlen
Probit analysis
Sprague Dawley)maleoral gavage

single dose: 40, 50, 60, 80, 100
observed first 6 hours then day 1, 7 and 14; hypoactivity, lacrimation,
mg/kg; dosing solution 50%
mydriasis, diarrhea, change in body weight; LD50 increased as the(w/v); 0.8-20 mL/kg dosing
concentration of the dosing solution increased
volume; doses in distilled water

animals acclimated for 2 weeks
before dosing; animals fasted
overnight; 5 male and 5 female rats
per dose; 10 rats/dose; 5 malerats/dose for this value

member companiesof the Industrial
Health Foundation

Gad SC, Powers WJ, Dunn BJ, Hoffman GM, Siino KM, Walsh RD. 1986. Acute toxicity of fourchromate salts. Proceedings of the Chromium Symposium, pp. 43-58.
G.D. Searle and Co., Skokie, IL

Sodium dichromate (Sodium
bichromate VI)

50

59.84

+/- 7.74(S.D.)

Gad and Weil (1982) Fischer 344 rats (Harlen
Probit analysis
Sprague Dawley)maleoral gavage

single dose: 40,60,80 mg/kg;
dosing solution: 10,5,1,0.5%(w/v); dosing vol: 0.4-8.0
mL/kg (40 mg/kg); 0.6-12
mL/kg (60 mg/kl); 0.8-16
mL/kg (80 mg/kg); doses indistilled water; 0.5% dose

observed first 6 hours then day 1, 7 and 14; hypoactivity, lacrimation,
mydriasis, diarrhea, change in body weight; LD50 increased as theconcentration of the dosing solution increased

animals acclimated for 2 weeks
before dosing; animals fasted
overnight; 5 animals/dose

member companiesof the Industrial
Health Foundation

Gad SC, Powers WJ, Dunn BJ, Hoffman GM, Siino KM, Walsh RD. 1986. Acute toxicity of fourchromate salts. Proceedings of the Chromium Symposium, pp. 43-58.
G.D. Searle and Co., Skokie, IL

Gad and Weil (1982) Fischer 344 rats (Harlen
Probit analysis
Sprague Dawley)maleoral gavage

single dose: 40,60,80 mg/kg;
dosing solution: 10,5,1,0.5%(w/v); dosing vol: 0.4-8.0
mL/kg (40 mg/kg); 0.6-12
mL/kg (60 mg/kl); 0.8-16
mL/kg (80 mg/kg); doses indistilled water; 1% dose

observed first 6 hours then day 1, 7 and 14; hypoactivity, lacrimation,
mydriasis, diarrhea, change in body weight; LD50 increased as theconcentration of the dosing solution increased

animals acclimated for 2 weeks
before dosing; animals fasted
overnight; 5 animals/dose

member companiesof the Industrial
Health Foundation

Gad SC, Powers WJ, Dunn BJ, Hoffman GM, Siino KM, Walsh RD. 1986. Acute toxicity of fourchromate salts. Proceedings of the Chromium Symposium, pp. 43-58.
G.D. Searle and Co., Skokie, IL

Gad and Weil (1982) Fischer 344 rats (Harlen
Probit analysis
Sprague Dawley)femaleoral gavage

single dose: 40,60,80 mg/kg;
dosing solution: 10,5,1,0.5%(w/v); dosing vol: 0.4-8.0
mL/kg (40 mg/kg); 0.6-12
mL/kg (60 mg/kl); 0.8-16
mL/kg (80 mg/kg); doses indistilled water; 1% dose

observed first 6 hours then day 1, 7 and 14; hypoactivity, lacrimation,
mydriasis, diarrhea, change in body weight; LD50 increased as theconcentration of the dosing solution increased

animals acclimated for 2 weeks
before dosing; animals fasted
overnight; 5 animals/dose

member companiesof the Industrial
Health Foundation

Gad SC, Powers WJ, Dunn BJ, Hoffman GM, Siino KM, Walsh RD. 1986. Acute toxicity of fourchromate salts. Proceedings of the Chromium Symposium, pp. 43-58.
G.D. Searle and Co., Skokie, IL

Sodium dichromate (Sodium
bichromate VI)

50

59.84

+/- 7.74(S.D.)

Sodium Dichromate (Sodium
Bichromate VI)

50

64.5

+/- 10.18(S.D.)

Sodium hypochlorite

8910
(from
HSDB)

8200

NA

NA

NA

NA

NA

NA

NA

12.5% hypochlorite solution

NA

Sodium Hypochlorite Toxicity Profile. 1990. British Industrial Biological Research Association(BIBRA).

Sodium hypochlorite

8910
(from
HSDB)

9360 - 11700

NA

NA

NA

NA

NA

NA

NA

12.5% hypochlorite solution

NA

Colgate-Palmolive. 1990. Internal Report: Investigation of the properties of the wash water inconnection with washing using "Klorin" bleach. Unpublished.

Sodium hypochlorite

8910
(from
HSDB)

>11800

NA

NA

NA

NA

NA

NA

NA

3.6% hypochlorite solution

NA

Colgate-Palmolive. 1990. Internal Report: Investigation of the properties of the wash water inconnection with washing using "Klorin" bleach. Unpublished.

H-34

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

Sodium hypochlorite

LD501
mg/kg

8910
(from
HSDB)

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

13000

NA

NA

Animal Information(stock, weight, age)

NA

GenderNA

Route and/or
Method ofExposureNA

Dose

Observations

Notes

5.25% hypochlorite solution

Reference
Substance Source

NA

NA

oral

5 mL/kg

22 rats died within 3 hour; 15 rats died after 3 hour; observed for 7
reference paprer in Japanese; English
days; signs of toxicity appeared from 5-15 minutes after administration summary and table/graph headers;of NaF: muscle weakness, salivation, diarrhea, lacrimation, tremor,
see paper for information aboutNA
convulsion, hypopnea, cynosis, urinary incontinence; most animals
regression coefficient of log dosedied within 24 hour after dosing
NED mortality curve

NA

Primary Reference

MSDS Chlorine Institute 1982

Sodium I fluoride

115

64(29 mg F/kg;
converted to mg
NaF/kg)60 - 69(95% CI)

Litchfield andWilcoxon method(1949); Bliss (1938)

rats; mean bw = 169 g; 3monthsSodium I fluoride

115

69(31 mg F/kg;
converted to mg
NaF/kg)55 - 84(CL)

Litchfield andWilcoxon method(1949)

Sprague-Dawley rats; meanbw and ranges 250 g (200- female
359 g); 90 days

stomach tube

NaF in aqueous solution (0.2 1.6 mL/dose)

mortality confined to 24 hour; when doses equal to or greater than the
LD50 were administered, half of the 250 g rats died within 3 hours

Sodium I fluoride

115

73(33 mg F/kg;
converted to mg
NaF/kg)66 - 80(95% CI)

Litchfield andWilcoxon method(1949); Bliss (1938)

rats; mean bw = 295 g; 3monthsmaleoral

3 mL/kg

6 rats died within 3 hour; 35 rats died after 3 hour; observed for 7 days; reference paprer in Japanese; English
signs of toxicity appeared from 5-15 minutes after administration ofsummary and table/graph headers;
NaF: muscle weakness, salivation, diarrhea, lacrimation, tremor,
see paper for information aboutNA
convulsion, hypopnea, cynosis, urinary incontinence; most animals
regression coefficient of log dosedied within 24 hour after dosing
NED mortality curve

Sodium I fluoride

115

80

+/- 5
(S.E.)

Winthrop logarithmic
probit graph paper;
Albino rats; 200- 300 g
Miller and Tainter(1944)

NA

oral; stomach tube

single dose; 25% solution; 22 - percentage mortality observed in 24 hour calculated, then LD50
288 mg/kg doses;
determined

Sodium I fluoride

115

84(38 mg F/kg;
converted to mg
NaF/kg)77 - 93(95% CI)

Litchfield andWilcoxon method(1949); Bliss (1938)

rats; mean bw = 60 g; 3
weeks

femaleoral

5 mL/kg

16 rats died within 3 hour; 32 rats died after 3 hour; observed for 7
reference paprer in Japanese; English
days; signs of toxicity appeared from 5-15 minutes after administration summary and table/graph headers;of NaF: muscle weakness, salivation, diarrhea, lacrimation, tremor,
see paper for information aboutNA
convulsion, hypopnea, cynosis, urinary incontinence; most animals
regression coefficient of log dosedied within 24 hour after dosing
NED mortality curve.

Sakama H. 1980. Toxicological studies of fluorine compounds. I. Acute toxicity of sodium fluoride torats and mice in relation to age, sex, animal genus, and administration route. Shika Gakuho. Journal of
Dentistry. 80: 1519. Tokyo Dental College, Japan.

Sodium I fluoride

115

107
(46 mg F/kg;
converted to mg
NaF/kg)95 - 110(95% CI)

Litchfield andWilcoxon method(1949); Bliss (1938)

rats; mean bw = 58 g; 3
weeks

male

oral

5 mL/kg

2 rats died within 3 hour; 32 rats died after 3 hour; observed for 7 days; reference paprer in Japanese; English
signs of toxicity appeared from 5-15 minutes after administration ofsummary and table/graph headers;
NaF: muscle weakness, salivation, diarrhea, lacrimation, tremor
see paper for information aboutNA
convulsion, hypopnea, cynosis, urinary incontinence; most animals
regression coefficient of log dosedied within 24 hour after dosing
NED mortality curve.

Sakama H. 1980. Toxicological studies of fluorine compounds. I. Acute toxicity of sodium fluoride torats and mice in relation to age, sex, animal genus, and administration route. Shika Gakuho. Journal of
Dentistry. 80: 1519. Tokyo Dental College, Japan.

Sodium I fluoride

115

115
106 - 126
Litchfield and(52 mg F/kg;(slope = 1.23 [1.06 - Wilcoxin method
converted to mg
1.43]; 95% CL) (1949)
NaF/kg)Sprague-Dawley rats; meanbw and ranges 150 g (112- female
184 g); 30-45 days

stomach tube

mortality confined to 24 hour; when doses > the LD50 wereadministered, one-third of the 150 g rats died within 7 hours; dose in
NaF in aqueous solution (0.2 mg F/kg and 24 hour mortality: 75-2/2 dead; 70-9/10 dead; 65-7/9
1.6 mL/dose); 30 - 100 mg F/kg
dead; 62-6/8 dead; 58-4/10 dead; 55-9/15 dead; 50-8/12 dead; 45-3/10
doses;
dead; 42-2/10 dead; 40-0/2 dead; 35-0/2 dead; salivation, diarrhea,
thirst, lethargy

Sodium I fluoride

115

115
(52 mg F/kg;
converted to mg
NaF/kg)108 - 119
Litchfield and(slope = 1.28 [1.0 - Wilcoxon method1.6]; 95% CL)(1949)

Sprague-Dawley rats; meanbw and ranges 80 g (50-108 femaleg); 30-45 days

stomach tube

fasted 24 hour before dosing; at least
mortality confined to 24 hour; when doses equal to or greater than theseven dose levels used for each
NaF in aqueous solution (0.2 - LD50 were administered, half of the 80 g rats died within 6 hours; dose
population; groups of 2 -12 rats; 63
1.6 mL/dose); 30 - 100 mg F/kg in mg F/kg and 24 hour mortality: 100-9/12 dead; 75-8/9 dead; 70-8/10
NA
rats used; 36 dead; detailed
doses;
dead; 60-8/10 dead; 50-2/10 dead; 40-2/10 dead; 30-0/2 dead;information from RTECS reference
salivation, diarrhea, thirst, lethargy(master thesis for de Lopez 1970)

DeLopez OH. 1970. Acute fluoride toxicity: plasma fluoride concentrations following acute oral
doses of sodium fluoride in the rat. Master of Science thesis.
Univ. of Rochester School of Med. And Dent., Rochester, NY (see DeLopez 1976)(RTECS REFERENCE)

Sodium I fluoride

115

119
(54 mg F/kg;
converted to mg
NaF/kg)108 - 119
Litchfield and(slope = 1.28 [1.0 - Wilcoxon method1.6]; 95% CL)(1949)

Sprague-Dawley rats; meanbw and ranges 80 g (50-108 femaleg); 30-45 days

stomach tube

fasted 24 hour before dosing; at least
mortality confined to 24 hour; when doses equal to or greater than theseven dose levels used for each
NaF in aqueous solution (0.2 - LD50 were administered, half of the 80 g rats died within 6 hours; dose
population; groups of 2 -12 rats; 63
1.6 mL/dose); 30 - 100 mg F/kg in mg F/kg and 24 hour mortality: 100-9/12 dead; 75-8/9 dead; 70-8/10
NA
rats used; 36 dead; detailed
doses
dead; 60-8/10 dead; 50-2/10 dead; 40-2/10 dead; 30-0/2 dead;information from RTECS reference
salivation, diarrhea, thirst, lethargy(master thesis for de Lopez 1970)

DeLopez OH, Smith FA, Hodge HC. 1976. Plasma fluoride concentrations in rats acutely poisoned
with sodium fluoride. Toxicol Appl Pharmacol 37:75-83.
Univ. of Rochester School of Med. And Dent., Rochester, NY

Sodium I fluoride

115

180

120 - 260
Thompson method;(these limits are +/Weil tables
1.96 S.D.)

Carworth-Wistar rats; 90120 g; 4-5 weeks

maleoral gastricintubation

in aqueous solution;
concentration intubated = 5
mg/mL; dosages arranged in a LD50 based on mortalities during a 14 day period
logarithmic series differing by afactor of 2

Litchfield andWilcoxon method(1949)

Sprague-Dawley rats; 152202 g

maleoral; intragastric

50 to 220 mg F/kg (111 - 486
mg NaF/kg) in water

femaleSodium I fluoride

115

189
(85.5 mg F/kg;
converted to mg
NaF/kg)#2: 170 -209
(95%CI)

Sodium I fluoride

115

200

NA

NA

rats

NA

Sodium I fluoride

115

223

NA

Probit analysis

Sprague-Dawley rats; 190315 g

malefasted 24 hour before dosing; at least
seven dose levels used for eachNA
population; groups of 8 -15 rats

98 rats usedNA

fasted 24 hour before dosing; 11 doselevels used; groups of 2 -15 rats; 90
rats used; 50 dead; detailedNA
information from RTECS reference(master thesis for de Lopez 1970)

non-fasted; groups of 5 rats; single
oral dose toxicity

Sakama H. 1980. Toxicological studies of fluorine compounds. I. Acute toxicity of sodium fluoride torats and mice in relation to age, sex, animal genus, and administration route. Shika Gakuho. Journal of
Dentistry. 80: 1519. Tokyo Dental College, Japan.

DeLopez OH, Smith FA, Hodge HC. 1976. Plasma fluoride concentrations in rats acutely poisoned
with sodium fluoride. Toxicol Appl Pharmacol 37:75-83.
Univ. of Rochester School of Med. And Dent., Rochester, NY

Sakama H. 1980. Toxicological studies of fluorine compounds. I. Acute toxicity of sodium fluoride torats and mice in relation to age, sex, animal genus, and administration route. Shika Gakuho. Journal of
Dentistry. 80: 1519. Tokyo Dental College, Japan.

Shourie KL, Hein JW, Hodge HC. 1950. Preliminary studies of the caries inhibiting potential andacute toxicity of sodium monofluorophosphate. J Dent Res 29:529-533.
Univeristy of Rochester, School of Medicine and Denistry, Rochester, NY.

DeLopez OH, Smith FA, Hodge HC. 1976. Plasma fluoride concentrations in rats acutely poisoned
with sodium fluoride. Toxicol Appl Pharmacol 37:75-83.
Univ. of Rochester School of Med. And Dent., Rochester, NY

Smyth HF Jr, Carpenter CP, Weil CS, Pozzani UC, Striegel JA, Nycum, JS. 1969. Range-finding
toxicity data: List VII. Am Ind Hyg Assoc J 30: 470-476.
Carnegie-Mellon University, Pittsburgh, PA (LD50 value)
reagent grade
Smyth HF Jr, Carpenter CP, Weil CS, Pozzani UC, Striegel JA. 1962. Range-finding toxicity data:
List VI. Am Ind Hyg Assoc J 23:95-107.
Mellon Institute of Industrial Research, Pittsburg, PA (experimental parameters)

number of deaths determined at 1, 2, 4, 8, 24 hour and daily thereafter;
20 rats dead at 24 hour; 26 rats dead at 14 days; monitored for 2 weeks fasted 18 hour before dosing; 10 day
but no deaths after 4 days; deaths/dose (mg/kg): 111-0/10, 122-0/10,
acclimatization before dosing; 8 rats >99.5% purity
134-1/10, 147-0/10, 162-0/10, 166-4/10, 183-4/10, 201-3/10, 221-6/10, in each dosage group; 80 rats used
243-8/10

Whitford GM, Birdsong-Whitford NL, et al. 1990. Acute oral toxicity of sodium fluoride andmonofluorophosphate separately or in combination in rats. Caries Res 24(2):121-126.
Medical College of Georgia, Augusta, GA; Dept. of Odonto-Stomatologie, Laboratoires Goupil SA,
Cachan, France.

oral; stomach tube NA

information from the laboratories ofabdominal distress, diarrhea, cyanosis, dyspnea, fibrillation of skeletal
Division of Pharmacology, U.S.
NAmuscles; onset within 6 hours
FDA.; fasted animals

Lehman AJ. 1951. Chemicals in Foods: a report to the association of food and drug officials on
current developments. Part II. Pesticides. Quarterly Bulletine (Association of Food and Drug Officialsof the United States). Vol.15:122-133. U.S. FDA

oral gavage

animals observed for mortality frequently during first 4 hour after
dosing; observed daily thereafter for 14 days

0.101 - 0.500 g NaF/kg bw

H-35

fasted 18 - 20 hour before dosing; 8rats per group; 48 total rats used;
Skare JA, Schrotei KR, Nixon GA. 1986. Lack of DNA-strand breaks in rat testicular cells after in
J.T. Baker Chemicalmortality confined to 24 hour after
vivo treatment with sodium fluoride. Mutat Res 170:85-92.
Co.
dosing except 3 animals died on day
The Proctor and Gamble Company, Cincinnati, OH
2, 3, and 5

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Sodium I fluoride

115

279
(126.3 mg F/kg;
converted to mg
NaF/kg)#1: 218 - 358(95%CI)

Sodium oxalate

11160

11160

NA

Sodium oxalate

11160

558.13(converted from
7.5 mL/kg 5%
oxalic acid)

Sodium oxalate

11160

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Litchfield andWilcoxon method(1949)

Sprague-Dawley rats; 152202 g

maleoral; intragastric

50 to 220 mg F/kg (111 - 486
mg NaF/kg) in water

number of deaths determined at 1, 2, 4, 8, 24 hour after dose and each fasted 18 hour before dosing; 10 day
day thereafter; 32% rats dead during 1st day; 23 rats dead at 14 days; acclimatization before dosing; 10>99.5% purity
monitored for 2 weeks but no deaths after 4 days; deaths/dose (mg/kg): rats in each dosage group; 50 rats
160- 1/10, 207- 4/10, 254-5/10, 330-6/10, 428-7/10
used

NA

rat

NA

oral

NA

NA

372 - 819

moving average(Thompson & Weil)

Sprague-Dawley; 200-300 g female

oral gastricintubation

5% aqueous solution; doses
arranged in a logarithmic series
LD50 based on mortalities during a 14 day period (assumed fromdiffering by a factor of 2
Smyth et al. 1962)(assumed from Smyth et al.
1962)

706.96(converted from
9.5 mL/kg 5%
oxalic acid)402 - 915

moving average(Thompson & Weil)

Sprague-Dawley; 200-300 g maleoral gastricintubation

Whitford GM, Birdsong-Whitford NL, et al. 1990. Acute oral toxicity of sodium fluoride andmonofluorophosphate separately or in combination in rats. Caries Res 24(2):121-126.
Medical College of Georgia, Augusta, GA; Dept. of Odonto-Stomatologie, Laboratoires Goupil SA,
Cachan, France.

Value derived from 7500 mg'kg fromRTECS for oxalic acid, which is aNA
typo. Original reference (Vernot et al1977) has 7.5 ml/kg)

EHP, Environmental Health Perspectives. (U.S. Government Printing Office, Supt of Documents,
Washington, DC 20402) No.1-1971. 106(Suppl). (RTECS REFERENCE)

non-fasted; groups of 5 rats; single
oral dose toxicity (assumed from
Smyth et al 1962); reported as 7.5
ml/kg of 5% oxalic acid

NA

Vernot EH, MacEwen JD, Haun CC, Kinkead ER. 1977. Acute toxicity and skin corrosion data forsome organic and inorganic compounds and aqueous solutions. Toxicology and Applied
Pharmacology 42:417-423. (Indicates methods of Smyth et al. 1962 were used.)non-fasted; groups of 5 rats; single
oral dose toxicity (assumed from
Smyth et al 1962); reported as 9.5
ml/kg of 5% oxalic acid

NA

Vernot EH, MacEwen JD, Haun CC, Kinkead ER. 1977. Acute toxicity and skin corrosion data forsome organic and inorganic compounds and aqueous solutions. Toxicology and Applied
Pharmacology 42:417-423. (Indicates methods of Smyth et al. 1962 were used.)NA

reference in RussianNA

Novikov JV, Plitman SE, et al. 1984. Selenium in water and its effect on the human body. Gigiena i
Sanitariya 49(9):66-68. (RTECS REFERENCE)

violent gastroenteritis, diarrhea, rice water stools,garlic breath,nervousness, CNS depression; onset within 15 min.

information from the laboratories ofDivision of Pharmacology, U.S.
NA
FDA.; fasted animals

5% aqueous solution; doses
arranged in a logarithmic series
LD50 based on mortalities during a 14 day period (assumed fromdiffering by a factor of 2
Smyth et al. 1962)(assumed from Smyth et al.
1962)

NA

Primary Reference

Sodium selenate

1.6

1.6

NA

NA

rats

NA

oral

Sodium selenate

1.6

5.98

NA

NA

rats

NA

oral; stomach tube NA

Strychnine

2.35

2.35

NA

mortality curvesadult white rats

female2.25 - 15 mg/kg dose; single
15, 10, 7.5, 6, 5mg/kg dose killed 90 rats (100% mortality); 4 mg/kg,
oral, stomach tube; dose; cmpd mixed in gum
17/18 rats dead (95%); 3 mg/kg, 20/27 rats dead (74%); 2.5 mg/kg
single dose
acacia and water; 1 mg/mL dose 19/27 rats dead (70%); 2.25 mg/kg, 7/18 rats dead (39%); 7.3 - 14.1
solution
minutes average time to death

180 rats used

Ward JC, Crabtree DG. 1942. Strychnine X. Comparative accuracies of stomach tube and
U.S.P IX Strychnine intraperitoneal injection methods of bioassay. Journal of the American Pharmaceutical Association,
alkaloid
Scientific Edition 31:113-115. U.S. Fish and Wildlife Service, Denver, CO (RTECSREFERENCE)

Strychnine

2.35

6.5

NA

mortality curvesadult white ratsmale

15 mg/kg, 16/18 rats dead (89% mortality); 10 mg/kg, 15/18 rats dead5 - 15 mg/kg dose; single dose;
oral, stomach tube;(83%); 7.5 mg/kg, 16/18 rats dead (89%); 6 mg/kg 6/18 rats deadcmpd mixed in gum acacia andsingle dose(33%); 5 mg/kg, 4/18 rats dead (39%); 10.8 - 19.5 minutes averagewater; 1 mg/mL dose solution
time to death

90 rats used

Ward JC, CrabtreeDG. 1942. Strychnine X. Comparative accuracies of stomach tube and
U.S.P IX Strychnine
intraperitoneal injection methods of bioassay. Journal of the American Pharmaceutical Association,
alkaloid
Scientific Edition 31:113-115. U.S. Fish and Wildlife Service, Denver, CO

Strychnine

2.35

16.2

NA

NA

rats

NA

oral, stomach tube;
NAsingle dose

Strychnine

2.35

25

NA

statistical formulabased on mortality
rates

wild Norway ratsNA

a number of individual doses oforal, stomach tube; a cmpd, each dose at a different
convulsions
single doseconcentration level, are given toan equal number of test animals

Litchfield and Fetig(1941)

wild Norway rats (trapped inmale and
Baltimore, MD); 134-579 g
female(ave = 298 g), adult

Lehman AJ. 1951. Chemicals in Foods: a report to the association of food and drug officials on
current developments. Part II. Pesticides. Quarterly Bulletine (Association of Food and Drug Officialsof the United States). Vol.15:122-133. U.S. FDA

NALehman AJ. 1951. Chemicals in Foods: a report to the association of food and drug officials on
current developments. Part II. Pesticides. Quarterly Bulletine (Association of Food and Drug Officialsof the United States). Vol.15:122-133. U.S. FDA

NA

NAPeardon DL, Kilbourn E, et al. 1972. New selective rodenticides. Soap Cosmet Chem Spec 48(12):6.
Rohm and Haas Company, Philadelphia, PA

GIBCO brand;
99.0% pure

Dieke SH, Richter CP. 1946. Comparative assays of rodenticides on wild Norway rats. I. Toxicity.
Publ Health Rep 61:672-679. Johns Hopkins Hospital, Baltimore, MD

tonic convulsions; deaths from medullary paralysis and exhaustion andNAusually occur within a 12 hour periodoral gavage

chemical suspended in 10%
acacia solution; received
appropriate doses in 1 mL per
100 g bw

rats survived from 6 - 72 hours

37 rats used (approx. equal numberof male/female); overnight fasting
before dosing; assays performed in
winter, repeated in summer; LD50
values from combined information;
final LD50 was higher than winter
LD50; attributed to not having
enough rats in winter.

NA

NA

reference is a review article inJapanese; this LD50 value assumed
to be from Peardon et al. 1972.

NA

Gekkan Yakuji. Pharmaceuticals Monthly. (Yakugyo Jihosha, Inaoka Bldg., 2-36 Jinbo-cho, Kanda,
Chiyoda-ku, Tokyo 101, Japan) V.1- 1959. 1980. (RTECS REFERENCE)

Thallium I sulfate

16

15.8

+/- 0.9(S.E.)

Thallium I sulfate

16

16

NA

NA

rats

NA

oral

Thallium I sulfate

16

16

NA

statistical formulabased on mortality
rates

wild Norway ratsNA

a number of individual doses oforal, stomach tube; a cmpd; each dose at a different
respiratory failure
single doseconc level given to equal
number of test animals

NA

NA

Peardon DL, Kilbourn E, et al. 1972. New selective rodenticides. Soap Cosmet Chem Spec 48(12):6.
Rohm and Haas Company, Philadelphia, PA

Thallium I sulfate

16

25

NA

NA

rats

NA

oral, stomach tube;
NAsingle dose

72 hour observation; most rats dead within this period

fasted animals

NA

Lehman AJ. 1951. Chemicals in Foods: a report to the association of food and drug officials on
current developments. Part II. Pesticides. Quarterly Bulletine (Association of Food and Drug Officialsof the United States). Vol.15:122-133. U.S. FDA

Trichloroacetic acid

NA

400

NA

NA

rats

NA

oral

NA

(source of information not provided) NA

Worthing CR, Walker SB, eds. 1987. Pesticide Manual. 8th edition. 765-766.

Trichloroacetic acid

NA

3320

fasted 18 hours before dosing; 65 rats
NAused; 43 of 65 deadWoodard G, Lange SW, Nelson KW, Calvery HO. 1941. The acute oral toxicity of acetic,
chloroacetic, dichloroacetic, and trichloroacetic acids. J Ind Hyg Toxicol 23(2):78-82.

3160 - 3480
(95% certainty; slope Bliss= 20.97)

rats (raised in themale andlaboratory); 150- 250 g; 70- female
oral intubation
100 days(mostly male)NAobserved for 6 days; passed into narcosis to seminarcosis and died or
single dose; acid adjusted withrecovered within 36 hours; dose in g/kg versus mortality: 2.594-0/5;
sodium hydroxide to pH range
3.000-3/10; 3.153 - 1/5; 3.400-5/10; 3.800-9/10; 3991-5/5; 4.200of 6 -7; 10 mL/kg dose volume
10/10; 4.600-10/10

H-36

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

Trichloroacetic acid

NA

5000

rats

male

oral

NA

NA

NA

NA

Farm Chemicals Handbook. 1992. Meister Pub., 37841 Euclid Ave., Willoughy, OH. p. C326.

Trichloroacetic acid

NA

5060

rats

female

oral

NA

NA

NA

NA

Farm Chemicals Handbook. 1992. Meister Pub., 37841 Euclid Ave., Willoughy, OH. p. C326.

Trichloroacetic acid

NA

8900

7000 - 9900

NArats; 220 +/- 40 gNAoral; intragastricNANA(source of information not provided) NA

Izmerov NF, Sanotsky IV, Sidorov KK. 1982. Toxicometric Parameters of Industrial Toxic Chemicals
under Single Exposure. International Register of Potentially Toxic Chemicals (IRPTC). United
Nations Environment Programme (UNEP). Centre of International Projects, GKNT. Moscow, Russia.

Triethylenemelamine

13

1

NA

NA

rats

NA

oral

NA

NA

Reference offers neither experimental
details nor the primary reference for NA
LD50. Value reported as "ca. 1"

Hayes WJ Jr. 1964. The toxicology of chemosterilants. Bulletin of the World Health Organization.
31:721-736. (RC's reference from 1983/84 RTECS.)

Triethylenemelamine

13

4

NA

Probit method

Sprague-Dawley rats; 190200 g

femaleoral

geometric progression of 14 for
20 rats used; 11 dead; observed for 14 days
dosing; in water or neat

non-fasted; 4 groups of 5 female; 20 Polysciences, Inc.
rats used
Warrington, PA

Thompson ED, Gibson DP. 1984. A method for determining the maximum tolerated dose for acute invivo cytogenetic studies. Food Chem Toxicol 22(8):665-76.
The Procter and Gamble Co., Cincinnati, OH

Triethylenemelamine

13

6.9

NA

Probit method

Sprague-Dawley rats; 190200 g

male

oral

geometric progression of 14 for
20 rats used; 9 dead; observed for 14 days
dosing; in water or neat

non-fasted; 4 groups of 5 male; 20
rats used

Thompson ED, Gibson DP. 1984. A method for determining the maximum tolerated dose for acute invivo cytogenetic studies. Food Chem Toxicol 22(8):665-76.
The Procter and Gamble Co., Cincinnati, OH

Triethylenemelamine

13

13

8 - 20(95% CL; slope =2.1)

Litchfield andWilcoxin (1949)

Wistar rats; 150- 350 g

male andfemaledissolved in isotonic saline
within 30 minutes of dosing;oral; stomach tube less than 5% weight ofinsoluble matter filtered out;
highest dose 500 mg/kg

Triphenyltin hydroxide

46

46.4

NA

NA

Fischer rats; 6 weeks

male andfemaleoral intubation

single dose followed by daily
doses up to 14 days

Triphenyltin hydroxide

46

156

115 - 208
(CL)

NA

rats

femaleoral

single dose; 80, 160, 315, or
630 mg/kg doses

Polysciences, Inc.
Warrington, PA

14 observation period; absence of acute toxicity signs

information not grouped according tosex since differences not evident; 6
NArats per dose; animals fasted
overnight

Philips FS, Thiersch JB. 1950. The nitrogen mustard-like actions of 2,4,6-tris(ethylenimino)-s-triazine
and other bis(ethylenimines). Journal of Pharmacology and Experimental Therapeutics 100:398-407.
Sloan Kettering Institute for Cancer Research, New York, NY (RTECS REFERENCE)

observed up to 14 days

NA

Carcinogenesis bioassay of environmental chemicals annual progress report NIH-NCI-E-C-72-3252.
5/13/71 -- 8/6/73 and Final report NIH-NCI-E-71-2146. Submitted to The National Cancer Institute,
National Institutes of Health, Bethesda, MD. 8/15/73 (revised 8/10/73). FM Garner (princ. investigat.)
Litton Bionetics, Inc., Bethesda, MD. (RTECS REFERENCE)

NA

observed for 19 days; toxicity develops slowly; toxic signs 2 days after
dose; deaths 5 - 9 days after initial dose; dose (mg/kg), number dead: fasted animals; 4 groups of 10 female
triphenyltin
80 - 1/10; 160 - 4/10; 315 - 10/10; 630 - 10/10; toxic signs included
rats each; each received one dose; 35hydroxide 96%
squatting, ataxy, bristled hair, blood-crusted adherent margins of the of 40 diedeyelid, decreased respiratory rate and poor general condition

triphenyltinhydroxide 80.0%

Pharma Forschung Toxikologie; Report 183/81; A 21593; Apr. 22, 1981; U.S. EPA, Office ofPesticide Programs; Health Effects Division; Tox Oneliners; MRID No. 00124210 and 00139030;
Hoechst Aktiengesellschaft; EPA Acc. No. 071364; EPA Chem. Code: 083601; Core Grade/Tox
Record No. minimum 005275

Products Safety Labs; T-1399; May 8, 1992; U.S. EPA, Office of Pesticide Programs; Health Effects
Division; Tox Oneliners; MRID No. 42265507; EPA Chem. Code: 083601; Core Grade/Tox Record
No. Guideline 009941, Jan. 5, 1993;

Triphenyltin hydroxide

46

160

NA

NA

rats

NA

oral

NA

NA

NA

Triphenyltin hydroxide

46

165

113 - 230
(CL)

NA

rats

male

oral

single dose; 80, 160, 315, or
630 mg/kg doses

observed for 19 days; toxic signs 2 days after dose; toxicity develops
slowly; deaths 5 - 13 days after initial dose; dose (mg/kg), number
dead: 160 - 6/10; 315 - 10/10; 630 - 9/10; toxic signs included
squatting, ataxy, bristled hair, blood-crusted adherent margins of theeyelid, decreased respiratory rate, dischourage of mucous feces, andpoor general condition

fasted animals; 4 groups of 10 male
triphenyltin
rats each; each received one dose; 25hydroxide 96%
of 40 diedPharma Forschung Toxikologie; Report 182/81; A 21353; Apr. 22, 1981; U.S. EPA, Office ofPesticide Programs; Health Effects Division; Tox Oneliners; MRID No. 00124209; Hoechst
Aktiengesellschaft; EPA Acc. No. 071364; EPA Chem. Code: 083601; Core Grade/Tox Record No.
minimum 005275, minimum 003116

Triphenyltin hydroxide

46

240

NA

NA

rats

male

oral

NA

NA

NA

triphenyltinhydroxide tech

U.S. EPA, Office of Pesticide Programs; Health Effects Division; Tox Oneliners; EPA Chem. Code:083601; Core Grade/Tox Record No. 001493

Triphenyltin hydroxide

46

313

232 - 422

NA

rats

male

oral

NA

NA

NA

triphenyltinhydroxide tech

Cannon Laboratories, Inc.; Jan. 31, 1978; U.S. EPA, Office of Pesticide Programs; Health Effects
Division; Tox Oneliners; MRID No. 099049; EPA Chem. Code: 083601; Core Grade/Tox Record No.
minimum 001492

Triphenyltin hydroxide

46

345

138 - 862

NA

rats

female

oral

NA

NA

NA

triphenyltinhydroxide tech

Cannon Laboratories, Inc.; Jan. 31, 1978; U.S. EPA, Office of Pesticide Programs; Health Effects
Division; Tox Oneliners; MRID No. 099049; EPA Chem. Code: 083601; Core Grade/Tox Record No.
minimum 001492

Triphenyltin hydroxide

46

360

NA

NA

rats

female

oral

NA

NA

NA

triphenyltinhydroxide tech

U.S. EPA, Office of Pesticide Programs; Health Effects Division; Tox Oneliners; EPA Chem. Code:083601; Core Grade/Tox Record No. 001493

Triphenyltin hydroxide

46

375

280 - 502

NA

rats

male

oral

NA

NA

NA

Duter WP (TPTH
47%)

Cannon Laboratories, Inc.; Feb. 23, 1978; U.S. EPA, Office of Pesticide Programs; Health Effects
Division; Tox Oneliners; MRID No. 099049; EPA Chem. Code: 083601; Core Grade/Tox Record No.
minimum 001492

Triphenyltin hydroxide

46

375

NA

rats

male andfemale

oral

NA

NA

NA

50% WP (Reg. No.
148-1195

U.S. EPA, Office of Pesticide Programs; Health Effects Division; Tox Oneliners; MRID No. 099049;
EPA Chem. Code: 083601; Core Grade/Tox Record No. minimum

Triphenyltin hydroxide

46

380

NA

rats

female

oral

NA

NA

NA

Duter WP (TPTH
47%)

Cannon Laboratories, Inc.; Feb. 23, 1978; U.S. EPA, Office of Pesticide Programs; Health Effects
Division; Tox Oneliners; MRID No. 099049; EPA Chem. Code: 083601; Core Grade/Tox Record No.
minimum 001492

288 - 502

H-37

In Vitro Acute Toxicity Test Methods BRD Appendix H1

November 2006
Rat and Mouse Oral LD50 Database

Rat OralReference Substance

LD501
mg/kg

Rat Oral LD502
LD50 Calculation
LD503
mg/kg
mg/kg (range)
Method4
Primary
Primary Reference Primary Reference
Reference

Animal Information(stock, weight, age)

GenderRoute and/or
Method ofExposure

Dose

Observations

Notes

Reference
Substance Source

Primary Reference

Triphenyltin hydroxide

46

720

520 - 920

NA

rats

female

oral

NA

NA

NA

Kansai Robamame
soin B A/F 1000B(Red Point)

Bio/dynamics, Inc.; 6584-81; Sept. 30, 1981; U.S. EPA, Office of Pesticide Programs; Health Effects
Division; Tox Oneliners; MRID No. 00086072; EPA Chem. Code: 083601; Core Grade/Tox Record
No. Guideline 001881

Triphenyltin hydroxide

46

830

580 - 1080

NA

rats

male andfemale

oral

NA

NA

NA

Kansai Robamame
soin B A/F 1000B(Red Point)

Bio/dynamics, Inc.; 6584-81; Sept. 30, 1981; U.S. EPA, Office of Pesticide Programs; Health Effects
Division; Tox Oneliners; MRID No. 00086072; EPA Chem. Code: 083601; Core Grade/Tox Record
No. Guideline 001881

Triphenyltin hydroxide

46

840

512 - 1378

NA

rats

unknown

oral

NA

NA

NA

Duter Flowable 30(TPTH 19.7%)

Cannon Laboratories, Inc.; 9E-6359; Nov. 13, 1979; U.S. EPA, Office of Pesticide Programs; Health
Effects Division; Tox Oneliners; MRID No. 00086591; EPA Chem. Code: 083601; Core Grade/Tox
Record No. minimum 001496

Triphenyltin hydroxide

46

1200

600 - 1800

NA

rats

male

oral

NA

NA

NA

Kansai Robamamesoln B A/F 1000B(Red Point)

Bio/dynamics, Inc.; 6584-81; Sept. 30, 1981; U.S. EPA, Office of Pesticide Programs; Health Effects
Division; Tox Oneliners; MRID No. 00086072; EPA Chem. Code: 083601; Core Grade/Tox Record
No. Guideline 001881

Valproic acid

670

670

oral intubation

2% in water

usual observaton time of 2 weeks; depression, scrawny appearance,
diarrhea; dead within 2 hour - 2 days

18 hours fasting; groups of 10 rats;
evenly divided between male andfemalecommerciallyavailable material

Jenner PM, Hagan EC, Taylor JM, Cook EL, Fitzhugh OG. 1964. Food flavorings and compounds ofrelated structure I. Acute Oral Toxicity. Fd Cosmet Toxicol 2:327-334.
U.S. Food and Drug Administration, Washington, D.C. (RTECS REFERENCE)

Valproic acid

670

1480

NA

NA

rats

male andfemale

oral

NA

NA

reference in French

NA

Deboeck AM. Valproic acid salt, its preparation and utilization. European Patent Office, Publication
No. EP 0078785A1. Application date 11/03/82.

Verapamil HCl

108

108

NA

NA

rats

NA

oral

NA

NA

NA

NA

Drugs in Japan (Ethical Drugs). (Yakugyo Jiho Co., Ltd., Tokyo, Japan). 1982.
(RTECS REFERENCE)

Verapamil HCl

108

114

97 - 135

Litchfield andWilcoxin (1949)

rats

NA

oral

NA

NA

reference in German

NA

Haas VH, Hartfelder G. 1962. A-Isopropyl-a-[(N-methyl-N-homoveratryl-g-amino-propyl]-3,4dimethoxyphenylacetonitrile, eine Substanz mit coronargefaferweiternden Eigenschaften 12:549-558.

Xylene4300

1537

1294 - 1781
(95% CL; slope =
9.6)

oral; intragastricintubation

single dose in aqueous solution(25%); doses = 1200, 1600,
2000 mg/kg; dose = 1.2 - 2.0
mL

16 dead; observed over 14-day recovery period; 1200 dose: lacrimation
and wet perineal area (1/10 dead); 1600 dose: tremors, salivation,
prostration, piloerection, lacrimation, wet perineal area, ataxia (7/10
3 groups of 10 rats each; date of testNAdead; death within 15 hours after dosing); 2000 dose: tremors, severe is 1979
fascicutations, ataxia, lacrimation, prostration, piloerection, lethargy,
wet and stained perineal area, weakness (8/10 dead)Xylene

4300

4300

NA

Xylene4300

8314

7716 - 8803
(95% CL)

Xylene4300

598 - 750
Litchfield and(95% CL; slope = 1.2 Wilcoxon method[1.0 - 1.4; 95% CL]) (1949)

Osborne-Mendel rats; young male andadult
femaleChR-CD; ave bw for eachFinney (1971) Probit
group = 253, 251, and 256 g; male
Analysisyoung adults

NAwhite rats; Wistar; 175- 250
maleg

ChR-CD; ave bw eachFinney (1971) Probit
group = 276, 258, 286, 262, male
Analysis256 g; young adults

8620
6465 - 11465
Litchfield and(10 mL/kg;(CL; reported as 7.5 - Wilcoxon method
density = 0.862)
13.3 mL/kg)(1949)

Long-Evans rats; 150-300 g malesingle dose in either olive oil orcorn oil solution emulsified
oral; stomach tube with aqueous solution of acacia; all surviving rats observed up to 2 weeks; 20 rats usedor undiluted; no more than 7 cc
administered
16 dead; observed over 14-day recovery period; 7500 dose: (3/10
dead); 8000 dose: (3/10 dead); 9000 dose: (6/10 dead); 9500 dose
(10/10 dead); salivation, lethargy, ruffled fur, diarrhea, respiratory
congestion, wet/bloody perineal areas

percent of isomers: o = 19; p = 24;NAm = 52

Wolfe MA, Rowe VK, McCollister DD, Hollingsworth RL, Oyen F. 1956. Toxicological studies ofcertain alkylated benzenes and benzene: experiments on laboratory animals. AMA Archives ofIndustrial Health. 14:387-397. The Dow Chemical Co. Midland, MI. (RTECS REFERENCE)

4 groups of 10 rats each; date of testNA
is 1975

from EPA TSCATS database; Oral LD50 test (1975), EPA Document No. 878221390 Fiche No.
OTS0215213; E.I Dupont DeNemours & Co., Inc./Haskell Labs

oral; intragastricintubation

single dose in corn oil (50%solution); doses = 7500, 8000,
9000, and 9500 mg/kg; dose =3.93-5.25 mL

oral; intragastricintubation

single dose; graded doses up to observed for 14 days; mortality values based on the number of animals ortho, meta, and para xylene; ethyl
25 mL/kg; undiluted samples
which died during this time; 6 rats per dose
benzenearomatic
concentrated fromcommercial sourceby an absorption
technique; 98%
aromatic.

Abbreviations: NA=Not available; CL=Confidence limit; CI=Confidence interval; SE=Standard error; UDP=Up-and-Down Procedure; TSCATS=Toxic Substances Control Act Test Submissions; RTECS=Registry of Toxic Effects of Chemical Substances; min=Minimum; HSDB=Hazardous Substances Data Bank (NLM 2001).
Gray cells highlight the rationale for exclusion of reference value.
1

RTECS® database value at the time of database search by NICEATM (2002). If rat oral LD50 was unavailable, rat oral LD50 from HSDB was used, or mouse oral LD50 from RTECS was used.
Value reported in the reference publication
Range (if provided in the reference publication)
4
Method reported from the reference publication
2
3

H-38

from EPA TSCATS database; Oral LD50 test (1979), EPA Document No. 878221390 Fiche No.
OTS0215213; E.I Dupont DeNemours & Co., Inc./Haskell Labs

Hine CH, Zuidema HH. 1970. The toxicological properties of hydrocarbon solvents. Industrial
Medicine. 39(5):39-44.

In Vitro Cytotoxicity Test Methods BRD Appendix H2

November 2006

Appendix H2

Evaluation of the Candidate Reference Oral LD50 Data

H-39

In Vitro Cytotoxicity Test Methods BRD Appendix H2[This Page Intentionally Left Blank]

H-40

November 2006

In Vitro Cytotoxicity Test Methods BRD Appendix H2

H.2

November 2006

Evaluation of the Candidate Reference Oral LD50 Data

The 491 LD50 values identified by the literature search consisted of 485 rat oral LD50 values
and six mouse oral LD50 values. Mouse oral LD50 values were used to determine reference
values for colchicine, epinephrine bitartrate, and propylparaben since rat oral LD50 values forthese three reference substances could not be located. Thirty rat oral LD50 values were
believed to be duplicates of other reported values because the LD50 values and theexperimental information matched exactly those cited by other publications from the sameauthor(s) or because the same animal data were used to calculate multiple LD50 values (e.g.,
to evaluate various methods of calculation).
Two rat oral LD50 values provided by RTECS® were incorrect, possibly due to typographicalerrors. For the value of 200 mg/kg for acetylsalicylic acid, RTECS® cited a review by
Diechmann (1969) that referred to a paper by Coldwell and Boyd (1966). Coldwell and Boyd(1966), however, actually reported an LD50 of 920 mg/kg. For sodium oxalate, RTECS® citeda review paper by Walum (1998) for an LD50 value of 11160 mg/kg. Although Walum(1998) provided no source, the LD50 is the same as that used in the MEIC study (Ekwall et al.
1998b). That LD50 was calculated from the LD50 for oxalic acid (Ekwall et al. 1998b) which
is 7500 mg/kg according to RTECS®. The source for this figure, however, provides a valueof 7.5 mL/kg of 5% oxalic acid (Vernot et al. 1977). Extrapolating this to sodium oxalate(MW=134.0 g/mole vs 90.04 g/mole for oxalic acid) yields an LD50 of 558 mg/kg.
After exclusion of the 30 duplicate values and the two erroneous values for acetylsalicylic
acid and sodium oxalate, 459 records remained for further evaluation. Figure H2-1 shows
the frequency of the number of LD50 values retrieved for the 72 reference substances. The
number of LD50 values identified for any one reference substance ranged from one to 29. The
highest frequency was two LD50 values per reference substance (14 reference substances).
The highest number of LD50 values retrieved for an individual reference substance(acetonitrile) was 29. A large number of LD50 values were also identified forhexachlorophene (21), ethylene glycol (19), and carbon tetrachloride (19). Only one LD50
value was identified for seven reference substances: aminopterin, digoxin, epinephrine
bitartrate, glutethimide, physostigmine, and propranolol HCl.

H-41

In Vitro Cytotoxicity Test Methods BRD Appendix H2

November 2006

Figure H2-1 Distribution of the Number of LD50 Values Per Reference substance
16
14
12
10
8
6
4
2
0
1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 19 21 29

Number of LD50 Values
Bars show number of reference substances with the noted number of LD50 values for the 459 oral LD50 values
remaining after the exclusion of 30 duplicate values and two erroneous values.

H.2.1
Protocols Used for the Candidate Reference Data
The LD50 data were collected using various protocols; however, information on the protocol
details was often incomplete due to limited documentation in the reports. The 459 remaining
data records exhibited the following characteristics:• 64% (293/459) specified the stock or strain of rodent used. The remaining
36% (167/459) that did not specify the stock/strain described rats as rats,
albino rats, white rats, rats of different strains, and mice were described asmice.
• 63% (290/459) included age or weight information for the rodents.
• 77% (354/459) specified the gender of the rodent.
• 66% (305/459) stated the method used to calculate the LD50.
• 48% (221/459) reported the number of rodents used at each dose and 47%(216/459) reported the total number of rodents used.
• 26% (118/459) specified the doses used.
• 14% (66/459) quantitatively specified the purity of the reference substanceused. Of the remaining records, 18% (83/459) described the purity
qualitatively using such terms as “technical grade,” “pure,” “reagent grade,”and “pharmaceutical grade,” 11% (51/459) named only the source of thereference substance, and 56% (259/459) provided no information on thereference substance.
• 13% (61/459) reported the deaths at each dose.

H-42

In Vitro Cytotoxicity Test Methods BRD Appendix H2

November 2006

Although many LD50 studies did not specify the strain or stock of rat used, the 293 studies
that provided this information indicated that Sprague-Dawley/CD rats were the strain mostfrequently used (see Figure H2-2). Wistar rats were also frequently used. Strains such as
Alderly Park, SD-JCL, THOM, Gunn, and HLA were the least frequently used. Of the six
mouse LD50 values, the strain was unspecified for two studies. The other four LD50 values
were obtained using CD-1, MS/Ae, dd, and B6D1F1(BDF1) mice.
Of the 354 studies that reported rodent gender, the most frequently used gender for bothrodents was male, which was used for 193 (55%) LD50 values. Female rodents were used for
104 (29%) LD50 values, both sexes were used for 55 (16%) LD50 values, and rodents ofunspecified gender were used for 104 (29%) LD50 values.
Figure H2-2 Distribution of Rat Stocks/Strains
Unspecified
Sprague-Dawley/CD
WistarShermanFischer
FDRL
Osborne-Mendel
Wild Norway
MJ
CFY
PortonHindustan Antibiotics
Long Evans
Alderly Park SPF
SD-JCL
THOM
Gunn
HLA

0

25

50

75

100

125

150

175

Number of Rat LD50
Bars show number of rat oral LD50 records for each rat strain for the 453 rat values remaining after the
exclusion of 30 duplicate values, two erroneous values, and six mouse values.

H-43

In Vitro Cytotoxicity Test Methods BRD Appendix H2

November 2006

The age of the rodents used for the acute oral lethality studies also varied. Of the 174 LD50
studies that reported age, the most frequently used age was 4-7 weeks, which was reported
for 42 (24%) LD50 values (see Figure H2-3). The majority of the reported ages were
descriptive. Forty-five (26%) LD50 values used rodents that were described as young, adults,
young adults, or older adults. Thirty (17%) LD50 studies used 8-12 week old rodents, whichis the age recommended by current oral acute toxicity test guidelines (OECD 2001a, c, d;
EPA 2002a). Twenty-three (13%) LD50 values were determined using rodents less than fourweeks of age, and 34 (20%) LD50 values were determined using rodents greater than 12
weeks old.
Figure H2-3 Distribution of Rat and Mouse Ages

50
45
40
35
30
25
20
15
10
5
0
< 4 weeks

4-7 weeks8-12 weeks> 12 weeks

Descriptive

Age
Bars show the number of rat and mouse LD50 records that report using animals of the specified ages.
Descriptive indicates that age was described qualitatively (e.g., adult, juvenile).

The duration of animal observation was not specified for 39% (179/459) of the LD50 reports.
Of the 280 (61%) studies that reported the duration of observation, 136 (48%) reported an
observation period of 14 days, which is recommended in the current oral acute toxicity test
guidelines (OECD 2001a, c, d; EPA 2002a). The second most commonly used observation
period was seven days, which was reported by 59 (21%) studies. Clinical signs were reported
in 30% (137/459) of the studies.
Of the 305 studies that reported the method used to calculate the LD50 value, the mostfrequently used were the graphical log-probit methods such as Litchfield and Wilcoxon(1949), with 99 (33%) LD50 values, and Miller and Tainter (1944), with 24 (8%) LD50
H-44

In Vitro Cytotoxicity Test Methods BRD Appendix H2

November 2006

values. The maximum likelihood probit method of Bliss (1938) and modifications were used
for the calculation of 46 (15%) LD50 values. An additional 36 (12%) LD50 values were
calculated using methods referred to in a general way as probit or log probit methods. The
moving average method, such as that of Thompson (1947) or Weil (1952), was cited for 57(19%) LD50 values. Thirteen (4%) LD50 values were described as being calculated by one
method or another (e.g., by Weil or Litchfield and Wilcoxon), or by methods that were
described generally, such as graphical or approximative. Some of the least frequently usedmethods were linear regression (six values), UDP (four values), and linear interpolation (onevalue). Estimates of variability such as confidence limits, standard error, or standard
deviation were included in 62% (283/459) of the LD50 reports, but only 6% (28/459)
included slopes.
H.2.2
Final Reference Values
Based on the study exclusion criteria described in Section 4.1.2, 73 (16%) of the 459 records
identified were excluded. Thirty-one LD50values were excluded because they were reported
as ranges, 21 were excluded because the rats were less than four weeks old, five were
excluded because the rats were feral, five were excluded because the rats were anesthetized,
and four were excluded because the reference substance administered was mixed with food.
Additionally, four LD50 values for copper sulfate pentahydrate were excluded because very
low purity (i.e., ≤20%) reference substance was used. Three LD50 values were excluded
because they were outliers at the 99% level (Dixon and Massey 1981) compared with the restof the values for the particular reference substance. These included one ethylene glycol valueof 17,800 mg/kg (range of the other 16 values=4000-9900 mg/kg), one meprobamate valueof 794 mg/kg (range of other six values=1286-1522 mg/kg), and one mercury chloride valueof 160 mg/kg (range of other 10 values=12-92 mg/kg). Appendix H-1 provides theindividual rationale for each LD50 value excluded by shading the cell that contains the reason
for exclusion.
Triethylenemelamine, trichloroacetic acid, and xylene had the largest confidence limits inproportion to the geometric means. The confidence limits for triethylenemelamine and xylene
were calculated from four LD50 values while those for trichloroacetic acid were calculatedwith five LD50 values. Nicotine and 2-propanol had the smallest confidence limits eventhough the number of values per reference substance were similar to that for the reference
substances with large confidence limits (nicotine N=4, 2-propanol N=6).

H-45

In Vitro Cytotoxicity Test Methods BRD Appendix H2[This Page Intentionally Left Blank]

H-46

November 2006

In Vitro Cytotoxicity Test Methods BRD Appendix I

November 2006

Appendix I

In Vitro NRU Data
I1

3T3 NRU Reference Substance Data............................................ I-3

I2

NHK NRU Reference Substance Data ........................................I-59

I3

3T3 NRU Positive Control (SLS) Data......................................I-111

I4

NHK NRU Positive Control (SLS) Data ...................................I-129

I-1

In Vitro Cytotoxicity Test Methods BRD Appendix I

November 2006

[This Page Intentionally Left Blank]

I-2

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

Appendix I1

3T3 NRU Reference Substance Data

I-3

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

[This Page Intentionally Left Blank]

I-4

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC ID

ACETAMINOPHEN
IIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 66.7, 44.4, 29.6,
19.8, 13.2, 8.78, 5.85
100, 66.7, 44.4, 29.6,
19.8, 13.2, 8.78, 5.85
100, 66.7, 44.4, 29.6,
19.8, 13.2, 8.78, 5.85

A1

RF

AA61HU

30.8

0.203

0.266

0.88%

2

6

0.9628B1DF

AA61HU

32.1

0.212

0.457

0.71%

3

5

0.9728B2DFAA61HU

54.8

0.363

0.402

4.77%

2

5

0.9221B3DF

AA61HU

43.3

0.286

0.356

1.85%

3

5

0.9794

AA61LR-A1

RF

AA61LR

66.8

0.442

0.253

4.38%

2

0

0.9619AA61LR-B1

DF

AA61LR

30.3

0.200

0.449

13.54%

5

3

0.9875

AA61LR-B2

DF

AA61LR

46.1

0.305

0.298

3.30%

4

4

0.9557

AA61LR-B3

DF

AA61LR

46.1

0.305

0.407

3.13%

4

4

0.9855

FAL.3T3.PY.A1.21.10.04

RF

AA61PY

62.1

0.411

0.212

1.41%

2

6

0.9541

FAL.3T3.PY.B1.26.11.04

DF

AA61PY

92.3

0.610

0.290

3.71%

4

2

0.9374

FAL.3T3.PY.B2.02.12.04

DF

AA61PY

57.1

0.378

0.194

4.85%

6

2

0.9518FAL.3T3.PY.B3.09.12.04

DF

AA61PY

49.1

0.325

0.416

1.16%

6

2

0.9672

RF

AA61GF

NA

NA

0.393

2.29%

0.0

7

0.031910000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

1.5

YES

range finder

SLS-A5SLS-B5

1.5

YES

SLS-B91.5

YES

SLS-B10ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
200, 136, 92.6, 63.0,
42.8, 29.1, 19.8, 13.5
200, 136, 92.6, 63.0,
42.8, 29.1, 19.8, 13.5
200, 136, 92.6, 63.0,
42.8, 29.1, 19.8, 13.5

10

RF

1.47

YES

range finder

SLS-14SLS-P38

1.47

YES

SLS-P39

1.47

YES

SLS-P42

FRAME
1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
500, 340, 231, 157, 107,
72.8, 49.6, 33.7
500, 340, 231, 157, 107,
72.8, 49.6, 33.7

10

RF

2.15

YES

range finder

FAL.3T3.SLS.21.10.04
FAL.3T3.SLS.26.11.04

1.47

YES

FAL.3T3.SLS.03.12.04

1.47

YES

FAL.3T3.SLS.09.12.04

10RF

range finder

SLS-A1

1.4

NO

VC1 ODs lower than VC2values; volatility issues.
% VC difference >15
VC1 removed from
subsequent analysis.

SLS-B6

1.4

YES

OD measured 15-16 hr late;orignial reading used wrong
OD wavelength; plate sealer SLS-B11
used; outliers removed by
SD; ppt in 1X C1-C4

1.2

YES

plate sealer used

SLS-B12

1.2

YES

plate sealer used; outliers
removed by SD

SLS-B13NANO

PC failed

10RFrange finder

10

RFrange finder

SLS-P3

1.47

NO%VC difference > 15

SLS-P38ACETONITRILE
IIVS
A1

B1

DF

AA61GF

18100

441.250.305

45.66%

2

3

0.8837

100000, 71429, 51020,
36443, 26031, 18593,
13281, 9486

B2

DF

AA61GF

10500

256.854

0.426

0.14%

4

1

0.9638

100000, 71429, 51020,
36443, 26031, 18593,
13281, 9486

B3

DF

AA61GF

8070

196.647

0.330

3.56%

6

2

0.9540B4DF

AA61GF

9420

229.449

0.336

0.05%

4

4

0.8516B5DF

AA61GF

NA

NA

NA

NA

NA

NA

NA

AA61PH-A1

RF

AA61PH

NA

NA

0.309

4.26%

0

0

NA

AA61PH-A2

RF

AA61PH

NA

NA

0.308

36.98%

2

3

NA

AA61PH-B1(sealer)DF

AA61PH

NA

NA

0.372

19.13%

52

NA

20000, 16667,13889,
11574, 9645, 8038, 6698,
5582
20000, 16667,13889,
11574, 9645, 8038, 6698,
5582
NA

ECBC

I-5

10000, 1000, 100,10, 1,
0.1, 0.01, 0.001
200000, 20000, 2000,
200, 20, 2, 0.2, 0.02
50000, 34014, 23139,
15740, 10708, 7284,
4955, 3371

SLS-B14
no toxicity detected

SLS-P1In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

AA61PH-B2 (sealer)DF

AA61PH

NA

NA

0.257

29.42%

6

1

NA

AA61PH-B3 (sealer)DF

AA61PH

6340

154.414

0.448

7.35%

6

2

0.9770AA61PH-B4 (sealer)DF

AA61PH

6580

160.2090.445

14.54%

4

2

0.9796

AA61PH-B5 (sealer)DF

AA61PH

6380

155.484

0.453

4.90%

5

3

0.9823

FAL3T3.PL.A1.22-01-04

RF

AA61PL

NA

NA

0.439

1.52%

0

3

0.0000FAL3T3.PL.B1.29-01-04

DF

AA61PL

56800

1382.569

0.404

17.45%

1

4

0.8826

FAL3T3.PL.B2.05-02-04

DF

AA61PL6920

168.5340.230

62.44%

2

2

0.9721

Exposure
Concentrations(ug/mL)7

50000, 34014, 23139,
15740, 10708, 7284,
4955, 3371
50000, 34014, 23139,
15740, 10708, 7284,
4955, 3371
40000, 27211, 18511,
12592, 8566, 5827, 3964,
2697
40000, 27211, 18511,
12592, 8566, 5827, 3964,
2697

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

1.47

NO

%VC difference > 15

1.47

YES

SLS-P41

1.47

YES

SLS-P43

1.47

YES

SLS-P45

Notes

PC ID

SLS-P39FRAME

FAL3T3.PL.B4.25-02-04

DF

AA61PL

NA

NA

0.331

71.55%

3

1

NA

FAL3T3.PL.B5.29-04-04

DF

AA61 PL

15200

371.267

0.327

2.12%

2

4

0.8985

FAL3T3.PL.B6.06-05-04

DF

AA61 PL9930

241.928

0.334

5.53%

3

5

0.9631FAL3T3.PL.B7.20/05/04

DF

AA61 PL

6490

158.0110.344

19.62%

2

4

0.8881FAL3T3.PL.B8.27/05/04

DF

AA61 PL

3940

95.871

0.354

7.19%

3

3

0.9226

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100000, 46512, 21633,
10062, 4680, 2177, 1012,
471
100000, 46512, 21633,
10062, 4680, 2177, 1012,
471
100000, 46512, 21633,
10062, 4680, 2177, 1012,
471
25000, 11628, 5408,
2516, 1170, 544, 253, 118
30000, 20408, 13883,
9444, 6425, 4371, 2973,
2023
30000, 13953, 6490,
3019, 1404, 653, 304,
141
30000, 13953, 6490,
3019, 1404, 653, 304,
141

10

RF

range finder

2.15

NO

%VC difference >15

volatility problem; C1
alkaline

FAL.3T3.SLS.22/01/04
FAL3T3.SLS.29-01-04

2.15

NO

PC failed; % VC
difference > 15

problem with reservoir
liners; volatility issue; VC1<<< VC2

FAL.3T3.SLS.5/02/04

2.15

NO

%VC difference >15;
possible volatility
problem

2.15

YES

1.47

YES

FAL.3T3.SLS.06/05/04

2.15

NO

%VC difference >15; SD having difficulty in using
possible volatility
plate covers for volatility
FAL.3T3.SLS.20/05/04
problemproblems

2.15

YES

FAL3T3.SLS.25.02.04
heated C1-C3 to dissolve

C1-C3 heated to dissolve

FAL.3T3.SLS.29/04/04

FAL.3T3.SLS.27/05/04

ACETYLSALICYLIC ACID
IIVS
A1

RF

AA61HM

480

2.662

0.371

2.14%

1

2

0.9294B1

DF

AA61HM

344

1.911

0.413

7.89%

5

3

0.9635B2DF

AA61HM

467

2.590

0.394

1.04%

4

4

0.9853B3DF

AA61HM

392

2.174

0.383

1.33%

4

4

0.9724

AA61ME-A1

RF

AA61ME

175

0.969

0.256

7.03%

1

6

0.7065

AA61ME-B1

DF

AA61ME

589

3.268

0.344

6.13%

2

2

0.9566

AA61ME-B2

DF

AA61ME

711

3.947

0.304

4.93%

2

6

0.9182

AA61ME-B3

DF

AA61ME

637

3.534

0.345

0.84%

2

4

0.9244

FAL.3T3.JA.A1.21.05.04

RF

AA61JA

1110

6.169

0.190

4.35%

0

1

0.5653

FAL.3T3.JA.B1.04.06.04

DF

AA61JA

1290

7.149

0.358

12.22%

2

6

0.98691000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 1333, 889, 593,
395, 263, 176, 117
2000, 1333, 889, 593,
395, 263, 176, 117
2000, 1333, 889, 593,
395, 263, 176, 117

10

RF

1.5

YES

range finder

SLS-A3
SLS-B5

1.5

YES

SLS-B91.5

YES

SLS-B10ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 930, 433, 201,93.6, 43.5, 20.2, 9.4
2000, 930, 433, 201,93.6, 43.5, 20.2, 9.4
2000, 930, 433, 201,93.6, 43.5, 20.2, 9.4

10

RF

2.15

YES

range finder

SLS-P9SLS-P30

2.15

YES

SLS-P32

2.15

YES

SLS-P34

10

RF

1.47

YES

FRAME

I-6

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1701, 1157, 787,
535, 364, 248, 169

range finder; nopoints 0 - 50%

FAL.3T3.SLS.21.05.04
FAL.3T3.SLS.04.06.04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

2500, 1701, 1157, 787,
535, 364, 248, 169
2500, 1701, 1157, 787,
535, 364, 248, 169

FAL.3T3.JA.B2.18.06.04

RF

AA61JA

1500

8.342

0.471

8.60%

1

5

0.9217

FAL.3T3.JA.B3.08.07.04

DF

AA61JA

912

5.061

0.262

0.73%

3

5

0.9499A1

RF

AA61JD

0.006

0.00001

0.449

1.25%

6

1

0.8361B1DF

AA61JD

0.006

0.00001

0.310

1.69%

4

4

0.8810B2DFAA61JD

0.004

0.00001

0.402

1.66%

5

3

0.8854B3DF

AA61JD

0.003

0.00001

0.461

0.11%

6

2

0.8529B4DF

AA61JD

0.005

0.00001

0.300

0.33%

5

1

0.8025

RF

AA61MB

0.012

0.000030.373

14.44%

6

2

0.6985

1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001

0.7532

1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001

Dilution

Acceptable

Factor8

Tests9

1.47

YES

outlier removed by SD from
FAL.3T3.SLS.18.06.04
VC1

1.47

YES

FAL.3T3.SLS.08.07.04

10

RF

1.5

YES

SLS-B1

1.5

YES

SLS-B2

1.5

NO

1.5

YES

10

RF

low r2; range finder

SLS-P410NO

low r2;% VC
difference > 15;
range finder

SLS-P5

2.15

YES

1.47

NO

1.47

YES

SLS-P11

1.47

YES

SLS-P14

Rationale for
Unacceptability

Notes

PC ID

AMINOPTERIN

IIVS

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
0.02, 0.013, 0.0089,
0.0059, 0.0040, 0.0026,
0.0018, 0.0012
0.02, 0.013, 0.0089,
0.0059, 0.0040, 0.0026,
0.0018, 0.0012
0.02, 0.013, 0.0089,
0.0059, 0.0040, 0.0026,
0.0018, 0.0012
0.02, 0.013, 0.0089,
0.0059, 0.0040, 0.0026,
0.0018, 0.0012

range finder

PC failed

SLS-A2

SLS-B3
SLS-B4ECBC
AA61MB-A1
AA61MB-A2

RF

AA61MB

0.014

0.00003

0.470

22.09%

6

1

0.1000, 0.0465, 0.0216,
0.0101, 0.0047, 0.0022,
0.0010, 0.0005
0.0200, 0.0136, 0.0093,
0.0063, 0.0043, 0.0029,
0.0020, 0.0013
0.0200, 0.0136, 0.0093,
0.0063, 0.0043, 0.0029,
0.0020, 0.0013
0.0200, 0.0136, 0.0093,
0.0063, 0.0043, 0.0029,
0.0020, 0.0013

AA61MB-B1

DF

AA61MB

0.007

0.00002

0.435

3.5%

4

1

0.8625SLS-P7

AA61MB-B2

DF

AA61MB

0.004

0.00001

0.400

5.46%

5

1

0.8409

AA61MB-B3

DF

AA61MB

0.005

0.00001

0.383

11.29%

5

1

0.8251

AA61MB-B4

DF

AA61MB

0.005

0.00001

0.544

7.46%

5

2

0.8840

A1PU190603

RF

AA61PU

0.146

0.00033

0.550

2.01%

6

0

0.64901000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001

10

RF

FAL.3T3.PU.A2.26.06.03

RF

AA61PU

NA

NA

0.446

4.4%

8

0

0.06693.50, 2.38, 1.62, 1.10,
0.75, 0.51, 0.35, 0.24

1.47

NO

FAL.3T3.PU.B1.03.07.03

DF

AA61PU

NA

NA

0.453

0.09%

8

0

NA

2.13

NO

FAL.3T3.B2.PU.10.07.03

DF

AA61PU

NA

NA

0.451

3.11%

8

0

0.00182.13

NOFAL.3T3.B7.PU.17.10.03

DF

AA61PU

0.005830.00001

0.302

10.79%

1

4

0.8196

2.5

YES

FAL.3T3.SLS.171003

FAL.3T3.B8.PU.30.10.03

DF

AA61PU0.01290.00003

0.361

0.21%

1

4

0.9443

2.2

YES

FAL.3T3.SLS.301003

FAL.3T3.B9.PU.31.10.03

DF

AA61PU

0.01660.00004

0.289

8.80%

2

2

0.86982.2

YES

FAL.3T3.SLS.301003(should be 311003)

PC failed

SLS-P9FRAME

I-70.100, 0.047, 0.022,
0.010, 0.005, 0.002,
0.001, 0.0005
0.100, 0.047, 0.022,
0.010, 0.005, 0.002,
0.001, 0.0005
0.010, 0.005, 0.002,
0.0010, 0.0005, 0.0002,
0.0001, 0.00005
0.022, 0.010, 0.005,
0.0022, 0.0010, 0.0005,
0.0002, 0.0001
0.046, 0.021, 0.010,
0.0046, 0.0022, 0.001,
0.0005, 0.0002

PC failed; no points
between 50 - 90%;
low r2no points between
10 - 50%; low r2;
range finder
PC failed; no points
between 10 - 50; r2
not available
no points between
50 - 90%; low r2

A1SLS190603
FAL.3T3.SLS.A2.26.06.
03
FAL.3T3.SLS.B1.03.07.
03
FAL.3T3.SLS.10.07.03

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9

10

RF

1.3

YES

SLS-B6
plates read 15-16 hr late;no points between 0 orignial reading used wrong SLS-B11
50%
OD wavelength

Rationale for
Unacceptability

Notes

PC ID

5-AMINOSALICYLIC ACID
IIVS
A1

RF

AA61GZ

NA

NA

0.448

0.95%

0

5

0.7520B1DF

AA61GZ

1360

8.872

0.447

3.00%

1

7

0.9462B2DF

AA61GZ

1610

10.520

0.451

0.98%

0

8

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1500, 1154, 888, 683,
525, 404, 311, 239

0.9642

1500, 1154, 888, 683,
525, 404, 311, 239

1.3

NO

range finder

SLS-A3B3DF

AA61GZ

1710

11.144

0.349

4.42%

2

6

0.9177

2500, 1786, 1276, 911,
651, 465, 332, 237

1.4

YES

B4

DF

AA61GZ

NA

NA

NA

NA

NA

NA

NA

NA

NA

NO

B5

DF

AA61GZ

1600

10.472

0.409

0.65%

2

6

0.9854

2500, 1786, 1276, 911,
651, 465, 332, 237

1.4

YES

AA61KD-A1

RF

AA61KD

NA

NA

0.318

12.19%

0

4

NA

10

RF

AA61KD-B1

DF

AA61KD

1530

10.024

0.709

2.06%

1

7

0.9218

1.47

YES

SLS-P46

AA61KD-B2

DF

AA61KD

1240

8.110

0.413

0.16%

2

6

0.9375

1.47

YES

SLS-P47

AA61KD-B3

DF

AA61KD

1630

10.642

0.386

0.26%

2

6

0.9711

1.47

YES

SLS-P49

FAL.3T3.PA.A1.21.05.04

RF

AA61PA

NA

NA

0.394

4.59%

0

1

0.565810RF

FAL.3T3.PA.B1.04.06.04

DF

AA61PA

1770

11.535

0.501

0.57%

1

7

0.9637

1.47

YES

FAL.3T3.SLS.04.06.04

FAL.3T3.PA.B2.18.06.04

DF

AA61PA2010

13.1230.491

14.06%

1

4

0.8978

1.47

YES

FAL.3T3.SLS.18.06.04

FAL.3T3.PA.B3.08.07.04

DF

AA61PA

2430

15.850

0.343

1.34%

1

4

0.86501.47

YES

FAL.3T3.SLS.08.07.04

ppt in 2X C1-C3
PC failed

SLS-B12
SLS-B14

ppt in 2X C1-C2

SLS-B15ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1701, 1157, 787,
535, 364, 248, 169
2500, 1701, 1157, 787,
535, 364, 248, 169
2500, 1701, 1157, 787,
535, 364, 248, 169

range finder

SLS-11

FRAME
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
2500, 1701, 1157, 787,
535, 364, 248, 169
2500, 1701, 1157, 787,
535, 364, 248, 169
2500, 1701, 1157, 787,
535, 364, 248, 169

range finder

FAL.3T3.SLS.21.05.04

AMITRIPTYLINE HCL
IIVS
A1

RF

AA61RF

5.45

0.017

0.327

1.25%

1

2

0.9939B1

DF

AA61RF

8.83

0.03

0.349

0.19%

2

5

0.9858B2DFAA61RF

8.35

0.03

0.344

1.92%

2

2

0.9464B3DF

AA61RF

6.24

0.02

0.357

0.02%

2

2

0.9701

AA61PR-A1

RF

AA61PR

10.6

0.034

0.352

8.18%

0

5

0.8920AA61PR-B1

DF

AA61PR

6.26

0.020

0.384

2.37%

2

4

0.9661

AA61PR-B2

DF

AA61PR

4.55

0.014

0.451

1.05%

2

5

0.9214

AA61PR-B3

DF

AA61PR

7.28

0.023

0.577

2.79%

2

4

0.9701

RF

AA61LE

12.9

0.041

0.463

1.62%

1

3

0.97391000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
25.0, 16.7, 11.1, 7.41,
4.94, 3.29, 2.19, 1.46
25.0, 16.7, 11.1, 7.41,
4.94, 3.29, 2.19, 1.46
25.0, 16.7, 11.1, 7.41,
4.94, 3.29, 2.19, 1.46

10

RF

1.5

YES

range finder

ppt in 2X C1 and 1X C1SLS-A1
SLS-B1

1.5

YES

SLS-B2

1.5

YES

SLS-B3ECBC
1000, 100,10, 1, 0.1,
0.01, 0.001, 0.0001
80.0, 37.2, 17.3, 8.05,
3.74, 1.74, 0.81, 0.38
15.0, 10.2, 6.94, 4.72,
3.21, 2.19, 1.49, 1.01
15.0, 10.2, 6.94, 4.72,
3.21, 2.19, 1.49, 1.01

10

RF

2.15

YES

range finder

ppt in 2X C1 and 1X C1SLS-P4SLS-P20

1.47

YES

SLS-P22

1.47

YES

SLS-P24

10

RF

FRAME
FAL.3T3.LE.A1.090104

I-8

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

range finder

ppt in 2X C1

FAL.3T3.SLS.09/01/04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

FAL3T3.LE.B1.16.01.04

DF

AA61 LE

10.4

0.033

0.500

7.09%

3

4

0.9391FAL3T3.LE.B2.23.01.04

DF

AA61LE

6.48

0.021

0.347

13.33%

52

0.9709Exposure
Concentrations(ug/mL)7

100, 46.5, 21.6, 10.1, 4.7,
2.2, 1.0, 0.5
50.0, 34.0, 23.1, 15.7,
10.7, 7.3, 5.0, 3.4

Dilution

Acceptable

Factor8

Tests9

2.15

YES

1.47

YES

FAL3T3.LE.B3.30.01.04

DF

AA61LE

NA

NA

0.262

13.73%

53NA

50.0, 34.0, 23.1, 15.7,
10.7, 7.3, 5.0, 3.4

FAL3T3.LE.B4.06-02-04

DF

AA61LE

6.70

0.021

0.325

5.48%

5

3

0.9586

50.0, 34.0, 23.1, 15.7,
10.7, 7.3, 5.0, 3.4

1.47

YES

1.50

0.008

0.409

2.18%

0

1

0.9861

100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001

10RF

0.9787100, 46.4, 21.6, 10.0,
4.64, 2.16, 1.00, 0.46

2.16

YES

2.16

YES

1.47

NO

Rationale for
Unacceptability

Notes

PC ID

FAL.3T3.SLS.16/01/04
FAL3T3.23-01-04
SD rejects thisexperimentserious NR crystal problem;
FAL.3T3.SLS.29/01/04
SD rejects this experimentpossible NR crystalspresent; blanks slightly
higher than usual

FAL.3T3.SLS.06/02/04

ARSENIC III TRIOXIDEIIVSA1 Preliminary

RF

B1

DF

AA61FX
AA61FX

3.17

0.016

0.529

5.69%

1

2

range finder

SLS-A1
not fully soluble at 200
ug/ml; part. observed at 100 SLS-B1
ug/ml
not fully soluble at 200
ug/ml; part. observed at 100 SLS-B2
ug/ml
not fully soluble at 200
ug/ml; part. observed at 100 SLS-B3
ug/ml

B2

DF

AA61FX

2.47

0.012

0.485

1.29%

1

2

0.9875100, 46.4, 21.6, 10.0,
4.64, 2.16, 1.00, 0.46

B3DF

AA61FX

6.63

0.034

0.599

6.19%

1

3

0.9597100, 46.4, 21.6, 10.0,
4.64, 2.16, 1.00, 0.46

2.16

YES

RF

AA61KU

18.3

0.093

0.414

3.18%

1

2

0.645625, 2.5, 0.25, 0.025,
0.0025,0.00025,
0.000025, 0.0000025

10RF

range finder

SLS-P1

1.47

NONo points between
50 and 90%; PC
failedSLS-P3

1.47

NO

PC failed

SLS-P4

1.47

YES

SLS-P5

1.47

YES

SLS-P7

1.47

YES

SLS-P9

1.47

YES

SLS-P12ECBC
ECBC-3T3-Ib-01
A1

AA61KU-

ECBC-3T3-Ib-02
AA61KU-B1

DF

AA61KU

2.39

0.012

0.340

0.32%

3

0

0.8812DF

AA61KU

2.57

0.013

0.405

4.55%

3

1

0.9221DF

AA61KU

3.07

0.016

0.777

7.74%

3

2

0.9511DF

AA61KU

2.53

0.013

0.419

0.20%

4

1

0.9580DF

AA61KU

2.74

0.014

0.606

3.92%

2

2

0.9663DF

AA61KU

1.28

0.006

0.393

6.66%

3

1

0.9680

1b3T3RF01FALNC

RF

AA61NC

6.85

0.035

0.426

3.32%

1

4

0.9380

1b3T3RF02FALNC

RF

AA61NC

2.77

0.014

0.543

8.42%

0

0

0.6786

1b3T3RF02FALNC

RF

AA61NC

1.48

0.007

0.247

12.73%

2

3

0.8760

1b3T3DF01FALNC

DF

AA61NC

0.328

0.002

0.669

4.88%

1

0

1b3T3DF02FALNC

DF

AA61NC

1.74

0.009

0.363

3.42%

3

0

ECBC-3T3-Ib-03
AA61KU-B2
ECBC-3T3-Ib-04
AA61KU-B3
ECBC-3T3-Ib-05
AA61KU-B4
ECBC-3T3-Ib-06
AA61KU-B5
ECBC-3T3-Ib-07
AA61KU-B6

50, 34, 23.1, 15.7, 10.7,
7.3, 5.0, 3.4
34.2, 23.2, 15.8, 10.8,
7.3, 5.0, 3.4, 2.3
25.0, 17.0, 11.6, 7.9, 5.4,
3.6, 2.5, 1.7
25.0, 17.0, 11.6, 7.9, 5.4,
3.6, 2.5, 1.7
25.0, 17.0, 11.6, 7.9, 5.4,
3.6, 2.5, 1.7
15.0, 10.2, 6.9, 4.7, 3.2,
2.2, 1.5, 1.0

FRAME100, 20, 4, 0.8, 0.16,
0.032, 0.0064, 0.00128
50, 34, 23.1, 15.7, 10.7,
7.3, 5, 3.4
10, 6.8, 4.63, 3.15, 2.14,
1.46, 0.99, 0.67

5RFrange finder

1.47

RF

range finder

1.47

RF

0.5431

24, 16.33, 11.11, 7.56,
5.14, 3.5, 2.38, 1.61

1.47

NO

0.951728.5, 19.39, 13.19, 8.97,
6.1, 4.15, 2.82, 1.92

1.47

NO

1.47

YES

NR crystals in plate

NR crystals in plate;
stopped after 1 h
No points between
50 & 90% viability; r2 Didn't reach 50% viability< 0.8
No points between
50 & 90% viability;
Didn't reach 50% viability
PC failedrange finder

1b3T3CTRFALSLS
12/17/02
1b3T3CTRFALSLS
1/7/03
1b3T3CTRFALSLS
1/8/03
1b3T3CTRFALSLS
1/14/03
1b3T3CTRFALSLS
1/15/03

1b3T3DF03FALNC

DF

AA61NC

1.05

0.005

0.742

0.84%

3

3

0.91637.000, 4.762, 3.239,
2.204, 1.499, 1.020,
0.694, 0.472

1b3T3DF04FALNC

DF

AA61NC

1.39

0.007

0.303

15.26%

2

4

0.95917, 4.76, 3.24, 2.20, 1.50,
1.02, 0.69, 0.47

1.47

NO

NR crystals in plate;
stopped after 1 h;
PC failed

1b3T3CTRFALSLS
1/28/03

1b3T3DF09FALNC

DF

AA61NC

1.25

0.006

0.624

1.40%

1

3

0.96717, 4.76, 3.24, 2.20, 1.50,
1.02, 0.69, 0.47

1.47

NO

PC failed

1b3T3CTRFALSLS
1/29/03I-9

1b3T3CTRFALSLS
1/21/03

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

1b3T3DF06FALNC

DF

AA61NC

0.984

0.005

0.569

0.76%

1

2

0.90991b3T3DF07FALNC

DF

AA61NC

1.00

0.005

0.639

1.80%

2

3

0.9303

1b3T3DF07(2)FALNC

DF

AA61NC

1.14

0.006

0.651

2.48%

2

2

0.9256

Exposure
Concentrations(ug/mL)7

2.500, 1.701, 1.157,
0.787, 0.535, 0.364,
0.248, 0.169
5.000, 3.401, 2.314,
1.574, 1.071, 0.728,
0.496, 0.337
7, 4.76, 3.24, 2.20, 1.50,
1.02, 0.69, 0.47

Dilution

Acceptable

Factor8

Tests9

1.47

YES

1b3T3CTRFALSLS
2/403

1.47

YES

1b3T3CTRFALSLS
2/5/03

1.47

YES

1b3T3CTRFALSLS
2/5/03

Rationale for
Unacceptability

Notes

PC ID

ATROPINE SULFATE
IIVS
A1

RF

AA61NE

50.4

0.072

0.391

0.62%

1

2

0.9941B1DF

AA61NE

63.8

0.092

0.485

4.11%

3

5

0.8808B2DF

AA61NE

71.1

0.102

0.374

1.70%

3

5

0.9230B3DFAA61NE

75.0

0.108

0.436

3.00%

2

6

0.907010000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
300, 167, 92.6, 51.4,
28.6, 15.9, 8.82, 4.90

10

RF

1.8

YES

300, 167, 92.6, 51.4,
28.6, 15.9, 8.82, 4.90

1.8

YES

300, 167, 92.6, 51.4,
28.6, 15.9, 8.82, 4.90

1.8

YES

range finder

SLS-A4
outlier removed by study director
SLS-B4
G11 in VC2 not used; rec'd
extra 100ul medium during
SLS-B7seeding process;SDremoved
SD note: response curves in
3 valid DF similar & don't
SLS-B8follow classic Hill response
curve

ECBC
AA61KX-A1

RF

AA61KX

87.9

0.127

0.390

11.37%

1

5

0.9664

AA61KX-B1

DF

AA61KX

31.3

0.045

0.510

7.40%

3

3

0.9452

AA61KX-B2

DF

AA61KX

43.4

0.062

0.465

9.34%

3

4

0.9483

AA61KX-B3

DF

AA61KX

87.5

0.126

0.686

5.74%

3

4

0.9275

FAL.3T3.FU.A1.10.09.04

RF

AA61FU

461

0.664

0.384

4.08%

1

0

0.9358FAL.3T3.FU.B1.16.09.04

DF

AA61FU

160

0.231

0.350

1.76%

5

3

0.9137

FAL.3T3.FU.B2.15.10.04

DF

AA61FU

153

0.221

0.342

2.06%

4

4

0.9807

FAL.3T3.FU.B3.28.10.04

DF

AA61FU

85.5

0.123

0.184

5.35%

5

3

0.9528

A1

RF

AA61LD979

15.842

0.433

3.67%

1

6

0.9184

B1

DF

AA61LD1090

17.571

0.403

0.04%

4

4

0.9456

10000,1000, 100, 10,1,0.1, 0.01, 0.001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

10

RF

2.15

YES

range finder

SLS-P13SLS-P31

2.15

YES

SLS-P33

2.15

YES

SLS-P35

10

RF

1.47

YES

1.47

YES

FAL.3T3.SLS.15.10.04

1.47

YES

FAL.3T3.SLS.28.10.04

FRAME
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 680, 463, 315, 214,
146, 99.1, 67.4
500, 340, 231, 157, 107,
72.8, 49.6, 33.7
500, 340, 231, 157, 107,
72.8, 49.6, 33.7

range finder; nopoints between 50 100%

FAL.3T3.SLS.10.09.04
outlier removed by SD

FAL.3T3.SLS.16.09.04

BORIC ACID
IIVS

B2
B3

DF

AA61LD685

11.087

0.486

2.50%

5

3

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
5000, 3125, 1953, 1221,
763, 477, 298, 186

10

RF

1.6

YES

range finder

SLS-A4SLS-B6

0.9462

5000, 3125, 1953, 1221,
763, 477, 298, 186

1.6

YES

plates read 15-16 hr late;orignial reading used wrong SLS-B11
OD wavelength

1.6

YES

SLS-B12

10

RF

DF

AA61LD1830

29.635

0.349

0.52%

2

4

0.91295000, 3125, 1953, 1221,
763, 477, 298, 186

RF

AA61JH

897

14.514

0.329

0.46%

2

6

0.8984

10000,1000, 100, 10,1,0.1, 0.01, 0.001

ECBC
AA61JH-A1

I-10

range finder

SLS-P12In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC ID

AA61JH-B1

DF

AA61JH

1150

18.570

0.477

1.66%

3

5

0.9684

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

YES

SLS-P31

AA61JH-B2

DF

AA61JH

1290

20.932

0.423

0.14%

4

4

0.9524

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

YES

SLS-P33

AA61JH-B3

DF

AA61JH

2050

33.098

0.691

5.22%

3

3

0.9571

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

YES

SLS-P35

RF

AA61GR

2000

32.270

0.394

4.32%

1

1

0.8608

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

1.47

YES

FRAME
FAL.3T3.GR.A1.10.09.04
FAL.3T3.GR.B1.16.09.04

DF

AA61GR

4320

69.7910.351

10.82%

2

3

0.863010000, 6803, 4628, 3148,
2142, 1457, 991, 674

FAL.3T3.GR.B2.23.09.04

DF

AA61GR

4450

71.912

0.336

3.84%

2

4

0.8582

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

FAL.3T3.GR.B3.14.10.04

DF

AA61GR

3190

51.618

0.319

3.58%

3

5

0.7925

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

A1

RF

AA61RL

29.2

0.118

0.387

12.48%

1

6

0.8879B1

DF

AA61RL

41.7

0.169

0.425

3.61%

3

5

0.8760B2

DF

AA61RL

44.9

0.18

0.332

5.19%

5

3

0.8920B3DF

AA61RL

44.6

0.18

0.332

3.79%

4

4

0.8775

AA61LH-A1

RF

AA61LH

97.3

0.395

0.360

3.64%

1

5

0.8554

AA61LH-B1

DF

AA61LH

57.3

0.233

0.293

3.70%

3

5

0.8885

AA61LH-B2

DF

AA61LH

44.6

0.181

0.385

6.29%

3

5

0.8764

AA61LH-B3

DF

AA61LH

19.4

0.079

0.463

0.58%

5

3

0.8778FAL.3T3.JE.A1.09/01/04

RF

AA61JE

38.7

0.156

0.677

5.72%

1

4

0.9065

FAL.3T3.JE.A2.16/01/04

DF

AA61JE

528

2.145

0.597

9.65%

1

7

0.7176

range finder

FAL.3T3.SLS.10.09.04
FAL.3T3.SLS.16.09.04
outlier removed bySD

FAL.3T3.SLS.23.09.04
FAL.3T3.SLS.14.10.04

BUSULFAN
IIVS100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49,
500, 278, 154, 85.7, 47.6,
26.5, 14.7, 8.17
500, 278, 154, 85.7, 47.6,
26.5, 14.7, 8.17

10

RF

1.4

YES

range finder

SLS-A1
SLS-B5

1.8

YES

SLS-B9

1.8

YES

plate sealer used

SLS-B10

ppt in 2X C1SLS-P4ECBC
1000, 100,10, 1, 0.1,
0.01, 0.001, 0.0001
500, 233, 108, 50.3, 23.4,
10.88, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.88, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.88, 5.06, 2.35

10

RF

2.15

YES

range finder

SLS-P18

2.15

YES

SLS-P20

2.15

YES

SLS-P21

10

RF

2.15

YES

FRAME

FAL.3T3.JE.B1.23/01/04

DF

AA61JE

234

0.952

0.361

10.07%

1

6

0.9558250, 25, 2.5, 0.25, 0.025,
0.0025, 0.00025,
0.000025
500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

2.15

YES

FAL.3T3.JE.B2.30/01/04

DF

AA61JE

NA

NA

0.266

3.00%

0

6

NA

500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

2.15

NO

FAL.3T3.JE.B3.06-02-04

DF

AA61JE

202

0.819

0.308

7.09%

1

7

0.8537500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

2.15

YES

I-11

range finder

FAL.3T3.SLS.09/01/04
FAL.3T3.SLS.16/01/04
morphological changes
seen at C5 but not noted in
NRU

FAL3T3.23-01-04

no points between 0serious NR crystal problem;
50; SD rejects thisFAL.3T3.SLS.29/01/04
SD rejects this experimentexperimentpossible NR crystalspresent; blanks slightly
FAL.3T3.SLS.06/02/04
higher than usual

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9

10

RF

1.5

YES

SLS-B1

1.5

YES

SLS-B2

1.5

NO

1.5

YES

10

RF

1.47

YES

SLS-P6

1.47

YES

SLS-P7

1.47

YES

SLS-P10

Rationale for
Unacceptability

Notes

PC ID

CADMIUM II CHLORIDE
IIVS
A1

RF

AA61NK

0.462

0.003

0.442

2.10%

1

3

0.9959B1DF

AA61NK

1.31

0.007

0.325

0.39%

2

5

0.9811B2DF

AA61NK

0.575

0.003

0.382

8.48%

3

4

0.9735B3DF

AA61NK

0.529

0.003

0.407

1.25%

4

3

0.9907B4DF

AA61NK

0.565

0.003

0.336

4.71%

3

4

0.9832

RF

AA61KR

0.620

0.003

0.346

0.53%

0

0

0.9671

100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
3.0, 2.0, 1.33, 0.889,
0.593, 0.395, 0.263,
0.176
3.0, 2.0, 1.33, 0.889,
0.593, 0.395, 0.263,
0.176
3.0, 2.0, 1.33, 0.889,
0.593, 0.395, 0.263,
0.176
3.0, 2.0, 1.33, 0.889,
0.593, 0.395, 0.263,
0.176

range finder

SLS-A1

PC failed

SLS-B3
SLS-B4ECBC
AA61KR-A1
AA61KR-B1

DF

AA61KR

0.514

0.003

0.542

7.85%

2

4

0.8434

AA61KR-B2

DF

AA61KR

0.530

0.003

0.496

3.06%

3

4

0.9625

AA61KR-B3

DF

AA61KR

0.406

0.002

0.389

3.87%

2

3

0.9474

A1JP190603

RF

AA61JP

0.973

0.005

0.523

1.02%

1

0

0.9777

FAL.3T3.JP.B1 .26.06.03

RF

AA61JP

0.547

0.003

0.463

3.71%

1

2

0.9748FAL.3T3.JP.B2.03.07.03

DF

AA61JP

0.817

0.004

0.364

4.50%

1

1

0.9422

FAL.3T3.B3.JP.10.07.03

DF

AA61JP

0.343

0.002

0.484

1.25%

2

2

0.9894

FAL.3T3.B4.JP.17.07.03

DF

AA61JP

0.309

0.002

0.549

0.47%

2

2

0.9837

A1

RF

AA61JM

176

0.905

0.439

6.89%

1

1

0.9381B1DF

AA61JM

183

0.941

0.510

0.72%

4

4

0.9939B2DF

AA61JM

208

1.073

0.379

8.66%

4

4

0.9793B3DF

AA61JM

183

0.944

0.452

1.60%

4

4

0.9857

AA61NU-A1

RF

AA61NU

119

0.613

0.457

8.10%

1

5

0.9548

AA61NU-B1

DF

AA61NU

130

0.668

0.469

0.04%

3

4

0.9366

AA61NU-B2

DF

AA61NU

148

0.760

0.539

2.01%

3

5

0.9798

AA61NU-B3

DF

AA61NU

122

0.631

0.543

0.37%

3

5

0.9791

1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
2.0, 1.361, 0.926, 0.630,
0.428, 0.291, 0.198,
0.135
2.0, 1.361, 0.926, 0.630,
0.428, 0.291, 0.198,
0.135
2.0, 1.361, 0.926, 0.630,
0.428, 0.291, 0.198,
0.135

no points between
10 -90%; range
finderSLS-P4

FRAME
1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
5.0, 3.4, 2.3, 1.5, 1.1, 0.7,
0.5, 0.3
3.0, 2.04, 1.39, 0.94,
0.64, 0.44, 0.30, 0.20
3.0, 2.04, 1.39, 0.94,
0.64, 0.44, 0.30, 0.20
3, 2.04, 1.39, 0.94, 0.64,
0.44, 0.30, 0.20

10

RF

1.47

YES

range finder

A1SLS190603

1.47

NO

1.47

YES

FAL.3T3.SLS.10.07.03

1.47

YES

FAL.3T3.SLS.17.07.03

FAL.3T3.SLS.A2.26.06.
03
FAL.3T3.SLS.B1.03.07.
03

PC failed

CAFFEINE
IIVS
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 625, 391, 244, 153,
95.4, 59.6, 37.3
1000, 625, 391, 244, 153,
95.4, 59.6, 37.3
1000, 625, 391, 244, 153,
95.4, 59.6, 37.3

10

RF

1.6

YES

1.6

YES

1.6

YES

range finder

SLS-A1
SLS-B4
outlier removed bySD

SLS-B7
SLS-B8ECBC

I-1210000, 1000, 100,10, 1,
0.1, 0.01, 0.001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7

10

RF

2.15

YES

range finder

SLS-P3SLS-P17

2.15

YES

SLS-P19

2.15

YES

SLS-P22

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests910

RF

Rationale for
Unacceptability

Notes

PC ID

FRAME
FAL.3T3.GW.A1.09/01/04
FAL.3T3.GW.A2.16.01.04
revised by NICEATM; bottom
set to 0 as constant
FAL.3T3.GW.B1.23.01.04
revised by NICEATM; bottom
set to 0 as constantRF

AA61GW

198

1.018

0.632

5.54%

1

6

0.8800DF

AA61GW

67.9

0.350

1.046

2.64%

6

2

0.9544

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
5000, 2325.6, 1081.7,
503.1, 234.0, 108.8, 50.6,
23.5

range finder

FAL.3T3.SLS.09/01/04

2.15

YES

FAL.3T3.SLS.16/01/04

5000, 1582, 501, 158,
50.1, 15.9, 5.02, 1.59

3.16

YES

FAL3T3.23-01-04

DF

AA61GW

228

1.174

0.562

0.19%

3

4

0.9827

FAL.3T3.GW.B2.30.01.04

DF

AA61GW

NA

NA

0.315

1.72%

2

4

NA

5000, 1587, 504, 160, 51,
16, 5.1, 1.6

3.15

NO

FAL.3T3.GW.B3.06-02-04

DF

AA61GW

176

0.907

0.460

3.57%

3

5

0.9731

5000, 1587, 504, 160, 51,
16, 5.1, 1.6

3.15

YES

AA61NB

NA

NA

0.281

6.74%

0

4

NA

10

RF

range finderNO

no points between 0 50%

SD rejects thisexperimentserious NR crystal problem;
FAL.3T3.SLS.29/01/04
SD rejects this experiment
FAL.3T3.SLS.06/02/04

CARBAMAZEPINE
IIVS
A1
B1

RF
DF

AA61NB

164

0.694

0.397

2.68%

B2

DF

AA61NB

88.7

0.375

0.381

8.51%

B3

DF

AA61NB

104

0.441

0.318

1.57%

B4

DF

AA61NB

NA

NA

NA

NA

B4 (should be B5)DF

AA61NB

82.6

0.350

0.403

5.68%

AA61LX-A1

RF

AA61LX

88.9

0.376

0.438

AA61LX-B1

DF

AA61LX

93.8

0.397

AA61LX-B2

DF

AA61LX

85.1

AA61LX-B3

DF

AA61LX

FAL.3T3.HD.A1.21.10.04

RF

FAL.3T3.HD.B1.11.11.04

DF

0

6

8

0.544710.91793

50.9379

NA

NA

NA

4

4

0.9465

9.61%

1

0

0.8266

0.601

3.55%

3

5

0.9413

0.360

0.614

1.82%

2

6

0.9155

70.1

0.297

0.314

9.73%

3

5

0.9105

AA61HD

107

0.451

0.190

1.01%

1

1

0.9436

AA61HD

217

0.917

0.217

2.35%

2

1

0.7684

10.0, 1.0, 0.1, 0.01,
0.001, 0.0001, 0.00001,
0.000001
50.0, 38.5, 29.6, 22.8,
17.5, 13.5, 10.4, 7.97
300, 250, 208, 174, 145,
121, 100, 83.7
200, 154, 118, 91.0, 70.0,
53.9, 41.4, 31.9NA200, 154, 118, 91.0, 70.0,
53.9, 41.4, 31.9

1.3

1.2

YES

1.3

YES

NA

NO

1.3

YES

SLS-A5SLS-B6
C1 data removed from Hill
analyses; plates read 15-16
SLS-B11hr late; orignial reading used
wrong OD wavelength
SLS-B13

PC failed

SLS-B14
SLS-B15ECBC100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
250, 170, 116, 78.7, 53.5,
36.4, 24.8, 16.9
250, 170, 116, 78.7, 53.5,
36.4, 24.8, 16.9
170, 116, 78.7, 53.5, 36.4,
24.8, 16.8, 11.5

10

RF

1.47

YES

range finder

SLS-16SLS-P45

1.47

YES

SLS-P46

1.47

YES

SLS-P48

10

RF

FRAME
1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
1000, 318, 101, 32.0,
10.2, 3.2, 1.0, 0.3

range finder

outliers removed by SD; ppt
FAL.3T3.SLS.21.10.04
in 1X C1 and 2X C1

3.15

YES

ppt in 2X C1-C2; &1X in C1 FAL.3T3.SLS.10.11.04

0.9861

1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7

2.15

YES

ppt in 2X C1-C2 & 1X in C1FAL.3T3.SLS.19.11.04
C2

1

0.9107

1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7

2.15

YES

ppt in 2X C1-C2 & 1X in C1FAL.3T3.SLS.25.11.04
C3;

FAL.3T3.HD.B3.18.11.04(should be B2 and 19.11.04)

DF

AA61HD

130

0.550

0.237

4.35%

3

2

FAL.3T3.HD.B3.18.11.04

DF

AA61HD

110

0.466

0.241

0.24%

3

CARBON TETRACHLORIDE
IIVS
A1

RF

AA61JK

NA

NA

0.349

15.06%

0

2

NA

B1

DF

AA61JK

NA

NA

0.391

6.82%

0

5

NA

I-13

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1923, 1479, 1138,
875, 673, 518, 398

10RF

range finder

ppt in 2X C1-C3

SLS-A2

1.3

NO

no points between 0ppt in 2X C1-C3
50%

SLS-B6

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC ID

B2

DF

AA61JK

NA

NA

0.394

7.97%

0

1

NA

2500, 1923, 1479, 1138,
875, 673, 518, 398

1.3

NO

no points between 0- ppt in 2X C1-C8; no toxicity
SLS-B950%detected
ppt in 2X C1-C8; someno points between 0toxicity detected; C1 has
SLS-B1050%
lower toxicity than C2

B3

DF

AA61JK

NA

NA

0.368

3.76%

0

5

NA

2500, 1923, 1479, 1138,
875, 673, 518, 398

1.3

NO

AA61NZ-A1

RF

AA61NZ

NA

NA

0.328

16.23%

06

NA1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10RF

range finder

SLS-P9

AA61NZ-A2

RF

AA61NZ

NA

NA

0.419

7.89%

0

1

NA

3000, 300, 30, 3, 0.3,
0.03, 0.003, 0.0003

10RF

range finder

SLS-P19

AA61NZ-B1

DF

AA61NZ

NA

NA

0.416

2.67%

0

8

NA

4500, 3719, 3074, 2540,
2099, 1735, 1434, 1185

1.21

NO

no points between 0 50%

SLS-P65

SLS-P67

ECBC

AA61NZ-B2

DF

AA61NZ

NA

NA

0.567

3.83%

0

7

NA

7000, 5785, 4781, 3951,
3266, 2699, 2230, 1843

1.21

NO

dilution factor is 1.21; nono points between 0 - points between 0-50%; test
50%
would pass due to dilution
factor

AA61NZ-B3

DF

AA61NZ

NA

NA

0.536

8.16%

0

7

NA

7000, 5785, 4781, 3951,
3266, 2699, 2230, 1843

1.21

NO

no points between 0 ppt in 2X C1 - C5; oily
50%

SLS-P73

RF

AA61HC

NA

NA

0.179

1.46%

0

1

NA

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10RF

range finder

FAL.3T3.SLS.30/04/04

NA

2500, 1701, 1157, 787,
535, 364, 248, 169

1.47

NO

NA

2500, 1701, 1157, 787,
535, 364, 248, 169

1.47

NO

0.5412

2500, 1701, 1157, 787,
535, 364, 248, 169

1.47

NO

1.21

NO

no points between 0 50%

10RF

range finder

1.8

YES

FRAME
FAL.3T3.HC.A1.30/04/04
FAL.3T3.HC.B1.06/05/0404
FAL.3T3.HC.B2.26.11.04
FAL.3T3.HC.B3.03.12.04
FAL.3T3.HC.B4.09.12.04

DF
DF
DF
DF

AA61HCAA61HCAA61HC

NA
NA
2430

NA
NA
15.776

0.218
0.253
0.179

2.75%
12.16%
3.22%

0
0
1

0
5
0

AA61HC

NA

NA

0.286

7.61%

0

6

NA

2500, 2066, 1708, 1411,
1166, 964, 797, 658

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

no points between 0 - SD ends testing and returns
FAL.3T3.SLS.06/05/04
100%
as non-toxic at 2500ug/ml
the toxicity curve appears
no points between 0 reversed; higher conc. less FAL.3T3.SLS.26.11.04
50%
toxic than lower conc.
no points between
FAL.3T3.SLS.03.12.04
50 - 100%
FAL.3T3.SLS.09.12.04

CHLORAL HYDRATE
IIVS
A1

RF

AA61FJ

56.2

0.340

0.469

87.75%

2

4

0.9868B1DF

AA61FJ

156

0.943

0.509

2.80%

2

6

0.9655B2DF

AA61FJ

193

1.165

0.336

2.36%

2

5

0.9653B3DF

AA61FJ

162

0.981

0.447

5.20%

2

6

0.9613

AA61KB-A1

RF

AA61KB

NA

NA

0.189

94.19%

3

0NA

AA61KB-A2

RF

AA61KB

107

0.648

0.295

0.25%

0

1

NA

AA61KB-B1

DF

AA61KB

160

0.965

0.474

0.63%

3

5

0.9590AA61KB-B2

DF

AA61KB

160

0.969

0.703

2.49%

3

5

0.9682

AA61KB-B3

DF

AA61KB

133

0.806

0.588

0.17%

3

5

0.9604500, 278, 154, 85.7, 47.6,
26.5, 14.7, 8.17
500, 278, 154, 85.7, 47.6,
26.5, 14.7, 8.17
500, 278, 154, 85.7, 47.6,
26.5, 14.7, 8.17

VC1 ODs < VC2 ODs; VC1
removed from subsequent SLS-A2
analysis; volatility issues.
SLS-B4

1.8

YES

outliers removed by SD;
plate sealer used

1.8

YES

plate sealer used

SLS-B810RFrange finder

probable volatility problem;
VC1 <<< VC2

SLS-P610RFrange finder

1.47

YES

SLS-P22

1.47

YES

SLS-P24

1.47

YES

SLS-P26

SLS-B7

ECBC

FRAME

I-14

10000,1000, 100, 10,1,0.1, 0.01, 0.001
100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
500, 340, 231, 157, 107,
72.8, 49.6, 33.7
500, 340, 231, 157, 107,
72.8, 49.6, 33.7
500, 340, 231, 157, 107,
72.8, 49.6, 33.7

SLS-P7

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

FAL.3T3.LK.A1.01/04/04

RF

AA61LK

711

4.300

0.271

69.44%

2

00.2684

FAL.3T3.LK.B1.29/04/04

DF

AA61LK

243

1.470

0.287

5.23%

2

2

0.9262

FAL.3T3.LK.B2.06/05/04

DF

AA61LK

265

1.605

0.313

8.07%

4

4

0.9706

FAL.3T3.LK.B3.20/05/04

DF

AA61LK

1450

8.739

0.347

12.28%

01

0.9010FAL.3T3.LK.B4.27/05/04

DF

AA61LK

215

1.302

0.412

4.39%

4

4

0.9575

Exposure
Concentrations(ug/mL)7

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
1000, 680, 463, 315, 214,
146, 99.1, 67.4
2000, 1361, 926, 630,
428, 291, 198, 135
1000, 680, 463, 315, 214,
146, 99.1, 67.4

Dilution

Acceptable

Factor8

Tests9

10

RF

2.15

YES

1.47

YES

1.47

NO

1.47

YES

Rationale for
Unacceptability

range finder

Notes

PC ID

possible volatility problem

FAL.3T3.SLS.01/04/04

NR crystals; highbackground

FAL.3T3.SLS.29/04/04
FAL.3T3.SLS.06/05/04

no points between 0 - curve very different
50%
compared to other curves

FAL.3T3.SLS.20/05/04
FAL.3T3.SLS.27/05/04

CHLORAMPHENICOL
IIVS
A1

RF

AA61GJ

98.9

0.306

0.323

27.15%

1

10.1298B1DF

AA61GJ

187

0.579

0.307

4.85%

2

6

0.9661B2DF

AA61GJ

148

0.458

0.421

0.91%

3

5

0.9649B3DF

AA61GJ

142

0.439

0.428

0.12%

3

5

0.9668B3 with plate coverDF

AA61GJ

171

0.529

0.345

4.49%

2

5

0.9683B4DF

AA61GJ

133

0.412

0.350

3.69%

3

5

0.9171

AA61JS-A1

RF

AA61JS

54.5

0.169

0.401

14.20%

1

4

0.7119AA61JS-B1

DF

AA61JS

88.5

0.274

0.440

20.33%

2

3

0.8484

AA61JS-B2

DF

AA61JS

39.1

0.121

0.461

1.90%

2

4

0.9618AA61JS-B3

DF

AA61JS

61.1

0.189

0.395

1.46%

3

4

0.8537

AA61JS-B4

DF

AA61JS

55.1

0.171

0.504

2.80%

3

4

0.9541

AA61JS-B5

DF

AA61JS

68.5

0.212

0.448

5.20%

3

4

0.9401

RF

AA61MU

568

1.758

0.550

1.44%

1

0

0.9021

100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
558, 310, 172, 95.7, 53.2,
29.5, 16.4, 9.11
558, 310, 172, 95.7, 53.2,
29.5, 16.4, 9.11
558, 310, 172, 95.7, 53.2,
29.5, 16.4, 9.11
558, 310, 172, 95.7, 53.2,
29.5, 16.4, 9.11
593, 329, 183, 102, 56.5,
31.4, 17.4, 9.69

10

RF

1.8

YES

range finder

SLS-A1

1.8

YES

1.8

NO

PC failed

SLS-B3

1.8

NO

PC failed

SLS-B3

1.8

YES

SLS-B1
SLS-B2

SLS-B4ECBC
1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
1000, 300, 100, 30, 10, 3,
1, 0.3
1000, 465.1, 216.3,
100.6, 46.8, 21.8, 10.1,
4.7
1000, 465.1, 216.3,
100.6, 46.8, 21.8, 10.1,
4.7
1000, 465.1, 216.3,
100.6, 46.8, 21.8, 10.1,
4.7
1000, 465.1, 216.3,
100.6, 46.8, 21.8, 10.1,
4.7

10

RF

low r2; range finder

SLS-P4

3.33

NO% VC difference >15; range finder

SLS-P6

2.15

YES

SLS-P7

2.15

YES

SLS-P10

2.15

YES

SLS-P14

2.15

YES

SLS-P15

10

RF

1.47

YES

2.15

NO

PC failed

1.47

NO

no points between
10 - 50%

2.15

YES

FAL.3T3.SLS.17.07.03

2.15

YES

FAL.3T3.SLS.25.07.03

FRAME
A1MU190603
FAL.3T3.MU.B1.26.06.03

DF

AA61MU

276

0.854

0.491

13.66%

5

3

0.8425

FAL.3T3.MU.B2.03.07.03

DF

AA61MU

520

1.609

0.306

3.63%

2

2

0.8810FAL.3T3.B3.MU.10.07.03

DF

AA61MU

NA

NA

0.486

1.00%

0

2

NA

DF

AA61MU

237

0.733

0.455

1.91%

3

2

0.9782DF

AA61MU

385

1.191

0.379

0.65%

2

2

0.9291DF

AA61MU

64.4

0.199

0.721

1.63%

4

4

0.8501DF

AA61MU

193

0.597

0.363

0.80%

4

4

0.9490FAL.3T3.B3.MU.17.07.03(should be B4?)
FAL.3T3.B4.MU.25.07.03(should be B5?)
FAL.3T3.B5.MU.070803(should be B6?)
FAL.3T3.MU.B7.120903

I-15

1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
1500, 1020, 690, 470,
320, 220, 150, 100
1250, 580, 270, 125,
58.5, 27.2, 12.6, 5.9
50.0, 34.0, 23.1, 15.7,
10.7, 7.3, 5.0, 3.4
2500, 1160, 540, 251,
117, 54.4, 25.3, 11.7
2500, 1160, 540, 251,
117, 54.4, 25.3, 11.7
2500, 1160, 540, 251,
117, 54.4, 25.3, 11.7
2500, 1162, 540, 251,
117, 54.4, 25.3, 11.7

2.15

NO

2.15

YES

PC failed; no points
between 50 - 90%;
range finder

PC failed

A1SLS190603
FAL.3T3.SLS.A2.26.06.
03
FAL.3T3.SLS.B1.03.07.
03
FAL.3T3.SLS.10.07.03

FAL.3T3.SLS.070803
FAL.3T3.SLS.120903

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC ID

CITRIC ACID

IIVS
A1

RF

AA61MH

1030

5.376

0.363

7.76%

1

2

0.8924

B1

DF

AA61MH

681

3.54

0.389

3.15%

2

3

0.9722B2DF

AA61MH

942

4.90

0.379

5.58%

1

4

0.9742B3DF

AA61MH

971

5.05

0.381

1.66%

1

4

0.9858RF

AA61HH

409

2.130

0.341

0.32%

2

5

0.92750.987910000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10000, 5556, 3086, 1715,
953, 529, 294, 163
10000, 5556, 3086, 1715,
953, 529, 294, 163
10000, 5556, 3086, 1715,
953, 529, 294, 163

10

RF

1.8

YES

range finder

SLS-A1
SLS-B1

1.8

YES

SLS-B2

1.8

YES

SLS-B3

10000, 1000, 100,10, 1,
0.1, 0.01, 0.001

10RF

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

ECBC
AA61HH-A1
AA61HH-B1

DF

AA61HH

598

3.115

0.299

2.62%

4

4

4651, 2163, 1006, 468,
218, 101, 47.1, 21.9
4651, 2163, 1006, 468,
218, 101, 47.1, 21.9

range finder

ppt in 1X C1SLS-P1YES

ppt in 1X C1SLS-P5

2.15

YES

ppt in 1X C1SLS-P8

2.15

YES

AA61HH-B2

DF

AA61HH

325

1.692

0.418

9.20%

4

2

0.9800AA61HH-B3

DF

AA61HH

497

2.585

0.423

1.95%

3

5

0.9732

RF

AA61RB

1050

5.489

0.557

1.11%

1

1

0.8824

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RFrange finder

FAL.3T3.SLS.080104

0.9467

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

NO

PC failed;

FAL.3T3.SLS.15/01/04

2.15

YES

SLS-P17

FRAME
FAL.3T3.RB.A1.08/01/04
FAL3T3.RB.A2.15-01-04

DF

AA61RB

668

3.479

0.730

5.04%

4

4

FAL3T3.RB.B1.22-01-04

DF

AA61RB

1080

5.617

0.411

1.38%

3

2

0.9403

5000, 2325.6, 1081.7,
503.1, 234.0, 108.8, 50.6,
23.5

FAL3T3.RB.B2.29-01-04

DF

AA61RB

1050

5.476

0.423

12.11%

4

4

0.9575

5000, 3401, 2314, 1574,
1071, 728, 496, 337

1.47

YES

FAL3T3.RB.B3.05.02.04

DF

AA61RB

345

1.797

0.344

4.03%

7

0

0.8104

5000, 3401, 2314, 1574,
1071, 728, 496, 337

1.47

NO

FAL3T3.RB.25-02-04

DF

AA61RB

1100

5.721

0.481

11.65%

4

4

0.9805

1.47

YES

FAL3T3.RB.B5.17.03.04

DF

AA61RB

1360

7.087

0.304

6.25%

2

2

0.9139

2.15

YES

A1

RF

AA61FL

0.027

0.0001

0.514

1.69%

5

1

0.9699

10

RF

B1

DF

AA61FL

0.028

0.0001

0.416

3.16%

4

4

0.9768

1.49

YES

SLS-B1B2DF

AA61FL

0.028

0.0001

0.527

2.34%

4

4

0.9809

1.49

YES

SLS-B2B3DF

AA61FL

0.037

0.0001

0.578

6.33%

3

2

0.9522

1.49

NO5000, 3401, 2314, 1574,
1071, 728, 496, 337
5000, 2325.6, 1081.7,
503.1, 234.0, 108.8, 50.6,
23.5

FAL.3T3.SLS.22/01/04

PC failed; no points
between 50-100

pH 3 fpr C1; SD suggests
high pH may be cause of
FAL3T3.SLS.29-01-04
toxicity for thisconcentration;
problem with reservoir
liners; SD incorrectly
determined 4 points
FAL.3T3.SLS.5/02/04
between 50-100 instead of 0
pointsdefinitive test B4

FAL3T3.SLS.25.02.04
FAL.3T3.SLS.17/03/04

COLCHICINE
IIVS

I-16

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
0.1, 0.067, 0.044, 0.030,
0.020, 0.013, 0.0088,
0.00590.1, 0.067, 0.044, 0.030,
0.020, 0.013, 0.0088,
0.00590.1, 0.067, 0.044, 0.030,
0.020, 0.013, 0.0088,
0.0059range finder

PC failed

SLS-A1

SLS-B3

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

B4

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9

Notes

PC ID

DF

AA61FL

0.028

0.0001

0.406

0.86%

4

2

0.95080.1, 0.067, 0.044, 0.030,
0.020, 0.013, 0.0088,
0.0059

1.49

YES

RF

AA61JZ

0.008

0.0000

0.369

3.91%

2

0

0.9383

10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001

10

RF

1.47

YES

SLS-P8

1.47

YES

SLS-P9

1.47

YES

SLS-P12

1.47

YES

SLS-P13

10

RF

2.15

RF

2.15

NO

2.15

YES

FAL.3T3.SLS.11.07.03

2.15

YES

FAL.3T3.SLS.18.07.03

2.15

YES

FAL.3T3.SLS.25.07.03

10RF

range finder

1.4

NO

% VC difference >
15

1.4

YES

SLS-B6ECBC
AA61JZ-A1

Rationale for
Unacceptability

AA61JZ-B2

DF

AA61JZ

0.023

0.0001

0.595

8.49%

6

2

0.8811

AA61JZ-B3

DF

AA61JZ

0.018

0.0001

0.494

0.43%

6

2

0.9020AA61JZ-B4

DF

AA61JZ

0.019

0.0001

0.549

0.68%

4

2

0.9658AA61JZ-B5

DF

AA61JZ

0.022

0.0001

0.664

1.90%

6

1

0.9584

FAL.3T3.A1.NW.200603

RF

AA61NW

0.088

0.0003

0.699

5.16%

6

0

0.4881

FAL.3T3.A2.NW.27.06.03

RF

AA61NW

NA

NA

0.519

0.16%

8

0

0.2194

FAL.3T3.B1.NW.04.07.03

DF

AA61NW

0.184

0.0006

0.503

2.71%

5

1

0.7952

FAL.3T3.B2.NW.11.07.03

DF

AA61NW

0.046

0.0001

0.532

4.41%

6

2

0.8093

FAL.3T3.B3.NW.18.07.03(recalculated to fit bottom)

DF

AA61NW

0.127

0.0004

0.481

5.60%

5

2

0.8882

FAL.3T3.B5.NW.25.07.03

DF

AA61NW

0.106

0.0003

0.397

3.23%

5

3

0.8590

0.016

0.496

4.40%

2

5

0.96470.200, 0.136, 0.093,
0.063,0.043, 0.029,
0.020, 0.013
0.136, 0.093, 0.063,
0.043, 0.029, 0.020,
0.013, 0.009
0.136, 0.093, 0.063,
0.043, 0.029, 0.020,
0.013, 0.009
0.136, 0.093, 0.063,
0.043, 0.029, 0.020,
0.013, 0.009

SLS-B4

PC failed; no points
between 50 - 90%;
range finder

SLS-P2

FRAME
10000,1000, 100, 10, 1.0,
0.1, 0.01, 0.001
2.50, 1.16, 0.54, 0.25,
0.12, 0.05, 0.025, 0.012
2.50, 1.16, 0.54, 0.25,
0.12, 0.05, 0.025, 0.012
2.50, 1.16, 0.54, 0.25,
0.12, 0.05, 0.025, 0.012
5.00, 2.33, 1.08, 0.50,
0.234, 0.109, 0.051,
0.024
2.50, 1.16, 0.54, 0.25,
0.12, 0.05, 0.025, 0.012

no points between
50 - 90%; low r2;
range finder
no points between
50 - 90%; low r2

FAL.3T3.SLS2.A1.2006
03
FAL.3T3.SLS.A2.27.06.
03

PC failed; low r2

FAL.3T3.SLS.04.07.03

CUPRIC SULFATE PENTAHYDRATE
IIVS
A1

RF

AA61LA

4.02

B1

DF

AA61LA

4.26

0.017

0.395

23.78%

3

1

0.6017B2

DF

AA61LA

4.58

0.018

0.463

1.04%

3

3

0.9765100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
10.0, 7.14, 5.10, 3.64,
2.60, 1.86, 1.33, 0.949
10.0, 7.14, 5.10, 3.64,
2.60, 1.86, 1.33, 0.949

SLS-A2excessive variability within
SLS-B4
treatment and cotrol groups

B3

DF

AA61LA

4.84

0.019

0.418

0.86%

3

3

0.9887

10.0, 7.14, 5.10, 3.64,
2.60, 1.86, 1.33, 0.949

1.4

YES

plates read 15-16 hr late;orignial reading used wrong SLS-B11
OD wavelength

B4DF

AA61LA

7.73

0.031

0.375

2.20%

1

2

0.8726

10.0, 7.14, 5.10, 3.64,
2.60, 1.86, 1.33, 0.949

1.4

YES

SLS-B12

AA61HX-A1

RF

AA61HX

50.7

0.203

0.461

2.51%

2

1

0.9661

AA61HX-B1

DF

AA61HX

86.3

0.346

0.604

0.57%

3

3

0.9913

AA61HX-B2

DF

AA61HX

81.7

0.327

0.668

3.02%

3

3

0.9623

AA61HX-B3

DF

AA61HX

80.2

0.321

0.447

5.54%

5

3

0.9336

ECBC

FRAME

I-17

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
400, 272, 185, 126, 85.7,
58.3, 39.6, 27.0
400, 272, 185, 126, 85.7,
58.3, 39.6, 27.0

10

RF

2.15

YES

range finder

ppt in 2X C1SLS-P8ppt in 2X C1

1.47

YES

SLS-P26
SLS-P28

1.47

YES

SLS-P29

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

FAL.3T3.LP.A1.21.05.04

RF

AA61LP

85.9

0.344

0.266

1.47%

2

0

0.5809FAL.3T3.LP.B1.04.06.04

DF

AA61LP

99.1

0.397

0.415

9.90%

6

1

0.9314

FAL.3T3.LP.B2.17/06/04

DF

AA61LP

204

0.816

0.492

0.27%

3

1

0.9641

FAL.3T3.LP.B3.09.07.04

DF

AA61LP

106

0.425

0.408

0.31%

5

0

0.9552

FAL.3T3.LP.B4.14.10.04

DF

AA61LP

101

0.404

0.304

1.01%

3

0

0.9749FAL.3T3.LP.B5.15.10.04

DF

AA61LP

138

0.552

0.303

7.50%

4

0

0.9352

FAL.3T3.LP.B6.21.10.04

DF

AA61LP

NA

NA

0.284

10.97%

700.0000FAL.3T3.LP.B7.28.10.04

DF

AA61LP

91.8

0.368

0.211

3.94%

4

1

0.9658FAL.3T3.LP.B8.04.11.04

DF

AA61LP

97.9

0.392

0.329

2.47%

5

2

0.9464

A1

RF

AA61GL0.08730.0003

0.403

1.70%

5

1

0.9733B1

DF

AA61GL

0.101

0.0004

0.500

4.13%

6

2

0.9567B2

DF

AA61GL

0.136

0.0005

0.363

2.02%

5

3

0.9053B3DF

AA61GL0.08870.0003

0.444

0.43%

6

2

0.9577

AA61KK-A1

RF

AA61KK

0.102

0.00040.377

10.52%

5

00.9586

AA61KK-B1

DF

AA61KK

0.11

0.0004

0.659

9.97%

5

3

0.9666

AA61KK-B2

DF

AA61KK

0.07670.0003

0.412

3.79%

5

3

0.9698

AA61KK-B3

DF

AA61KK

0.187

0.0007

0.553

9.02%

4

4

0.9535

RF

AA61PF

1.89

0.0067

0.435

1.28%

4

2

0.9465

Exposure
Concentrations(ug/mL)7

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 680, 463, 315, 214,
146, 99.1, 67.4
500, 340, 231, 157, 107,
72.8, 49.6, 33.7
500, 340, 231, 157, 107,
72.8, 49.6, 33.7
500, 340, 231, 157, 107,
72.8, 49.6, 33.7
1000, 680, 463, 315, 214,
146, 99.1, 67.4
500, 413, 342, 282, 233,
193, 159, 132
250, 207, 171, 141, 117,
96.4, 79.7, 65.8
250, 207, 171, 141, 117,
96.4, 79.7, 65.8

Dilution

Acceptable

Factor8

Tests9

10

RF

1.47

YES

1.47

YES

1.47

NO

1.47

NO

1.47

NO

Rationale for
Unacceptability

Notes

range finder; nopoints 50 - 100%

PC ID

FAL.3T3.SLS.21.05.04
FAL.3T3.SLS.04.06.04
FAL.3T3.SLS.17.06.04

no points between
50 - 100%no points between
50 - 100%no points between
50 - 100%no points between
50 - 100%

FAL.3T3.SLS.09.07.04
FAL.3T3.SLS.14.10.04
outlier removed bySD

FAL.3T3.SLS.15.10.04

1.21

NO

1.21

YES

FAL.3T3.SLS.21.10.04

1.21

YES

10

RF

1.8

YES

1.8

YES

1.8

YES

10

RF

2.15

YES

SLS-P37

2.15

YES

SLS-P40

2.15

YES

SLS-P4110RFrange finder

FAL.3T3.SLS.10.09.04

3.15

NO

no points between
50 - 100%

FAL.3T3.SLS.16.09.04

3.15

YES

3.15

NO

3.15

YES

FAL.3T3.SLS.28.10.04
outlier removed bySD

FAL.3T3.SLS.04.11.04

CYCLOHEXIMIDE
IIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2.00, 1.11, 0.617, 0.343,
0.191, 0.106, 0.059,
0.033
2.00, 1.11, 0.617, 0.343,
0.191, 0.106, 0.059,
0.033
2.00, 1.11, 0.617, 0.343,
0.191, 0.106, 0.059,
0.033

range finder

SLS-A4SLS-B4
outlier removed bySD

SLS-B7
SLS-B8ECBC
10000,1000, 100, 10,1,0.1, 0.01, 0.001
3.00, 1.40, 0.649,
0.302,0.140, 0.065,
0.030, 0.014
3.00, 1.40, 0.649,
0.302,0.140, 0.065,
0.030, 0.014
3.00, 1.40, 0.649,
0.302,0.140, 0.065,
0.030, 0.014

range finder

SLS-P13FRAME
FAL.3T3.PF.A1.10.09.04
FAL.3T3.PF.B1.16.09.04

DF

AA61PF0.07960.0003

0.334

6.16%

8

0

0.9819FAL.3T3.PF.B2.15.10.04

DF

AA61PF

1.12

0.0040

0.333

0.40%

4

2

0.8800FAL.3T3.PF.28.10.04

DF

AA61PF0.009460.0000

0.272

2.42%

8

0

0.9126

FAL.3T3.PF.B4.04.11.04

DF

AA61PF

0.221

0.0008

0.282

7.83%

5

2

0.9566

I-18

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
465, 148, 46.9, 14.9, 4.7,
1.5, 0.476, 0.151
50.0, 15.9, 5.04, 1.60,
0.508, 0.161, 0.0512,
0.016250.0, 15.9, 5.04, 1.60,
0.508, 0.161, 0.0512,
0.016250.0, 15.9, 5.04, 1.60,
0.508, 0.161, 0.0512,
0.0162

FAL.3T3.SLS.15.10.04
no points between 0 outlier removed bySD
50%

FAL.3T3.SLS.28.10.04
FAL.3T3.SLS.04.11.04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

FAL.3T3.PF.B5.11.11.04

Assay Substance
ID
Type1

DF

AA61PF

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

IC50(ug/mL)

IC50(mM)

Mean VC

0.601

0.0021

0.266

5.33%

4

2

0.9235

OD2

R2 6

Exposure
Concentrations

Dilution

Acceptable

Factor8

Tests9

3.15

YES

10

RF

1.6

YES

ppt in 2X C1 and 1X C1SLS-B5

1.6

YES

ppt in 2X C1-C4; ppt in 1X
C1

SLS-B91.6

YES

ppt in 1X C1-C2

SLS-B10

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10RF

range finder(ug/mL)7

50.0, 15.9, 5.04, 1.60,
0.508, 0.161, 0.0512,
0.0162

Rationale for
Unacceptability

Notes

outlier removed bySD

PC ID

FAL.3T3.SLS.10.11.04

DIBUTYL PHTHALATEIIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 62.5, 39.1, 24.4,
15.3, 9.54, 5.96, 3.73
100, 62.5, 39.1, 24.4,
15.3, 9.54, 5.96, 3.73
100, 62.5, 39.1, 24.4,
15.3, 9.54, 5.96, 3.73

range finder

ppt in 2X C1-C2 and 1X C1SLS-A3
C2

A1

RF

AA61FD

13.5

0.048

0.371

2.38%

2

1

0.9701B1DF

AA61FD

19.5

0.070

0.474

7.57%

4

2

0.9692B2DF

AA61FD

20.4

0.073

0.393

4.01%

4

4

0.9786B3DF

AA61FD

22.2

0.080

0.338

1.43%

4

4

0.9749AA61JX-A1

RF

AA61JX

127

0.458

0.245

7.41%

0

2

0.9266

AA61JX-B1

DF

AA61JX

NA

NA

0.643

4.39%

NA

NA

NA

300, 204, 139, 94.4, 64.2,
43.7, 29.7, 20.2

1.47

NO

odd toxicity curve;
toxicity curve goes up at the
couldn't accurately
SLS-P44higher concentrations
calculate ICx values

AA61JX-B2

DF

AA61JX

NA

NA

0.551

4.45%

N/A

N/A

NA

300, 204, 139, 94.4, 64.2,
43.7, 29.7, 20.2

1.47

NO

odd toxicity curve;
toxicity curve goes up at the
couldn't accurately
SLS-P46higher concentrations
calculate ICx values

AA61JX-B3

DF

AA61JX

NA

NA

0.627

6.38%

0

8

NA

1.47

NO

PC failed; no points
between 0 - 50%

AA61JX-B4

DF

AA61JX

19.8

0.071

0.491

8.85%

5

3

0.84501.47

YES

SLS-P63ECBC

60.0, 40.8, 27.8, 18.9,
12.8, 8.74, 5.95, 4.05
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7

ppt in 2X C1 and 1X C1;
SLS-P10higher than usual blank OD

SLS-P60

AA61JX-B5

DF

AA61JX

27.7

0.099

0.442

3.39%

3

5

0.7470100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7

1.47

YES

1X C1-C4 have small
globules; highest conc. (C7, SLS-P67
C8) less toxicity than C3-C6

AA61JX-B6

DF

AA61JX

22.9

0.082

0.342

4.56%

4

3

0.9178100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7

1.47

YES

1X C1-C4 have small
globules; highest conc. (C7, SLS-P69
C8) less toxicity than C3-C4

RF

AA61MK

104

0.372

0.225

1.44%

1

1

0.7617

10

RF

FRAME
FAL.3T3.MK.A1.21.05.04
FAL.3T3.MK.B1.04.06.04

DF

AA61MK

306

1.100

0.429

4.08%

3

5

0.8027

FAL.3T3.MK.B2.17.06.04

DF

AA61MK

74.6

0.268

0.410

0.20%

5

3

0.9555

FAL.3T3.MK.B3.09.07.04

DF

AA61MK

190

0.683

0.304

0.47%

4

4

0.9592

FAL.3T3.MK.B4.25.11.04

DF

AA61MK

192

0.689

0.319

2.64%

3

5

0.9167

RF

AA61NP

8.66

0.039

0.341

83.42%

1

1

0.9677

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 680, 463, 315, 214,
146, 99.1, 67.4
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2000, 930, 433, 201,93.6, 43.5, 20.3, 9.42
2000, 930, 433, 201,93.6, 43.5, 20.3, 9.42

range finder

ppt in 1X C1-C2

FAL.3T3.SLS.21.05.04

ppt in 1X C1-C8 and 2X C1FAL.3T3.SLS.04.06.04
C2
ppt in 1X C1-C4 and 2X C1FAL.3T3.SLS.17.06.04
C2
ppt in 1X C1-C8 and 2X C1FAL.3T3.SLS.09.07.04
C2
ppt in 2X C1; ppt in 1X C1FAL.3T3.SLS.25.11.04
C3;

1.47

YES

2.15

YES

2.15

YES

2.15

YES

10

RF

1.8

YES

SLS-B1

1.8

YES

SLS-B2

DICHLORVOS
IIVS
A1
B1

DF

AA61NP

16.9

0.076

0.347

8.46%

3

5

0.9602B2DF

AA61NP

17.3

0.078

0.321

0.23%

3

3

0.9593I-19

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
70.0, 38.9, 21.6, 12.0,
6.67, 3.70, 2.06, 1.14
70.0, 38.9, 21.6, 12.0,
6.67, 3.70, 2.06, 1.14

range finder

VC1 Ods < VC2 ODs; VC1
removed from subsequent
analysis; volatility issues.

SLS-A3

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC ID

DF

AA61NP

20.7

0.093

0.366

4.92%

3

2

0.973370.0, 38.9, 21.6, 12.0,
6.67, 3.70, 2.06, 1.14

1.8

YES

AA61PZ-A1

RF

AA61PZ

NA

NA

0.121

98.38%

30NA1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10RF

range finder

AA61PZ-A2 (sealer)RF

AA61PZ

13.7

0.062

0.473

4.53%

0

5

0.946110RFrange finder

SLS-P43

AA61PZ-B1 (sealer)DF

AA61PZ

NA

NA

0.242

11.12%

3

4

NA

2.15

NO

PC failed

SLS-P50

AA61PZ-B2 (sealer)DF

AA61PZ

NA

NA

0.256

6.09%

4

4

NA

2.15

NO

PC failed

SLS-P52

AA61PZ-B3 (sealer)DF

AA61PZ

12.1

0.055

0.503

12.85%

2

5

0.9711

2.15

YES

AA61PZ-B4 (sealer)DF

AA61PZ

NA

NA

0.322

25.27%

2

5

NA

2.15

NO

AA61PZ-B5 (sealer)DF

AA61PZ

5.90

0.027

0.298

7.56%

3

4

0.9166

2.15

YES

AA61PZ-B6 (sealer)DF

AA61PZ

11.1

0.050

0.421

3.01%

3

4

0.9466

2.15

NO

B3

SLS-B3ECBC

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

probable volatility problem;
VC1 <<< VC2; higher thanusual blank OD

SLS-P10SLS-P54
PC failed; % VC
difference > 15

SLS-P56
SLS-P58

PC failed

SLS-P60
C1 conc. seems to interact
with NR; toxicity curve going
SLS-P62
in opposite direction at this
point

DF

AA61PZ

11.5

0.052

0.347

2.26%

2

5

0.9275100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

2.15

YES

FAL.3T3.HS.A1.21.05.04

RF

AA61HS

57.8

0.262

0.119

90.83%

2

00.1864

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10

RF

FAL.3T3.HS.B1.04.06.04

DF

AA61HS

35.0

0.158

0.371

5.60%

3

3

0.9832

1.47

YES

FAL.3T3.SLS.04.06.04

FAL.3T3.HS.B2.18.06.04

DF

AA61HS

30.9

0.140

0.685

1.85%

3

4

0.9772

1.47

YES

FAL.3T3.SLS.18.06.04

FAL.3T3.HS.B3.08.07.04

DF

AA61HS

32.5

0.147

0.209

11.98%

2

2

0.9328

1.47

YES

FAL.3T3.SLS.08.07.04

276

1.242

0.232

5.28%

1

1

0.1408

10

RF

1.8
1.8
1.8

AA61PZ-B7 (sealer)

FRAME

100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74

range finder; % VC
difference > 15; nopoints between 50 100%

volatility problem

FAL.3T3.SLS.21.05.04

DIETHYL PHTHALATE
IIVS
A1

RF

AA61NX

B1

DF

AA61NX

135

0.607

0.369

9.08%

3

2

0.9536B2

DF

AA61NX

97.1

0.437

0.338

6.11%

4

3

0.9853B3DF

AA61NX

87.1

0.392

0.342

4.96%

5

3

0.9870AA61GA-A1

RF

AA61GA

115

1.086

0.230

6.44%

1

1

0.9260AA61GA-B1

DF

AA61GA

119

0.536

0.323

6.29%

4

4

0.9776

AA61GA-B2

DF

AA61GA

68.1

0.306

0.324

4.70%

5

3

0.9414

AA61GA-B3

DF

AA61GA

69.5

0.313

0.552

0.35%

5

3

0.9527

FAL.3T3.KZ.A1.10.09.04

RF

AA61KZ

148

0.666

0.259

12.82%

1

20.7507

FAL.3T3.KZ.B1.16.09.04

DF

AA61KZ

176

0.791

0.239

15.05%

3

3

0.9712

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3

the solvent controls treatedwith 1% DMSO instead of
0.5%

SLS-A4YES

ppt in 2X C1-C2

SLS-B5YES

ppt in 2X C1-C2

SLS-B9YES

ppt in 2X C1-C3

SLS-B10

range finder

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 930, 433, 201,93.6, 43.5, 20.2, 9.4
2000, 930, 433, 201,93.6, 43.5, 20.2, 9.4
2000, 930, 433, 201,93.6, 43.5, 20.2, 9.4

10

RF

2.15

YES

range finder

SLS-P12SLS-P32

2.15

YES

SLS-P34

2.15

YES

SLS-P36

FRAME

I-20

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7

10

RF

2.15

YES

range finder

ppt in 2X C1

FAL.3T3.SLS.10.09.04

outlier removed bySD; ppt in
FAL.3T3.SLS.16.09.04
2X C1-C2

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

FAL.3T3.KZ.B2.15.10.04

DF

AA61KZ

160

0.720

0.244

1.62%

3

5

0.9759FAL.3T3.KZ.B3.28.10.04

DF

AA61KZ

104

0.469

0.185

4.87%

3

3

0.9759RF

AA61MF

310

0.398

0.350

0.21%

1

1

0.9022

Exposure
Concentrations(ug/mL)7

1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7

Dilution

Acceptable

Factor8

Tests92.15

YES

outlier removed by SD

FAL.3T3.SLS.15.10.04

2.15

YES

ppt in 2X C1-C2

FAL.3T3.SLS.28.10.04

10RF

ppt in 2X C1 and in 1X C1SLS-A3

ppt in 1X C1-C3 & 2X C1;
SD removed C1 & C2 from
PRISM to get Hill analysis;
ppt in 1X C1 and C2
caused upswing in the
toxicity curve

SLS-B5

Rationale for
Unacceptability

Notes

PC ID

DIGOXIN

IIVS
A1

B1

DF

AA61MF

269

0.344

0.427

6.41%

B2

DF

AA61MF

NA

NA

0.308

9.13%

B3

DF

AA61MF

NA

NA

NA

NA

B4

DF

AA61MF

365

0.467

0.296

2.70%

B5

DF

AA61MF

1500

1.925

0.335

4.20%

1

3

0.8853

0

3

NA

NA

NA

NA

0

4

0.54360

40.3342

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

1000, 588, 346, 204, 120,
70.4, 41.4, 24.4

1.7

YES

1.5

NO

NA

NO

1000, 556, 309, 171, 95,
53, 29.4, 16.3

1.8

YES

1000, 556, 309, 171, 95,
53, 29.4, 16.3

1.8

NO

400, 267, 178, 119, 79.0,
52.7, 35.1, 23.4
NArange finder

no points between 0 ppt in 1X C1SLS-B1350%
PC failed
SLS-B14
ppt in 2X C1 and 1X C1-C2;
SD removed C1 & C2 from
PRISM analyses; no points SLS-B15
left between 0-50% viability;
SD accepts test
ppt in 2X C1; ppt in 1X C1no points between 0 - C2; SD ends testing of
SLS-B1650%
chemical; solubility limits
have been reachedECBC
AA61PP-A1

RF

AA61PP

123

0.157

0.238

3.43%

1

4

0.8888

AA61PP-B1

DF

AA61PP

NA

NA

0.344

6.81%

0

6

NA

AA61PP-B2

DF

AA61PP

475

0.609

0.463

6.42%

2

3

0.8877

AA61PP-B3

DF

AA61PP

204

0.261

0.452

4.45%

5

3

AA61PP-B4

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
400, 272, 185, 126, 85.7,
58.3, 39.6, 27.0

10

RF

1.47

NO

400, 272, 185, 126, 85.7,
58.3, 39.6, 27.0

1.47

YES

0.6366

1000, 680, 463, 315, 214,
146, 99, 67

1.47

YES

1000, 680, 463, 315, 214,
146, 99, 67

1.47

YES

10

RF

2.15

YES

DF

AA61PP

373

0.478

0.452

10.52%

2

6

0.6692

FAL.3T3.HN.A1.27/05/04

RF

AA61HN

918

1.176

0.381

7.78%

1

0

0.6117

FAL.3T3.HN.B1.04/06/04
FAULT

DF

AA61HN

873

1.118

0.419

6.70%

1

2

0.8308ppt in 1X C1 and 2X C1;
SLS-P10higher than usual blank OD
no points between 0 - crystals in !x C1-C2; not like
SLS-P40
50%
NR crystals
crystals in 1X C1-C2; notlike NR crystals; C1 toxicity SLS-P42
less than C2
ppt in 2X C1; ppt in 1X C1C5 (large chemical crystals SLS-P72
in wells);
ppt in 2X C1-C2; ppt in 1X
SLS-P74
C1-C4
range finder

FRAME

FAL.3T3.HN.B2.18/06/04

RF

AA61HN

387

0.496

0.451

4.84%

NA

NA

0.53991000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
750, 347, 162, 75.0, 35.0,
16.3, 7.6, 3.5
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
1500, 698, 325, 151,
70.2, 32.7, 15.2, 7.1
2000, 930, 433, 201,93.6, 43.5, 20.3, 9.42

2.15

NO

2.15

NO

2.15

NO

FAL.3T3.HN.B3.09/07/04

DF

AA61HN

75900

97.141

0.317

1.92%

0

6

0.8417

FAL.3T3.HN.B4.16/07/04

DF

AA61HN

NA

NA

0.262

0.86%

0

4

0.3528FAL.3T3.HN.B5.17.09.04

DF

AA61HN

NA

NA

0.304

0.27%

0

4

NA

500, 340, 231, 157, 107,
72.8, 49.6, 33.7

1.47

NO

FAL.3T3.HN.B6.23.09.04

DF

AA61HN

582

0.745

0.310

3.38%

2

2

0.6844

1000, 680, 463, 315, 214,
146, 99.1, 67.4

1.47

YES

I-21

range finder; nopoints 50 - 100%

ppt in 1X C1-C3

FAL.3T3.SLS.27/05/04

ppt in 1X C1-C3; dilution
factor not provided

FAL.3T3.SLS.04.06.04

can't determine
ppt in 2X C1 and ppt in 1X
which points are true
C1-C3
toxicity points
no points between 0 - ppt in 2X C1 and ppt in 1X
50%
C1-C5
no points between 0 - ppt in 2X C1-C2 and ppt in
50%
1X C1-C4

FAL.3T3.SLS.18.06.04
FAL.3T3.SLS.09.07.04
FAL.3T3.SLS.16.07.04

problem with stimulation of
no points between 0 - NRU; toxicity increases thenFAL.3T3.SLS.17.09.04
50%
as conc. rises NRU also
rises & IC50 not reachedoutlier removed by SD; ppt
in 2X C1-C2; ppt in 1X C1C4

FAL.3T3.SLS.23.09.04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

FAL.3T3.HN.B7.14.10.04

Assay Substance
ID
Type1

DF

AA61HN

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

IC50(ug/mL)

IC50(mM)

Mean VC

1220

1.568

0.322

4.77%

1

7

0.4589OD2

R2 6

Exposure
Concentrations(ug/mL)7

1000, 680, 463, 315, 214,
146, 99.1, 67.4

Dilution

Acceptable

Factor8

Tests9

1.47

YES

10

RF

1.5

YES

SLS-B4

1.5

YES

SLS-B7

1.5

YES

SLS-B8

10

RF

1.47

YES

SLS-P42

1.47

YES

SLS-P43

1.47

YES

SLS-P45

Rationale for
Unacceptability

Notes

PC ID

outlier removed bySD; ppt in
FAL.3T3.SLS.14.10.04
2X C1-C3; ppt in 1X C1-C6

DIMETHYLFORMAMIDEIIVS
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
20000, 13333, 8889,
5926, 3951, 2634, 1756,
1171
20000, 13333, 8889,
5926, 3951, 2634, 1756,
1171
20000, 13333, 8889,
5926, 3951, 2634, 1756,
1171

A1

RF

AA61FN6870

93.990

0.392

1.04%

1

5

0.7331

range finder

SLS-A5B1DF

AA61FN

5060

69.196

0.485

3.39%

4

4

0.9915B2DF

AA61FN

4940

67.621

0.375

8.71%

4

4

0.9900B3DFAA61FN

4700

64.281

0.413

5.07%

4

4

0.9892

AA61MW-A1

RF

AA61MW

6410

87.717

0.522

4.15%

1

2

0.9137

AA61MW-B1

DF

AA61MW

4750

65.025

0.522

3.30%

4

4

0.9866

AA61MW-B2

DF

AA61MW

5680

77.639

0.697

1.26%

3

5

0.9610

AA61MW-B3

DF

AA61MW

5600

76.574

0.616

0.92%

4

4

0.9830RF

AA61KF

8990

123.050

0.315

7.39%

1

0

0.3085

10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001

10

RF

2.15

YES

FAL.3T3.SLS.10.11.04

2.15

NO

ppt in 2X C1; concentraton% VC difference >15 range may be off by factor of FAL.3T3.SLS.19.11.04
10; C1 probably 50000

2.15

YES

ppt in 2X C1

FAL.3T3.SLS.25.11.04

2.15

YES

ppt in 2X C1

FAL.3T3.SLS.26.11.04

10

RF

1.4

YES

SLS-B4

1.4

YES

SLS-B7

1.4

YES

SLS-B8ECBC
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348

range finder

SLS-15FRAME
FAL.3T3.KF.A1.21.10.04
FAL.3T3.KF.B1.11.11.04

DF

AA61KF

5180

70.8080.276

14.58%

4

2

0.964950000, 23256, 10817,
5031, 2340, 1088, 506,
236

FAL.3T3.KF.B2.18.11.04

DF

AA61KF

673

9.206

0.305

26.13%

3

20.9507

5000, 2326, 1082, 503,
234, 109, 50.6, 23.5

FAL.3T3.KF.B3.25.11.04

DF

AA61KF

6080

83.192

0.382

0.74%

2

3

0.9630FAL.3T3.KF.B4.26.11.04

DF

AA61KF

5190

70.971

0.381

9.84%

2

3

0.8958

0.013

0.448

3.20%

2

4

0.986250000, 23256, 10817,
5031, 2340, 1088, 506,
236
50000, 23256, 10817,
5031, 2340, 1088, 506,
236

range finder; nopoints between 50100%

FAL.3T3.SLS.21.10.04

DIQUAT DIBROMIDE MONOHYDRATE
IIVS
A1

RF

AA61GN

4.65

B1

DF

AA61GN

3.83

0.011

0.485

2.39%

3

4

0.9675B2

DF

AA61GN

6.04

0.017

0.353

0.24%

2

3

0.9379B3DF

AA61GN

6.31

0.017

0.442

3.25%

2

4

0.9544ECBC

I-22

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10.0, 7.14, 5.10, 3.64,
2.60, 1.86, 1.33, 0.949
10.0, 7.14, 5.10, 3.64,
2.60, 1.86, 1.33, 0.949
10.0, 7.14, 5.10, 3.64,
2.60, 1.86, 1.33, 0.949

range finder

SLS-A3

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

AA61KS-A1

RF

AA61KS

5.48

0.015

0.301

6.79%

2

1

0.9864

AA61KS-B1

DF

AA61KS

3.47

0.010

0.518

5.60%

4

3

0.9823

AA61KS-B2

DF

AA61KS

3.26

0.009

0.423

8.46%

4

3

0.9818

AA61KS-B3

DF

AA61KS

4.89

0.013

0.721

3.07%

5

3

0.9904

FAL.3T3.NV.A1.21.05.04

RF

AA61NV

9.05

0.025

0.484

4.80%

2

0

0.9320FAL.3T3.NV.B1.04.06.04

DF

AA61NV

76.7

0.212

0.468

11.19%

1

1

0.7598

Exposure
Concentrations(ug/mL)7

10000,1000, 100, 10,1,0.1, 0.01, 0.001
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

range finder

PC ID

10

RF

2.15

YES

SLS-P11SLS-P31

2.15

YES

SLS-P33

2.15

YES

SLS-P35

FRAME

FAL.3T3.NV.B2.18.06.04

DF

AA61NV

20.4

0.056

0.720

0.86%

8

0

0.9479FAL.3T3.NV.B3.08.07.04

DF

AA61NV

NA

NA

0.370

4.61%

6

0

NA

FAL.3T3.NV.B4.16.07.04

DF

AA61NV

11.1

0.031

0.384

6.76%

2

1

0.8922

RF

AA61FC

95.1

0.346

0.255

12.80%

2

1

0.4754

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 46.5, 21.6, 10.1, 4.7,
2.2, 1.01, 0.47
100, 31.6, 10.0, 3.2, 1.0,
0.3, 0.100, 0.032

10

RF

1.47

YES

1.21

YES

2.15

NO

3.16

YES

10

RF

1.6

YES

range finder

FAL.3T3.SLS.21.05.04
FAL.3T3.SLS.04.06.04
C8 % viability < 20; usedlowest dilution factor; passeven though not enoughpoints between 0-100%

no points between
50 - 100%

FAL.3T3.SLS.18.06.04

FAL.3T3.SLS.08.07.04
FAL.3T3.SLS.16.07.04

DISULFOTON
IIVS
A1
B1

DF

AA61FC

25.4

0.093

0.437

5.32%

5

3

0.9601B2DF

AA61FC

46.3

0.169

0.269

7.62%

3

4

0.9111B3DF

AA61FC

NA

NA

NA

NA

NA

NA

NA

B4

DF

AA61FC

138

0.504

0.294

0.57%

1

7

0.9243B5DF

AA61FC

31.8

0.116

0.259

11.99%

5

3

0.9540RF

AA61NY

NA

NA

0.247

1.18%

0

6

NA

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

range finder

the solvent controls weretreated with 1% DMSO,
rather than 0.5%;
ppt in 2X C1-C4; outlier
removed by SD

SLS-A4200, 125, 78.1, 48.8,
30.5, 19.1, 11.9, 7.45
200, 125, 78.1, 48.8,
30.5, 19.1, 11.9, 7.45
NA200, 125, 78.1, 48.8,
30.5, 19.1, 11.9, 7.45
200, 125, 78.1, 48.8,
30.5, 19.1, 11.9, 7.45

1.6

YES

NA

NO

1.6

YES

ppt in 2X C1SLS-B15

1.6

YES

ppt in 2x C1-C4

SLS-B16

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10RF

ppt in 2X C1-C2

SLS-P51

ppt in 2X C1-C4
PC failed

SLS-B5SLS-B13
SLS-B14ECBC
AA61NY-A1
AA61NY-B1

DF

AA61NY

155

0.564

0.379

1.45%

3

4

0.9199

AA61NY-B2

DF

AA61NY

NA

NA

0.356

4.07%

0

8

NA

AA61NY-B3

DF

AA61NY

54.6

0.199

0.398

0.71%

4

4

0.9654

AA61NY-B4

DF

AA61NY

201

0.734

0.406

2.30%

1

7

RF

AA61LC

1070

3.914

0.258

10.84%

03500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

2.15

YES

2.15

NO

1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

2.15

YES

0.8792

1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

2.15

YES

0.39891000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10

RF

FRAME
FAL.3T3.LC.A1.10.09.04

I-23

range finder; PC
failed; no points
between 0-50%

ppt in 2X C1-C4; ppt in 1X
C1-C3
no points between 0 - small pieces of chemical in50%
1X C1-C4
small globules in 1X C1-C5& 2X C1-C3; SD removed
C1 from PRISM analysis;
C1 toxicity < C2
small globules in 1X C1-C6;
ppt in 2X C1-C5; SDremoved C1 & C2 from
PRISM analysis; C1 & C2
toxicity < C3range finder; nopoints between 0 -50%

ppt in 1X C1SLS-P67
SLS-P69

SLS-P72

SLS-P74

FAL.3T3.SLS.10.09.04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

Dilution

Acceptable

Factor8

Tests9

2.15

YES

2.15

NO

2.15

NO

FAL.3T3.LC.B1.16.09.04

DF

AA61LC

11200

40.793

0.254

2.23%

1

6

0.8311

FAL.3T3.LC.B2.15.10.04

DF

AA61LC

NA

NA

0.257

1.49%

0

8

0.4810FAL.3T3.LC.B3.19.11.04

DF

AA61LC

NA

NA

0.260

13.68%

06

0.6459A1

RF

AA61HZ

1.3

0.003

0.366

49.45%

1

6

0.9673100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001

10

RF

B1

DF

AA61HZ

5.35

0.013

0.397

1.30%

3

5

0.920730.0, 16.7, 9.26, 5.14,
2.86, 1.59, 0.882, 0.490

1.8

YES

B2

DF

AA61HZ

13.6

0.033

0.261

20.27%

3

3

0.9195

1.8

NO

B3

DF

AA61HZ

NA

NA

NA

NA

NA

NA

NA

NA

NO

2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

Rationale for
Unacceptability

Notes

PC ID

outlier removed bySD; ppt in
1X C1 and 2X C1-C5; IC50
FAL.3T3.SLS.16.09.04
out of synch with otherIC50s
no points between 0 ppt in 1X C1-C6;
50%
no points between 0 - ppt in 1X C1-C6; ppt in 2X
50%
C1-C3

FAL.3T3.SLS.15.10.04
FAL.3T3.SLS.19.11.04

ENDOSULFAN
IIVS

B4

DF

AA61HZ

1.64

0.004

0.302

42.29%

6

2

0.7300B5DFAA61HZ

2.52

0.006

0.256

3.03%

6

2

0.6745B6DF

AA61HZ

2.95

0.007

0.256

14.77%

5

2

0.7624

RF

AA61LG

NA

NA

0.237

18.26%

22NA

50.0, 27.8, 15.4,8.57,
4.76, 2.65, 1.47, 0.817
NA50.0, 27.8, 15.4,8.57,
4.76, 2.65, 1.47, 0.817
50.0, 27.8, 15.4,8.57,
4.76, 2.65, 1.47, 0.817
50.0, 27.8, 15.4,8.57,
4.76, 2.65, 1.47, 0.817

range finder

VC1 Ods < VC2 ODs; VC1
removed from subsequent
analysis; ppt in 2X C1 and1X C1;volatility issues

SLS-A2

ppt in 2X C1; outlier
SLS-B5
removed by SD; plate sealer% VC difference >15
PC failed% VC difference >15

ppt in 2X C1; very high OD
value in VC1

SLS-B13

SLS-B14
ppt in 2X C1-C2; low ODs in
VC1; used VC2 value for
SLS-B15
viability calculations

1.8

NO

1.8

YES

ppt in 2X C1-C2

SLS-B16

1.8

YES

ppt in 2X C1-C2

SLS-B1710RF

ppt in 2X C1SLS-P51

2.15

NO

ppt in 2X C1SLS-P55

2.15

NO

ppt in 2X C1SLS-P56

2.15

YES

2.15

YES

2.15

YES

10

RF

2.15

ECBC
AA61LG-A1
AA61LG-B1

DF

AA61LG

NA

NA

0.383

25.75%

5

2

NA

AA61LG-B2 (sealer)DF

AA61LG

NA

NA

0.445

5.26%

7

1

NA

AA61LG-B3 (sealer)DF

AA61LG

4.15

0.010

0.217

8.65%

3

5

0.9066

AA61LG-B4 (sealer)DF

AA61LG

2.98

0.007

0.319

13.07%

3

5

0.8831

AA61LG-B5 (sealer)DF

AA61LG

8.68

0.021

0.338

4.57%

2

6

0.9264

FAL.3T3.PW.A1.01/04/04

RF

AA61PW

52500

128.974

0.209

16.91%

02

0.3175

FAL.3T3.PW.B1.29/04/04(should be A2)

DF

AA61PW

0.249

0.001

0.261

24.71%

2

5

0.4825

FAL.3T3.PW.B2.29/04/04(should be B1)

DF

AA61PW

22.9

0.056

0.241

29.31%

2

6

0.3954

FAL.3T3.PW.B2.20/05/04

DF

AA61PW

32.7

0.080

0.324

10.51%

1

7

0.3827

FAL.3T3.PW.B3.27/05/04

DF

AA61PW

6.47

0.016

0.444

1.54%

6

2

0.7075

FAL.3T3.PW.B4.17/06/04

DF

AA61PW

11.2

0.028

0.396

5.49%

7

1

0.7541

FAL.3T3.PW.B5.24/06/04

DF

AA61PW

10.4

0.026

0.408

5.45%

1

6

0.8455100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
80.0, 37.2, 17.3, 8.05,
3.74, 1.74, 0.81, 0.38
60.0, 27.9, 13.0, 6.04,
2.81, 1.31, 0.61, 0.28
30.0, 14.0, 6.49, 3.02,
1.40, 0.65, 0.30, 0.14
30.0, 14.0, 6.49, 3.02,
1.40, 0.65, 0.30, 0.14
30.0, 14.0, 6.49, 3.02,
1.40, 0.65, 0.30, 0.14

range finder; PC
failed; % VC
difference > 15
% VC difference >15
PC failed

SLS-P58
SLS-P63
ppt in 2X C1-C3

SLS-P64

range finder

possible volatility problem

FAL.3T3.SLS.01/04/04

NO%VC difference > 15

NR crystals; highbackground

FAL.3T3.SLS.29/04/04

1.47

NO

%VC difference > 15 possible volatility problem

1.47

YES

FAL.3T3.SLS.20/05/04

1.47

YES

FAL.3T3.SLS.27/05/04

1.47

YES

FAL.3T3.SLS.17.06.04

2.15

YES

FAL.3T3.SLS.24.06.04

FRAME

I-24

10.0, 1.0, 0.1, 0.01,
0.001, 0.0001, 0.00001,
0.000001,
10.0, 4.65, 2.16, 1.01,
0.468, 0.218, 0.101,
0.047
50.0, 34.0, 23.1, 15.7,
10.7, 7.3, 5.0, 3.4
50.0, 34.0, 23.1, 15.7,
10.7, 7.3, 5.0, 3.4
50.0, 34.0, 23.1, 15.7,
10.7, 7.3, 5.0, 3.4100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

FAL.3T3.SLS.07/05/04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC ID

EPINEPHRINE BITARTRATE
IIVS
A1

RF

AA61LT

34.4

0.103

0.460

5.31%

0

2

0.9689B1

DF

AA61LT

61.8

0.185

0.429

3.46%

1

6

0.8482B2DF

AA61LT

65.5

0.196

0.413

3.71%

0

6

0.8365

B3DF

AA61LT

62.8

0.188

0.388

2.42%

2

6

0.8693

AA61HW-A1

RF

AA61HW

25.4

0.076

0.280

0.36%

2

1

0.9466

AA61HW-B1

DF

AA61HW

58.5

0.175

0.682

5.06%

1

6

0.8963

AA61HW-B2

DF

AA61HW

46.8

0.140

0.582

3.32%

2

6

0.9135

AA61HW-B3

DF

AA61HW

49.3

0.148

0.440

2.56%

1

6

0.9306

RF

AA61RK

37.2

0.112

0.361

17.45%

300.8041

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49,

10

RF

1.4

YES

range finder

SLS-A2
SLS-B1

100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49,

1.4

YES

SMT accepts this test in
spite of no points between 0- SLS-B2
50%; agreed to on 8/12/04

100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49,

1.4

YES

SLS-B3

ECBC
10000,1000, 100, 10,1,0.1, 0.01, 0.001
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74

10

RF

1.47

YES

range finder

SLS-P5SLS-P23

1.47

YES

SLS-P27

1.47

YES

SLS-P2910RFrange finder

2.15

NO

no points between
50 - 100%

1.21

YES

1.21

YES

1.21

YES

10

RF

FRAME
FAL.3T3.RK.A1.01/04/04

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
200, 165, 137, 113, 93.3,
77.1, 63.7, 52.7
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3

FAL.3T3.RK.B1.29/04/04

DF

AA61RK

79.4

0.238

0.349

2.51%

1

0

0.9283

FAL.3T3.RK.B2.06/05/04

DF

AA61RK

70.5

0.211

0.341

4.84%

2

1

0.9573

FAL.3T3.RK.B3.20/05/04

DF

AA61RK

62.2

0.187

0.407

6.36%

1

0

0.9364

FAL.3T3.RK.B4.27/05/04

DF

AA61RK

57.4

0.172

0.490

12.09%

2

1

0.8531RF

AA61FH

NA

NA

0.416

0.36%

0

8

NA

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

0.9319100000, 71429, 51020,
36443, 26031, 18593,
13281, 9486

100000, 71429, 51020,
36443, 26031, 18593,
13281, 9486

possible volatility problem

FAL.3T3.SLS.01/04/04
FAL.3T3.SLS.29/04/04
FAL.3T3.SLS.06/05/04

lowest dilution factor used;
SMT will accept this test

FAL.3T3.SLS.20/05/04
FAL.3T3.SLS.27/05/04

ETHANOL
IIVS
A2

B1

DF

AA61FH

12500

270.7580.154

83.09%

4

1

B2

DF

AA61FH

7140

155.089

0.400

8.34%

4

0

0.9518B3DF

AA61FH

5200

112.871

0.388

2.54%

8

0

0.8605B4DF

AA61FH

6760

146.751

0.384

4.75%

6

2

0.8518B5DF

AA61FH

NA

NA

NA

NA

NA

NA

NA

B6

DF

AA61FH

6070

131.699

0.458

1.89%

4

4

0.9316B7DF

AA61FH

6410

139.182

0.322

8.21%

4

3

0.9515I-25

20000, 16667, 13889,
11574, 9645, 8038, 6698,
5582
20000, 16667, 13889,
11574, 9645, 8038, 6698,
5582
NA
20000, 14286, 10204,
7289, 5206, 3719, 2656,
1897
20000, 14286, 10204,
7289, 5206, 3719, 2656,
1897

range finder

SLS-A3NO% VC difference >15

VC1 ODs < VC2 ODs; VC1
removed from subsequent
analysis; volatility issues.

1.4

NO

no points between
50--100%

ppt in 1X C1-C3; plates read
15-16 hr late; orignial
reading used wrong OD
SLS-B11wavelength; outliers
removed by SD; plate sealer
used

1.2

NO

no points between
50--100%;

plate sealer used

SLS-B12

1.2

YES

plate sealer used; outliers
removed bySD

SLS-B13

NA

NO

1.4

YES

1.4

YES

1.4

PC failed

SLS-B6

SLS-B14
SLS-B15
plate sealer used; outliers
removed by SD

SLS-B16

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests910RFrange finder

no toxicity detected

SLS-P610RFrange finder

probable volatility problem;
VC1 <<< VC2

SLS-P8

1.47

NO% VC difference >15

volatility problem

SLS-P37

1.47

NO

no points between
50 - 100%

1.21

NO

no points between
50 - 100%

Rationale for
Unacceptability

Notes

PC ID

ECBC
AA61JU-A1

RF

AA61JU

NA

NA

0.322

0.67%

0

2

NA

AA61JU-A2

RF

AA61JU

NA

NA

0.193

57.54%

2

2

NA

AA61JU-B1(sealer)DF

AA61JU

NA

NA

0.134

49.27%

6

1

NA

AA61JU-B2 (sealer)DF

AA61JU

NA

NA

0.255

10.33%

4

0NA

AA61JU-B3(sealer)DF

AA61JU

NA

NA

0.218

19.55%

80NA

AA61JU-B4 (sealer)DF

AA61JU

NA

NA

0.234

15.04%

7

0NA

AA61JU-B5 (sealer)DF

AA61JU

NA

NA

0.250

9.95%

7

1

NA

AA61JU-B6 (sealer)DF

AA61JU

5400

117.107

0.556

6.34%

3

5

0.8953

AA61JU-B7 (sealer)DF

AA61JU

6300

136.6410.478

17.05%

3

5

0.9477

AA61JU-B8 (sealer)DF

AA61JU

4860

105.580

0.389

3.72%

3

5

0.9188AA61JU-B9 (sealer)DF

AA61JU7310

158.702

0.416

6.29%

2

6

0.8826

AA61JU-B10 (sealer)DF

AA61JU

3910

84.836

0.393

5.15%

4

4

0.9316

RF

AA61PC

NA

NA

0.224

10.66%

01

0.000010000,1000, 100, 10,1,0.1, 0.01, 0.001
100000, 10000, 1000,
100, 10, 10, 0.1, 0.01
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
68027, 46277, 31481,
21416, 14568, 9910,
6742, 4586
40000, 33058, 27321,
22579, 18660, 15422,
12745, 10533
30000, 24793, 20490,
16934, 13995, 11566,
9559, 7900
20000, 16529, 13660,
11289, 9330, 7711, 6373,
5267
15000, 10204, 6942,
4722, 3212, 2185, 1487,
1011
15000, 10204, 6942,
4722, 3212, 2185, 1487,
1011
15000, 10204, 6942,
4722, 3212, 2185, 1487,
1011
15000, 10204, 6942,
4722, 3212, 2185, 1487,
1011
15000, 10204, 6942,
4722, 3212, 2185, 1487,
1011

SLS-P39
SLS-P47
dilution factor is 1.21; nopoints between 50-100%;
test would pass due todilution factor

1.21

NO

PC failed

1.21

NO

PC failed

SLS-P52

1.47

NO

PC failed

SLS-P60

1.47

NO% VC difference >15

SLS-P62

1.47

YES

SLS-P64

1.47

YES

SLS-P66

1.47

YES

SLS-P6810RF

range finder

FAL.3T3.SLS.30/04/04

1.47

NO

no points between 0 50%; %VC
difference > 15

FAL.3T3.SLS.06/05/04

1.47

NO

%VC difference >15; volatility problem

FAL.3T3.SLS.20/05/04

1.47

NO

%VC difference >15; volatility problem

FAL.3T3.SLS.27/05/04

1.47

NO

no points between 0 100%

FAL.3T3.SLS.17.06.04

1.47

YES

FAL.3T3.SLS.24.06.04

1.47

YES

FAL.3T3.SLS.08.07.04

1.47

YES

FAL.3T3.SLS.16.07.04

SLS-P50

FRAME
FAL.3T3.PC.A1.30/04/04
FAL.3T3.PC.B1.06/05/04

DF

AA61PC

NA

NA

0.190

26.31%

05

0.7166

FAL.3T3.PC.B2.20/05/04

DF

AA61PC

14200

308.732

0.223

34.53%

3

3

0.8898FAL.3T3.PC.B2.27/05/04
should be B3

DF

AA61PC

8300

180.1280.412

19.58%

4

3

0.9538FAL.3T3.PC.B4.17/06/04

DF

AA61PC

44000

954.073

0.462

10.31%

00

0.9212

FAL.3T3.PC.B5.24/06/04

DF

AA61PC

7110

154.377

0.311

6.43%

6

2

0.9785

FAL.3T3.PC.B6.08.07.04

DF

AA61PC

9480

205.8650.234

14.05%

4

4

0.8796

FAL.3T3.PC.B7.16.07.04

DF

AA61PC

8670

188.184

0.308

13.82%

4

4

0.9668

I-26

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
50000, 34014, 23139,
15741, 10708, 7284,
4955, 3371
50000, 34014, 23139,
15741, 10708, 7284,
4955, 3371
50000, 34014, 23139,
15741, 10708, 7284,
4955, 3371
50000, 34014, 23139,
15741, 10708, 7284,
4955, 3371
50000, 34014, 23139,
15741, 10708, 7284,
4955, 3371
40000, 27211, 18511,
12592, 8566, 5827, 3964,
2697
40000, 27211, 18511,
12592, 8566, 5827, 3964,
2697

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9

10

RF

1.78

YES

SLS-B1

1.78

YES

SLS-B2

1.78

YES

SLS-B310RFrange finder

SLS-P110RFNo points between
10 and 50%; r2 <0.8; PC failed; range
finderSLS-P3

1.47

NO

VC difference >15%: PC failed

SLS-P4

1.47

YES

SLS-P5

1.47

YES

SLS-P7

1.47

YES

SLS-P9

1.47

NO

1.47

YES

Rationale for
Unacceptability

Notes

PC ID

ETHYLENE GLYCOL
IIVS
A1 PreliminaryRF

AA61HR

15700

252.899

0.430

9.87%

0

1

0.5803B1

DF

AA61HR

27100

436.534

0.489

7.90%

2

2

0.9878B2DFAA61HR

22400

360.825

0.505

4.97%

2

3

0.9713B3DF

AA61HR

28200

454.253

0.573

5.77%

2

5

0.9449RF

AA61LM

13000

209.4070.288

17.62%

03

0.05128

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100000, 56250, 31600,
17800, 10000, 5600,
3160, 1770
100000, 56250, 31600,
17800, 10000, 5600,
3160, 1770
100000, 56250, 31600,
17800, 10000, 5600,
3160, 1770

range finder

SLS-A1ECBC
ECBC-3T3-Ib-01
A1

AA61LM-

ECBC-3T3-Ib-02
AA61LM-A2

RF

AA61LM

18000

289.9480.238

13.45%

03

0.7979ECBC-3T3-Ib-03
AA61LM-B1

DF

AA61LM

21200

341.495

0.408

19.53%

3

2

0.9087

ECBC-3T3-Ib-04
AA61LM-B2

DF

AA61LM

19200

309.278

0.839

4.60%

3

3

0.9718ECBC-3T3-Ib-05
AA61LM-B3

DF

AA61LM

16100

259.343

0.445

8.06%

3

3

0.9290

ECBC-3T3-Ib-06
AA61LM-B4

DF

AA61LM

19900

320.554

0.554

2.47%

3

3

0.9186

ECBC-3T3-Ib-07
AA61LM-B5

DF

AA61LM

16500

265.7860.480

16.31%

3

3

0.9611

ECBC-3T3-Ib-08
AA61LM-B6

DF

AA61LM

18100

291.559

0.529

1.25%

3

3

0.9695

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100000, 68000, 46300,
31500, 21400, 14600,
9910, 6740
100000, 68000, 46300,
31500, 21400, 14600,
9910, 6740
100000, 68000, 46300,
31500, 21400, 14600,
9910, 6740
100000, 68000, 46300,
31500, 21400, 14600,
9910, 6740
100000, 68000, 46300,
31500, 21400, 14600,
9910, 6740
100000, 68000, 46300,
31500, 21400, 14600,
9910, 6740

VC difference > 15%

SLS-P12SLS-P13

FRAME
A1

1b3T3RF01FALPD

RF

AA61PD

NA

NA

0.527

11.89%

0

0

NA

A2

1b3T3RF02FALPD

RF

AA61PD

34800

560.567

0.449

6.05%

1

0

0.9623

1b3T3DF01FALPD

DF

AA61PD

34200

550.902

0.443

1.22%

2

3

0.9645

1b3T3DF02FALPD

DF

AA61PD

36500

587.9510.612

12.90%

2

5

0.9340

1b3T3DF03FALPD

DF

AA61PD

40500

652.3840.306

12.08%

1

4

0.8911

1b3T3DF04FALPD

DF

AA61PD

27200

438.1440.489

11.17%

2

5

0.9232

1b3T3DF05FALPD

DF

AA61PD

41700

671.714

0.463

6.48%

1

5

0.9483

1b3T3DF06FALPD

DF

AA61PD

23600

380.1550.557

13.34%

4

3

0.8834

I-27

985, 98.5, 9.9, 1.0, 0.1,
0.0099, 0.0010, 0.0001
263510, 52702, 10540.4,
2108.1, 421.6, 84.3, 16.9,
3.4
182500, 124150, 85460,
57450, 39080, 26590,
18090, 12304
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
85300, 58027, 39474,
26853, 18268, 12427,
8454, 5751
100000, 68100, 46100,
31500, 21400, 14600,
9900, 6700
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742

RFrange finder

1b3T3CRTFALSLS
12/4/02

NO

no points between
50 anad 100%

1b3T3CRTFALSLS
12/10/02

NO

PC failed

1b3T3CRTFALSLS
12/17/02

YESNO

NR crystals in plate
NR crystals in plate;
stopped after 1 h;
PC failed

YES

1b3T3CRTFALSLS
1/8/03
1b3T3CRTFALSLS
1/14/03

YES
NO

1b3T3CRTFALSLS
1/7/03

PC failed

1b3T3CRTFALSLS
1/15/03
1b3T3CRTFALSLS
1/21/03

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

1b3T3DF07FALPD

Assay Substance
ID
Type1

DF

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

IC50(ug/mL)

IC50(mM)

Mean VC

AA61PD

39300

633.054

0.281

12.56%

230.8509OD2

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9100000, 68100, 46100,
31500, 21400, 14600,
9900, 6700

Rationale for
Unacceptability

Notes

PC ID

1b3T3CRTFALSLS
2/26/03YES

FENPROPATHRIN
IIVS
A1

RF

AA61HY

15.3

0.044

0.359

2.00%

2

6

0.9682B1DF

AA61HY

17.7

0.051

0.454

5.40%

4

4

0.9881B2DF

AA61HY

18.1

0.05

0.362

1.43%

4

3

0.9827B3DF

AA61HY

14.4

0.04

0.371

0.16%

5

3

0.9848

AA61LJ-A1

RF

AA61LJ

29.5

0.084

0.290

2.90%

2

2

0.8956

AA61LJ-B1

DF

AA61LJ

20.3

0.058

0.316

1.48%

6

2

AA61LJ-B2

DF

AA61LJ

22.3

0.064

0.254

6.77%

3

AA61LJ-B3

DF

AA61LJ

25.1

0.072

0.471

3.22%

FAL.3T3.PT.A1.080104

RF

AA61PT

142

0.405

0.407

FAL3T3.PT.A2.15-01-04

DF

AA61PT

54.7

0.157

FAL3T3.PT.B1.22-01-04

DF

AA61PT

59.7

FAL3T3.PT.B2.29-01-04

DF

AA61PT

69.0

FAL3T3.PT.B3.05.02.04

DF

AA61PT

21.6

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 62.5, 39.1, 24.4,
15.3, 9.54, 5.96, 3.73
100, 62.5, 39.1, 24.4,
15.3, 9.54, 5.96, 3.73
100, 62.5, 39.1, 24.4,
15.3, 9.54, 5.96, 3.73

range finder

ppt in 2X C1-C2 and 1X C1SLS-A1
C2
ppt in 2X C1-C3; ppt in 1X
SLS-B5
C1-C3
ppt in 2X C1-C4; ppt in 1X
SLS-B9C1-C3
ppt in 2X C1-C4; ppt in 1X
SLS-B10
C1-C2

10

RF

1.6

YES

1.6

YES

1.6

YES

1000, 100,10, 1, 0.1,
0.01, 0.001, 0.0001

10RF

0.9404150, 102, 69.4, 47.2,
32.1, 21.9, 14.9, 10.1

1.47

YES

4

0.937960.0, 40.8, 27.8, 18.9,
12.9, 8.7, 6.0, 4.1

1.47

YES

2

5

0.927460.0, 40.8, 27.8, 18.9,
12.9, 8.74, 5.95, 4.05

1.47

YES

9.41%

1

4

0.663910RFrange finder

0.386

2.12%

5

3

0.92032.15

NO

PC failed;

0.171

0.310

1.66%

5

3

0.8978

2.15

YES

well B7 outlier; no cells; not
FAL.3T3.SLS.080104
removed by SD
ppt in 1X C1-C5; ppt in 2X
FAL.3T3.SLS.15/01/04
C1-C5
ppt in 1X C1-C6 and 2X C1FAL.3T3.SLS.22/01/04
C5

0.198

0.362

7.84%

3

2

0.9594

2.15

YES

0.062

0.259

4.89%

4

1

0.9415

2.15

NO

PC failed;

2.15

YES

ppt in 1X C1-C2; ppt in 2X
FAL3T3.SLS.29-01-04
C1-C4
problem with reservoir
liners; ppt in 1X C1-C5 & 2X FAL.3T3.SLS.5/02/04
C1-C4
ppt in 1X C1-C3 & 2X C1FAL3T3.SLS.25.02.04
C2

2.15

YES

10

RF

range finderNO

no points between 050%

ECBC
range finder

ppt in 2X C1-C2 and 1X C1SLS-P2
C2
slope & IC50 similar for B1,
B2, and B3; ppt does not
SLS-P6appear to be a factor;ppt in2X C1-C3; ppt in 1X C1-C3
slope & IC50 similar for B1,
B2, and B3; ppt does not
SLS-P7
appear to be a factor;ppt in2X C1-C3
slope & IC50 similar for B1,
B2, and B3; ppt does not
SLS-16appear to be a factor

FRAME

FAL3T3.PT.B4.25-02-04

DF

AA61 PT

29.8

0.085

0.523

3.33%

4

2

0.9173

FAL3T3.PT.B5.17.03.04

DF

AA61PT

10.9

0.031

0.238

10.23%

3

3

0.8792

AA61RE

NA

NA

0.403

3.68%

0

4

NA

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 680, 465, 216, 100,
46.5, 21.6, 10.1
1000, 680, 465, 216, 100,
46.5, 21.6, 10.1
500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

ppt in 1X C1-C4 & 2X C1C4

FAL.3T3.SLS.17/03/04

GIBBERELLIC ACID
IIVS
A1
B1

B2

RF
DF

DF

AA61RE

AA61RE

13300

7830

38.32222.618

0.557

0.457

0.22%

1.39%

0

1

8

7

0.41820.9631

I-28

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
5000, 3846, 2959, 2276,
2276, 1751, 1347, 1036,
797
10000, 7692, 5917, 4552,
3501, 2693, 2072, 1594

1.3

1.3

YES

SLS-A4SLS-B6
plates read 15-16 hr late;orignial reading used wrong
SLS-B11
OD wavelength; ppt in 2X
C1-C3

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations

Dilution

Acceptable

Factor8

Tests910000, 7692, 5917, 4552,
3501, 2693, 2072, 1594

1.3

YES

(ug/mL)7

Rationale for
Unacceptability

Notes

PC ID

ppt in 2X C1-C4; outlier
removed bySD because well
SLS-B12didn't receive 50 ul of growth
medium during refeeding

B3

DF

AA61RE

6840

19.745

0.340

7.57%

2

6

0.9288B4DF

AA61RE

NA

NA

NA

NA

NA

NA

NA

NA

NA

NO

10000, 7692, 5917, 4552,
3501, 2693, 2072, 1594

1.3

YES

10

RF

range finder

SLS-P14

1.21

NO

no points between 0 50%

SLS-P47

1.47

YES

ppt in 2X C1SLS-P65

1.47

YES

ppt in 2X C1-C4; ppt in 1X
C1

SLS-P67

1.47

YES

ppt in 2X C1-C4

SLS-P69

B5

DF

AA61RE

8300

23.958

0.413

2.36%

1

7

0.8974RF

AA61FR

NA

NA

0.472

0.90%

0

7

NA

PC failed

SLS-B14
ppt in 2X C1-C3

SLS-B15ECBC
AA61FR-A1
AA61FR-B1
AA61FR-B2

DFDF

AA61FR
AA61FR

NA
9020

NA
26.028

0.385
0.430

3.16%
3.16%

0
1

8
7

NA
0.8611

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
5000, 4132, 3415, 2822,
2333, 1928, 1593, 1317
10000, 6803, 4628, 3148,
2142, 1457, 991, 674
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348

AA61FR-B3

DF

AA61FR

7820

22.566

0.436

1.89%

2

5

0.9515

AA61FR-B4

DF

AA61FR

7240

20.914

0.356

2.99%

3

4

0.9605

FAL.3T3.GY.A1.10.09.04

RF

AA61GY

78.3

0.226

0.293

5.52%

2

0

0.90081000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10

RF

FAL.3T3.GY.B1.16.09.04

DF

AA61GY

NA

NA

0.317

23.98%

000.00001000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7

2.15

NO

FAL.3T3.GY.B2.15.10.04

DF

AA61GY

NA

NA

0.286

4.02%

0

4

0.00001.47

NO

FAL.3T3.GY.B3.25.11.04

DF

AA61GY

NA

NA

0.342

6.74%

0

2

0.0000

1.47

NO

A1

RF

AA61NN

80.5

0.371

0.294

7.00%

2

6

0.9499B1DF

AA61NN

139

0.640

0.374

6.19%

3

5

0.9421B2DF

AA61NN

119

0.548

0.263

2.36%

4

4

0.9536B3DF

AA61NN

NA

NA

NA

NA

NA

NA

NA

B4

DF

AA61NN

122

0.561

0.350

9.49%

4

4

0.9580B5DFAA61NN

121

0.558

0.339

0.00%

4

4

0.9484

AA61FE-A1

RF

AA61FE

256

1.177

0.486

2.55%

1

7

0.9256

AA61FE-B1

DF

AA61FE

160

0.736

0.605

10.75%

5

3

0.9842

AA61FE-B2

DF

AA61FE

174

0.800

0.575

4.13%

5

3

0.9784

AA61FE-B3

DF

AA61FE

167

0.767

0.256

3.42%

5

3

0.9456

RF

AA61KY

508

2.339

0.227

5.39%

1

1

0.8073

FRAME

5000, 3401, 2314, 1574,
1071, 728, 496, 337
5000, 3401, 2314, 1574,
1071, 728, 496, 337

range finder; nopoints between 50 100%no points between 0 100%; %VC
difference >15
no points between 0 outlier removed by SD
50%
no points between 0 50%

FAL.3T3.SLS.10.09.04
FAL.3T3.SLS.16.09.04
addendum lists incorrect
PC
FAL.3T3.SLS.15.10.04
FAL.3T3.SLS.25.11.04

GLUTETHIMIDE
IIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
500, 313, 195, 122, 76.3,
47.7, 29.8, 18.6
500, 313, 195, 122, 76.3,
47.7, 29.8, 18.6NA500, 313, 195, 122, 76.3,
47.7, 29.8, 18.6
500, 313, 195, 122, 76.3,
47.7, 29.8, 18.6

10

RF

1.6

YES

range finder

ppt in 2X C1SLS-A5

1.6

YES

NA

NO

1.6

YES

SLS-B15

1.6

YES

SLS-B17

SLS-B6outliers removed bySD
PC failed

SLS-B13
SLS-B14

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 680, 463, 315, 214,
146, 99, 67
1000, 680, 463, 315, 214,
146, 99, 67
1000, 680, 463, 315, 214,
146, 99, 67

10

RF

1.47

YES

1.47

YES

1.47

YES

10

RF

range finder

SLS-16
ppt in 2X C1-C2; appear
oily
ppt in 2X C1-C2; appear
oily

SLS-P44SLS-P46
SLS-P48

FRAME
FAL.3T3.KY.A1.21.10.04

I-29

1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001

range finder

ppt in 2X C1

FAL.3T3.SLS.21.10.04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

FAL.3T3.KY.B1.11.11.04

DF

AA61KY

303

1.396

0.268

4.20%

3

5

0.9424

FAL.3T3.KY.B2.19.11.04

DF

AA61KY

262

1.208

0.207

3.18%

2

5

0.9086

FAL.3T3.KY.B3.25.11.04

DF

AA61KY

288

1.327

0.350

10.56%

2

5

0.7829A1

RF

AA61JF

NA

NA

0.402

3.00%

0

4

0.5520B1DFAA61JF

38200

414.75

0.453

1.93%

3

5

0.9665

Exposure
Concentrations(ug/mL)7

1000, 680, 463, 315, 214,
146, 99.1, 67.4
1000, 680, 463, 315, 214,
146, 99.1, 67.4
1000, 680, 463, 315, 214,
146, 99.1, 67.4

Dilution

Acceptable

Factor8

Tests91.47

YES

ppt in 2X C1

FAL.3T3.SLS.10.11.04

1.47

YES

ppt in 2X C1

FAL.3T3.SLS.19.11.04

1.47

YES

10

RF

1.4

YES

SLS-B6

1.4

YES

plates read 15-16 hr late;orignial reading used wrong SLS-B11
OD wavelength

1.4

YES

outlier removed bySD

10

RF

range finder

SLS-P110RFrange finder

SLS-P3

1.47

YES

SLS-P17

1.47

YES

SLS-P23

1.47

NO

1.47

YES

10

RF

no points between 0straight line; no toxicity
50; range finder

FAL.3T3.SLS.080104

3.16

NO

PC failed;

FAL.3T3.SLS.15/01/04

3.16

YES

FAL.3T3.SLS.22/01/04

1.47

YES

little variation in curve; no
acidity at C1; morpholog.
score didn't match NRU
FAL3T3.SLS.29-01-04
which was lower than
expected; affect lysosomes?

1.47

NO

1.47

YES

FAL3T3.SLS.25.02.04

1.47

YES

FAL.3T3.SLS.17/03/04

Rationale for
Unacceptability

Notes

PC ID

FAL.3T3.SLS.25.11.04

GLYCEROL

IIVS

B2

DF

AA61JF

28800

313.175

0.460

1.18%

4

4

0.9609B3DF

AA61JF

16500

178.973

0.392

5.33%

4

2

0.9540AA61HG-A1

RF

AA61HG

31800

344.975

0.345

4.28%

0

4

0.3823

AA61HG-A2

RF

AA61HG

1870

20.314

0.446

5.59%

1

2

0.9208

AA61HG-B1

DF

AA61HG

23400

254.558

0.471

0.04%

4

4

0.9245

AA61HG-B2

DF

AA61HG

18800

204.544

0.434

1.94%

4

3

0.9732

AA61HG-B3

DF

AA61HG

11600

125.8310.341

18.42%

6

2

0.9815

AA61HG-B4

DF

AA61HG

17800

193.102

0.642

0.19%

4

4

0.9798FAL.3T3.RA.A1.08/01/04

RF

AA61RA

NA

NA

0.777

13.65%08

NA

FAL3T3.RA.A2.15-01-04

DF

AA61RA

11400

123.819

0.717

1.03%

2

6

0.6816

FAL3T3.RA.B1.22-01-04

DF

AA61RA

5710

62.057

0.447

3.38%

3

5

0.9498

FAL3T3.RA.B2.29-01-04

DF

AA61RA

71800

779.449

0.481

1.42%

1

7

0.9674

FAL3T3.RA.B3.05.02-04

DF

AA61RA

18900

205.016

0.370

3.33%

4

4

0.8908

FAL3T3.RA.B4.25-02-04

DF

AA61RA

49200

534.303

0.513

2.62%

2

3

0.9772

FAL3T3.RA.B5.17-03-04

DF

AA61RA

28800

313.175

0.438

7.92%

4

4

0.9627

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100000, 71429, 51020,
36443, 26031, 18593,
13281, 9486
100000, 71429, 51020,
36443, 26031, 18593,
13281, 9486
100000, 71429, 51020,
36443, 26031, 18593,
13281, 9486

range finder

SLS-A1

SLS-B12ECBC
10000, 1000, 100,10, 1,
0.1, 0.01, 0.001
10000, 1000, 100,10, 1,
0.1, 0.01, 0.001
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
68027, 46277, 31481,
21416, 14568, 9910,
6742, 4586

%VC difference > 15

SLS-P30
SLS-P36

FRAME

I-30

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100000, 31646, 10014,
3169, 1003, 317, 100,
31.8
100000, 31646, 10014,
3169, 1003, 317, 100,
31.8
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742

PC failed;problem with reservoir liners FAL.3T3.SLS.5/02/04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9

10

RF

1.3

YES

1.4

YES

NA

NO

1.4

YES

Rationale for
Unacceptability

Notes

PC ID

HALOPERIDOL
IIVS
A1

RF

AA61LW

7.60

0.020

0.290

0.23%

0

1

0.4600B1

DF

AA61LW

5.98

0.016

0.399

1.50%

3

5

0.9242B2DF

AA61LW

5.69

0.015

0.318

4.32%

4

4

0.9350B3DF

AA61LW

NA

NA

NA

NA

NA

NA

NA

B4

DF

AA61LW

4.73

0.013

0.358

6.35%

3

4

0.9252

AA61JC-A1

RF

AA61JC

3.45

0.009

0.346

9.78%

2

5

0.9328AA61JC-B1

DF

AA61JC

5.01

0.013

0.454

8.40%

3

4

0.9612

AA61JC-B2

DF

AA61JC

4.89

0.013

0.320

12.12%

4

4

0.8878AA61JC-B3

DF

AA61JC

6.07

0.016

0.433

1.12%

2

5

0.9620RF

AA61PM

NA

NA

0.373

3.11%

0

1

0.0000100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
10.0, 7.69, 5.92, 4.55,
3.50, 2.69, 2.07, 1.59
20.0, 14.3, 10.2, 7.29,
5.21, 3.72, 2.66, 1.90
NA20.0, 14.3, 10.2, 7.29,
5.21, 3.72, 2.66, 1.90

range finder

the solvent controls weretreated with 1% DMSO,SLS-A4rather than 0.5%.; ppt in 1X
C1-C2
SLS-B5SLS-B13

PC failed

SLS-B14
SLS-B15ECBC100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
20.0, 13.6, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.6, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.6, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35

10

RF

1.47

YES

range finder

ppt in 2X C1 and 1X C1SLS-P14SLS-P38

1.47

YES

SLS-P39

1.47

YES

SLS-P42

10

RF

1.47

NO

2.15

NO

1.47
1.47
1.47

FRAME
FAL.3T3.PM.A1.10.09.04
FAL.3T3.PM.B1.16.09.04

DF

AA61PM

NA

NA

0.269

3.91%

0

0

0.0000FAL.3T3.PM.B2.23.09.04

DF

AA61PM

10.1

0.027

0.199

6.98%

1

0

0.8164

FAL.3T3.PM.B3.14.10.04

DF

AA61PM

8.75

0.023

0.232

1.04%

2

2

0.9504

FAL.3T3.PM.B4.21.10.04

DF

AA61PM

7.60

0.020

0.251

12.27%

3

1

0.9286

FAL.3T3.PM.B5.04.11.04

DF

AA61PM

7.63

0.020

0.190

12.15%

3

3

0.9797100, 10, 1.0, 0.1, 0.01,
0.001, 0.0001, 0.00001
250, 170, 116, 78.7, 53.5,
36.4, 24.8, 16.9
25.0, 11.6, 5.4, 2.5, 1.2,
0.544, 0.253, 0.118
25.0, 17.0, 11.6, 7.87,
5.35, 3.64, 2.48, 1.69
25.0, 17.0, 11.6, 7.87,
5.35, 3.64, 2.48, 1.69
25.0, 17.0, 11.6, 7.87,
5.35, 3.64, 2.48, 1.69

range finder; nopoints between 0 -50%
no points between
50 - 100%no points between
50 - 100%

ppt in 1X C1

FAL.3T3.SLS.10.09.04

ppt in 2X C1

FAL.3T3.SLS.16.09.04

ppt in 2X C1

FAL.3T3.SLS.23.09.04

YES

ppt in 2X C1

FAL.3T3.SLS.14.10.04

YES

ppt in 2X C1

FAL.3T3.SLS.21.10.04

YES

outlier removed bySD

FAL.3T3.SLS.04.11.04

ppt in 2X C1SLS-A1HEXACHLOROPHENE
IIVS
A1

RF

AA61JN

3.21

0.008

0.353

1.04%

1

2

0.9799B1DF

AA61JN

2.90

0.01

0.440

0.93%

5

3

0.9582B2DF

AA61JN

3.39

0.01

0.367

0.61%

4

4

0.9595B3DF

AA61JN

2.88

0.01

0.341

3.03%

5

3

0.9868

AA61ND-A1

RF

AA61ND

9.47

0.023

0.329

9.12%

2

2

0.9700AA61ND-B1

DF

AA61ND

7.81

0.019

0.293

3.27%

3

3

0.9653

AA61ND-B2

DF

AA61ND

3.70

0.009

0.426

9.89%

3

3

0.9878

AA61ND-B3

DF

AA61ND

3.56

0.009

0.371

3.77%

5

3

0.9882

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
20.0, 12.5, 7.81, 4.88,
3.05, 1.91, 1.19, 0.745
20.0, 12.5, 7.81, 4.88,
3.05, 1.91, 1.19, 0.745
20.0, 12.5, 7.81, 4.88,
3.05, 1.91, 1.19, 0.745

10

RF

1.6

YES

range finder

SLS-B5

1.6

YES

SLS-B9

1.6

YES

SLS-B10

10

RF

2.15

YES

SLS-P18

2.15

YES

SLS-P20

2.15

YES

SLS-P21

ECBC

FRAME

I-31

1000, 100,10, 1, 0.1,
0.01, 0.001, 0.0001
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

range finder

ppt in 2X C1-C2 and ppt in
1X C1

SLS-P4

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

FAL.3T3.HB.A1.09/01/04

RF

AA61HB

9.80

0.024

0.387

8.80%

1

4

0.9858FAL.3T3.HB.B1.16.01.04

DF

AA61 HB

7.35

0.018

0.558

6.11%

4

3

0.9833

FAL.3T3.HB.B2.23.01.04

DF

AA61HB

4.59

0.011

0.393

5.57%

3

3

0.8846

Exposure
Concentrations(ug/mL)7

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

100, 47.0, 22.0, 10.0,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

Dilution

Acceptable

Factor8

Tests9

10

RF

2.15

YES

2.15

YES

FAL.3T3.HB.B3.30.01.04

DF

AA61HB

NA

NA

0.264

12.04%

2

6

NA100, 47.0, 22.0, 10.0,
4.68, 2.18, 1.01, 0.47

FAL.3T3.HB.B4.06-02-04

DF

AA61HB

4.10

0.010

0.455

3.82%

5

3

0.9631

100, 47.0, 22.0, 10.0,
4.68, 2.18, 1.01, 0.47

2.15

YES

RF

AA61FW

1710

18.9400.443

13.41%

1

2

0.8766

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

1.5

YES

2.15

NO

Rationale for
Unacceptability

range finder

Notes

PC ID

did'nt dissolve properly; top
2 conc. prepared from stock& C2 from C1. C3 prepared
FAL.3T3.SLS.09/01/04
by diluting stock and C4-8
from the respective C3-7
(from SD); ppt at 100 ug/mL.
FAL.3T3.SLS.16/01/04
FAL3T3.23-01-04

SD rejects thisexperimentserious NR crystal problem;
FAL.3T3.SLS.29/01/04
SD rejects this experimentpossible NR crystalspresent; blanks slightly
higher than usual

FAL.3T3.SLS.06/02/04

LACTIC ACID
IIVS
A1

range finder

B1

DF

AA61FW

3020

33.525

0.447

0.32%

1

2

0.905010000, 6667, 4444, 2963,
1975, 1317, 878, 585

B2

DF

AA61FW

3210

35.594

0.371

3.03%

0

5

0.9595

10000, 6667, 4444, 2963,
1975, 1317, 878, 585

1.5

NO

no points between 050%

B3

DF

AA61FW

2770

30.787

0.422

6.41%

0

5

0.9166

10000, 6667, 4444, 2963,
1975, 1317, 878, 585

1.5

NO

no points between 050%

B4DF

AA61FW

2840

31.577

0.494

1.43%

2

5

0.8914B5DF

AA61FW

2510

27.821

0.349

3.18%

2

5

0.8772

RF

AA61NL

1890

20.9590.260

14.18%

1

1

0.8301

5000, 4167,
3472,2894,2411, 2009,
1674, 1395
5000, 4167,
3472,2894,2411, 2009,
1674, 1395

SLS-A2plate sealer used

SLS-B4SLS-B7
SLS-B8

1.2

YES

plates read 15-16 hr late;orignial reading used wrong SLS-B11
OD wavelength

1.2

YES

outliers removed bySD

SLS-B12

10000,1000, 100, 10,1,0.1, 0.01, 0.001

10RF

ppt in 1X C1SLS-P5

1.47

YES

SLS-P26ECBC
AA61NL-A1

range finder

AA61NL-B1

DF

AA61NL2630

29.199

0.587

4.77%

3

5

0.9427

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

AA61NL-B2

DF

AA61NL

2940

32.687

0.526

1.28%

3

5

0.9463

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

SLS-P27

1.47

YES

SLS-P29

10RF

AA61NL-B3

DF

AA61NL3260

36.172

0.441

1.38%

3

4

0.966010000, 6803, 4628, 3148,
2142, 1457, 991, 674

RF

AA61JT

5750

63.881

0.314

3.27%

1

0

0.7232

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

2.15

YES

FRAME
FAL.3T3.JT.A1.01/04/04
FAL.3T3.JT.B1.29/04/04

DF

AA61JT3000

33.294

0.315

0.03%

2

2

0.9638

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

FAL.3T3.JT.B2.07/05/04

DF

AA61JT

3590

39.8450.361

17.30%

4

2

0.975910000, 7519, 5653, 4251,
3196, 2403, 1807, 1358

1.33

NO

FAL.3T3.JT.B3.20/05/04

DF

AA61JT

4100

45.538

0.377

2.39%

4

1

0.973010000, 7519, 5653, 4251,
3196, 2403, 1807, 1358

1.33

YES

I-32

range finder

FAL.3T3.SLS.01/04/04
NR crystals; highbackground%VC difference > 15 possible volatility problem

FAL.3T3.SLS.29/04/04
FAL.3T3.SLS.07/05/04
FAL.3T3.SLS.20/05/04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9DF

AA61JT

3360

37.271

0.363

1.72%

4

4

0.895010000, 7519, 5653, 4251,
3196, 2403, 1807, 1358

1.33

YES

A1

RF

AA61PJ

15.9

0.055

0.403

35.64%

1

7

0.9488100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001

10

RF

B1

DF

AA61PJ

39.0

0.134

0.403

5.91%

2

4

0.9245100, 55.6, 30.9, 17.1,
9.53, 5.29, 2.94, 1.63

1.8

YES

B2

DF

AA61PJ

51.2

0.176

0.244

10.19%

3

4

0.9211500, 278, 154, 85.7, 47.6,
26.5, 14.7, 8.17

1.8

YES

B3

DF

AA61PJ

NA

NA

NA

NA

NA

NA

NA

B4

DF

AA61PJ

35.2

0.121

0.239

1.50%

4

3

0.9526B5DF

AA61PJ

288

0.989

0.251

0.40%

1

5

0.8492B6DF

AA61PJ

38.8

0.133

0.324

4.98%

4

3

0.8974

AA61FK-A1

RF

AA61FK

38.9

0.134

0.191

14.03%

2

6

0.9093

AA61FK-B1

DF

AA61FK

42.9

0.147

0.242

3.72%

3

5

0.9082

AA61FK-B2

DF

AA61FK

262

0.902

0.340

0.96%

2

6

0.8636

AA61FK-B3

DF

AA61FK

71.0

0.244

0.240

5.46%

3

4

0.8190FAL.3T3.KN.A1.27/05/04

RF

AA61KN

37.1

0.127

0.252

24.49%

2

2

0.7351

FAL.3T3.KN.B1.04/06/04

DF

AA61KN

125

0.431

0.363

11.01%

3

50.7052

FAL.3T3.JT.B4.27/05/04

Rationale for
Unacceptability

Notes

PC ID

FAL.3T3.SLS.27/05/04

LINDANE
IIVS

NA500, 278, 154, 85.7, 47.6,
26.5, 14.7, 8.17
500, 278, 154, 85.7, 47.6,
26.5, 14.7, 8.17
500, 278, 154, 85.7, 47.6,
26.5, 14.7, 8.17

range finder

VC1 ODs < VC2 ODs; VC1
removed from subsequent
analysis; ppt in 2X C1;
volatility issues.
ppt in 2X C1-C2; plate
sealer used

SLS-A3
SLS-B5SD removed C1 from Hillfunction due to upswing in
response curve; C1 toxicity< SLS-B13
C2-C4; plate sealer used;
ppt in 2X C1-C4

NA

NO

1.8

YES

ppt in 2X C1-C5; ppt in 1X
C1-C3

SLS-B15

1.8

YES

ppt in 2X C1-C3

SLS-B16

1.8

YES

ppt in 2X C1-C6

SLS-18

10

RF

2.15

YES

2.15

YES

2.15

YES

10

RF

3.16

YES

PC failed

SLS-B14

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35

range finder

ppt in 2X C1-C2; ppt in 1X
C1; higher than usual blank SLS-P10
OD
ppt in 2X C1-C3; ppt in 1X
SLS-P65
C1-C2
ppt in 2X C1-C3; ppt in 1X
SLS-P66
C1
ppt in 2X C1-C3; ppt in 1X
C1-C3; SD removed data for SLS-P69
C1 from PRISM analysis

FRAME

FAL.3T3.KN.B2.18/06/04

DF

AA61KN

45.5

0.156

0.404

11.01%

4

00.8725

FAL.3T3.KN.B3.24.06.04

DF

AA61KN

153

0.528

0.355

17.86%

3

1

0.9198FAL.3T3.KN.B4.08.07.04

DF

AA61KN

308

1.060

0.250

11.89%

1

7

0.7219FAL.3T3.KN.B5.09.07.04

DF

AA61KN

303

1.041

0.333

4.48%

2

6

0.7443

FAL.3T3.KN.B6.16.07.04

DF

AA61KN

329

1.131

0.238

6.21%

2

3

0.9111

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 791, 250, 79.2,
25.1, 7.9, 2.5, 0.8
1500, 475, 150, 47.5,
15.0, 4.76, 1.51, 0.48
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
500, 340, 231, 157, 107,
72.8, 49.6, 33.7
500, 340, 231, 157, 107,
72.8, 49.6, 33.7
500, 340, 231, 157, 107,
72.8, 49.6, 33.7

3.16

NO

2.15

NO

1.47

YES

1.47

YES

1.47

YES

range finder; % VC
difference > 15

no points between
50 - 100%% VC difference >15

ppt in 1X C1; volatility problemFAL.3T3.SLS.27/05/04
odd graph; ppt in 2X C1-C4
FAL.3T3.SLS.04.06.04
and ppt in 1X C1
ppt in 1X C1-C3 and 2X C1FAL.3T3.SLS.18.06.04
C2
volatility problem; ppt in 2X
FAL.3T3.SLS.24.06.04
C1-C3
ppt in 2X C1-C3 and ppt in
FAL.3T3.SLS.08.07.04
1X C1-C3
ppt in 2X C1-C5 and ppt in
FAL.3T3.SLS.09.07.04
1X C1-C3
ppt in 2X C1-C4 and ppt in
FAL.3T3.SLS.16.07.04
1X C1-C2

LITHIUM I CARBONATE
IIVS
A1

RF

AA61RN

625

8.459

0.557

5.35%

0

2

-0.1197

B1

DF

AA61RN877

11.869

0.378

2.23%

0

6

0.1322

I-33

100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
300, 214, 153, 109,78.1,55.8, 39.8, 28.5

10RF

range finder

SLS-A1

1.4

NO

no points between
0.1 - 50%; low r2

SLS-B1In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

300, 214, 153, 109,78.1,55.8, 39.8, 28.5
300, 214, 153, 109,78.1,55.8, 39.8, 28.5
300, 214, 153, 109,78.1,55.8, 39.8, 28.5

Dilution

Acceptable

Factor8

Tests9

1.4

NO

1.4

NO

1.4

NO

Rationale for
Unacceptability

Notes

No points between
0.1 - 50%; low r2
no points 0.1- 50%;
PC failedno points between
0.1 - 50%; low r2

PC ID

B2

DF

AA61RN

NA

NA

0.499

7.37%

0

4

0.2402B3DF

AA61RN

2.74

0.037

0.573

2.02%

0

3

-0.0036

SLS-B2B4DF

AA61RN

NA

NA

0.500

8.09%

0

5

NA

AA61RR-A1

RF

AA61RR

NA

NA

0.363

7.10%

0

0

0.2245100, 10, 1.0, 0.1, 0.01,
0.001, 0.0001, 0.00001

10

RFAA61RR-A2

RF

AA61RR

561

7.592

0.387

11.51%

03

0.2234500, 50, 5.0, 0.5, 0.05,
0.005, 0.0005

10NO

AA61RR-B1

DF

AA61RR

656

8.878

0.574

2.50%

1

5

0.7540750, 510.2, 347.1, 236.1,
160.6, 109.3, 74.3, 50.6

1.47

NO

low r2

AA61RR-B2

DF

AA61RR762

10.313

0.568

2.56%

1

5

0.7590750, 510.2, 347.1, 236.1,
160.6, 109.3, 74.3, 50.6

1.47

NO

low r2

AA61RR-B3

DF

AA61RR

574

7.768

0.545

0.11%

2

6

0.88641102.5, 750, 510.2, 347.1,
236.1, 160.6, 109.3, 74.3

1.47

YES

AA61RR-B4

DF

AA61RR

630

8.526

0.608

3.32%

2

4

0.95611102.5, 750, 510.2, 347.1,
236.1, 160.6, 109.3, 74.3

1.47

YES

ppts. In C1-C3

SLS-P15

AA61RR-B5

DF

AA61RR

498

6.740

0.195

1.42%

2

5

0.91761102.5, 750, 510.2, 347.1,
236.1, 160.6, 109.3, 74.3

1.47

YES

ppts. In C1-C3

SLS-P16

FAL.3T3.A1.RM.200603

RF

AA61RM

28200

381.648

0.729

6.72%

0

0

0.2031

100, 10, 1.0, 0.1, 0.01,
0.001, 0.0001, 0.00001

10RF

no points between
10 - 90%; low r2;
range finder

FAL.3T3.RM.B1.04.07.03

DF

AA61RM

0.002

0.000

0.509

0.53%

0

0

-0.3160

250, 170, 115.7, 78.7,
53.5, 36.4, 24.8, 16.9

1.47

NO

PC failed; no points
between 10 - 90%

NA250, 170, 115.7, 78.7,
53.5, 36.4, 24.8, 16.9

1.47

NO

250, 170, 115.7, 78.7,
53.5, 36.4, 24.8, 16.9

1.47

NO

SLS-B3
SLS-B4ECBC
no points between
10 - 90%; low r2;
range finder
no points between
10 - 50%; low r2;
range finder

SLS-P1SLS-P3cloudy stock solution

SLS-P6SLS-P8SLS-P10FRAME

FAL.3T3.B2.RM.11.07.03

DF

AA61RM

NA

NA

0.490

2.31%

0

0

FAL.3T3.SLS2.A1.2006
03
straight cytotoxicity line;
can't perform proper
calculations

PC failed; no points
between 10 - 90%
no points between
10 - 90%; No
toxicity

FAL.3T3.SLS.04.07.03
FAL.3T3.SLS.11.07.03

FAL.3T3.B3.RM.18.07.03

DF

AA61RM

NA

NA

0.517

2.17%

0

0

NA

FAL.3T3.SLS.18.07.03

FAL.3T3.RM.B4.070803

DF

AA61RM

24.7

0.334

0.738

5.09%

0

8

0.6965

1000, 680, 462, 314, 214,
145, 99.1, 67.4

1.47

NO

PC failed; no points
between 0 & 50%
viability; cytotoxicity
curve goes in
opposite direction

FAL.3T3.SLS.070803

FAL.3T3.RM.B5.080803

DF

AA61RM

1190

16.1050.474

18.96%

1

7

0.2883

1000, 680, 462, 314, 214,
145, 99.1, 67.4

1.47

NO

PC failed; low r2; %VC difference > 15

FAL.3T3.SLS.080803

A1

RF

AA61LS

390

1.786

0.329

5.97%

1

7

0.9290range finder

B1

DF

AA61LS

395

1.811

0.544

1.09%

3

5

0.9490B2

DF

AA61LS

385

1.762

0.367

1.27%

3

5

0.9715

B3DF

AA61LS

377

1.726

0.381

5.07%

3

5

0.9719MEPROBAMATE
IIVS

ECBC

I-34

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 1111, 617, 343,
191, 106, 58.8, 32.7
2000, 1111, 617, 343,
191, 106, 58.8, 32.7
2000, 1111, 617, 343,
191, 106, 58.8, 32.7

10

RF

1.8

YES

SLS-A1

1.8

YES

SLS-B9

1.8

YES

SLS-B10

ppt in 2X C1SLS-B5

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

AA61RJ-A1

RF

AA61RJ

283

1.297

0.266

3.58%

1

5

0.8633

AA61RJ-B1

DF

AA61RJ

309

1.416

0.336

9.11%

2

6

0.8967

AA61RJ-B2

DF

AA61RJ

344

1.577

0.285

3.34%

3

4

0.9449AA61RJ-B3

DF

AA61RJ

407

1.866

0.345

0.70%

3

5

0.8884

FAL.3T3.HV.A1.080104

RF

AA61HV

798

3.655

0.505

5.60%

1

1

0.8944

FAL3T3.HV.A2.15-01-04

DF

AA61HV

1030

4.720

0.526

6.07%

2

6

0.9564

Exposure
Concentrations(ug/mL)7

1000, 100,10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 930, 433, 201,93.6, 43.5, 20.2, 9.4
2000, 930, 433, 201,93.6, 43.5, 20.2, 9.4
2000, 930, 433, 201,93.6, 43.5, 20.2, 9.4

Dilution

Acceptable

Factor8

Tests9

10

RF

2.15

YES

2.15

YES

2.15

YES

Rationale for
Unacceptability

Notes

range finder

PC ID

SLS-P2SLS-P6SLS-P8outlier not removed fromfrom C6

SLS-P18

FRAME

FAL3T3.HV.B1.22-01-04

DF

AA61HV

984

4.508

0.311

13.54%

2

5

0.7904

FAL3T3.HV.B2.29-01-04

DF

AA61HV

904

4.139

0.377

3.07%

3

5

0.9632

FAL3T3.HV.B3.05.02.04

DF

AA61HV

80

0.366

0.341

11.28%

8

00.5764

FAL.3T3.HV.B4.25.02.04

DF

AA61HV

927

4.246

0.437

3.66%

3

5

0.9673

FAL3T3.HV.B5.17.03.04

DF

AA61HV

692

3.169

0.378

0.13%

4

4

0.9275

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1701, 1157, 787,
535, 364, 248, 169
2500, 1701, 1157, 787,
535, 364, 248, 169
2500, 1701, 1157, 787,
535, 364, 248, 169
2500, 1701, 1157, 787,
535, 364, 248, 169

10RF

range finder

2.15

NO

PC failed;

FAL.3T3.SLS.080104
FAL.3T3.SLS.15/01/04
solubility a problem above
2500 ug/ml

2.15

YES

1.47

YES

FAL.3T3.SLS.22/01/04

1.47

NO

1.47

YES

FAL3T3.SLS.25.02.04

1.47

YES

FAL.3T3.SLS.17/03/04

10

RF

1.8

YES

SLS-B1

1.8

YES

SLS-B2

1.8

YES

SLS-B310RFrange finder
range finder

FAL3T3.SLS.29-01-04
PC failed; no points
between 50-100

problem with reservoir liners FAL.3T3.SLS.5/02/04

MERCURY II CHLORIDE
IIVS
A1

RF

AA61MX

1.21

0.004

0.316

58.59%

1

4

0.9661B1DF

AA61MX

3.39

0.012

0.320

2.63%

1

6

0.9147B2

DF

AA61MX

3.50

0.013

0.311

5.10%

1

1

0.9564B3DF

AA61MX

3.63

0.013

0.346

7.05%

2

5

0.9477

AA61KP-A1

RF

AA61KP

NA

NA

0.152

58.91%

2

2

0.9275

AA61KP-A2

RF

AA61KP

1.43

0.005

0.373

3.96%

0

1

0.9241

AA61KP-B1

DF

AA61KP

3.26

0.012

0.278

2.28%

2

1

0.8937

AA61KP-B2

DF

AA61KP

3.61

0.013

0.353

5.88%

2

5

0.9465

AA61KP-B3

DF

AA61KP

3.48

0.013

0.384

6.51%

2

5

0.9682

FAL.3T3.HA.A1.080104

RF

AA61HA

4.11

0.015

0.399

9.97%

1

0

0.9558

FAL3T3.HA.A2.15-01-04

DF

AA61HA

6.77

0.025

0.363

6.52%

2

6

0.8549FAL3T3.HA.B1.22-01-04

DF

AA61HA

5.71

0.021

0.371

3.49%

1

6

0.8036

FAL3T3.HA.B2.29-01-04

DF

AA61HA

7.98

0.029

0.481

1.42%

1

5

0.9674

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
10.0, 5.56, 3.09, 1.71,
0.953, 0.529, 0.294,
0.163
10.0, 5.56, 3.09, 1.71,
0.953, 0.529, 0.294,
0.163
10.0, 5.56, 3.09, 1.71,
0.953, 0.529, 0.294,
0.163

range finder

VC1 ODs < VC2 ODs; VC1
removed from subsequent
analysis; volatility issues.

SLS-A1

ECBC
1000, 100,10, 1, 0.1,
0.01, 0.001, 0.0001
10, 1, 0.1, 0.01, 0.001,
0.0001, 0.00001,
0.000001
10.0, 4.65, 2.16, 1.01,
0.47, 0.22, 0.10, 0.05
10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

low ODs for VC1; ppt in C1

SLS-P2

10

RF

2.15

YES

SLS-P4SLS-P18

1.47

YES

SLS-P20

1.47

YES

SLS-P2110RF

no points between
50-100 range finder

1.3

NO

PC failed;

1.3

YES

FAL.3T3.SLS.22/01/04

1.3

YES

FAL3T3.SLS.29-01-04

FRAME

I-35

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
10.0, 7.7, 5.9, 4.6, 3.5,
2.7, 2.1, 1.6
10.0, 7.7, 5.9, 4.6, 3.5,
2.7, 2.1, 1.6
10.0, 7.7, 5.9, 4.6, 3.5,
2.7, 2.1, 1.6

ppt in 1000ug/ml

FAL.3T3.SLS.080104
FAL.3T3.SLS.15/01/04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

10.0, 7.7, 5.9, 4.6, 3.5,
2.7, 2.1, 1.6
10, 7.63, 5.83, 4.45, 3.40,
2.59, 1.98, 1.51

FAL3T3.HA.B3.05.02.04

DF

AA61HA

0.967

0.004

0.380

7.96%

8

0

0.8305

FAL3T3.HA.B4.17.03.04

DF

AA61HA

4.28

0.016

0.223

2.28%

3

5

0.9519A1

RF

AA61FZ

NA

NA

0.256

7.10%

0

6

NA

B1

DF

AA61FZ

NA

NA

0.380

5.76%

0

2

0.4933B2DF

AA61FZ

NA

NA

0.284

2.14%

0

1

NA

B3

DF

AA61FZ

NA

NA

NA

NA

NA

NA

NA

B4

DF

AA61FZ

NA

NA

0.400

2.42%

0

2

NA

RF

AA61MJ

NA

NA

0.443

6.04%

0

3

NA

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

NA

3000, 2479, 2049, 1693,
1400, 1157, 956, 790

Dilution

Acceptable

Factor8

Tests9

1.3

NO

1.31

YES

Rationale for
Unacceptability

PC failed; no points
between 50-100

Notes

PC ID

problem with reservoir liners FAL.3T3.SLS.5/02/04
FAL.3T3.SLS.17/03/04

METHANOL
IIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1923, 1479, 1138,
875, 673, 518, 398
2500, 1923, 1479, 1138,
875, 673, 518, 398
NA2500, 1923, 1479, 1138,
875, 673, 518, 398

10

RF

1.3

NO

range finder

SLS-A5

1.3

NO

NA

NO

1.3

NO

10

RF

range finder

1.21

NO

no points between 0 - no toxicity detected; need
SLS-P4450%
larger conc; dilut. factor 1.21

1.21

NO

1.21

NO

10

RF

1.47

NO

1.21

NO

no points between 0 50%
no points between 0 50%
PC failedno points between 0 50%

SLS-B9SLS-B13
SLS-B14
SLS-B15ECBC
AA61MJ-A1
AA61MJ-B1

DF

AA61MJ

NA

NA

0.709

5.19%

0

8

AA61MJ-B2

DF

AA61MJ

NA

NA

0.512

2.61%

0

7

NA

AA61MJ-B3

DF

AA61MJ

NA

NA

0.375

14.56%

0

0

NA

RF

AA61RG

NA

NA

0.203

7.09%

0

0

NA

3500, 2893, 2391, 1976,
1633, 1349, 1115, 922
3500, 2893, 2391, 1976,
1633, 1349, 1115, 922

SLS-16no points between 0 no toxicity was detected
50%
no points between 0 no toxicity was detected
100%

SLS-P72
SLS-P74

FRAME
FAL.3T3.RG.A1.21.10.04
FAL.3T3.RG.B1.04.11.04

DF

AA61RG

NA

NA

0.175

6.75%

0

0

NA

FAL.3T3.RG.B2.25.11.04

DF

AA61RGNA

329085

0.258

0.36%

0

0

-1.2340DF

AA61RG

NA

NA

0.263

5.11%

0

0

NA

A1

RF

AA61HL

339

2.089

0.457

7.49%

0

5

0.9490B1DF

AA61HL

422

2.600

0.539

5.98%

1

2

0.9929B2DF

AA61HL

508

3.133

0.392

3.15%

2

0

0.8900B3DFAA61HL

513

3.162

0.469

2.05%

2

1

0.9111

FAL.3T3.RG.B3.26.11.04

1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
2500, 1701, 1157, 787,
535, 264, 248, 169
2500, 2066, 1708, 1411,
1166, 964, 797, 658
2500, 2066, 1708, 1411,
1166, 964, 797, 658

1.21

range finder; nopoints between 0100%
no points between 0100%
no points between 0100%

FAL.3T3.SLS.21.10.04
FAL.3T3.SLS.04.11.04
FAL.3T3.SLS.25.11.04

NO

no toxicity detected; 1.21
no points between 0- dilut. factor doesn't affect100%
acceptability; outlier
removed bySD

FAL.3T3.SLS.26.11.04

range finder

SLS-A4

NICOTINE
IIVS

B4

DF

AA61HL

415

2.558

0.440

1.63%

3

2

0.9953

AA61NA-A1

RF

AA61NA

NA

NA

0.410

19.57%

05NA

AA61NA-B1

DF

AA61NA

NA

NA

0.532

3.40%

3

5

NA

AA61NA-B2 (sealer)DF

AA61NA

292

1.803

0.603

4.02%

3

5

0.8541

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 714, 510, 364, 260,
186, 133, 94.9
1000, 714, 510, 364, 260,
186, 133, 94.9
1000, 714, 510, 364, 260,
186, 133, 94.9

10

RF

1.4

YES

1.4

NO

1.4

YES

SLS-B8YES

plates read 15-16 hr late;orignial reading used wrong SLS-B11
OD wavelength

SLS-B4no points between
50 - 100%

SLS-B7

1000, 833, 694, 579, 482,
402, 335, 279

1.2

10000,1000, 100, 10,1,0.1, 0.01, 0.001
500, 413, 342, 282, 233,
193, 159, 132
500, 413, 342, 282, 233,
193, 159, 132

10RF

range finder

SLS-P49

1.21

NO

PC failed

SLS-P53

1.21

YES

ECBC

I-36

SLS-P54

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

AA61NA-B3 (sealer)DF

AA61NA

NA

NA

0.399

9.58%

4

4

NA

AA61NA-B4 (sealer)DF

AA61NA

325

2.004

0.451

5.32%

3

5

0.7971

AA61NA-B5 (sealer)DF

AA61NA

199

1.227

0.536

5.08%

5

3

0.8836

RF

AA61KL

582

3.589

0.402

9.59%

1

0

0.9633

Exposure
Concentrations(ug/mL)7

500, 413, 342, 282, 233,
193, 159, 132
500, 413, 342, 282, 233,
193, 159, 132
500, 413, 342, 282, 233,
193, 159, 132

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC failed

PC ID

1.21

NO

1.21

YES

SLS-P56
SLS-P58

1.21

YES

SLS-P62

10

RF

FRAME
FAL.3T3.KL.A1.10.09.04
FAL.3T3.KL.B1.16.09.04

DF

AA61KL

460

2.838

0.375

1.07%

3

0

0.9720FAL.3T3.KL.B2.23.09.04

DF

AA61KL

481

2.964

0.356

3.30%

4

2

0.9817

FAL.3T3.KL.B3.14.10.04

DF

AA61KL

499

3.076

0.359

4.34%

2

1

0.9323

FAL.3T3.KL.B4.04.11.04

DF

AA61KL

255

1.574

0.227

6.96%

6

2

0.9486

A1

RF

AA61GD

14.1

0.055

0.454

0.61%

1

1

0.9683B1

DF

AA61GD

7.91

0.031

0.491

0.08%

6

2

0.9744B2DF

AA61GD

22.7

0.088

0.386

6.81%

3

5

0.9777B3DF

AA61GD

39.4

0.153

0.478

2.70%

2

6

0.9759

AA61MP-A1

RF

AA61MP

11.9

0.046

0.345

1.23%

1

1

0.9870

AA61MP-B1

DF

AA61MP

23.6

0.092

0.654

5.58%

2

5

0.9673

AA61MP-B2

DF

AA61MP

13.1

0.051

0.632

7.30%

3

5

0.9128

AA61MP-B3

DF

AA61MP

27.1

0.105

0.622

7.05%

2

5

0.9779FAL.3T3.HP.A1.01/04/04

RF

AA61HP

62.4

0.243

0.396

1.11%

3

0

0.8909FAL.3T3.HP.B1.29/04/04

DF

AA61HP

39.8

0.155

0.275

10.84%

1

00.8164

FAL.3T3.HP.B2.07/05/04

DF

AA61HP

7.35

0.029

0.347

7.92%

4

2

0.9791

FAL.3T3.HP.B3.20/05/04

DF

AA61HP

40.2

0.156

0.360

0.93%

4

1

0.9192

FAL.3T3.HP.B4.27/05/04

DF

AA61HP

27.0

0.105

0.425

2.86%

4

4

0.9183

RF

AA61PS

50.6

0.174

0.402

5.64%

1

3

0.9458

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 680, 463, 315, 214,
146, 99.1, 67.4
1000, 826, 683, 565, 467,
386, 319, 263
1000, 826, 683, 565, 467,
386, 319, 263
750, 620, 512, 423, 350,
289, 239, 197

1.47

NO

1.21

YES

1.21

YES

1.21

YES

range finder; nopoints between 50 100%no points between
50 - 100%

FAL.3T3.SLS.10.09.04
FAL.3T3.SLS.16.09.04
outlier removed by SD

FAL.3T3.SLS.23.09.04
FAL.3T3.SLS.14.10.04

outlier removed by SD

FAL.3T3.SLS.04.11.04

PARAQUATIIVS

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
86.8 ,54.3, 33.9, 21.2,
13.3, 8.28, 5.18, 3.24
100, 62.5, 39.1, 24.4,
15.3, 9.54, 5.96, 3.73
100, 62.5, 39.1, 24.4,
15.3, 9.54, 5.96, 3.73

10

RF

1.6

YES

range finder

SLS-A2
SLS-B4

1.6

YES

SLS-B7

1.6

YES

SLS-B8ECBC
10000,1000, 100, 10,1,0.1, 0.01, 0.001
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

10

RF

2.15

YES

range finder

SLS-P5SLS-P23

2.15

YES

SLS-P26

2.15

YES

SLS-P27

FRAME
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 75.2, 56.5, 42.5,
32.0, 24.0, 18.1, 13.6
100, 75.2, 56.5, 42.5,
32.0, 24.0, 18.1, 13.6

10RF

range finder

2.15

NO

no points between
50 - 100%

2.15

YES

1.33

YES

1.33

YES

10

RF

FAL.3T3.SLS.01/04/04
NR crystals; highbackground

FAL.3T3.SLS.29/04/04
FAL.3T3.SLS.07/05/04
FAL.3T3.SLS.20/05/04

outlier removed by SD

FAL.3T3.SLS.27/05/04

ppt in 2X C1-C3

SLS-A3SD removed C1 & C2 from
Hill function in PRISM due
to upswing in response
curve

SLS-B5

PARATHION
IIVS
A1

B1

DF

AA61PS

20.7

0.071

0.435

5.34%

3

3

0.8956I-37

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
300, 167, 92.6, 51.4,
28.6, 15.9, 8.82, 4.90

1.8

YES

range finder

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

B2

Assay Substance
ID
Type1

DF

AA61PS

IC50(ug/mL)

IC50(mM)

Mean VC

27.5

0.095

0.348

OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

13.21%

5

3

R2 6

0.9431DF

AA61PS

17.9

0.062

0.353

5.03%

3

3

0.9864

AA61MD-A1

RF

AA61MD

NA

NA

0.329

7.11%

2

6

NA

AA61MD-B1

DF

AA61MD

18

0.062

0.648

8.54%

3

5

0.9126

AA61MD-B2

DF

AA61MD

13.6

0.047

0.418

10.70%

3

5

0.8929

AA61MD-B3

DF

AA61MD

36.4

0.125

0.321

10.09%

2

5

0.9559FAL.3T3.KE.A1.28.05.04

RF

AA61KE

407

1.399

0.396

3.60%

1

4

0.9626

FAL.3T3.KE.B1.04.06.04

DF

AA61KE

51.9

0.178

0.330

7.50%

5

3

0.7298FAL.3T3.KE.B2.18.06.04

DF

AA61KE

NA

0.121

0.714

6.99%

3

0

0.8485

FAL.3T3.KE.B3.09.07.04

DF

AA61KE

123

0.423

0.283

6.74%

2

6

0.9136

FAL.3T3.KE.B4.16.07.04

DF

AA61KE

247

0.847

0.263

3.84%

2

3

0.8273B3Exposure
Concentrations(ug/mL)7

300, 167, 92.6, 51.4,
28.6, 15.9, 8.82, 4.90
300, 167, 92.6, 51.4,
28.6, 15.9, 8.82, 4.90

Dilution

Acceptable

Factor8

Tests91.8

YES

ppt in 2X C1-C4

SLS-B91.8

YES

ppt in 2X C1-C4 & 1X C1C2; SD removed C1 & C2from Hill function in PRISM
due to ppts & flat response
curve

SLS-B1010RF

ppt in 1X C1 & 2X C1-C2(cloudy)

SLS-P50

2.15

YES

2.15

YES

2.15

YES

Rationale for
Unacceptability

Notes

PC ID

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

range finder; PC
failedSLS-P55
ppt in 2X C1SLS-P57
SLS-P59

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

10

RF

2.15

YES

range finder

2.15

NO

2.15

YES

ppt in 2X C1

FAL.3T3.SLS.09.07.04

2.15

YES

ppt in 2X C1-C2; ppt in 1X
C1-C2

FAL.3T3.SLS.16.07.04

no points between
50 - 100%

ppt in 1X C1-C2

FAL.3T3.SLS.28.05.04

ppt in 2X C1-C4; ppt in 1X
FAL.3T3.SLS.04.06.04
C1-C2
ppt in 2X C1-C2; tox.curve
going in wrong direction; SD
FAL.3T3.SLS.18.06.04
suggests ppt is cause of bad
curve

PHENOBARBITAL
IIVS
A1

RF

AA61FG

77.4

0.333

0.283

14.30%

1

5

0.9228B1DF

AA61FG

813

3.500

0.359

8.28%

0

8

0.4992B2DF

AA61FG

379

1.633

0.342

1.76%

3

5

0.9762B2 (should be B3)

DF

AA61FG

624

2.686

0.302

13.14%

2

6

0.8659B4DF

AA61FG

NA

NA

NA

NA

NA

NA

NA

B5

DF

AA61FG

497

2.138

0.335

2.24%

3

5

0.9744B6DF

AA61FG

405

1.742

0.302

1.43%

3

5

0.9775

AA61KV-A1

RF

AA61KV

351

1.510

0.324

3.98%

1

2

0.9078

AA61KV-B1

DF

AA61KV

624

2.686

0.405

1.63%

3

5

0.9793

AA61KV-B2

DF

AA61KV

505

2.173

0.412

8.99%

3

5

0.7926

AA61KV-B3

DF

AA61KV

773

3.327

0.410

6.44%

2

6

0.9504

RF

AA61NJ

796

3.428

0.169

1.18%

1

0

0.5114

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
200, 125, 78.1, 48.8,
30.5, 19.1, 11.9, 7.45
1000, 714, 510, 364, 260,
186, 133, 94.9
1000, 714, 510, 364, 260,
186, 133, 94.9
NA1000, 714, 510, 364, 260,
186, 133, 94.9
1000, 714, 510, 364, 260,
186, 133, 94.9

10RFrange finder

1.6

NO

no points between 0 50%

SLS-A5SLS-B6
plates read 15-16 hr late;orignial reading used wrong SLS-B11
OD wavelength

1.4

YES

1.4

YES

NA

NO

1.4

YES

SLS-B15

1.4

YES

SLS-B16

outliers removed bySD
PC failed

SLS-B13
SLS-B14

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1500, 1020, 694, 472,
321, 219, 149, 101
1500, 1020, 694, 472,
321, 219, 149, 101
1500, 1020, 694, 472,
321, 219, 149, 101

10

RF

1.47

YES

range finder

SLS-P68
SLS-P70

1.47

YES

SLS-P71

1.47

YES

SLS-P72

10

RF

FRAME
FAL.3T3.NJ.A1.21.10.04

I-38

1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001

range finder; nopoints between 50100%

FAL.3T3.SLS.21.10.04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

FAL.3T3.NJ.B1.11.11.04

DF

AA61NJ

435

1.871

0.311

4.08%

4

4

0.8929FAL.3T3.NJ.B2.25.11.04

DF

AA61NJ

832

3.582

0.295

9.31%

2

2

0.8514

FAL.3T3.NJ.B3.26.11.04

DF

AA61NJ

912

3.927

0.204

1.06%

2

2

0.9435

A1

RF

AA61PG

3.12

0.033

0.418

100.04%2

40.9933B1

DF

AA61PG

NA

NA

0.465

1.52%

0

3

0.5739B2

DF

AA61PG

54.8

0.583

0.422

2.43%

3

5

0.9767

Exposure
Concentrations(ug/mL)7

2000, 1361, 926, 630,
428, 291, 198,135
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

Dilution

Acceptable

Factor8

Tests91.47

YES

ppt in 2X C1

FAL.3T3.SLS.10.11.04

2.15

YES

ppt in 2X C1

FAL.3T3.SLS.25.11.04

2.15

YES

ppt in 2X C1

FAL.3T3.SLS.26.11.04

10RFrange finder

VC1 ODs < VC2 ODs; VC1
removed from subsequent
analysis; volatility issues.

SLS-A3

1.6

NO

no points between 0 plate sealer used
50%

SLS-B4

1.6

YES

plate sealer used

SLS-B6

1.6

NO

plates read 15-16 hr late;orignial reading used wrong
OD wavelength; VC2 used SLS-B11
to calculate viability;
volatility problem

1.6

YES

NA

NO

1.6

YES

10

RF

range finder

10

RFrange finder

2.15

YES

2.15

NO

PC failed

SLS-P50

2.15

NO

PC failed

SLS-P52

2.15

YES

2.15

NO

2.15

YES

Rationale for
Unacceptability

Notes

PC ID

PHENOL
IIVS

B3

DF

AA61PG

33.6

0.357

0.392

34.71%B4

DF

AA61PG

65.9

0.700

0.337

1.73%

B5

DF

AA61PG

NA

NA

NA

NA

B6

DF

AA61PG

53.6

0.569

0.411

3.44%

AA61FV-A1

RF

AA61FV

NA

NA

0.140

AA61FV-A2 (sealer)RF

AA61FV

56.0

0.595

AA61FV-B1(sealer)DF

AA61FV

50.6

AA61FV-B2 (sealer)DF

AA61FVNA

AA61FV-B3 (sealer)DF

AA61FV

AA61FV-B4 (sealer)DF

AA61FV-B5 (sealer)
AA61FV-B6 (sealer)

4

40.99253

50.9669

NA

NA

NA

3

5

0.977599.80%

4

0

NA

0.430

3.64%

2

1

0.8997

0.537

0.305

4.48%

4

4

0.9861

NA

0.280

0.51%

5

3

NA

NA

NA

0.341

7.61%

4

4

NA

AA61FV

60.8

0.646

0.552

2.48%

3

3

0.9615DF

AA61FV

NA

NA

0.354

3.58%

4

4

NA

DF

AA61FV

39.1

0.415

0.416

4.85%

4

4

0.9808FAL.3T3.MS.A1.21.05.04

RF

AA61MS

10.4

0.110

0.176

99.85%

2

1

0.4657

FAL.3T3.MS.B1.17/06/04

DF

AA61MS

NA

NA

0.387

40.08%

2

1

NA

FAL.3T3.MS.B2.24/06/04

DF

AA61MS

375

3.984

0.154

18.61%

1

4

0.9472

FAL.3T3.MS.B3.08.07.04

DF

AA61MS

142

1.504

0.308

26.58%

4

1

0.9369FAL.3T3.MS.B4.09.07.04

DF

AA61MS

37.8

0.401

0.301

25.71%

3

2

0.5823

FAL.3T3.MS.B5.16.07.04

DF

AA61MS

110

1.168

0.360

7.07%

3

2

0.9794

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
20.0, 12.5, 7.81, 4.88,
3.05, 1.91, 1.19, 0.745
200, 125, 78.1, 48.8,
30.5, 19.1, 11.9, 7.45
200, 125, 78.1, 48.8,
30.5, 19.1, 11.9, 7.45
200, 125, 78.1, 48.8,
30.5, 19.1, 11.9, 7.45
NA200, 125, 78.1, 48.8,
30.5, 19.1, 11.9, 7.45

% VC difference >15

SLS-B12
PC failed

SLS-B14
SLS-B15

ECBC
10000,1000, 100, 10,1,0.1, 0.01, 0.001
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 465, 216, 101,
46.8, 21.8, 10.1
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35

probable volatility problem;
VC1 <<< VC2

SLS-11SLS-P45
SLS-P47

SLS-P54
PC failed

SLS-P56
SLS-P58

FRAME

FAL.3T3.MS.B6.17.09.04

DF

AA61MS

124

1.322

0.530

17.30%

3

2

0.9579FAL.3T3.MS.B7.23.09.04

DF

AA61MS

126

1.335

0.313

7.13%

3

2

0.9717

I-39

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 317, 100, 31.7,
10.0, 3.2, 1.0, 0.31000, 317, 100, 31.7,
10.0, 3.2, 1.0, 0.3
1500, 698, 325, 151,
70.2, 32.7, 15.2, 7.1
1500, 698, 325, 151,
70.2, 32.7, 15.2, 7.1
1500, 698, 325, 151,
70.2, 32.7, 15.2, 7.1
1500, 698, 325, 151,
70.2, 32.7, 15.2, 7.1
1500, 698, 325, 151,
70.2, 32.7, 15.2, 7.1

10

RF

3.16

NO

3.16

NO

2.15

NO

2.15

NO

2.15

YES

2.15

NO

2.15

YES

range finder% VC difference >15% VC difference >15% VC difference >15% VC difference >15

volatility problem

FAL.3T3.SLS.21.05.04
FAL.3T3.SLS.17.06.04

used plate sealer; volatility
problem

FAL.3T3.SLS.24.06.04
FAL.3T3.SLS.08.07.04
FAL.3T3.SLS.09.07.04
FAL.3T3.SLS.16.07.04% VC difference >15

did not use plate sealer

FAL.3T3.SLS.17.09.04

outlier removed bySD

FAL.3T3.SLS.23.09.04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

FAL.3T3.MS.B8.14.10.04

DF

AA61MS

116

1.231

0.234

27.97%

4

2

0.9535

FAL.3T3.MS.B9.21.10.04

DF

AA61MS

77.3

0.821

0.339

13.82%

4

3

0.9581

Exposure
Concentrations(ug/mL)7

1500, 698, 325, 151,
70.2, 32.7, 15.2, 7.1
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7

Dilution

Acceptable

Factor8

Tests9

2.15

NO

2.15

YES

Rationale for
Unacceptability

Notes% VC difference >15

volatility problem

PC ID

FAL.3T3.SLS.14.10.04
FAL.3T3.SLS.21.10.04

PHENYLTHIOUREA
IIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
300, 188, 117, 73.2, 45.8,
28.6, 17.9, 11.2
1000, 625, 391,244, 153,
95.4, 59.6, 37.3

10

RF

1.6

YES

1.6

YES

NA

NA

NA

NO

2

0.95801000, 625, 391,244, 153,
95.4, 59.6, 37.3

1.6

YES

2

6

NA

10

RF

range finder; PC
failedSLS-P51

14.50%

4

4

NA

2.15

NO

PC failed

SLS-P53

11.02%

4

4

0.9747

2.15

YES

SLS-P55

0.601

9.45%

5

3

0.9428

2.15

YES

SLS-P57

0.215

0.374

8.69%

4

4

0.97302.15

YES

SLS-P59

34.0

0.223

0.302

7.47%

2

1

0.9382

AA61JB

164

1.075

0.320

7.72%

3

5

0.8392DF

AA61JB

288

1.891

0.388

2.92%

2

3

0.9514DF

AA61JB

264

1.736

0.250

6.43%

2

6

0.8568RF

AA61NF

673

2.444

0.262

11.85%

1

4

0.9016

A1

RF

AA61PV

49.4

0.325

0.369

1.67%

2

3

0.8971B1DF

AA61PV

113

0.741

0.446

4.65%

3

4

0.9548B2DF

AA61PV

83.9

0.552

0.262

5.15%

6

2

0.9737B3DF

AA61PV

NA

NA

NA

NA

NA

NA

B4

DF

AA61PV

70.0

0.460

0.335

8.85%

6

AA61LN-A1

RF

AA61LNNA#VALUE!

0.284

5.42%

AA61LN-B1

DF

AA61LNNA#VALUE!

0.350

AA61LN-B2

DF

AA61LN

48.6

0.320

0.613

AA61LN-B3

DF

AA61LN

9.11

0.060

AA61LN-B4

DF

AA61LN

32.7

FAL.3T3.JB.A1.27/05/04

RF

AA61JB

FAL.3T3.JB.B1.04/06/04

DF

FAL.3T3.JB.B2.18/06/04
FAL.3T3.JB.B3.08.07.04

range finder

SLS-A3
SLS-B5SLS-B13

PC failed

SLS-B14
SLS-B15ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 791, 250, 79.2,
25.1, 7.9, 2.5, 0.82500, 791, 250, 79.2,
25.1, 7.9, 2.5, 0.82500, 791, 250, 79.2,
25.1, 7.9, 2.5, 0.8

10

RF

3.16

YES

range finder
ppt in 2X C1

FAL.3T3.SLS.27/05/04
FAL.3T3.SLS.04.06.04

3.16

YES

ppt in 2X C1

FAL.3T3.SLS.18.06.04

3.16

YES

ppt in 2X C1

FAL.3T3.SLS.08.07.04

10RF

range finder

SLS-A5

1.4

NO

no points between
50 - 100%

SLS-B5

1.6

YES

1.6

YES

NA

NO

1.6

YES

SLS-B15

1.6

YES

SLS-B16

10

RF

1.47

YES

PHYSOSTIGMINE
IIVS
A1
B1

DF

AA61NF

75.9

0.275

0.517

10.55%

2

00.8901B2DF

AA61NF

30.1

0.109

0.411

6.64%

2

4

0.9115B3DF

AA61NF

19.8

0.072

0.338

7.64%

2

3

0.9406B4DF

AA61NF

NA

NA

NA

NA

NA

NA

NA

B5

DF

AA61NF

16.0

0.058

0.350

0.98%

3

2

0.9600B6DFAA61NF

15.8

0.057

0.365

1.30%

4

2

0.9575

AA61FT-A1

RF

AA61FT

NA

NA

0.281

7.43%

2

4

NA

AA61FT-B1

DF

AA61FT

42.8

0.155

0.678

8.75%

2

6

0.9263

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 714, 510, 364, 260,
186, 133, 94.9
200, 125, 78.1, 48.8,
30.5, 19.1, 11.9, 7.45
200, 125, 78.1, 48.8,
30.5, 19.1, 11.9, 7.45
NA200, 125, 78.1, 48.8,
30.5, 19.1, 11.9, 7.45
200, 125, 78.1, 48.8,
30.5, 19.1, 11.9, 7.45

SLS-B6outliers removed bySD
PC failed

SLS-B13
SLS-B14

ECBC

I-40

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
80.0, 54.4, 37.0, 25.2,
17.1, 11.7, 7.93, 5.39

range finder; PC
failedSLS-P51
SLS-P55

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

AA61FT-B2

DF

AA61FT

13.0

0.047

0.592

9.27%

5

3

0.8332

AA61FT-B3

DF

AA61FT

28.8

0.105

0.354

8.67%

5

3

0.9265RF

AA61GT

34.4

0.125

0.217

3.30%

1

0

0.9835

Exposure
Concentrations(ug/mL)7

80.0, 54.4, 37.0, 25.2,
17.1, 11.7, 7.93, 5.39
80.0, 54.4, 37.0, 25.2,
17.1, 11.7, 7.93, 5.39

Dilution

Acceptable

Factor8

Tests9

1.47

YES

SLS-P57

1.47

YES

SLS-P59

10

RF

1.33

YES

ppt in 2X C1-C2;

FAL.3T3.SLS.25.11.04

1.33

YES

ppt in 2X C1-C2;

FAL.3T3.SLS.26.11.04

1.33

NO

1.33

YES

FAL.3T3.SLS.09.12.04

Rationale for
Unacceptability

Notes

PC ID

FRAME
FAL.3T3.GT.A1.21.10.04

1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
100, 75.2, 56.5, 42.5,
32.0, 24.0, 18.1, 13.6
100, 75.2, 56.5, 42.5,
32.0, 24.0, 18.1, 13.6
100, 75.2, 56.5, 42.5,
32.0, 24.0, 18.1, 13.6
100, 75.2, 56.5, 42.5,
32.0, 24.0, 18.1, 13.6

range finder; nopoints between 50100%

FAL.3T3.SLS.21.10.04

FAL.3T3.GT.B1.25.11.04

DF

AA61GT

38.2

0.139

0.344

4.78%

4

2

0.9738FAL.3T3.GT.B2.26.11.04

DF

AA61GT

35.7

0.130

0.167

1.60%

4

3

0.6701

FAL.3T3.GT.B3.02.12.04

DF

AA61GT

77.1

0.280

0.179

5.82%

0

1

0.2009FAL.3T3.GT.B4.09.12.04

DF

AA61GT

39.5

0.144

0.286

6.43%

2

2

0.9799

611

8.196

0.457

25.09%

1

1

0.8205

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

SLS-A1

1.8

YES

SLS-B1
SLS-B2no points between 0most values above 125%50%

FAL.3T3.SLS.02.12.04(RB)

POTASSIUM I CHLORIDE
IIVS
A1

RF

AA61FF

B1

DF

AA61FF

4150

55.667

0.394

5.13%

2

6

0.9627

10000, 5556, 3086, 1715,
953, 529, 294, 163

B2

DF

AA61FF

3660

49.095

0.536

1.54%

2

5

0.9837

10000, 6667, 4444, 2963,
1975, 1317, 878, 585

1.5

YES

B3

DF

AA61FF3230

43.327

0.561

1.06%

2

5

0.9387

10000, 6667, 4444, 2963,
1975, 1317, 878, 585

1.5

NO

B4

DF

AA61FF

3320

44.534

0.442

4.82%

2

4

0.9856

10000, 6667, 4444, 2963,
1975, 1317, 878, 585

1.5

YES

AA61KM-A1

RF

AA61KM

2160

28.974

0.424

3.92%

0

0

0.8877

10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001

10RF

AA61KM-B1

DF

AA61KM

3140

42.119

0.607

0.88%

1

4

0.8821

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

SLS-P6

AA61KM-B2

DF

AA61KM

4060

54.460

0.552

4.78%

1

1

0.9805

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

SLS-P8

AA61KM-B3

DF

AA61KM

3160

42.388

0.526

0.98%

1

3

0.9435

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

SLS-P10

AA61KM-B4

DF

AA61KM

3080

41.315

0.676

1.49%

1

4

0.9563

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

SLS-P13

FAL.3T3.A1.MY.200603

RF

AA61MY

1290

17.304

0.745

1.93%

0

1

0.958010000,1000, 100, 10, 1.0,
0.1, 0.01, 0.001

10RF

FAL.3T3.MY.A2.27.06.03

RF

AA61MY

9440

126.626

0.511

2.94%

0

2

0.7401

6000, 4080, 2780, 1890,
1280, 874, 595, 405

1.47

NO

FAL.3T3.MY.B1.04.07.03

DF

AA61MY4470

59.960

0.551

2.97%

0

4

0.951420000, 13600, 9260,
6300, 4280, 2910, 1980,
1350

1.47

NO

PC failed

SLS-B3
SLS-B4ECBC
no points between
10 - 90%; range
finderSLS-P1

FRAME

I-41

no points between
10 - 50%; range
finderno points between
10 - 50%; low r2;
range finder
PC failed; no points
between 10 - 50%

FAL.3T3.SLS2.A1.2006
03
FAL.3T3.SLS.A2.27.06.
03
FAL.3T3.SLS.04.07.03

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

15000, 10204, 6942,
4722, 3212, 2185, 1487,
1011
15000, 10204, 6942,
4722, 3212, 2185, 1487,
1011

Dilution

Acceptable

Factor8

Tests9

1.47

YES

FAL.3T3.SLS.11.07.03

1.47

YES

FAL.3T3.SLS.18.07.03
FAL.3T3.SLS.25.07.03

Rationale for
Unacceptability

Notes

PC ID

FAL.3T3.B2.MY.11.07.03

DF

AA61MY4350

58.350

0.583

0.25%

1

4

0.9622

FAL.3T3.B3.MY.18.07.03

DF

AA61MY4760

63.850

0.499

0.50%

2

2

0.9202

FAL.3T3.B4.MY.25.07.03

DF

AA61MY

4740

63.581

0.478

6.48%

1

2

0.9631

10000, 7519, 5633, 4251,
3196, 2403, 1807, 1350

1.33

YES

FAL.3T3.B5.MY.070803

DF

AA61MY3440

46.144

1.263

6.60

3

5

0.9364

10000, 6802, 4627, 3148,
2141, 1456, 991, 674

1.47

NO

PC failed

FAL.3T3.SLS.070803

FAL.3T3.B6.MY.080803

DF

AA61MY

1160

15.560

0.432

11.91

5

2

0.6458

10000, 6802, 4627, 3148,
2141, 1456, 991, 674

1.47

NO

PC failed; low r2

FAL.3T3.SLS.080803

FAL.3T3.MY.B7.120903

DF

AA61MY1920

25.755

0.629

1.58

4

2

0.9144

10000, 7519, 5633, 4251,
3196, 2403, 1807, 1358

1.33

YES

FAL.3T3.SLS.120903

FAL.3T3.MY.B8.180903

DF

AA61MY3450

46.278

0.367

6.74

3

5

0.8706

10000, 7519, 5633, 4251,
3196, 2403, 1807, 1358

1.33

YES

FAL.3T3.SLS.180903

25.5

0.392

0.116

99.22%

1

5

0.9238

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

1.5

YES

1.5

YES

SLS-B7

1.5

YES

SLS-B8POTASSIUM CYANIDE
IIVS
A1

RF

AA61KW

B1

DF

AA61KW

19.8

0.304

0.403

7.47%

3

3

0.9494

B2

DF

AA61KW

18.9

0.291

0.366

0.25%

5

3

0.9756

B3DF

AA61KW

17.9

0.275

0.408

5.75%

5

3

0.9767

AA61MN-A1

RF

AA61MN

421

6.461

0.085

0.69%

1

6

0.9516

AA61MN-B1 (sealer)DF

AA61MN

NA

NA

0.125

6.06%

7

0

NA

AA61MN-B2 (sealer)DF

AA61MN

NA

NA

0.434

3.69%

0

8

NA

AA61MN-B3 (sealer)DF

AA61MN

19.6

0.301

0.325

1.90%

3

5

0.9619

AA61MN-B4 (sealer)DF

AA61MN

13.9

0.213

0.435

9.17%

3

5

0.9485

AA61MN-B5 (sealer)DF

AA61MN

12.5

0.192

0.446

0.73%

3

5

0.8689FAL.3T3.GP.A1.21.10.04

RF

AA61GP

153

2.357

0.029

97.12%

000.9807

FAL.3T3.GP.B1.11.11.04

DF

AA61GP

219

3.360

0.203

10.87%

8

00.8961

FAL.3T3.GP.B2.26.11.04

DF

AA61GP

253

3.884

0.184

5.76%

2

6

0.3284

FAL.3T3.GP.B3.09.12.04

DF

AA61GP

172

2.638

0.195

22.57%

6

1

0.6436

FAL.3T3.GP.B4.10.12.04

DF

AA61GP

106

1.634

0.236

5.84%

4

2

0.5610100, 66.7, 44.4, 29.6,
19.8, 13.2, 8.78, 5.85
100, 66.7, 44.4, 29.6,
19.8, 13.2, 8.78, 5.85
100, 66.7, 44.4, 29.6,
19.8, 13.2, 8.78, 5.85

range finder

VC1 ODs < VC2 ODs;VC1
removed from subsequent
analysis; volatility issues.

SLS-A5plate sealer used

SLS-B4ECBC
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1500, 1020, 694, 472,
321, 219, 149, 101
200, 136, 92.6, 63.0,
42.8, 29.1, 19.8, 13.5
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

10

RF

1.47

NO

range finder

ppt in 1X C1no points between
ppt in 1X C1-C5
50-100%; PC failed
no points between 0 50%

SLS-P49
SLS-P52

1.47

NO

2.15

YES

SLS-P62

2.15

YES

2.15

YES

10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001

10RFrange finder; nopoints between 0100%

ppt in 1X C11000, 826, 683, 565, 467,
386, 319, 263

1.21

NO

no points between
50-100%ppt in 1X C1-C8; viability not
FAL.3T3.SLS.10.11.04
above 50% for any conc

1.21

YES

SLS-P64
ppt in 2X C1-C2; ppt in 1X
C1-C5

SLS-P66
SLS-P68

FRAME

I-42

500, 413, 342,282,
233,193,159, 132
500, 413, 342,282,
233,193,159, 132
500, 376, 283, 213, 160,
120, 90.3, 67.9

1.21

NO

1.33

YES

FAL.3T3.SLS.21.10.04

FAL.3T3.SLS.26.11.04% VC difference >15

FAL.3T3.SLS.09.12.04
FAL.3T3.SLS.10.12.04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

FAL.3T3.GP.B5.15.12.04

Assay Substance
ID
Type1

DF

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

IC50(ug/mL)

IC50(mM)

Mean VC

AA61GP

117

1.804

0.126

2.18%

4

4

0.6827

5.01%

0

1

0.9614

OD2

R2 6

Exposure
Concentrations(ug/mL)7

500, 376, 283, 213, 160,
120, 90.3, 67.9

Dilution

Acceptable

Factor8

Tests9

1.33

YES

10

RF

1.4

YES

1.4

NO

1.4

YES

SLS-B8YES

plates read 15-16 hr late;orignial reading used wrong SLS-B11
OD wavelength

Rationale for
Unacceptability

Notes

PC ID

FAL.3T3.SLS.15.12.04

PROCAINAMIDE HCL
IIVS
A1

RF

AA61ML

406

1.492

0.421

B1

DF

AA61ML

453

1.666

0.485

3.11%

1

1

0.9213B2

DF

AA61ML

485

1.786

0.400

0.77%

1

0

0.8992B3DF

AA61ML

528

1.944

0.453

4.01%

1

1

0.8702B4DF

AA61ML

511

1.878

0.457

3.83%

3

1

0.9248

AA61KC-A1

RF

AA61KC

363

1.336

0.365

3.67%

0

1

0.9503

AA61KC-B1

DF

AA61KC

406

1.495

0.499

11.41%

3

3

0.9929

AA61KC-B2

DF

AA61KC

412

1.516

0.392

0.00%

4

1

0.9682

AA61KC-B3

DF

AA61KC

383

1.409

0.528

4.19%

3

1

0.9813

RF

AA61GV

550

2.022

0.582

11.63%

2

00.8758

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 714, 510, 364, 260,
186, 133, 94.9
1000, 714, 510, 364, 260,
186, 133, 94.9
1000, 714, 510, 364, 260,
186, 133, 94.9
1000, 833, 694, 579, 482,
402, 335, 279

1.2

range finder

SLS-A4SLS-B4no points between
50-100%outliers removed bySD

SLS-B7

ECBC
10000,1000, 100, 10,1,0.1, 0.01, 0.001
1000, 680, 463, 315, 214,
146, 99.1, 67.4
800, 661, 546, 452, 373,
308, 255, 211
800, 661, 546, 452, 373,
308, 255, 211

10

RF

1.47

YES

range finder

SLS-P13SLS-P37

1.21

YES

SLS-P40

1.21

YES

SLS-P41

10

RF

FRAME
FAL.3T3.GV.A1.10.09.04
FAL.3T3.GV.B1.16.09.04

DF

AA61GV

423

1.555

0.367

5.86%

4

0

0.8061

FAL.3T3.GV.B2.23.09.04

DF

AA61GV

433

1.591

0.405

3.52%

1

1

0.4667

FAL.3T3.GV.B3.14.10.04
FAL.3T3.GV.B4.04.11.04

DF

AA61GV

426

1.566

0.340

7.26%

3

1

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 752, 565, 425, 320,
240, 181, 136
500, 413, 342, 282, 233,
193, 159, 132

1.33

NO

1.21

YES

range finder; nopoints between 50 100%no points between
50 - 100%

FAL.3T3.SLS.10.09.04
outlier removed by SD

FAL.3T3.SLS.16.09.04
FAL.3T3.SLS.23.09.04

C5-C8 show % viabilities>144%; outlier removed
FAL.3T3.SLS.14.10.04
bySD
4 concentrations with values
FAL.3T3.SLS.04.11.04>150%

0.5102750, 620, 512, 423, 350,
289, 239, 197

1.21

YES

750, 620, 512, 423, 350,
289, 239, 197

1.21

YES

10

RF

range finder

SLS-A110RFrange finder

SLS-A1

1.6

NO% VC difference >15

SLS-B1

1.6

NO% VC difference >15

SLS-B1

1.6

NO% VC difference >15

SLS-B2

1.6

NO

no points between
50 - 99.9%

SLS-B2DF

AA61GV

435

1.599

0.238

1.58%

3

1

0.4580A1

RF

AA61GC

NA

NA

0.486

1.76%

0

8

NA

A1 with plate coverRF

AA61GC4380

72.879

0.421

6.01%

1

6

0.82572-PROPANOL

IIVS

B1

DF

AA61GC

11000

183.0280.082

89.61%

3

1

0.8759B1 with plate coverDF

AA61GC6280

104.493

0.216

15.53%

4

1

0.9691B2DF

AA61GC9620

160.067

0.098

87.70%

3

1

0.9420B2 with plate coverDF

AA61GC3160

52.579

0.404

2.68%

5

0

0.9710I-43

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100000, 62500, 39063,
24414, 15259, 9537,
5960, 3725
100000, 62500, 39063,
24414, 15259, 9537,
5960, 3725
100000, 62500, 39063,
24414, 15259, 9537,
5960, 3725
100000, 62500, 39063,
24414, 15259, 9537,
5960, 3725

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

B3

DF

AA61GC

11200

186.3560.223

61.18%

4

2

0.8927B3 with plate coverDF

AA61GC4280

71.215

0.525

6.06%

5

1

0.9764

Exposure
Concentrations(ug/mL)7

50000, 33333, 22222,
14815, 9877, 6584, 4390,
2926
50000, 33333, 22222,
14815, 9877, 6584, 4390,
2926
20500, 14643, 10459,
7471, 5336, 3812, 2723,
1945

Dilution

Acceptable

Factor8

Tests91.5

NO% VC difference >15; PC failed

SLS-B3

1.5

NO

PC failed

SLS-B3

1.4

NO% VC difference >15; no points
between 0.1 - 50%;
low r2

SLS-B4

1.4

NO% VC difference >15

SLS-B4

1.4

YES

SLS-B5

1.4

YES

SLS-B6

Rationale for
Unacceptability

Notes

PC ID

B4

DF

AA61GC

16600

276.206

0.230

22.95%

06

0.6865B4 with plate coverDF

AA61GC

4690

78.0370.418

15.64%

4

3

0.9516B5 with plate coverDF

AA61GC

3940

65.557

0.432

3.99%

5

3

0.9607B6 with plate coverDF

AA61GC

4260

70.882

0.344

2.04%

5

3

0.9911B7 with plate coverDF

AA61GC5860

97.504

0.344

9.77%

4

4

0.618620000, 14286, 10204,
7289, 5206, 3719, 2656,
1897

1.4

NO

B8 with plate coverDF

AA61GC4130

68.719

0.452

0.86%

5

3

0.939920000, 14286, 10204,
7289, 5206, 3719, 2656,
1897

1.4

YES

B8 with DYNEX plate cover for research onlyDF

AA61GC

3210

53.411

0.347

1.34%

6

2

0.936920000, 14286, 10204,
7289, 5206, 3719, 2656,
1897

1.4

NO

for research; gives
lower OD values
than the EXCEL
plate sealers

SLS-B8AA61JL-A1

RF

AA61JL

NA

NA

0.405

7.48%

0

0

NA

10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001

10RF

no points between
high volatility
10 - 90; range finder

SLS-P1

AA61JL-A2

RF

AA61JL

NA

NA

0.19

62.97%

1

2

NA

10

RF

% VC difference >15; range finderhigh volatility

SLS-P3

AA61JL-B1

DF

AA61JL

NA

NA

0.133

75.92%

3

2

NA

1.47

NO

PC failed; % VC
difference > 15high volatility

SLS-P9AA61JL-B2

DF

AA61JL

NA

NA

0.119

75.18%

4

1

NA

1.47

NO

PC failed; % VC
difference > 15high volatility

SLS-P11

AA61JL-B3 sealer

DF

AA61JL

NA

NA

0.256

30.03

4

1

NA

1.47

NO

% VC difference >15high volatility

SLS-P17

AA61JL-B4 sealer

DF

AA61JL

NA

NA

0.446

19.53

7

1

NA

1.47

NO

% VC difference >15high volatility

SLS-P19

1.47

NO% VC difference >15; no points
between 0 -50 %SLS-P20

1.47

NO% VC difference >15

SLS-P20

1.47

NO

1.47

NO

20500, 14643, 10459,
7471, 5336, 3812, 2723,
1945
20000, 14286, 10204,
7289, 5206, 3719, 2656,
1897
20000, 14286, 10204,
7289, 5206, 3719, 2656,
1897

low r2; study director
also rejected due toexcessive well towell variability

SLS-B7

SLS-B8ECBCDF

AA61JL

NA

NA

0.204

46.32

0

4

NA

DF

AA61JL

NA

NA

0.117

67.16

5

2

NA

DF

AA61JL

NA

NA

0.475

15.59

5

3

NA

DF

AA61JL

NA

NA

0.373

31.51

5

2

NA

AA61JL-B6
AA61JL-B5 sealer
AA61JL-B7 sealer
AA61JL-B8 sealer

I-44

100000, 10000, 1000,
100, 10, 1.0, 0.1, 0.01
50000, 34014, 23139,
15740, 10707, 7284,
4955, 3370
50000, 34014, 23139,
15740, 10707, 7284,
4955, 3370
50000, 34014, 23139,
15740, 10707, 7284,
4955, 3370
34014, 23139, 15740,
10707, 7284, 4955, 3370,
2293
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348% VC difference >15: no r2% VC difference >15

SLS-P21
SLS-P21

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

15000, 10204, 6942,
4722, 3212, 2185, 1487,
1011
15000, 10204, 6942,
4722, 3212, 2185, 1487,
1011
15000, 10204, 6942,
4722, 3212, 2185, 1487,
1011

Dilution

Acceptable

Factor8

Tests9

1.47

YES

SLS-P22

1.47

YES

SLS-P23

1.47

NO

Rationale for
Unacceptability

Notes

PC ID

DF

AA61JL

2440

40.599

0.324

0.26

5

3

0.9415DF

AA61JL2780

46.256

0.214

11.21

5

3

0.9572DF

AA61JL2710

45.092

0.171

16.20

5

3

0.9661

A1NG190603

RF

AA61NG> 10,000

NA

0.965

0.22%

0

8

0.0127

10000,1000, 100, 10, 1.0,
0.1, 0.01, 0.001

10RF

F_L.3T3.NG.A2.26.06.03

RF

AA61NG

11700

194.6760.251

42.34%

02

0.7469100000,10000,1000, 100,
10, 1.0, 0.1, 0.01

10

RF

FAL.3T3.NG.B1.03.07.03

DF

AA61NG

92500

1539.101

0.404

12.52%

02

0.57062.15

NO

no points between
10 - 50; r2 too low

FAL.3T3.B2.NG.10.07.03

DF

AA61NG

NA

NA

0.157

56.97%

NA

NA

NA

1.47

NO

% VC difference >15; range finder

1.47

NO

No points between 0& 50%viability; lowr2; %VC difference >
15

1.47

YES

1.47

NO

Mineral oil

FAL.3T3.SLS.180903

1.47

NO

%VC difference > 15

FAL.3T3.SLS.190903

1.47

NO

Mineral oil

FAL.3T3.SLS.190903

1.47

YES

1.47

NO

1.47

YES

AA61JL-B9 sealer
AA61JL-B10

sealer

AA61JL-B11 sealer

FRAME

50000, 23256, 10817,
3031, 2340, 1088, 506,
235
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
50000, 34014, 23139,
15741, 10708, 7284,
4955, 3371

FAL.3T3.NG.B3.120903

DF

AA61NG

34900

580.699

0.251

42.34

0

2

0.7468FAL.3T3.NG.B5.180903 plate
sealerDF

AA61NG

3900

64.892

0.417

3.46

4

1

0.9517

FAL.3T3.NG.B5.180903
mineral oil

DF

AA61NG

5940

98.835

0.366

8.45

5

2

0.9380FAL.3T3.NG.B6.190903 plate
sealerDF

AA61NG

4570

76.040

0.258

17.26

5

1

0.8993

FAL.3T3.NG.B6.190903
mineral oil

DF

AA61NG

4740

78.869

0.384

7.46

5

2

0.9301

FAL.3T3.NG.B7.25.09.03
plate sealer

DF

AA61NG

4130

68.719

0.347

10.58

3

4

0.9244

FAL.3T3.NG.B8.25.09.03
mineral oil

DF

AA61NG

4220

70.216

0.361

7.83

4

4

0.9513

FAL.3T3.NG.B8-03-10-03
plate sealer

DF

AA61NG

3880

64.559

0.510

2.39

5

3

0.9519RF

AA61GU

19.3

0.065

0.320

5.15%

0

1

0.9764

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10

RF

1.79

YES

1.78

NO

50000, 34014, 23139,
15741, 10708, 7284,
4955, 3371
50000, 34014, 23139,
15741, 10708, 7284,
4955, 3371
50000, 34014, 23139,
15741, 10708, 7284,
4955, 3371
50000, 34014, 23139,
15741, 10708, 7284,
4955, 3371
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348% VC difference >15

SLS-P24

PC failed; no points
between 10 -50%;
low r2; range finder
VC difference
greater than 15%; nopoints between 10 50; r2 too low; range
finderA1SLS190603

FAL.3T3.SLS.A2.26.06.
03
FAL.3T3.SLS.B1.03.07.
03high volatility

FAL.3T3.SLS.10.07.03

FAL.3T3.SLS.120903

FAL.3T3.SLS.180903

FAL.3T3.SLS.250903
Mineral oil

FAL.3T3.SLS.250903
FAL.3T3.SLS.031003

PROPRANOLOLIIVS
A1 Preliminary

B1

DF

AA61GU

21.1

0.071

0.384

7.68%

1

1

0.9906

1000, 559.5, 313.0,
175.0, 98.0, 54.5, 30.6,
17.0 [IIVS retested; used
wrong dilution scheme]

B2

DF

AA61GU

19.7

0.067

0.386

4.75%

0

1

0.9834100, 56.2, 31.6, 17.8,
10.0, 5.63, 3.16, 1.78

I-45

range finder

SLS-A1

SLS-B1
No points between
10 and 50%

SLS-B2

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

B3

DF

AA61GU

13.6

0.046

0.484

4.31%

1

4

0.9443B4DF

AA61GU

18.3

0.062

0.444

0.43%

1

3

0.9816B5DF

AA61GU

18.2

0.062

0.319

0.94%

1

2

0.9927

RF

AA61KH

17.5

0.059

0.279

6.15%

0

1

0.8598DF

AA61KH

11.4

0.039

0.204

1.02%

2

2

0.9384DF

AA61KH

16.2

0.055

0.249

4.55%

1

2

0.9601DF

AA61KH

12.2

0.041

0.476

16.18%

2

4

0.8629DF

AA61KH

11.3

0.038

0.297

4.17%

2

4

0.9493DF

AA61KH

8.90

0.030

0.474

9.70%

2

3

0.8932DF

AA61KH

18.7

0.063

0.306

3.70%

2

2

0.9475DF

AA61KH

15.6

0.053

0.311

11.73%

2

2

0.9549Exposure
Concentrations(ug/mL)7

100, 56.2, 31.6, 17.8,
10.0, 5.63, 3.16, 1.78
100, 56.2, 31.6, 17.8,
10.0, 5.63, 3.16, 1.78
100, 56.2, 31.6, 17.8,
10.0, 5.63, 3.16, 1.78

Dilution

Acceptable

Factor8

Tests9

1.78

YES

1.78

NO

1.78

YES

Rationale for
Unacceptability

Notes

PC ID

SLS-B3
PC failed

SLS-B4SLS-B5ECBC
ECBC-3T3-Ib-01
A1
ECBC-3T3-Ib-02
AA61KH-B1
ECBC-3T3-Ib-03
AA61KH-B2
ECBC-3T3-Ib-04
AA61KH-B3
ECBC-3T3-Ib-05
AA61KH-B4
ECBC-3T3-Ib-06
AA61KH-B5
ECBC-3T3-Ib-07
AA61KH-B6
ECBC-3T3-Ib-08
AA61KH-B7

AA61KH-

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 68, 46.3, 31.5, 21.4,
14.6, 9.9, 6.7
100, 68, 46.3, 31.5, 21.4,
14.6, 9.9, 6.7
50, 34, 23.1, 15.7, 10.7,
7.3, 5.0, 3.4
50, 34, 23.1, 15.7, 10.7,
7.3, 5.0, 3.4
50, 34, 23.1, 15.7, 10.7,
7.3, 5.0, 3.4
50, 34, 23.1, 15.7, 10.7,
7.3, 5.0, 3.4
50, 34, 23.1, 15.7, 10.7,
7.3, 5.0, 3.4

10RF

range finder

SLS-P1

1.47

NO

PC failed

SLS-P3

1.47

NO

PC failed

SLS-P4

1.47

NO

VC difference > 15%

SLS-P5

1.47

YES

SLS-P7

1.47

YES

SLS-P9

1.47

YES

SLS-P12

1.47

YES

SLS-P1310RFrange finder

FRAME
A1

1b3T3RF01FALNM

RF

AA61NM

57.7

0.195

0.413

12.84%

000.9454A2

1b3T3RF02FALNM

RF

AA61NM

0.022

0.000

0.479

8.47%

1

3

0.9694

1b3T3DF02FALNM

DF

AA61NM

19.8

0.067

0.350

6.58%

1

3

0.8123

1b3T3DF02FALNM

DF

AA61NM

23.1

0.078

0.477

10.31%

1

1

0.8691

1b3T3DF02FALNM

DF

AA61NM

23.9

0.081

0.220

13.61%

02

0.8821

1b3T3DF05FALNM

DF

AA61NM

13.8

0.047

0.449

8.47%

1

3

0.9401

1b3T3DF06FALNM

DF

AA61NM

33.3

0.113

0.300

11.67%

1

2

0.8052

1b3T3DF07FALNM

DF

AA61NM

8.80

0.030

0.538

9.69%

1

5

0.9020

1b3T3DF08FALNM A2650

DF

AA61NM

15.2

0.051

0.223

5.91%

1

4

0.89791b3T3DF09FALNM

DF

AA61NM

22.2

0.075

0.582

4.98%

1

0

0.9438

1b3T3DF10FALNM

DF

AA61NM

8.36

0.028

0.426

12.59%

4

3

0.8917

1b3T3DF11FALNM

DF

AA61NM

18.5

0.063

0.227

13.72%

1

4

0.6461

AA61PX

19.4

0.108

0.451

0.06%

1

2

0.96591000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
35, 23.81, 16.20, 11.02,
7.50, 5.10, 3.47, 2.36
35, 23.81, 16.20, 11.02,
7.50, 5.10, 3.47, 2.36
35, 23.81, 16.20, 11.02,
7.50, 5.10, 3.47, 2.36
35.000, 23.810, 16.197,
11.018, 7.495, 5.099,
3.469, 2.360
35, 23.81, 16.20, 11.02,
7.50, 5.10, 3.47, 2.36
35, 23.81, 16.20, 11.02,
7.50, 5.10, 3.47, 2.36
35, 23.81, 16.20, 11.02,
7.50, 5.10, 3.47, 2.36
35.000, 23.810, 16.197,
11.018, 7.495, 5.099,
3.469, 2.360
35.000, 23.810, 16.197,
11.018, 7.495, 5.099,
3.469, 2.360
35, 23.81, 16.20, 11.02,
7.50, 5.10, 3.47, 2.36

10RFrange finder

1.47

NO

PC failed

1.47

YES

NR crystals in plate
NR crystals in plate;
stopped after 1 h; nopoint between 10 &50% viability; PC
failed

1b3T3CRTFALSLS
12/4/02
1b3T3CRTFALSLS
12/10/02
1b3T3CRTFALSLS
12/17/02
1b3T3CRTFALSLS
1/7/03
1b3T3CRTFALSLS
1/8/03

1.47

NO

1.47

YES

1.47

NO

1.47

YES

1.47

NO

NR crystals in plate;
stopped after 1 h;
PC failed

1b3T3CRTFALSLS
1/28/03

1.47

NONo points between
50 & 90% viability

1b3T3CRTFALSLS
2/4/03

1.47

YES

1.47

NO

r2< 0.8

Nonmonotonic curve.

1b3T3CRTFALSLS
2/26/0310RF

range finder

ppt in 2X C1SLS-A2

1b3T3CRTFALSLS
1/14/03
1b3T3CRTFALSLS
1/15/03
1b3T3CRTFALSLS
1/21/03

PC failed

1b3T3CRTFALSLS
2/5/03PROPYLPARABEN
IIVS
A1

RF

I-46

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

B1

DF

AA61PX

19.2

0.106

0.445

3.97%

3

4

0.9395B2

DF

AA61PX

17.0

0.094

0.354

9.68%

4

4

0.9707B3DF

AA61PX

15.0

0.083

0.368

7.51%

4

4

0.9675

AA61PK-A1

RF

AA61PK

22.9

0.127

0.231

2.13%

1

2

0.9271

AA61PK-B1

DF

AA61PK

18.2

0.101

0.561

6.97%

4

4

0.9538

AA61PK-B2

DF

AA61PK

19.8

0.110

0.543

6.49%

4

4

0.9827

AA61PK-B3

DF

AA61PK

21.8

0.121

0.367

17.33%

3

5

0.9431

AA61PK-B4

DF

AA61PK

24.6

0.137

0.341

7.63%

2

5

0.9812

FAL.3T3.HT.A1.01/04/04

RF

AA61HT

73.5

0.408

0.229

5.88%

2

1

0.8795

FAL.3T3.HT.B1.29/04/04

DF

AA61HT

41.3

0.229

0.193

7.55%

1

4

0.7787

FAL.3T3.HT.B2.07/05/04

DF

AA61HT

45.3

0.251

0.278

8.04%

4

2

0.9762

FAL.3T3.HT.B3.20/05/04

DF

AA61HT

68.7

0.381

0.332

10.50%

3

3

0.9633

Exposure
Concentrations(ug/mL)7

100, 62.5, 39.1, 24.4,
15.3, 9.54, 5.96, 3.73
100, 62.5, 39.1, 24.4,
15.3, 9.54, 5.96, 3.73
100, 62.5, 39.1, 24.4,
15.3, 9.54, 5.96, 3.73

Dilution

Acceptable

Factor8

Tests91.6

YES

C8 removed from PRISM by
SD due to the upswing of
SLS-B5the response curve at that
conc.

1.6

YES

SLS-B9

1.6

YES

SLS-B10

Rationale for
Unacceptability

Notes

PC ID

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
215, 100, 46.5, 21.6,
10.1, 4.68, 2.18, 1.01
215, 100, 46.5, 21.6,
10.1, 4.68, 2.18, 1.01
215, 100, 46.5, 21.6,
10.1, 4.68, 2.18, 1.01
215, 100, 46.5, 21.6,
10.1, 4.68, 2.18, 1.01

10

RF

2.15

YES

2.15

YES

2.15

NO

2.15

YES

range finder

ppt in 2X C1SLS-P7SLS-P26
SLS-P28% VC difference >15

SLS-P30
SLS-P32

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

10

RF

2.15

YES

range finder

ppt in 2X C1-C2

FAL.3T3.SLS.01/04/04

NR crystals; highbackground

2.15

YES

FAL.3T3.SLS.29/04/04
FAL.3T3.SLS.07/05/04

2.15

YES

FAL.3T3.SLS.20/05/04

SODIUM ARSENITE
IIVS
A1

RF

AA61MV

0.454

0.003

0.368

14.66%

2

3

0.9583

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

B1

DF

AA61MV

0.745

0.006

0.418

11.53%

3

3

0.97543.00, 2.00, 1.33, 0.889,
0.593, 0.395, 0.263,
0.176

1.5

YES

SD removed 7 data points
from PRISM analysis;
considered them outliers
SLS-B4even though EXCEL macros
did not identify as such

B2

DF

AA61MV

0.755

0.006

0.414

5.16%

3

4

0.9674

1.5

YES

SLS-B7B3DF

AA61MV

0.548

0.004

0.464

4.40%

4

4

0.9506

1.5

YES

SLS-B8

AA61KA-A1

RF

AA61KA

0.483

0.004

0.506

2.78%

3

3

0.994010

RF

AA61KA-B1

DF

AA61KA

0.482

0.004

0.565

4.68%

4

4

0.9795

2.15

YES

SLS-P41

AA61KA-B2

DF

AA61KA

0.528

0.004

0.739

1.30%

2

4

0.9661

2.15

YES

SLS-P43

AA61KA-B3

DF

AA61KA

0.477

0.004

0.617

1.99%

2

4

0.9795

2.15

YES

SLS-P43

RF

AA61GS

1.11

0.009

0.254

4.69%

0

3

0.985810RF

3.00, 2.00, 1.33, 0.889,
0.593, 0.395, 0.263,
0.176
3.00, 2.00, 1.33, 0.889,
0.593, 0.395, 0.263,
0.176

range finder

SLS-A5ECBC
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10.0, 4.65, 2.16, 1.01,
0.468, 0.218, 0.101,
0.047
10.0, 4.65, 2.16, 1.01,
0.468, 0.218, 0.101,
0.047
10.0, 4.65, 2.16, 1.01,
0.468, 0.218, 0.101,
0.047

range finder

SLS-16FRAME
FAL.3T3.GS.A1.21.10.04

I-47

10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001

range finder; nopoints between 050%

ppt in 2X C1

FAL.3T3.SLS.21.10.04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 4.65, 2.16, 1.01,
0.468, 0.218, 0.101,
0.047
10.0, 4.65, 2.16, 1.01,
0.468, 0.218, 0.101,
0.047
10.0, 4.65, 2.16, 1.01,
0.468, 0.218, 0.101,
0.047

Dilution

Acceptable

Factor8

Tests9

1.47

NO

2.15

YES

2.15

YES

2.15

YES

Rationale for
Unacceptability

Notes

no points between
50-100%ppt in 1X C1-C8

PC ID

FAL.3T3.GS.B1.11.11.04

DF

AA61GS

0.678

0.005

0.731

1.75%

8

0

0.9745

FAL.3T3.SLS.10.11.04

FAL.3T3.GS.B2.25.11.04

DF

AA61GS

0.872

0.007

0.381

2.01%

3

1

0.9740FAL.3T3.GS.B3.26.11.04

DF

AA61GS

1.07

0.008

0.299

7.88%

1

2

0.9795

FAL.3T3.GS.B4.02.12.04

DF

AA61GS

2.38

0.018

0.232

12.64%

2

2

0.9073

AA61PE

3400

58.249

0.474

1.86%

1

6

0.968010000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RF

0.9548

10000, 7143, 5102, 3644,
2603, 1859, 1328, 949

1.4

YES

SLS-B4

1.5

YES

SLS-B7

1.5

YES

SLS-B8FAL.3T3.SLS.25.11.04
outliers removed bySD

FAL.3T3.SLS.26.11.04
FAL.3T3.SLS.02.12.04(SW)

SODIUM CHLORIDE
IIVS
A1
B1

RF
DF

AA61PE

5160

88.367

0.496

1.02%

2

6

20000, 13333, 8889,
5926, 3951, 2634, 1756,
1171
20000, 13333, 8889,
5926, 3951, 2634, 1756,
1171

range finder

SLS-A3B2

DF

AA61PE

5120

87.557

0.391

6.63%

3

3

0.9651B3DF

AA61PE

4350

74.352

0.450

5.91%

2

4

0.9484RF

AA61JW

4140

70.842

0.365

0.96%

1

6

0.9393

10000,1000, 100, 10,1,0.1, 0.01, 0.001

10

RF

1.47

YES

SLS-27ECBC
AA61JW-A1

range finder

SLS-11

AA61JW-B1

DF

AA61JW

5050

86.355

0.538

7.42%

2

6

0.9446

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

AA61JW-B2

DF

AA61JW

4720

80.777

0.449

8.39%

2

6

0.9401

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

SLS-29AA61JW-B3

DF

AA61JW

4600

78.757

0.519

5.06%

2

6

0.936910000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

SLS-P31RF

AA61FM

3540

60.574

0.396

0.86%

1

4

0.9371

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RFrange finder

FAL.3T3.SLS.21.05.04

2.15

NO

% VC difference >15

FAL.3T3.SLS.04.06.04

FRAME
FAL.3T3.FM.A1.21.05.04
FAL.3T3.FM.B1.04.06.04

DF

AA61FM

3010

51.5570.452

18.08%

2

3

0.813820000, 9302, 4327, 2012,
936, 435, 202, 94.2

FAL.3T3.FM.B2.17.06.04

DF

AA61FM

4500

76.964

0.538

0.15%

2

6

0.972820000, 9302, 4327, 2012,
936, 435, 202, 94.2

2.15

YES

FAL.3T3.SLS.17.06.04

FAL.3T3.FM.B3.08.07.04

DF

AA61FM

4010

68.595

0.322

7.57%

2

4

0.961820000, 9302, 4327, 2012,
936, 435, 202, 94.2

2.15

YES

FAL.3T3.SLS.08.07.04

FAL.3T3.FM.B4.09.07.04

DF

AA61FM

4520

77.320

0.384

3.06%

2

3

0.755620000, 9302, 4327, 2012,
936, 435, 202, 94.2

2.15

YES

FAL.3T3.SLS.09.07.04

FAL.3T3.FM.B5.16.07.04

DF

AA61FM

5470

93.603

0.399

4.36%

1

3

0.936120000, 9302, 4327, 2012,
936, 435, 202, 94.2

2.15

YES

FAL.3T3.SLS.16.07.04

I-48

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC

0.002

0.380

OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9

10

RF

1.5

YES

SLS-B4

1.5

YES

SLS-B7

1.5

YES

SLS-B8

10

RF

2.15

YES

SLS-P32

2.15

YES

SLS-P34

2.15

YES

SLS-P36

Rationale for
Unacceptability

Notes

PC ID

SODIUM DICHROMATE DIHYDRATE
IIVS
A1

RF

AA61FP

0.642

5.57%

1

1

0.9860B1DFAA61FP

0.548

0.002

0.502

2.42%

5

3

0.9910B2DFAA61FP

0.527

0.002

0.435

0.54%

4

4

0.9751B3DF

AA61FP

0.455

0.002

0.449

3.30%

5

3

0.9931

RF

AA61NT

0.561

0.002

0.291

2.92%

2

1

0.985010000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
3.00, 2.00, 1.33, 0.889,
0.593, 0.395, 0.263,
0.176
2.47, 1.65, 1.10, 0.733,
0.489, 0.326, 0.217,
0.145
3.00, 2.00, 1.33, 0.889,
0.593, 0.395, 0.263,
0.176

range finder

SLS-A4ECBC
AA61NT-A1

10000,1000, 100, 10,1,0.1, 0.01, 0.001
6.00, 2.79, 1.30, 0.604,
0.281, 0.131, 0.061,
0.028
6.00, 2.79, 1.30, 0.604,
0.281, 0.131, 0.061,
0.028
6.00, 2.79, 1.30, 0.604,
0.281, 0.131, 0.061,
0.028

range finder

SLS-P12

AA61NT-B1

DF

AA61NT

0.555

0.002

0.438

5.78%

4

4

0.9835

AA61NT-B2

DF

AA61NT

0.550

0.002

0.409

9.10%

4

4

0.9713

AA61NT-B3

DF

AA61NT

0.703

0.002

0.654

1.90%

3

5

0.9871

FAL.3T3.HK.A1.10.09.04

RF

AA61HK

0.871

0.003

0.496

11.79%

5

00.971010000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RFrange finder; nopoints between 50 100%

FAL.3T3.SLS.10.09.04

FAL.3T3.HK.B1.16.09.04

DF

AA61HK

NA

NA

0.343

4.69%

0

0

NA

10.0, 4.7, 2.2, 1.0, 0.5,
0.2, 0.101, 0.047

2.15

NO

no points between 0 100%

FAL.3T3.SLS.16.09.04

1.47

YES

FAL.3T3.SLS.23.09.04

1.33

YES

FAL.3T3.SLS.14.10.04

1.33

YES

FAL.3T3.SLS.04.11.04

FRAME

FAL.3T3.HK.B2.23.09.04

DF

AA61HK

0.388

0.001

0.367

3.41%

2

1

0.9713

FAL.3T3.HK.B3.14.10.0404

DF

AA61HK

0.864

0.003

0.340

3.67%

1

7

0.9167

FAL.3T3.HK.B4.04.11.04

DF

AA61HK

0.719

0.002

0.265

8.67%

2

3

0.7857

1.00, 0.680, 0.463, 0.315,
0.214, 0.146, 0.099,
0.067
1.00, 0.752, 0.565, 0.425,
0.320, 0.240, 0.181,
0.136
1.00, 0.752, 0.565, 0.425,
0.320, 0.240, 0.181,
0.136

SODIUM I FLUORIDE
IIVS
A1

RF

AA61HF

59.2

1.410

0.526

0.64%

1

3

0.9854B1

DF

AA61HF

86.7

2.065

0.391

0.28%

2

4

0.9788B2DFAA61HF

75.5

1.798

0.512

5.46%

3

3

0.9857B3DF

AA61HF

71.4

1.700

0.541

9.13%

2

2

0.9894B4DF

AA61HF

83.8

1.996

0.465

2.42%

3

3

0.9676

RF

AA61MG

61.7

1.469

0.361

7.05%

0

0

0.956910000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3

10

RF

1.8

YES

SLS-B1

1.8

YES

SLS-B2

1.8

NO

1.8

YES

10

RF

range finder

PC failed

SLS-A1

SLS-B3
SLS-B4ECBC
AA61MG-A1

I-49

10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001

PC failed; no points
between 10 - 90%;
range finder

SLS-P2

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

AA61MG-B1

DF

AA61MG

59.7

1.422

0.597

4.34%

1

2

0.9567

AA61MG-B2

DF

AA61MG

56.8

1.353

0.566

1.90%

3

4

0.9553

AA61MG-B3

DF

AA61MG

67.5

1.608

0.522

6.32%

3

2

0.9336

FAL.3T3.A1.RH.200603

RF

AA61RH

208

4.954

0.716

0.48%

1

0

0.9733

FAL.3T3.B1.RH.27.06.03

DF

AA61RH

102

2.429

0.425

2.23%

2

1

0.9119FAL.3T3.B2.RH.04.07.03

DF

AA61RH

85.9

2.046

0.568

0.12%

2

1

0.9438FAL.3T3.B3.RH.11.07.03

DF

AA61RH

76.0

1.810

0.575

3.23%

2

1

0.9762

FAL.3T3.B4.RH.18.07.03

DF

AA61RH

110

2.620

0.552

4.70%

2

1

0.9301

Exposure
Concentrations(ug/mL)7

200, 136.1, 92.6, 63.0,
42.8, 29.1, 19.8, 13.5
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3

Dilution

Acceptable

Factor8

Tests9

1.47

YES

SLS-P6

1.21

YES

SLS-P7

1.21

YES

SLS-P10

10

RF

1.47

YES

Rationale for
Unacceptability

Notes

PC ID

FRAME
10000,1000, 100, 10, 1.0,
0.1, 0.01, 0.001
150, 102.0, 69.4, 47.2,
32.1, 21.8, 14.9, 10.1
300, 204, 139, 94.4, 64.2,
43.7, 29.7, 20.2
300, 204, 139, 94.4, 64.2,
43.7, 29.7, 20.2
300, 204, 139, 94.4, 64.2,
43.7, 29.7, 20.2

no points between
50 - 90%; range
finder

FAL.3T3.SLS2.A1.2006
03
FAL.3T3.SLS.A2.27.06.
03

1.47

NO

1.47

YES

PC failed

FAL.3T3.SLS.04.07.03
FAL.3T3.SLS.11.07.03

1.47

YES

FAL.3T3.SLS.18.07.03

SODIUM HYPOCHLORITEIIVS
VC1 ODs < VC2 ODs; VC1
removed from subsequent
analysis; volatility issues.

A1

RF

AA61RD

310

4.171

0.414

28.60%

1

4

0.9878

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RFrange finder

B1

DF

AA61RD

NA

NA

0.464

1.53%

0

5

NA

1000, 667, 444, 296, 198,
132, 87.8, 58.5

1.5

NO

no points between 0 50%

B1 (should be B2)

DF

AA61RD

1110

14.866

0.425

2.24%

2

1

0.9708

10000, 7143, 5102, 3644,
2603, 1859, 1328, 949

1.4

YES

plate sealer used

B3

DF

AA61RD1600

21.537

0.446

5.64%

3

2

0.981010000, 7143, 5102, 3644,
2603, 1859, 1328, 949

1.4

YES

plates read 15-16 hr late;orignial reading used wrong
SLS-B11
OD wavelength; plate sealer
used

B4

DF

AA61RD2170

29.187

0.404

9.58%

2

6

0.8825

1.4

YES

B5

DF

AA61RD

3140

42.188

0.431

0.64%

1

3

0.9519

1.4

YES

AA61HE-A1

RF

AA61HE

NA

NA

0.241

44.19%

1

10.000010

AA61HE-A2

RF

AA61HE

600

8.057

0.409

0.71%

1

1

0.6930AA61HE-B1

DF

AA61HE

728

9.777

0.418

6.56%

2

6

0.9476

AA61HE-B2

DF

AA61HE80210.769

0.550

3.94%

3

5

0.8389AA61HE-B3

DF

AA61HE940

12.624

0.603

3.31%

2

5

0.9363

RF

AA61LU1060

14.295

0.483

0.62%

0

1

4000, 2857, 2041, 1458,
1041, 744, 531, 379
4000, 2857, 2041, 1458,
1041, 744, 531, 379

SLS-A1
SLS-B4SLS-B6

SLS-B12plate sealer used

SLS-B15

ECBC
10000, 1000, 100,10, 1,
0.1, 0.01, 0.001
1000, 100,10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 826, 683, 565, 467,
386, 319, 263
1210, 1000, 826, 683,
565, 467, 386, 319
1210, 1000, 826, 683,
565, 467, 386, 319

RFrange finder

SLS-P310RFrange finder

SLS-P17

1.21

YES

SLS-P19

1.21

YES

SLS-P22

1.21

YES

SLS-P23

0.9323

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RF

0.7288

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

YES

FAL.3T3.SLS.16/01/04

2.15

YES

FAL3T3.23-01-04

1.47

YES

FRAME
FAL.3T3.LU.A1.09/01/04
FAL3T3.LU.A2.16.01.04

DF

AA61LU

391

5.250

0.897

4.13%

3

5

FAL3T3.LU.B1.23.01.04

DF

AA61LU

1090

14.696

0.505

7.27%

1

2

0.9546

5000, 2325.6, 1081.7,
503.1, 234.0, 108.8, 50.6,
23.5

FAL3T3.LU.B2.30.01.04

DF

AA61LU935

12.5660.401

11.07%

3

20.9787

5000, 3401, 2314, 1574,
1071, 728, 496, 337

I-50

range finder

FAL.3T3.SLS.09/01/04

steep toxicity curve; will
adjust concentrations for B3 FAL.3T3.SLS.29/01/04
to 2500 ug/ml (1.47 dil)

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

FAL3T3.LU.B3.06-02-04

Assay Substance
ID
Type1

DF

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

IC50(ug/mL)

IC50(mM)

Mean VC

AA6 LU923

12.3930.361

18.60%

1

3

0.9557

OD2

R2 6

Exposure
Concentrations(ug/mL)7

5000, 3401, 2314, 1574,
1071, 728, 496, 337

Dilution

Acceptable

Factor8

Tests9

1.47

NO%VC difference >15;
VC1 ODs lower than VC2
possible volatility
ODs
problem

FAL.3T3.SLS.06/02/04

range finder

SLS-A5

Rationale for
Unacceptability

Notes

PC ID

SODIUM OXALATE
IIVS
A1

RF

AA61GX

24.9

0.186

0.341

1.34%

1

4

0.9800B1DF

AA61GX

19.7

0.147

0.435

3.04%

5

3

0.9762B2DF

AA61GX

37.9

0.283

0.472

1.09%

3

5

0.9774B3DF

AA61GX

80.2

0.598

0.349

13.14%

1

4

0.9617B4DF

AA61GX

60.1

0.449

0.509

1.26%

2

6

0.9495

AA61LZ-A1

RF

AA61LZ

55.3

0.413

0.544

0.17%

1

4

0.9689AA61LZ-B1

DF

AA61LZ

49.9

0.372

0.455

3.70%

3

5

0.9871

AA61LZ-B2

DF

AA61LZ

54.0

0.403

0.527

6.96%

3

5

0.9578

AA61LZ-B3

DF

AA61LZ

22.2

0.166

0.450

3.43%

3

3

0.9836

RF

AA61RC

74.6

0.557

0.291

1.60%

3

0

0.9198100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
100, 66.7, 44.4, 29.6,
19.8, 13.2, 8.78, 5.85
100, 66.7, 44.4, 29.6,
19.8, 13.2, 8.78, 5.85
100, 66.7, 44.4, 29.6,
19.8, 13.2, 8.78, 5.85
100, 66.7, 44.4, 29.6,
19.8, 13.2, 8.78, 5.85

10

RF

1.5

YES

1.5

YES

1.5

YES

1.5

YES

10

RF

2.15

YES

2.15

YES

2.15

YES

10

RF

ppt in 1X C1 and 2X C1

ppt in 2X C1; ppt in 1X C1SLS-B6
C3
ppt in 2X C1-C4; plates read
15-16 hr late; orignial
SLS-B11reading used wrong OD
wavelength
ppt in 2X C1-C2; ppt in 1X
SLS-B12C1-C4
ppt in 2X C1SLS-B15ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

range finder

ppt in 2X C1; ppt in 1X in C1SLS-15
C2
ppt in 2X C1-C2; ppt in 1X
SLS-P65
C1-C5
ppt in 2X C1-C2; ppt in 1X in
SLS-P66
C1-C5
ppt in 2X C1-C2; ppt in 1X
SLS-P68
C1-C6

FRAME
FAL.3T3.RC.A1.21.10.04
FAL.3T3.RC.B1.11.11.04

DF

AA61RC

28.8

0.215

0.471

13.15%

500.8505

FAL.3T3.RC.B2.25.11.04

DF

AA61RC

34.5

0.258

0.369

4.78%

1

1

0.8807

FAL.3T3.RC.B3.26.11.04

DF

AA61RC

37.3

0.279

0.309

3.13%

1

1

0.9655

FAL.3T3.RC.B4.02.12.04

DF

AA61RC

235

1.753

0.282

2.19%

1

0

0.2212

FAL.3T3.RC.B5.09.12.04

DF

AA61RC

21.1

0.157

0.380

8.96%

2

2

0.8788

10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001
500, 340, 231, 157, 107,
72.8, 49.6, 33.7
250, 116, 54.1, 25.2,
11.7,5.44, 2.53, 1.18
250, 116, 54.1, 25.2,
11.7,5.44, 2.53, 1.18
250, 116, 54.1, 25.2,
11.7,5.44, 2.53, 1.18
250, 116, 54.1, 25.2,
11.7,5.44, 2.53, 1.18

1.47

NO

2.15

YES

2.15

YES

2.15

NO

2.15

YES

range finder; nopoints between 50100%
no points between
50-100%ppt in 2X C1; ppt in 1X in C1FAL.3T3.SLS.21.10.04
C3
ppt in 1X C1-C8

FAL.3T3.SLS.10.11.04

ppt in 2X C1; ppt in 1X in C1FAL.3T3.SLS.25.11.04
C4
ppt in 2X C1; ppt in 1X in C1FAL.3T3.SLS.26.11.04
C4;
no points between
50-100%C7 gives > 200% viability;
FAL.3T3.SLS.02.12.04
ppt in 2X C1 and 1X C1-C2 (RB)ppt in 2X C1; ppt in 1X in C1FAL.3T3.SLS.09.12.04
C4

SODIUM SELENATE
IIVS
A1

RF

AA61FS

39.2

0.208

0.540

3.31%

1

2

0.9909B1DF

AA61FS

42.3

0.224

0.407

0.61%

5

3

0.9884B2DF

AA61FS

35.2

0.186

0.507

2.69%

6

2

0.9874B3DF

AA61FS

40.0

0.212

0.504

9.65%

5

2

0.9879B4DF

AA61FS

32.1

0.170

0.458

0.02%

5

2

0.9884

AA61LF-A1

RF

AA61LF

6.04

0.032

0.438

0.99%

1

2

0.9663

AA61LF-B1

DF

AA61LF

13.6

0.072

0.537

2.38%

3

2

0.9271

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
300, 200, 133, 89, 59.3,
39.5, 26.3, 17.6
300, 200, 133, 89, 59.3,
39.5, 26.3, 17.6
300, 200, 133, 89, 59.3,
39.5, 26.3, 17.6
300, 200, 133, 89, 59.3,
39.5, 26.3, 17.6

10

RF

1.5

YES

1.5

YES

1.5

NO

1.5

YES

10

RF

1.47

YES

range finder

SLS-A1
SLS-B1
SLS-B2

PC failed

SLS-B3
SLS-B4

ECBC

I-51

10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001
100, 68.1, 46.3, 31.5,
21.4, 14.6, 9.9, 6.8

PC failed; range
finderSLS-P2SLS-P6

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

AA61LF-B2

DF

AA61LF

13.8

0.073

0.597

3.18%

4

2

0.9754

AA61LF-B3

DF

AA61LF

10.8

0.057

0.569

3.10%

3

2

0.9626

FAL.3T3.A1.NS.200603

RF

AA61NS

221

1.170

0.670

1.17%

0

0

0.9739FAL.3T3.B1.NS.27.06.03

DF

AA61NS

62.4

0.330

0.497

3.76%

1

1

0.8042

FAL.3T3.B2.NS.04.07.03

DF

AA61NS

52.6

0.278

0.525

2.86%

2

1

0.9189FAL.3T3.B3.NS.11.07.03

DF

AA61NS

57.7

0.305

0.555

5.84%

2

1

0.9734

FAL.3T3.B4.NS.17.07.03

DF

AA61NS

42.4

0.224

0.666

2.83%

2

1

0.9758

A1

RF

AA61JY

77.8

0.233

0.337

1.56%

1

0

0.8728B1DF

AA61JY

89.7

0.268

0.489

1.52%

1

3

0.8961B2DF

AA61JY

80.2

0.240

0.355

6.46%

1

2

0.8383B3DF

AA61JY

80.7

0.241

0.434

7.93%

1

2

0.9277

RF

AA61NR

NA

NA

0.317

12.60%

1

4

NA

Exposure
Concentrations(ug/mL)7

100, 68.1, 46.3, 31.5,
21.4, 14.6, 9.9, 6.8100, 68.1, 46.3, 31.5,
21.4, 14.6, 9.9, 6.8

Dilution

Acceptable

Factor8

Tests9

1.47

YES

SLS-P8

1.47

YES

SLS-P10

10

RF

1.47

YES

Rationale for
Unacceptability

Notes

PC ID

FRAME
10000,1000, 100, 10, 1.0,
0.1, 0.01, 0.001
120, 81.6, 55.5, 37.8,
25.7, 17.5, 11.9, 8.1
200, 136, 92.6, 63, 42.8,
29.2, 19.8, 13.5
200, 136, 92.6, 63, 42.8,
29.2, 19.8, 13.5
200, 136, 92.6, 63, 42.8,
29.2, 19.8, 13.5

no points between
10 - 90%; range
finder

FAL.3T3.SLS2.A1.2006
03
FAL.3T3.SLS.A2.27.06.
03

1.47

NO

1.47

YES

PC failed

FAL.3T3.SLS.04.07.03
FAL.3T3.SLS.11.07.03

1.47

YES

FAL.3T3.SLS.17.07.03

STRYCHNINE

IIVS100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49

10

RF

1.4

YES

1.4

YES

1.4

YES

10

RF

range finder

SLS-A5SLS-B4SLS-B7
ppt in 2X C1; slight film of
powder on medium surface

SLS-B8ppt in 2X C1SLS-P49

ECBC
AA61NR-A1
AA61NR-B1

DF

AA61NR

NA

NA

0.431

6.47%

8

0

NA

AA61NR-B2

DF

AA61NR

452

1.351

0.526

5.34%

2

6

0.8969

AA61NR-B3

DF

AA61NR

418

1.249

0.461

0.27%

2

5

0.9559

AA61NR-B4

DF

AA61NR

298

0.891

0.410

5.55%

1

6

0.8163

FAL.3T3.FY.A1.21.10.04

RF

AA61FY

133

0.397

0.362

10.70%

1

00.5214

FAL.3T3.FY.B1.25.11.04

DF

AA61FY

108

0.322

0.436

8.15%

5

2

0.8455

FAL.3T3.FY.B2.26.11.04

DF

AA61FY

118

0.352

0.289

2.16%

5

2

0.9110500, 50.0, 5.0, 0.5, 0.05,
0.005, 0.0005, 0.00005
800, 661, 546, 452, 373,
308, 255, 211
800, 544, 370, 252, 171,
117, 79.3, 53.9
800, 544, 370, 252, 171,
117, 79.3, 53.9
800, 544, 370, 252, 171,
117, 79.3, 53.9

range finder
no points between
50 - 100%

ppt in 2X C1-C7; dilution is
1.21 but no points havegreater than 50% viability
ppt in 2X C1-C4; ppt in 1X
C1

1.21

NO

SLS-P65

1.47

YES

1.47

YES

ppt in 2X C1-C2

SLS-P68

1.47

YES

ppt in 2X C1; ppt in 1X C1SLS-P70

10

RF

1.21

YES

1.21

YES

SLS-P66

FRAME

FAL.3T3.FY.B3.02.12.04

DF

AA61FY

NA

NA

0.258

2.30%

0

0

NA

FAL.3T3.FY.B4.09.12.04

DF

AA61FY

147

0.440

0.350

0.00%

4

3

0.7540250, 25, 2.5, 0.25, 0.025,
0.0025, 0.00025,
0.000025
250, 207, 171, 141, 117,
96.4, 79.7, 65.8
250, 207, 171, 141, 117,
96.4, 79.7, 65.8
250, 207, 171, 141, 117,
96.4, 79.7, 65.8
250, 207, 171, 141, 117,
96.4, 79.7, 65.8

1.21

NO

1.21

YES

10

RF

1.6

YES

range finder; nopoints between 50100%

FAL.3T3.SLS.21.10.04
FAL.3T3.SLS.25.11.04
steep toxicity curve

no points between 0- no toxicity values less than100%
140% viability

FAL.3T3.SLS.26.11.04
FAL.3T3.SLS.02.12.04(SW)
FAL.3T3.SLS.09.12.04

THALLIUM I SULFATE
IIVS
A1

RF

AA61KJ

7.74

0.015

0.407

4.94%

2

3

0.9809B1

DF

AA61KJ

5.31

0.011

0.466

2.22%

6

2

0.9348I-52

100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
50.0, 31.3, 19.5, 12.2,
7.63, 4.77, 2.98, 1.86

range finder

SLS-A1
SLS-B4

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

B2

DF

AA61KJ

8.29

0.02

0.357

10.53%

4

4

0.9392B3DF

AA61KJ

5.22

0.01

0.454

0.66%

5

3

0.9603

RF

AA61PB

5.41

0.011

0.362

9.63%

3

5

0.9706

Exposure
Concentrations(ug/mL)7

50.0, 31.3, 19.5, 12.2,
7.63, 4.77, 2.98, 1.86
50.0, 31.3, 19.5, 12.2,
7.63, 4.77, 2.98, 1.86

Dilution

Acceptable

Factor8

Tests9

1.6

YES

1.6

YES

10

RF

range finder

SLS-P49

2.15

NO

PC failed

SLS-P53

2.15

YES

SLS-P54

2.15

YES

SLS-P57

2.15

YES

SLS-P59

10

RF

2.15

YES

Rationale for
Unacceptability

Notes

outlier removed bySD

PC ID

SLS-B7
SLS-B8ECBC
AA61PB-A1
AA61PB-B1

DF

AA61PB

NA

NA

0.509

7.59%

6

2

NA

AA61PB-B2

DF

AA61PB

3.46

0.007

0.703

7.58%

5

3

0.9831

AA61PB-B3

DF

AA61PB

2.12

0.004

0.539

11.54%

6

2

0.9629

AA61PB-B4

DF

AA61PB

2.86

0.006

0.399

3.57%

3

5

0.9627

FAL.3T3.GB.A1.09/01/04

RF

AA61GB

0.015

0.000

0.664

4.29%

1

3

0.9201FAL3T3.GB.A2.16.01.04

DF

AA61GB

2.01

0.004

0.861

8.61%

7

1

0.8562500, 50.0, 5.0, 0.5, 0.05,
0.005, 0.0005, 0.00005
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

FRAME

FAL3T3.GB.B1.23.01.04

DF

AA61GB

13.6

0.027

0.552

1.48%

3

3

0.9318

FAL3T3.GB.B2.30.01.04

DF

AA61GB

27.1

0.054

0.422

2.70%

3

3

0.9382

FAL3T3.GB.B3.06-02-04

DF

AA61 GB

10.9

0.022

0.412

3.80%

3

5

0.96480.1, 0.01, 0.001, 0.0001,
0.00001, 0.000001,
0.0000001, 0.00000001
250.0, 116.0, 54.1, 25.2,
11.8, 5.4, 2.5, 1.2
250, 79.1, 25.0, 7.9, 2.5,
0.8, 0.25, 0.08
500, 158.7, 50.4, 16.0,
5.1, 1.6, 0.5, 0.2500, 158.7, 50.4, 16.0,
5.1, 1.6, 0.5, 0.2

3.16

YES

3.15

YES

3.15

YES

range finder

FAL.3T3.SLS.09/01/04
FAL.3T3.SLS.16/01/04
difficult to get above 250
ug/ml; unlikely to reach
100% toxicity
slow increase in toxicity;reached 90% toxicity;

FAL3T3.23-01-04
FAL.3T3.SLS.29/01/04
FAL.3T3.SLS.06/02/04

TRICHLOROACETIC ACID
IIVS
A1

RF

AA61MR

637

3.897

0.387

5.74%

2

1

0.9378B1DF

AA61MR

861

5.269

0.510

3.85%

3

5

0.9807B2

DF

AA61MR

873

5.343

0.351

6.44%

3

5

0.9556B3DF

AA61MR

670

4.100

0.423

0.22%

4

4

0.9652

AA61KT-A1

RF

AA61KT

977

5.981

0.403

6.66%

2

2

0.9703

AA61KT-B1

DF

AA61KT

859

5.257

0.408

5.82%

4

3

AA61KT-B2

DF

AA61KT

NA

NA

0.585

1.42%

1

AA61KT-B3

DF

AA61KT

767

4.696

0.491

0.48%

AA61KT-B4

DF

AA61KT

661

4.043

0.403

FAL.3T3.GH.A1.10.09.04

RF

AA61GH

1380

8.428

FAL.3T3.GH.1.16.09.04

DF

AA61GH

1240

7.564

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
3000, 2000, 1333, 889,
593, 395, 263, 176
3000, 2000, 1333, 889,
593, 395, 263, 176
3000, 2000, 1333, 889,
593, 395, 263, 176

10

RF

1.5

YES

range finder

ppt in 1X C1SLS-A4

outlier removed bySD

1.5

YES

SLS-B4SLS-B7

1.5

YES

SLS-B8

10000,1000, 100, 10,1,0.1, 0.01, 0.001

10RF

0.9878

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

YES

0

NA

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

NO

4

4

0.989010000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

0.04%

4

4

0.9878

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

0.459

10.90%

1

2

0.9027

0.394

2.96%

2

3

0.9170ECBC
range finder

ppt in 1X C1SLS-P13

ppt in 1X C1SLS-P33

SD rejected test; ppt in 1X
C1

SLS-P35

YES

ppt in 1X C1SLS-P37

2.15

YES

ppt in 1X C1SLS-P40

10RF

ppt in 1X C1

FAL.3T3.SLS.10.09.04

2.15

YES

no points between
50 - 100%; closest
point is 100.0%

FRAME

I-53

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
5000, 2326, 1082, 503,
234, 109, 50.6, 23.5

range finder

FAL.3T3.SLS.16.09.04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

5000, 2326, 1082, 503,
234, 109, 50.6, 23.5
5000, 2326, 1082, 503,
234, 109, 50.6, 23.5

Dilution

Acceptable

Factor8

Tests92.15

YES

FAL.3T3.SLS.15.10.04

2.15

YES

FAL.3T3.SLS.28.10.04

10RF

range finder
no points between
50 - 100%

FAL.3T3.GH.B2.15.10.04

DF

AA61GH

1140

6.962

0.302

14.14%

2

3

0.9396

FAL.3T3.GH.B3.28.10.04

DF

AA61GH

1280

7.830

0.188

9.65%

2

2

0.90915900

44.240

0.312

5.85%

1

6

0.6051

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

1.3

NO

Rationale for
Unacceptability

Notes

PC ID

1,1,1-TRICHLOROETHANE
IIVS
A1

RF

AA61KG

B1

DF

AA61KG

9710

72.7460.446

10.58%

1

00.9158

10000, 7692, 5917, 4552,
3501, 2693, 2072, 1594

B2

DF

AA61KG

9840

73.758

0.474

5.24%

1

6

0.742010000, 8333, 6944, 5787,
4823, 4019, 3349, 2791

1.2

YES

B3

DF

AA61KG

10000

75.303

0.355

0.14%

0

4

0.8872

10000, 8333, 6944, 5787,
4823, 4019, 3349, 2791

1.2

YES

B4

DF

AA61KG

9640

72.246

0.490

2.52%

1

2

0.9252

10000, 8333, 6944, 5787,
4823, 4019, 3349, 2791

1.2

YES

RF

AA61JV

NA

NA

0.565

0.77%

0

4

NA

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

1.21

NO

SLS-A5
ppt in 2X C1SLS-B6

ppt in 2X C1; plates read 1516 hr late; orignial reading
SLS-B11
used wrong OD wavelength
ppt in 2X C1; passes
no points between 0 because of 1.2 dilution
50%;
factorppt in 2X C1SLS-B12
SLS-B15ECBC
AA61JV-A1

range finder

AA61JV-B1 (sealer)DF

AA61JV

NA

NA

0.621

5.88%

0

8

NA

30000, 24793, 20490,
16934, 13995, 11566,
9559, 7900

AA61JV-B2 (sealer)DF

AA61JV

41100

308.185

0.353

2.97%

3

5

0.652550000, 41322, 34151,
28224, 23325, 19277,
15932, 13167

1.21

YES

AA61JV-B3 (sealer)DF

AA61JV

NA

NA

0.448

5.01%

?

?

NA

50000, 41322, 34151,
28224, 23325, 19277,
15932, 13167

1.21

NO

can't properly
determine points
between 0 - 100%

RF

AA61PN

NA

NA

0.315

6.33%

0

0

0.000010000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001

10RFrange finder; nopoints between 0100%

1.47

YES

PC failed; no points
between 0 - 50%

SLS-15dilution factor is 1.21; nopoints between 0-50%; test
would pass due to dilution
factor; ppt in 2X C4
ppt in 2X C1-C5; chemical
made pipets sticky andcorrosive to the reservoir"roller coaster" toxicity
curve; chemical physicallyintereacted with plastic
pipets; ppt in 2X C1-C8(oily)SLS-P61SLS-P64

SLS-P73

FRAME
FAL.3T3.PN.A1.21.10.04
FAL.3T3.PN.B1.04.11.04

DF

AA61PN

18400

137.661

0.285

9.94%

1

2

0.6655

FAL.3T3.PN.B2.19.11.04

DF

AA61PN

20600

154.458

0.278

4.29%

2

0

0.7843

FAL.3T3.PN.B3.25.11.04

DF

AA61PN

22000

165.125

0.365

1.64%

1

2

0.6250FAL.3T3.PN.B4.26.11.04

DF

AA61PN

24000

179.809

0.331

2.57%

2

4

0.17040.214

0.00100.338

10.51%

2

4

0.959125000, 17007, 11569,
7870, 5354, 3642, 2478,
1686
25000, 20661, 17075,
14112, 11663, 9639,
7966, 6583
25000, 20661, 17075,
14112, 11663, 9639,
7966, 6583
25000, 20661, 17075,
14112, 11663, 9639,
7966, 6583

1.21

YES

1.21

YES

1.21

YES

10

RF

FAL.3T3.SLS.21.10.04
ppt in 1X C1no points between
50-100%FAL.3T3.SLS.04.11.04

test passes because lowest
dilution factor used (1.21);
FAL.3T3.SLS.19.11.04
ppt in 2X C1-C2
ppt in 2X C1; ppt in 1X C1;
C8 concentration shows
FAL.3T3.SLS.25.11.04
high toxicity
ppt in 2X C1-C4;

FAL.3T3.SLS.26.11.04

ppt in 2000ug/ml stock in
DMSO

SLS-A2

TRIETHYLENEMELAMINE
IIVS
A1

RF

AA61MT

I-54

10.0, 1.0, 0.1, 0.01,
0.001, 0.0001, 0.00001,
0.000001

range finder

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

1.00, 0.625, 0.391, 0.244,
0.153, 0.095, 0.060,
0.037
2.00, 1.11, 0.617, 0.343,
0.191, 0.106, 0.059,
0.033
2.00, 1.11, 0.617, 0.343,
0.191, 0.106, 0.059,
0.033

Dilution

Acceptable

Factor8

Tests9

1.6

YES

SLS-B5

1.8

YES

SLS-B9

1.8

YES

SLS-B10

Rationale for
Unacceptability

Notes

PC ID

B1

DF

AA61MT

0.223

0.0011

0.497

2.36%

4

4

0.9169B2

DF

AA61MT

0.127

0.0006

0.377

3.14%

5

3

0.9339B3DF

AA61MT

0.156

0.0008

0.321

8.67%

5

3

0.9469

AA61GE-A1 revised by

RF

AA61GE

0.2

0.0010

0.256

6.24%

2

5

0.938910, 1, 0.1, 0.01, 0.001,
0.0001, 0.00001,
0.000001

10RF

range finder

AA61GE-B1

DF

AA61GE

0.117

0.00060.424

19.49%

5

3

0.91784.00, 1.86, 0.685, 0.402,
0.187, 0.087, 0.40, 0.019

2.15

NO

% VC difference >15

AA61GE-B2

DF

AA61GE

0.07660.0004

0.375

6.15%

6

2

0.93394.00, 1.86, 0.685, 0.402,
0.187, 0.087, 0.40, 0.019

2.15

YES

SLS-P21

AA61GE-B3

DF

AA61GE

0.09510.0005

0.599

3.52%

2

6

0.95944.00, 1.86, 0.685, 0.402,
0.187, 0.087, 0.40, 0.019

2.15

YES

SLS-P24

AA61GE-B4

DF

AA61GE

0.08610.00040.563

11.19%

2

6

0.9512

4.00, 1.86, 0.685, 0.402,
0.187, 0.087, 0.40, 0.019

2.15

YES

SLS-P26

RF

AA61LB

2.83

0.0138

0.270

4.91%

1

1

0.7626

10

RF

ECBC
SLS-P9ppt in 2X C1SLS-P19

FRAME
FAL.3T3.LB.A1.01/04/04
FAL.3T3.LB.B1.29/04/04

DF

AA61LB

1.44

0.0071

0.289

3.08%

3

3

0.8508FAL.3T3.LB.B2.07/05/04

DF

AA61LB

1.72

0.0084

0.269

3.01%

7

1

0.9859FAL.3T3.LB.B3.20/05/04

DF

AA61LB

1.19

0.0058

0.336

5.70%

4

3

0.9404

10.0, 1.0, 0.1, 0.01,
0.001, 0.0001, 0.00001,
0.000001,
50.0, 23.3, 10.8, 5.03,
2.34, 1.09, 0.506, 0.235
25.0, 17.0, 11.6, 7.9, 5.4,
3.6, 2.5, 1.7
25.0, 11.6, 5.4, 2.5, 1.2,
0.5, 0.3, 0.1

range finder

FAL.3T3.SLS.01/04/04
NR crystals; highbackground

2.15

YES

1.47

YES

FAL.3T3.SLS.29/04/04
FAL.3T3.SLS.07/05/04

2.15

YES

FAL.3T3.SLS.20/05/04

TRIPHENYLTIN HYDROXIDE
IIVS
A1

RF

AA61JR

0.013

0.00004

0.456

8.54%

0

1

0.9726

10.0, 1.0, 0.1, 0.01,
0.001, 0.0001, 0.00001,
0.000001

10

RF

B1

DF

AA61JR

0.02060.00006

0.434

2.34%

3

4

0.95760.100, 0.0625, 0.0391,
0.0244, 0.0153, 0.00954,
0.00596, 0.00373

1.6

YES

SLS-B5B2DF

AA61JR

0.005470.00001

0.371

8.48%

3

1

0.95690.100, 0.0625, 0.0391,
0.0244, 0.0153, 0.00954,
0.00596, 0.00373

1.6

YES

SLS-B9B3DF

AA61JR

0.01840.00005

0.367

0.57%

3

4

0.90730.100, 0.0625, 0.0391,
0.0244, 0.0153, 0.00954,
0.00596, 0.00373

1.6

YES

SLS-B10

AA61LL-A1

RF

AA61LL

0.01320.00004

0.297

4.91%

1

2

0.9825

10

RF

AA61LL-B1

DF

AA61LL

0.02580.00007

0.569

0.10%

2

6

0.9539

2.15

YES

range finder

SLS-A2ECBC

I-55

10, 1, 0.1, 0.01, 0.001,
0.0001, 0.00001,
0.000001
0.100, 0.0465, 0.0216,
0.0101, 0.0047, 0.0022,
0.0010, 0.0005

range finder

SLS-P7SLS-P24

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

0.100, 0.0465, 0.0216,
0.0101, 0.0047, 0.0022,
0.0010, 0.0005
0.100, 0.0465, 0.0216,
0.0101, 0.0047, 0.0022,
0.0010, 0.0005

Dilution

Acceptable

Factor8

Tests92.15

YES

SLS-P26

2.15

YES

SLS-P28

10

RF

2.15

YES

FAL.3T3.SLS.29/04/04

2.15

YES

FAL.3T3.SLS.07/05/04

2.15

YES

FAL.3T3.SLS.20/05/04

Rationale for
Unacceptability

Notes

PC ID

AA61LL-B2

DF

AA61LL0.02960.00008

0.519

1.61%

2

5

0.9359AA61LL-B3

DF

AA61LL0.02120.00006

0.486

8.28%

2

6

0.9428FAL.3T3.GG.A1.01/04/04

RF

AA61GG

0.01430.00004

0.267

9.46%

3

5

0.9563

FAL.3T3.GG.B1.29/04/04

DF

AA61GG

0.002860.00001

0.239

5.43%

1

1

0.9869FAL.3T3.GG.B2.07/05/04

DF

AA61GG

0.03140.00009

0.340

1.06%

1

1

0.7735

FAL.3T3.GG.B3.20/05/04

DF

AA61GG

0.04380.00012

0.367

1.82%

2

6

0.8325

A1

RF

AA61MZ

665

4.614

0.415

7.61%

1

2

0.8257

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10

RF

B1

DF

AA61MZ

574

3.981

0.353

10.44%

3

4

0.67492000, 1333, 889, 593,
395, 263, 176, 117

1.5

YES

B2

DF

AA61MZ

NA

NA

0.372

15.70%

04

NA2000, 1333, 889, 593,
395, 263, 176, 117

1.5

NO

B3

DF

AA61MZ

NA

NA

0.354

4.99%

0

6

NA

1.5

NO

B4

DF

AA61MZ

NA

NA

0.366

1.91%

0

3

NA

1.5

NO

no points between 0ppt in 2X C1-C3
50%

SLS-18RF

AA61JJ

723

5.012

0.252

3.67%

1

3

0.831910RFrange finder

SLS-P9FRAME
10.0, 1.0, 0.1, 0.01,
0.001, 0.0001, 0.00001,
0.000001,
0.100, 0.047, 0.022,
0.010, 0.0047, 0.0022,
0.0010, 0.0005
0.100, 0.0233, 0.0108,
0.0050, 0.0023, 0.0011,
0.0005, 0.0002
0.100, 0.047, 0.022,
0.010, 0.0047, 0.0022,
0.0010, 0.0005

range finder

FAL.3T3.SLS.01/04/04

VALPROIC ACID
IIVS

2000, 1333, 889, 593,
395, 263, 176, 117
2000, 1333, 889, 593,
395, 263, 176, 117

range finder

small immiscible droplets
initially coated insides of
SLS-A2
dilution tube in the highest
2X solution
ppt in 2X C1-C2; test article
adherred to glass pipettes
SLS-B6upon transference to the 8well reservoir

no points between 050%; %VC
ppt in 2X C1
difference >15; notoxicity detected
no points between 0ppt in 2X C1-C4
50%

SLS-B9SLS-B10ECBC
AA61JJ-A1

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

AA61JJ-B1

DF

AA61JJ

624

4.325

0.537

3.43%

4

2

0.9027

2000, 1361, 926, 630,
428, 291, 198, 135

1.47

YES

highest 2X solution clear,oily, & orange; DMSO <0.5%; no diff. in JJ-B2 & JJ- SLS-P26
B3 when compared to JJ-B1(no ppt)

AA61JJ-B2

DF

AA61JJ

519

3.598

0.433

11.56%

3

4

0.8624

2000, 1361, 926, 630,
428, 291, 198, 135

1.47

YES

ppt in 2X C1 - C5; oily; nodiff. in JJ-B2 & JJ-B3 when SLS-P28
compared to JJ-B1 (no ppt)

AA61JJ-B3

DF

AA61JJ

499

3.460

0.379

5.14%

4

4

0.92402000, 1361, 926, 630,
428, 291, 198, 135

1.47

YES

ppt in 2X C1 - C5; no diff. in
JJ-B2 & JJ-B3 when
SLS-P30
compared to JJ-B1 (no ppt)

FAL.3T3.GK.A1.01/04/04

RF

AA61GK

NA

NA

0.280

5.02%

0

1

NA

10

RF

FAL.3T3.GK.B1.29/04/04

DF

AA61GK1660

11.535

0.228

7.54%

1

2

0.7855

2.15

YES

FAL.3T3.SLS.29/04/04

FAL.3T3.GK.B2.07/05/04

DF

AA61GK1760

12.219

0.284

7.69%

1

2

0.4313

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

2.15

YES

FAL.3T3.SLS.07/05/04

FRAME

I-56

range finder

FAL.3T3.SLS.01/04/04

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC ID

DF

AA61GK2000

13.837

0.337

0.94%

1

2

0.5501

2500, 1701, 1157, 787,
535, 364, 248, 169

1.47

YES

A1

RF

AA61NH

38.1

0.078

0.266

2.04%

0

0

0.5147100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001

10

RF

B1

DF

AA61NH

35.9

0.073

0.480

1.13%

1

2

0.9635

1.4

YES

SLS-B5B2DF

AA61NH

43.7

0.089

0.352

7.48%

2

2

0.97501.4

YES

SLS-B9B3DF

AA61NH

37.1

0.075

0.359

12.81%

1

5

0.9378

1.4

YES

SLS-B10

AA61LY-A1

RF

AA61LY

15.1

0.031

0.287

1.45%

0

6

0.9401

AA61LY-B1

DF

AA61LY

26.7

0.054

0.347

12.36%

3

4

0.9375

AA61LY-B2

DF

AA61LY

38.3

0.078

0.523

5.85%

2

4

0.9789AA61LY-B3

DF

AA61LY

31.6

0.064

0.444

15.05%

3

4

0.9643

FAL.3T3.MC.A1.10.09.04

RF

AA61MC

62.8

0.128

0.369

12.62%

2

00.9133

FAL.3T3.MC.B1.16.09.04

DF

AA61MC

48.1

0.098

0.277

7.34%

0

1

FAL.3T3.MC.B2.23.09.04

DF

AA61MC

23.1

0.047

0.201

2.68%

3

FAL.3T3.MC.B3.14.10.04.04

DF

AA61MC

32.7

0.067

0.268

12.64%

FAL.3T3.MC.B4.21.10.04

DF

AA61MC

36.1

0.073

0.169

FAL.3T3.MC.B5.04.11.04

DF

AA61MC

34.9

0.071

A1

RF

AA61MANA

B1

DF

AA61MA

B2

DF

B3

FAL.3T3.GK.B3.20/05/04

FAL.3T3.SLS.20/05/04

VERAPAMIL HCL
IIVS100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49

range finder

solvent controls treated with
1% DMSO, rather than
SLS-A40.5%.

ECBC1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74

10

RF

1.47

YES

range finder

ppt in 2X C1SLS-P14

1.47

YES

1.47

YES

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10RF

0.9557100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74

1.47

NO

0

0.8298100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3

1.21

NO

2

1

0.9323

1.47

YES

ppt in 2X C1

FAL.3T3.SLS.14.10.04

0.30%

1

1

0.1575

1.47

YES

ppt in 2X C1; very highviability values for C3-C7

FAL.3T3.SLS.21.10.04

0.199

8.03%

2

1

0.6920

1.47

YES

NA

0.415

0.04%

0

1

NA

728

6.855

0.371

3.21%

5

3

0.9121

AA61MA

809

7.621

0.371

4.51%

5

3

0.9567DF

AA61MA

635

5.984

0.311

4.85%

6

2

0.9597

AA61GM-A1

RF

AA61GM

NA

NA

0.232

5.68%

0

5

NA

AA61GM-B1

DF

AA61GM

NA

NA

0.754

7.45%

0

8

NA

AA61GM-B2

DF

AA61GM

NA

NA

0.624

4.21%

0

7

NA

SLS-P34
SLS-P36potential volatility problem

SLS-P38

FRAME

50.0, 34.0, 23.1, 15.7,
10.7, 7.28, 4.96, 3.37
50.0, 34.0, 23.1, 15.7,
10.7, 7.28, 4.96, 3.37
75.0, 51.0, 34.7, 23.6,
16.1, 10.9, 7.43, 5.06

range finder; nopoints between 50 - ppt in 2X C1 and 1X C1
FAL.3T3.SLS.10.09.04
100%
no points between 0 ppt in 2X C1
FAL.3T3.SLS.16.09.04
50%
1.21 dilution factor doesn't
affect outcome since nono points between
values > 50% viability; ppt in FAL.3T3.SLS.23.09.04
50 - 100%
2X C1; outlier removed bySD

FAL.3T3.SLS.04.11.04

XYLENE
IIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1923, 1479, 1138,
875, 673, 518, 398
2500, 1923, 1479, 1138,
875, 673, 518, 398
2500, 1923, 1479, 1138,
875, 673, 518, 398

10

RF

1.3

YES

1.3

YES

1.3

YES

range finder

ppt in 2X C1SLS-A3
SLS-B6

ppt in 2X C1SLS-B9SLS-B10

ECBC

I-57

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
3000, 2479, 2049, 1693,
1400, 1157, 956, 790
4000, 3306, 2732, 2258,
1866, 1542, 1275, 1053

10

RF

1.21

NO

1.21

NO

range finder
PC failed; no points test could pass due tobetween 0 - 50%
dilution factor
no points between 0 - test could pass due to50%
dilution factor

SLS-12SLS-P61SLS-P63

In Vitro Cytotoxicity Test Methods BRD Appendix I1

November 2006

3T3 NRU Reference Substance Data

Experiment ID
3T3 Cells

AA61GM-B3

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6DFAA61GM

NA

NA

0.553

15.44%

2

6

NA

FAL.3T3.JG.A1.28.05.04

RF

AA61JG

NA

NA

0.327

3.31%

0

3

0.6108FAL.3T3.JG.B1.04.06.04

DF

AA61JG

NA

NA

0.250

0.50%

0

0

NA

FAL.3T3.JG.B2.17.06.04

DF

AA61JG

NA

NA

0.448

0.74%

0

NA

NA

FAL.3T3.JG.B2.24.06.04(should be B3)DF

AA61JG

NA

NA

0.396

9.40%

0

5

0.1548

Exposure
Concentrations(ug/mL)7

4000, 3306, 2732, 2258,
1866, 1542, 1275, 1053

Dilution

Acceptable

Factor8

Tests91.21

NO

can't properly
determine points
between 0 - 100%

range finder

Rationale for
Unacceptability

Notes

PC ID

roller coaster toxicity curve;
SLS-P73
ppt in 2X C1-C8 (oily)

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

10

RF

2.15

NO

2.15

NO

2.15

NO

FAL.3T3.SLS.28.05.04

no points between 0 ppt in 2X C1-C3;
FAL.3T3.SLS.04.06.04
100%
no points between 0 - ppt in 2X C1-C3; toxicity did
FAL.3T3.SLS.17.06.04
100%
not reach 50%
no points between 0 - no toxicity detected; SD
FAL.3T3.SLS.24.06.04
50%
ends testing

Abbreviations: ppt=Precipitate; SD=Study Director; RF=Range Finder; DF=Definitive Test; PC=Positive Control; C1 - C8=Concentration series applied to the the cells. C1 is the highest concentration and C8 is lowest; NA=Not Available; RC=Registry of Cytotoxicity; 2X=Two times the concentration applied to the cells; VC=Vehicle Control;
R2=Coefficient of Determination; OD=Optical Density; ID=Identification. Substance ID was the code assigned by the chemical distributor (BioReliance Corp.). Experiment ID and PC ID are test identification numbers assigned by the cytotoxicity testing laboratory.
1

Range finder or definitive test
Mean OD value for all VC wells in test plate
Difference of right and left VC column of wells in the test plate4% Viability values between 0 and 50% viability; test acceptance criterion. Phase Ib used the range of 10 -50%.
5% Viability values between 50 and 100% viability; test acceptance criterion. Phase Ib used the range of 50 - 90%.
6
Calculated value from the Prism® software
7
Reference substance concentrations applied to the cells
8
Step-wise dilution factor used to determine reference substance exposure concentrations
9
Determination for whether test meets or doesn’t meet test acceptance criteria; not applied to RF tests
Shaded boxes identify values that do not meet the specific test acceptance criteria
2
3

I-58

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

Appendix I2

NHK NRU Reference Substance Data

I-59

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

[This Page Intentionally Left Blank]

I-60

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC

1450

9.560

0.525

OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9

10

RF

1.4

YES

SLS-B12-N041022B

1.4

YES

SLS-B13-N041029B

1.4

YES

SLS-B14-N041030A

Rationale for
Unacceptability

Notes

PC ID

ACETAMINOPHEN
IIVS
A1

RF

AA61HU

0.11%

0

1

0.5444B1

DF

AA61HU

541

3.576

0.678

1.54%

5

3

0.9557B2DF

AA61HU

661

4.370

0.622

9.36%

5

3

0.9738B3DF

AA61HU

512

3.384

0.777

0.82%

5

3

0.9526

AA61LR-A1

RF

AA61LR

196

1.299

0.972

0.43%

1

6

0.8186

AA61LR-B1

DF

AA61LR

467

3.086

0.731

1.13%

3

5

0.9694

AA61LR-B2

DF

AA61LR

586

3.877

0.704

2.81%

3

4

0.9642

AA61LR-B3

DF

AA61LR

621

4.106

1.019

4.94%

3

4

0.9495

FAL.NHK.PY.A1.24.09.04

RF

AA61PY

137

0.907

0.578

8.76%

1

3

0.6981

FAL.NHK.PY.B1.01.10.04

DF

AA61PY

1130

7.489

1.026

8.47%

2

5

0.9753

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1786, 1276, 911,
651, 465, 332, 237
2500, 1786, 1276, 911,
651, 465, 332, 237
2500, 1786, 1276, 911,
651, 465, 332, 237

range finder; nopoints between 0 50%

SLS-A5-N040401A

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
4000, 1861, 865, 403,
187, 87.1, 40.5, 18.8
4000, 1861, 865, 403,
187, 87.1, 40.5, 18.8
4000, 1861, 865, 403,
187, 87.1, 40.5, 18.8

10

RF

2.15

YES

range finder

SLS-P19
SLS-P41

2.15

YES

SLS-P43

2.15

YES

SLS-P45FRAME

FAL.NHK.PY.B2.07.10.04

DF

AA61PY

421

2.783

0.575

3.20%

4

3

0.6590FAL.NHK.PY.B3.05.11.04

DF

AA61PY

541

3.576

0.418

10.47%

3

1

0.9335

FAL.NHK.PY.B4.10.11.04

DF

AA61PY

380

2.514

1.156

1.74%

3

5

0.7537

RF

AA61GF

43700

1063.376

0.479

4.37%

0

4

0.5946
0.9841

1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

10RF

range finder

2.15

NO

PC failed

FAL.NHK.SLS.24.09.03
FAL.NHK.SLS.01.10.04

2.15

YES

C1 shows high toxicity;
should this point be
removed & new calc. be
made?

2.15

YES

outlier removed by SD

2.15

YES

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RFrange finder; nopoints between 0 50%

200000, 111111, 61728,
34294, 19052, 10584,
5880, 3267

1.8

NO

%VC difference >15

2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

FAL.NHK.SLS.07.10.03
FAL.NHK.SLS.05.11.04
FAL.NHK.SLS.10.11.04

ACETONITRILE
IIVS
A1
B1

DF

AA61GF

6810

165.8390.494

99.86%

3

2

B2

DF

AA61GF

9730

236.966

0.624

3.54%

3

2

0.9960B3DFAA61GF9230

224.743

0.693

4.62%

3

2

0.9964B4DF

AA61GF

8910

217.114

0.605

5.04%

3

3

0.9878

AA61PH-A1

RF

AA61PH

NA

NA

0.635

1.57%

0

5

NA

AA61PH-A2

RF

AA61PH

NA

NA

0.231

97.27%

3

1

NA

AA61PH-B1

DF

AA61PH

22600

551.679

0.911

13.28%

1

3

0.8640200000, 111111, 61728,
34294, 19052, 10584,
5880, 3267
200000, 111111, 61728,
34294, 19052, 10584,
5880, 3267
40000, 25000,15625,
9766, 6104, 3815, 2384,
1490

SLS-A1-N040317B
Left VC was removed from
calc. due to volatility

SLS-B8-N040819A

SLS-B10-N040903A

SLS-B11-N040904H

1.8

YES

plate seal used; SD
removed top dose fromanalysis since only 4 wellsof 8 were treated

1.8

YES

plate seal used

1.6

YES

10

RF

range finder

no toxicity detected

SLS-P110RFrange finder

probable volatility problem

SLS-P3

2.15

YES

SLS-B12-N041022B

ECBC

I-61

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
200000, 20000, 2000,
200, 20, 2, 0.2, 0.02
50000, 23256, 10817,
5031, 2340, 1088, 506,
235

SLS-P7

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7
50000, 34014, 23139,
15740, 10708, 7284,
4955, 3371
50000, 34014, 23139,
15740, 10708, 7284,
4955, 3371
50000, 34014, 23139,
15740, 10708, 7284,
4955, 3371
40000, 27211, 18511,
12592, 8566, 5827, 3964,
2697

Dilution

Acceptable

Factor8

Tests9

1.47

NO

1.47

YES

SLS-P17

1.47

YES

SLS-P18

1.47

YES

SLS-P24

Rationale for
Unacceptability

AA61PH-B2

DF

AA61PH

31800

775.6880.865

21.14%

1

5

0.8532

AA61PH-B3(sealer)DF

AA61PH

7110

173.255

0.561

4.36%

6

2

0.9839

AA61PH-B4(sealer)DF

AA61PH

7050

171.667

0.643

1.06%

5

2

0.9812

AA61PH-B5

DF

AA61PH

6710

163.564

0.484

0.05%

5

2

0.9783

FAL.NHK.PL.A1.18.02.04

RF

AA61PL

NA

NA

0.107

11.79%00NA10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RFrange finder

FAL.NHK.PL.26.02.04

RF

AA61PL8220

200.3030.138

32.31%

1

00.4136

100000, 10000, 1000,
100, 10, 1, 0.1, 0.01

10RF

range finder

FAL.NHK.PL.B1.25.03.04

DF

AA61PL

8790

214.135

0.502

3.22%

1

2

0.933825000, 7937, 2520, 800,
254, 80.6, 25.6, 8.12

3.15

YES

FAL.NHK.PL.B3.26.03.04

DF

AA61PL7480

182.258

0.549

4.16%

2

0

0.842825000, 7911, 2504, 792,
251, 79.3, 25.1, 7.9

3.16

NO

FAL.NHK.PL.B4.25.04.04

DF

AA61PL

12400

302.473

0.860

5.09%

1

1

0.937125000, 11628, 5408,
2516, 1170, 544, 253, 118

2.15

YES

Notes

%VC difference > 15 possible volatility problem

PC ID

SLS-P9FRAME

FAL.NHK.PL.B5.28.04.04

DF

AA61PL8020

195.293

0.909

6.73%

0

1

0.810925000, 7937, 2520, 800,
254, 80.6, 25.6, 8.12

3.15

NO

FAL.NHK.PL.B5.19.08.04(rb)should be B6

DF

AA61PL

10800

262.233

0.266

7.45%

2

0

0.539525000, 11628, 5408,
2516, 1170, 544, 253, 118

2.15

NO

FAL.NHK.PL.B6.20.08.04
should be B7

DF

AA61PL9270

225.781

0.824

2.53%

2

2

0.955925000, 11628, 5408,
2516, 1170, 544, 253, 118

2.15

YES

no values calculated by
PRISM; % viability are
FAL.NHK.SLS.18.02.04"nonsense" values
chem. needs to be tested athigh conc. but have volatility FAL.NHK.SLS/NB.26.02
problems even w/plate.03
sealerdid SD use plate film cover? FAL.NHK.SLS.25.03.03

no points between
50-100%wrong solvent reported but
correct one used (correction
FAL.NHK.SLS.26.03.04
by SD); pts between 50 100% but several > 100%
FAL.NHK.SLS.25.04.04

wells D3,D4,E3,E4 data
removed by SD after
no points between 0- NICEATM recomm. to
FAL.NHK.SLS.28.04.03
50%
review potential outliers;
revised data eliminates point
between 0-50% and test
PC failed; no points
FAL.NHK.SLSbetween 50-100%
RB.19.08.04
FAL.NHK.SLS.20.08.04

ACETYLSALICYLIC ACID
IIVS
A1

RF

AA61HM

552

3.064

0.748

3.52%

1

4

0.9540B1DF

AA61HM

509

2.826

0.653

1.76%

5

3

0.9836B2

DF

AA61HM

596

3.306

0.599

5.27%

4

4

0.9664B3DF

AA61HM

438

2.428

0.607

3.62%

5

3

0.9107

AA61ME-A1

RF

AA61ME

631

3.501

0.916

2.80%

1

7

0.9492

AA61ME-B1

DF

AA61ME

614

3.406

0.765

3.36%

3

5

0.9409

AA61ME-B2

DF

AA61ME

653

3.624

0.791

2.60%

3

5

0.9719

AA61ME-B3

DF

AA61ME

627

3.477

0.983

0.71%

3

5

0.9596

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 1429, 1020, 729,
521, 372, 266, 190
2000, 1429, 1020, 729,
521, 372, 266, 190
2000, 1429, 1020, 729,
521, 372, 266, 190

10

RF

1.4

YES

range finder

SLS-A3-N040331A
SLS-B8-N040819A

1.4

YES

SLS-B9-N040820A

1.4

YES

SLS-B10-N040903A

10

RF

1.47

YES

SLS-P53

1.47

YES

SLS-P54

1.47

YES

SLS-P56

ECBC

FRAME

I-62

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1500, 1020, 694, 472,
321, 219, 149, 101
1500, 1020, 694, 472,
321, 219, 149, 101
1500, 1020, 694, 472,
321, 219, 149, 101

range finder

ppt in 2X C2 and 1X C1SLS-P14

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

FAL.NHK.JA.A1.14.05.04

RF

AA61JA

340

1.889

0.764

4.39%

1

2

0.9410FAL.NHK.JA.B1.08.10.04

DF

AA61JA

719

3.993

0.722

0.54%

2

3

0.9913

FAL.NHK.JA.B2.22.10.04

DF

AA61JA

778

4.318

0.715

2.72%

3

5

0.9753

FAL.NHK.JA.B3.28.10.04

DF

AA61JA

586

3.253

0.635

3.07%

4

4

0.9817

A2

RF

AA61JD

1480

3.360

0.809

5.29%

0

6

0.7064B1

DF

AA61JD

561

1.274

0.476

6.77%

2

6

0.9289B2DF

AA61JD

661

1.501

0.328

4.35%

2

6

0.9353B3DF

AA61JD

986

2.239

0.34

6.44%

0

5

0.9305

AA61MB-A1

RF

AA61MB

627

1.424

0.566

1.64%

1

3

0.8101

AA61MB-B1

DF

AA61MB

962

2.184

1.042

1.45%

1

7

0.7701

AA61MB-B2

DF

AA61MB

718

1.630

0.914

0.84%

3

5

0.8326

AA61MB-B3

DF

AA61MB

1080

2.452

0.778

2.61%

1

7

0.7956

AA61MB-B4

DF

AA61MB

944

2.143

0.904

5%

3

5

0.7754

RF

AA61PU

NA

NA

1.355

3.29%

0

8

0.03730.2733

Exposure
Concentrations(ug/mL)7
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1701, 1157, 787,
535, 364, 248, 169
2500, 1701, 1157, 787,
535, 364, 248, 169

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

range finder

PC ID

10

RF

2.15

YES

FAL.NHK.SLS.14.05.03
FAL.NHK.SLS.08.10.03

1.47

YES

FAL.NHK.SLS.22.10.04
(MO)

1.47

YES

FAL.NHK.SLS.28.10.04

AMINOPTERIN

IIVS

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 714, 510, 364, 260,
186, 133, 94.9
1000, 714, 510, 364, 260,
186, 133, 94.9
1000, 714, 510, 364, 260,
186, 133, 94.9

10

RF

1.40

YES

range finder

SLS-A2

1.40

YES

1.40

NO

No points 0-50%

10RFrange finder

1.47

NO

low r2

1.21

YES

SLS-P10

1.21

YES

SLS-P12

1.21

YES

SLS-P20

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10RF

range finder

solution is yellow and maybind to the cells thus
affecting NRU

FAL.NHK.SLS.30.07.03

1000, 680, 463, 314.8,
214.1, 145.6, 99.1, 67.4

1.47

NO

low r2

biphasic response

FAL.NHK.SLS.07.08.03

NO

PC failed; no points
SD rejects this assay; can't
between 0 - 50%; noexplain the variability of cell FAL.NHK.SLS.13.08.03
r2; %VC difference >growth in the wells
15

1.47

NO

PC failed

1.47

YES

1.47

YES

2.15

NO

no points between
50 - 100%; low r2

2.15

NO

low r2; no points
between 0-50%

1.47

YES

1.47

NO

low r2

FAL.NHK.SLS.24.10.03

2.15

NO

low r2

FAL.NHK.SLS.07.11.03

evidence of precipitate at
highest dose
evidence of precipitate at
highest dose
evidence of precipitate at
highest dose

SLS-B1
SLS-B2
SLS-B3ECBC
1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
1000, 680.3, 462.8,
314.8, 214.2, 145.7, 99.1,
67.4
1200, 991.7, 819.6,
677.4, 559.8, 462.7,
382.4, 316.0
1200, 991.7, 819.6,
677.4, 559.8, 462.7,
382.4, 316
1200, 991.7, 819.6,
677.4, 559.8, 462.7,
382.4, 316.0

range finder

SLS-P4SLS-P8FRAME
FAL.NHK.PU.30.07.03
FAL.NHK.PU.B1.07.08.03

DF

AA61PU

516

1.172

0.245

10.54%

2

6

FAL.NHK.PU.B2.13.08.03

DF

AA61PU

NA

NA

0.722

30.35%

07

NA

FAL.NHK.PU.B3.23.08.03

DF

AA61PU

366

0.831

0.408

5.58%

3

5

0.8213

FAL.NHK.PU.B4.28.08.05

DF

AA61PU

593

1.346

0.470

8.87%

2

6

0.7804

FAL.NHK.PU.B5.05.09.03

DF

AA61PU

515

1.169

0.217

7.60%

2

6

0.7145

FAL.NHK.PU.B6.01.10.03

DF

AA61PU

NA

NA

1.373

5.40%

0

8

0.0149FAL.NHK.PU.B6.19.10.03

DF

AA61PU

157

0.356

0.170

1.73%

0

7

0.4794

FAL.NHK.PU.B7.23.10.03

DF

AA61PU

526

1.194

0.236

3.75%

2

6

0.6618

FAL.NHK.PU.B8.24.10.03

DF

AA61PU

9950

22.591

0.869

1.69%

1

7

0.2607

FAL.NHK.PU.B9.07.11.03

DF

AA61PU

5400

12.260

0.385

2.23%

1

7

0.1515I-63

1000, 680, 463, 314.8,
214.1, 145.6, 99.1, 67.4
1000, 680, 463, 314.8,
214.1, 145.6, 99.1, 67.4
1000, 680, 463, 314.8,
214.1, 145.6, 99.1, 67.4
1000, 680, 463, 314.8,
214.1, 145.6, 99.1, 67.4
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7100, 46.5, 21.6, 10.1, 4.7,
2.2, 1.0, 0.47
1000, 680, 463, 314.8,
214.1, 145.6, 99.1, 67.4
1000, 680, 463, 314.8,
214.1, 145.6, 99.1, 67.4
2000, 930, 433, 201, 94,
44, 20.2, 9.4

1.47

FAL.NHK.SLS.230803
challenging chemical; SMT
accepts this test
challenging chemical; SMT
accepts this test

FAL.NHK.SLS.280803
FAL.NHK.SLS.050903
FAL.NHK.SLS.01.10.03

SD worked with wrong
dilution range; wanted tostart at 1000
challenging chemical; SMT
accepts this test

FAL.NHK.SLS.19.10.03
FAL.NHK.SLS.23.10.03

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9

10

RF

1.6

YES

1.6

YES

1.6

YES

10

RF

2.15

YES

SLS-P32

1.47

YES

SLS-P34

1.47

YES

SLS-P36

Rationale for
Unacceptability

Notes

PC ID

5-AMINOSALICYLIC ACID
IIVS
A1

RF

AA61GZ

93.1

0.608

0.631

0.67%

1

0

0.8972B1DF

AA61GZ

41.7

0.272

0.548

2.71%

6

2

0.9682B2DF

AA61GZ

47.3

0.309

0.557

3.54%

5

2

0.9749B3DF

AA61GZ

57.3

0.374

0.438

9.57%

3

3

0.9328

AA61KD-A1

RF

AA61KD

NA

NA

0.856

3.85%

1

4

NA

AA61KD-B1

DF

AA61KD

34.8

0.228

0.529

0.76%

4

1

0.9692

AA61KD-B2

DF

AA61KD

32.4

0.212

0.539

0.94%

5

2

0.9214

AA61KD-B3

DF

AA61KD

22.5

0.147

0.401

3.53%

6

2

0.9529FAL.NHK.PA.A1.14.05.04

RF

AA61PA

35.6

0.232

0.784

2.17%

2

0

0.8834

FAL.NHK.PA.B1.19.08.04 rb

DF

AA61PA

62.1

0.406

0.234

1.25%

6

2

0.7433

FAL.NHK.PA-NB.B2.25.08.04

DF

AA61PA

127

0.830

0.988

1.33%

2

3

0.8882

FAL.NHK.PA.17.09.04

DF

AA61PA

54.3

0.355

0.705

2.54%

2

1

FAL.NHK.PA.B4.30.09.04

DF

AA61PA

53.3

0.348

0.753

2.27%

3

AA61RF

10.3

0.033

0.516

5.22%

0

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
500, 313, 195, 122, 76.3,
47.7, 29.8, 18.6
500, 313, 195, 122, 76.3,
47.7, 29.8, 18.6
200, 125, 78.1, 48.8,
30.5, 19.1, 11.9 ,7.45

range finder

SD did not use data fromthe highest dose in Hill
analyses due to the effectsof the ppts; ppt in 2X C1 &1X C1SLS-A3-N040331A

SLS-B1-N040423A
SLS-B2-N040424A
flattening of the curve at
35% viability

SLS-B3-N040506A

ppt in 1X C1SLS-P12ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
150, 102, 69.4, 47.2,
32.1, 21.9, 14.9, 10.1
150, 102, 69.4, 47.2,
32.1, 21.9, 14.9, 10.1

range finder

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
500, 340, 231, 157, 108,72.8, 50.0, 33.7
500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

10RF

range finder

ppt in 1X C1; NR taken up
by C1 ppt

1.47

NO

PC failed

2.15

YES

FAL.NHK.SLS.25.08.04

0.8385

1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

2.15

YES

outlier removed by SD; ppt
in C1; interference with NRU
in C1-C3 conc.; SD consider FAL.NHK.SLS.17.09.04
removing C1-C3 data from
PRISM analyses?

20.9753500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

2.15

YES

toxicity curve begins to riseat high concentrations;maybe affecting NRU;
outlier removed by SD

1

0.9945

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10

RF

1.8

YES

SLS-B1-N040423A

1.8

YES

SLS-B2-N040424A

1.8

YES

SLS-B3-N040506A

FAL.NHK.SLS.14.05.03
FAL.NHK.SLSRB.19.08.04

FAL.NHK.SLS.30.09.03

AMITRIPTYLINE HCL
IIVS
A1

RF

B1

DF

AA61RF

10.1

0.032

0.543

3.51%

2

3

0.9878B2DFAA61RF

10.6

0.034

0.636

2.41%

2

3

0.9899B3DF

AA61RF

12.1

0.039

0.496

1.03%

2

2

0.9713

AA61PR-A1

RF

AA61PR

7.64

0.024

0.518

3.91%

2

3

0.9625

AA61PR-B1

DF

AA61PR

12.4

0.040

0.647

4.74%

2

3

0.9678

AA61PR-B2

DF

AA61PR

13.0

0.042

0.921

1.85%

3

3

0.9817

AA61PR-B3

DF

AA61PR

6.94

0.022

0.648

2.47%

3

4

0.9710100, 55.6, 30.9, 17.1,
9.53, 5.29, 2.94, 1.63
100, 55.6, 30.9, 17.1,
9.53, 5.29, 2.94, 1.63
100, 55.6, 30.9, 17.1,
9.53, 5.29, 2.94, 1.63

range finder; nopoints between 0 50%

SLS-A1-N040317B

ECBC

FRAME

I-64

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

10

RF

2.15

YES

range finder

ppt in 2X C1SLS-P4SLS-P21

2.15

YES

SLS-P23

2.15

YES

SLS-P24

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

FAL.NHK.LE.A1.13.02.03

RF

AA61LE

6.52

0.021

0.114

4.66%

2

2

0.8453

FAL.NHK.LE.A2.20.02.03

DF

AA61LE

3.08

0.010

0.213

0.12%

3

3

0.9449FAL.NHK.LE.B1.27.02.04new

DF

AA61LE

13.6

0.043

0.548

1.40%

3

4

0.9200FAL.NHK.LE.B3.19.03.04

DF

AA61LE

6.04

0.019

0.528

4.71%

3

5

0.9296

5.16

0.026

0.585

3.78%

1

0

0.9828

Exposure
Concentrations(ug/mL)7

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
50, 34.0, 23.1, 15.7, 10.7,
7.28, 4.96, 3.37
50.0, 23.3, 10.8, 5.03,
2.34, 1.09, 0.51, 0.24

Dilution

Acceptable

Factor8

Tests9

10

RF

2.15

YES

1.47

YES

2.15

YES

10

RF

2.15

YES

2.15

YES

Rationale for
Unacceptability

range finder

Notes

PC ID

SD rejected due to bacterial
contamination in some
FAL.NHK.SLS.13.02.03
plates in test series; ppt in
2X C1
FAL.NHK.SLS.20.02.03
file corrected by SD

FAL.NHK.SLS.27.02.03
FAL.NHK.SLS.19.03.03

ARSENIC III TRIOXIDEIIVSPreliminaryRF

AA61FX

B1

DF

AA61FX

26.4

0.133

0.487

0.24%

2

2

0.9238B2DF

AA61FX

22.5

0.114

0.633

7.02%

2

1

0.9682B3
B4DF

AA61FX

22.5

0.114

0.817

7.11%

2

0

100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
100, 46.4, 21.6, 10, 4.64,
2.16, 1.00, 0.46
100, 46.4, 21.6, 10, 4.64,
2.16, 1.00, 0.46

range finder

Preliminary
SLS-B1
SLS-B20.9900100, 46.4, 21.6, 10, 4.64,
2.16, 1.00, 0.46

No points between
50 & 90%

2.15

NO100, 46.4, 21.6, 10, 4.64,
2.16, 1.00, 0.46

2.15

YES

10

RF

1.47

YES

SLS-P3

1.47

YES

SLS-P4

1.47

YES

SLS-P5

1.47

YES

SLS-P7

SLS-B3DF

AA61FX

13.9

0.070

0.826

6.84%

1

1

0.9850SLS-B4

RF

AA61KU

32.2

0.163

0.811

7.13%

0

1

-0.8980DF

AA61KU

4.51

0.023

0.978

2.63%

3

1

0.9577DF

AA61KU

7.76

0.039

1.200

2.58%

3

1

0.9757DF

AA61KU

8.11

0.041

1.080

5.57%

3

2

0.8912DF

AA61KU

10.7

0.054

1.086

3.26%

2

1

0.9369A1 1b/NHKRF1/FAL/NC

RF

AA61NC

1.49

0.008

0.160

0.52%

1

1

0.656012.5, 2.5, 0.5, 0.1, 0.02,
0.004, 0.00080, 0.00016

5RF

range finder

A1
1b/NHKCTR1/FAL/SLS

A2 1b/NHKRF2/FAL/NC

RF

AA61NC

3.01

0.015

0.685

10.17%

4

4

0.516412.5, 8.5, 5.78, 3.93,
2.67, 1.82, 1.23, 0.84

1.47

NO

low r2

A2
1b/NHKCTR2/FAL/SLS

A3 1b/NHK/DF2/FAL/NC

DF

AA61NC

0.00016

0.000

0.051

18.01%

00-0.9880

10, 6.8, 4.6, 3.14, 2.13,
1.45, 0.98, 0.67

1.47

NO

VC difference >15%; no points
between 10 & 90%;

1.47

NO

NO

ECBC
ECBC-NHK-Ib-01
AA61KU-A1
ECBC-NHK-Ib-02
AA61KU-B1
ECBC-NHK-Ib-03
AA61KU-B2
ECBC-NHK-Ib-04
AA61KU-B3
ECBC-NHK-Ib-05
AA61KU-B4

25, 2.5, 0.25, 0.025,
0.0025,0.00025,
0.000025, 0.0000025
50, 34, 23.1, 15.7, 10.7,
7.3, 5.0, 3.4
25, 17.0, 11.6, 7.87, 5.35,
3.64, 2.48, 1.69
25, 17.0, 11.6, 7.87, 5.35,
3.64, 2.48, 1.69
25, 17.0, 11.6, 7.87, 5.35,
3.64, 2.48, 1.69

range finder

SLS-P2FRAME

R2 < 0.8; PC failed

NR crystal problems; useddifferent medium; % viability
A3 1b/NHK/CTR4/FAL/
values are negative; PRISM
curve below 0

No point between 50 NR crystal problems; usedmedium not normally used

A4 1b/NHK/DF3/FAL/NC

DF

AA61NC

0.502

0.003

0.144

1.97%

5

0

0.7012

10, 6.8, 4.6, 3.14, 2.13,
1.45, 0.98, 0.67

A5 1b/NHK/DF4/FAL/NC

DF

AA61NC

NA

NA-0.00383.48%

00NC

10, 6.8, 4.6, 3.14, 2.13,
1.45, 0.98, 0.67

1.47

0.892910, 6.8, 4.6, 3.14, 2.13,
1.45, 0.98, 0.67

1.47

YES

A6 1b/NHK/CTR7/FAL

0.885515, 10.2, 6.93, 4.72, 3.21,
2.18, 1.48, 1.01

1.47

YES

A8 1b/NHK/CTR9/FAL

A6 1b/NHK/DF5/FAL/NC
A8 1b/NHK/DF7/FAL/NC

DF
DF

AA61NC
AA61NC

2.95
6.26

0.015
0.032

1.145
0.740

11.51%
2.23%

2
1

3
2

I-65

& 90%; R2 < 0.8

VC difference >15%; no points
between 10& 90%;
no R2 or ICx; PC
failed

A4 1b/NHK/CTR5/FAL

NR crystal problems; useddifferent medium; OD valuesof test wells no different than A5 1b/NHK/CTR6/FAL
background ODs; negativevalues for VC

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

A9 1b/NHK/DF8/FAL/NC

DF

AA61NC

6.25

0.032

0.798

9.28%

1

6

0.7381

A10 1b/NHK/DF9/FAL/NC

DF

AA61NC

1.29

0.007

1.108

3.81%

4

1

0.8550A11
1b/NHK/DF10/FAL/SLS//NC

DF

AA61NC

1.54

0.008

1.439

0.51%

4

1

0.8443

A12 1b/NHK/DF11/FAL/NC
1b/NHK/DF4/FAL/NC

DF
DF

AA61NC

1.88

0.010

0.459

1.00%

5

2

Exposure
Concentrations(ug/mL)7
15, 10.2, 6.93, 4.72, 3.21,
2.18, 1.48, 1.01
15, 10.2, 6.93, 4.72, 3.21,
2.18, 1.48, 1.01
15, 10.2, 6.93, 4.72, 3.21,
2.18, 1.48, 1.01

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

1.47

NO

R2 < 0.8; PC failed

1.47

YES

no outliers
removed outliers from VCs

1.47

YES

0.8901

15, 10.2, 6.93, 4.72, 3.21,
2.18, 1.48, 1.01

1.47

YES

15, 10.2, 6.93, 4.72, 3.21,
2.18, 1.48, 1.01

1.47

YES

AA61NC

1.36

0.007

0.755

1.17%

4

1

0.8346

Notes

PC ID

A9 1b/NHK/CTR10/FAL
A10
1b/NHK/CTR11/FAL
A11
1b/NHK/CTR12/FAL
A12
1b/NHK/CTR13/FAL/SLS
1b/NHK/CTR14/FAL/SLS

ATROPINE SULFATE
IIVS
A1

RF

AA61NE

91.6

0.132

0.544

0.93%

2

1

0.9667B1DF

AA61NE

106

0.152

0.578

5.65%

5

3

0.9599B2DF

AA61NE

64.6

0.093

0.492

0.17%

5

3

0.9862B3DF

AA61NE

78.9

0.114

0.705

3.13%

5

3

0.9915

AA61KX-A1

RF

AA61KX

57.5

0.083

0.549

2.70%

3

2

0.9435

AA61KX-B1

DF

AA61KX

79.4

0.114

0.798

3.96%

4

4

0.9761

AA61KX-B2

DF

AA61KX

97.5

0.140

0.673

1.08%

3

5

0.9491

AA61KX-B3

DF

AA61KX

79.4

0.114

0.675

2.42%

4

2

0.9655

FAL.NHK.FU.A1.28.07.04

RF

AA61FU

33.3

0.048

0.059

10.09%

3

3

0.7561

FAL.NHK.FU.B1.11.08.04

DF

AA61FU

202

0.291

0.809

8.32%

3

3

0.9333

FAL.NHK.FU-NB.B2.25.08.04

DF

AA61FU

80.7

0.116

1.010

3.32%

6

2

0.9459FAL.NHK.FU.B3.27.08.04

DF

AA61FU

30.4

0.044

0.526

4.53%

5

1

0.9696

A1

RF

AA61LD724

11.717

0.536

2.15%

1

1

0.9101B1DF

AA61LD

455

7.359

0.583

4.16%

4

4

0.9594

B2

DF

AA61LD

460

7.444

0.541

3.17%

4

4

0.9778B3DF

AA61LD

476

7.705

0.553

4.25%

4

4

0.9713

AA61JH-A1

RF

AA61JH

449

7.258

0.449

0.45%

2

2

0.9280AA61JH-B1

DF

AA61JH

598

9.678

0.690

6.95%

4

4

0.9413

AA61JH-B2

DF

AA61JH

371

5.995

0.736

3.27%

4

3

0.9757

AA61JH-B3

DF

AA61JH

350

5.660

0.438

3.54%

4

4

0.9848

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3

10

RF

1.8

YES

1.8

YES

1.8

YES

range finder

SLS-A4-N040331N
SLS-B4-N040513C
SLS-B5-N040514B
outlier removed by SD

SLS-B6-N040716A

ECBC
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
800, 372, 173, 80.5, 37.4,
17.4, 8.1, 3.8
800, 372, 173, 80.5, 37.4,
17.4, 8.1, 3.8
800, 372, 173, 80.5, 37.4,
17.4, 8.1, 3.8

10

RF

2.15

YES

range finder

SLS-P16
SLS-P30

2.15

YES

SLS-P40

2.15

YES

SLS-P42

FRAME
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10000, 4651, 2163, 1006,
468, 218, 101, 47
5000, 2326, 1082, 503,
234, 109, 50.6, 23.6
5000, 2326, 1082, 503,
234, 109, 50.6, 23.6

10

RF

2.15

YES

range finder

FAL.NHK.SLS.28.07.04
FAL.NHK.SLS.11.08.04

2.15

YES

FAL.NHK.SLS.25.08.04

2.15

YES

FAL.NHK.SLS.27.08.04

BORIC ACID
IIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1563, 977, 610,
381, 238, 149, 93
2500, 1563, 977, 610,
381, 238, 149, 93
2500, 1563, 977, 610,
381, 238, 149, 93

10

RF

1.6

YES

range finder
ppt in 1X C1SLS-A4-N040331N
SLS-B8-N040819A

1.6

YES

ppt in 1X C1SLS-B9-N040820A

1.6

YES

SLS-B10-N040903A

ECBC

FRAME

I-66

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
6000, 2791, 1298, 604,
281, 131, 60.7, 28.3
6000, 2791, 1298, 604,
281, 131, 60.7, 28.3
6000, 2791, 1298, 604,
281, 131, 60.7, 28.3

10

RF

2.15

YES

range finder

SLS-P17
SLS-P32

2.15

YES

SLS-P35

2.15

YES

SLS-P37

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

FAL.NHK.GR.A1.28.07.04

RF

AA61GR

1020

16.474

0.055

0.90%

1

1

0.6145

FAL.NHK.GR.B1.11.08.04

DF

AA61GR

592

9.568

0.739

0.12%

4

4

0.9157

FAL.NHK.GR-NB.B2.25.08.04

DF

AA61GR851

13.766

0.943

0.07%

4

4

0.9741

FAL.NHK.GR.B3.27.08.04

DF

AA61GR

107

1.733

0.534

8.67%

6

2

0.9607

RF

AA61RL

1150

4.683

0.500

10.83%

03

0.5430

Exposure
Concentrations(ug/mL)7
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10000, 4651, 2163, 1006,
468, 218, 101, 47
10000, 4651, 2163, 1006,
468, 218, 101, 47
10000, 4651, 2163, 1006,
468, 218, 101, 47

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

range finder

PC ID

10

RF

2.15

YES

FAL.NHK.SLS.28.07.04
FAL.NHK.SLS.11.08.04

2.15

YES

FAL.NHK.SLS.25.08.04

2.15

YES

FAL.NHK.SLS.27.08.04

10

RF

1.8

YES

SLS-B12-N041022B

1.5

YES

SLS-B113-N041029B

1.5

YES

SLS-B14-N041030A

BUSULFAN
IIVS
A1
B1

DF

AA61RL

274

1.113

0.732

7.46%

2

4

0.9237B2

DF

AA61RL

317

1.287

0.598

3.83%

2

5

0.9721B3DF

AA61RL

348

1.414

0.792

2.36%

2

6

0.9429AA61LH-A1

RF

AA61LH

NA

NA

0.624

3.53%

0

7

NA

AA61LH-B1

DF

AA61LH

217

0.882

1.103

1.81%

1

7

0.6962

AA61LH-B2

DF

AA61LH

211

0.856

0.792

1.88%

2

6

0.8550AA61LH-B3

DF

AA61LH

332

1.347

1.344

2.99%

1

7

0.6216

FAL.NHK.JE.A1.13.02.03

RF

AA61JE

29.8

0.121

0.152

15.63%

1

2

0.7100FAL.NHK.JE.A2.20.02.03

DF

AA61JE

171

0.694

0.195

6.46%

2

3

0.6939FAL.NHK.JE.B1.27.02.04

DF

AA61JE

142

0.575

0.622

3.35%

2

6

0.8940FAL.NHK.JE.B2.19.03.03

DF

AA61JE

490

1.988

0.573

1.40%

1

6

0.8387100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
750, 417, 231, 129, 71.4,
39.7, 22.1, 12.3
500, 333, 222, 148, 98.8,
65.8, 43.9, 29.3
500, 333, 222, 148, 98.8,
65.8, 43.9, 29.3

range finder; nopoints between 0 50%

SLS-A1-N040317B

ECBC
100, 10, 1, 0.1, 0.01,
0.001, 0.0001,0.00001
800, 372, 173, 80.5, 37.4,
17.4, 8.10, 3.77
800, 372, 173, 80.5, 37.4,
17.4, 8.10, 3.77
800, 372, 173, 80.5, 37.4,
17.4, 8.10, 3.77

10

RF

2.15

YES

range finder
ppt in 2X C1SLS-P4SLS-P47

2.15

YES

ppt in 2X C1SLS-P48

2.15

YES

ppt in 2X C1SLS-P51

10

RF

2.15

YES

2.15

YES

FAL.NHK.SLS.27.02.03

2.15

YES

FAL.NHK.SLS.19.03.03

FRAME
250, 25, 2.5, 0.25, 0.025,
0.0025, 0.00025
250, 116.3, 54.1, 25.2,
11.7, 5.4, 2.5, 1.2
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35

range finder

SD rejected due to bacterial
contamination in some of
FAL.NHK.SLS.13.02.03
the plates in this test series
DF since conc. series isdifferent from A1 RF

FAL.NHK.SLS.20.02.03

CADMIUM II CHLORIDE
IIVS
A2

RF

AA61NK

2.05

0.011

0.841

4.19

2

2

0.9692B1DF

AA61NK

1.84

0.010

0.444

6.37

5

3

0.9906B2

DF

AA61NK

1.72

0.009

0.344

6.83

3

3

0.9819B3DF

AA61NK

2.02

0.011

0.338

4.78

2

2

0.9738

AA61KR-A1

RF

AA61KR

1.75

0.010

0.492

0.22

3

3

0.9218

AA61KR-B1

DF

AA61KR

2.31

0.013

0.918

6.16

4

3

0.9738

AA61KR-B3

DF

AA61KR

3.29

0.018

0.749

0.44

2

2

0.9446

AA61KR-B5

DF

AA61KR

1.16

0.006

0.143

12.96

2

3

0.8299100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
10, 6.7, 4.4, 3.0, 2.0, 1.3,
0.88, 0.59
10, 6.7, 4.4, 3.0, 2.0, 1.3,
0.88, 0.59
10, 6.7, 4.4, 3.0, 2.0, 1.3,
0.88, 0.59

10

RF

1.47

YES

range finder

SLS-A2
SLS-B1

1.47

YES

SLS-B2

1.47

YES

SLS-B3ECBC

I-67

1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
100, 46.5, 21.6, 10.1, 4.7,
2.2, 1.0, 0.47
8.00, 5.44, 3.70, 2.52,
1.71, 1.17, 0.793, 0.539
8.00, 5.44, 3.70, 2.52,
1.71, 1.17, 0.793, 0.539

10

RF

2.15

YES

range finder

SLS-P4SLS-P8

1.47

YES

SLS-P12

1.47

YES

SLS-P15

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

AA61KR-B6

DF

AA61KR

2.57

0.014

0.867

2.57

3

3

0.9730AA61KR-B7

DF

AA61KR

1.66

0.009

0.507

6.37

3

4

0.9495

FAL.NHK.JP.A1.30.07.03

RF

AA61JP

1.71

0.009

1.263

6.60

3

5

0.9364

FAL.NHK.JP.B1.07.08.03

DF

AA61JP

0.722

0.004

0.253

4.61

4

0

0.9034

FAL.NHK.JP.B2.13.08.03

DF

AA61JP

NA

NA

0.219

9.58

0

3

NA

Exposure
Concentrations(ug/mL)7
8.00, 5.44, 3.70, 2.52,
1.71, 1.17, 0.793, 0.539
8.00, 5.44, 3.70, 2.52,
1.71, 1.17, 0.793, 0.539

Dilution

Acceptable

Factor8

Tests9

1.47

YES

SLS-P16

1.47

YES

SLS-P18

Rationale for
Unacceptability

Notes

PC ID

FRAME

FAL.NHK.JP.B3.23.08.03

DF

AA61JP

2.19

0.012

0.384

4.86

2

6

0.9507

FAL.NHK.JP.B4.28.08.03

DF

AA61JP

2.96

0.016

0.504

7.31

1

1

0.8321

FAL.NHK.JP.B5.05.09.03

DF

AA61JP

0.553

0.003

0.180

4.62

3

2

0.8972

FAL.NHK.JP.B6.01.10.03

DF

AA61JP

2.46

0.013

1.289

6.38

2

6

0.4951

FAL.NHK.JP.B6.15.10.03(should be B7?)DF

AA61JP

2.12

0.012

0.482

1.44

2

4

0.9753

A1

RF

AA61JM

390

2.008

0.440

7.52%

2

3

0.9708B1DF

AA61JM

565

2.909

0.489

3.92%

3

4

0.9805

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
12.0, 8.2, 5.6, 3.2, 2.6,
1.8, 1.2, 0.8
3.0, 2.04, 1.39, 0.94,
0.64, 0.44, 0.3, 0.2
5.0, 3.401, 2.314, 1.574,
1.071, 0.728, 0.496,
0.337
5.0, 3.401, 2.314, 1.574,
1.071, 0.728, 0.496,
0.337
5.0, 3.401, 2.314, 1.574,
1.071, 0.728, 0.496,
0.337
5.0, 3.401, 2.314, 1.574,
1.071, 0.728, 0.496,
0.337
5.0, 3.401, 2.314, 1.574,
1.071, 0.728, 0.496,
0.337

10

RF

1.47

NO

range finder

FAL.NHK.SLS.30.07.03

1.47

NO

1.47

NO

1.47

YES

FAL.NHK.SLS.280803

1.47

YES

FAL.NHK.SLS.050903

1.47

NO

1.47

YES

No points between
FAL.NHK.SLS.07.08.03
50 & 100% viability
PC failed; no points SD rejects this assay; can't
between 0 - 50%; no explain the variability of cell FAL.NHK.SLS.13.08.03
r2;
growth in the wells
PC failed

FAL.NHK.SLS.230803

low r2

FAL.NHK.SLS.01.10.03
FAL.NHK.SLS.15.10.03

CAFFEINE
IIVS

B2

DF

AA61JM

578

2.977

0.554

4.28%

4

4

0.9817B3DF

AA61JM

579

2.984

0.456

2.91%

3

3

0.9762

RF

AA61NU

221

1.137

0.469

5.83%

2

3

0.9546

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10000, 4545, 2066, 939,
427, 194, 88.2, 40.1
10000, 4545, 2066, 939,
427, 194, 88.2, 40.1
10000, 4545, 2066, 939,
427, 194, 88.2, 40.1

10

RF

2.2

YES

range finder

SLS-A1-N040317B
SLS-B1-N040423A

2.2

YES

two phase dose response
curve

2.2

YES

ppt in 1X C210000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

2.15

YES

SLS-P7

SLS-B2-N040424A
SLS-B3-N040506A

ECBC
AA61NU-A1

range finder

SLS-P3

AA61NU-B1

DF

AA61NU

1070

5.492

1.065

6.83%

1

7

0.91402000, 930, 433,201,93.6,
43.5, 20.2, 9.4

AA61NU-B2

DF

AA61NU

824

4.244

1.076

0.91%

4

4

0.9433

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

YES

SLS-P9

AA61NU-B3

DF

AA61NU

558

2.876

0.777

7.01%

4

4

0.959010000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

YES

SLS-P11

FAL.NHK.GW.A1.13.02.03

RF

AA61GW

340

1.753

0.189

12.28%

2

2

0.8133

FAL.NHK.GW.A2.13.02.03

DF

AA61GW

553

2.849

0.247

2.26%

3

4

0.9267

FAL.NHK.GW.B1.27.02.04

DF

AA61GW

794

4.090

0.456

0.75%

2

2

0.9523

FAL.NHK.GW.B3.18.03.04

DF

AA61GW

427

2.197

0.522

9.68%

3

5

0.9542

FRAME

I-68

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10000, 3175, 1008, 320,
102, 32.2, 10.2, 3.25
10000, 3175, 1008, 320,
102, 32.2, 10.2, 3.25
10000, 3175, 1008, 320,
102, 32.2, 10.2, 3.25

10

RF

3.15

YES

range finder

FAL.NHK.SLS.13.02.03

3.15

YES

FAL.NHK.SLS.27.02.03

3.15

YES

FAL.NHK.SLS.18.03.03

DF because conc. series isdifferent from A1 RF

FAL.NHK.SLS.20.02.03

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9

10

RF

1.6

YES

1.6

NO

1.6

YES

SLS-B14-N041030A

1.6

YES

SLS-B15-N041110A

10

RF

2.15

YES

ppt in 1X C1SLS-P41

2.15

YES

ppt in 2X C1 and 1X C1SLS-P43

2.15

YES

ppt in 2X C1 and 1X C1SLS-P45

Rationale for
Unacceptability

Notes

PC ID

CARBAMAZEPINE
IIVS
A1

RF

AA61NB

NA

NA

0.575

4.51%

0

1

NA

B1

DF

AA61NB

67.3

0.285

0.698

0.74%

1

7

0.9759B2DF

AA61NB

88.3

0.374

0.609

1.12%

0

5

0.8732B3DF

AA61NB

57.8

0.245

0.726

1.01%

1

5

0.9378B4DF

AA61NB

66.5

0.282

0.691

8.74%

3

5

0.9237

AA61LX-A1

RF

AA61LX

40.7

0.17240

0.827

3.59%

1

4

0.9327

AA61LX-B1

DF

AA61LX

56.5

0.239

0.669

1.51%

3

4

0.9784

AA61LX-B2

DF

AA61LX

71.9

0.304

0.693

3.27%

3

3

0.9477

AA61LX-B3

DF

AA61LX

70.0

0.296

1.100

2.84%

2

5

0.9566

FAL.NHK.HD.A1.24.09.04

RF

AA61HD

594

2.515

0.292

5.56%

1

2

-0.5440

FAL.NHK.HD.B1.01.10.04

DF

AA61HD

187

0.78983

1.037

6.43%

2

5

0.9721

FAL.NHK.HD.B2.07.10.04

DF

AA61HD

58.2

0.24634

0.631

2.15%

4

4

0.9855

FAL.NHK.HD.B3.05.11.04

DF

AA61HD

71.3

0.30167

0.521

2.51%

4

4

0.9236

10, 1, 0.1, 0.01, 0.001,
0.0001, 0.00001,
0.000001
75.0, 46.9, 29.3, 18.3,
11.4, 7.15, 4.47, 2.79
75.0, 46.9, 29.3, 18.3,
11.4, 7.15, 4.47, 2.79
75.0, 46.9, 29.3, 18.3,
11.4, 7.15, 4.47, 2.79
200, 125, 78.1, 48.8,
30.5, 19.1, 11.9, 7.45

range finder; nopoints between 0 50%

SLS-A5-N040401A
SLS-B12-N041022B

no points between 0 50%

SLS-B113-N041029B

ECBC
200, 20, 2, 0.2, 0.02,
0.002, 0.0002, 0.00002
400, 186, 86.5, 40.2,
18.7, 8.71, 4.05, 1.88
400, 186, 86.5, 40.2,
18.7, 8.71, 4.05, 1.88
400, 186, 86.5, 40.2,
18.7, 8.71, 4.05, 1.88

range finder

SLS-P19FRAME

FAL.NHK.HD.B4.10.11.04

DF

AA61HD628

2.65789

1.114

4.71%

3

5

0.9316

1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 8870, 756, 658,
572, 497, 432, 376

10RF

range finder

2.15

NO

PC failed

FAL.NHK.SLS.24.09.03

2.15

YES

ppt In 1X C1-C2

FAL.NHK.SLS.07.10.03

2.15

YES

ppt In 1X C1-C2

FAL.NHK.SLS.05.11.04

1.15

YES

ppt In 1X C1-C2; ppt in 2X
C1-C2

FAL.NHK.SLS.10.11.04

FAL.NHK.SLS.01.10.04

CARBON TETRACHLORIDE
IIVS
A1

RF

AA61JK

NA

NA

0.627

0.48%

0

0

NA

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10RF

range finder; nopoints between 0 100%B1DF

AA61JK

1540

10.023

0.679

3.90%

0

2

0.7803

2500, 1389, 772, 429,
238, 132, 73.5, 40.8

1.8

NO

SD removed highest doseno points between 0 - from Hill analyses due to ppt
SLS-B12-N041022B
50%
and upswing in response
curve; ppt in 2X C1-C8

B2DF

AA61JK

NA

NA

0.634

6.32%

0

2

NA

1.8

NO

B3

DF

AA61JK

NA

NA

0.755

0.42%

0

1

NA

1.8

NO

RF

AA61NZ

NA

NA

0.844

3.30%

0

3

NA

3000, 300, 30, 3, 0.3,
0.03, 0.003, 0.0003

10

RF

1.21

NO

2500, 1389, 772, 429,
238, 132, 73.5, 40.8
2500, 1389, 772, 429,
238, 132, 73.5, 40.8

no points between 0 ppt in 2X C1-C4
50%
no points between 0 ppt in 2X C1-C4
50%

SLS-A2-N040320B

SLS-B113-N041029B
SLS-B14-N041030A

ECBC
AA61NZ-A1
AA61NZ-B1

DF

AA61NZ

NA

NA

0.642

0.54%

0

4

NA

4500, 3719, 3074, 2540,
2099, 1735, 1434, 1185

AA61NZ-B2

DF

AA61NZ

NA

NA

0.770

0.36%

NA

N/A

NA

7000, 5785, 4781, 3951,
3266, 2699, 2230, 1843

1.21

NO

AA61NZ-B3

DF

AA61NZ

NA

NA

0.668

1.36%

6

1

NA

7000, 5785, 4781, 3951,
3266, 2699, 2230, 1843

1.21

NO

FRAME

I-69

range finder; nopoints between 0 50%
no points between 0 ppt in 2X C1- C5
50%
ppt in 2X C1-C5; chemical
globules in 1X C1-C4; plate
SD rejects
columns C6 and C7 show
no cells were plated
can't properly"roller coaster" toxicity
determine points
curve; ppt in 2X C1-C8;
between 0 - 100%
outliers removed by SD

SLS-P13SLS-P52

SLS-P56

SLS-P59

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

FAL.NHK.HC.A1.25.04.04

RF

AA61HC

NA

NA

0.920

2.74%

0

0

NA

FAL.NHK.HC.B1.11.06.04

DF

AA61HC

NA

NA

1.044

2.28%

0

8

NA

FAL.NHK.HC.B2.25.06.04

DF

AA61HC

1380

8.953

1.023

7.07%

0

2

0.8467

Exposure
Concentrations(ug/mL)7
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

Dilution

Acceptable

Factor8

Tests9

10

RF

2.15

NO

2.15

NO

1.21

NO

FAL.NHK.HC.B3.19.08.04 nb

DF

AA61HC

NA

NA

0.419

8.26%

0

7

0.00002500, 2066, 1708, 1411,
1166, 964, 797, 658

FAL.NHK.HC.B4.20.08.04

DF

AA61HC

NA

NA

0.739

2.93%

0

1

0.00002500, 2066, 1708, 1411,
1166, 964, 797, 658

1.21

NO

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

2.2

YES

2.2

YES

2.2

YES

Rationale for
Unacceptability

Notes

range finder ; nopoints between 0 100%
PC failed; no points
between 0 - 50%
no points between 0 50%
curve unacceptable;
no points between 0 no toxicity detectedd
50% would be
acceptable due tocurve unacceptable;
no points between 0 no toxicity detected; outliers
50% would be
removed by SD
acceptable due to
1.21 dilution

PC ID

FAL.NHK.SLS.25.04.04
FAL.NHK.SLS.11.06.04
FAL.NHK.SLS.25.06.04
FAL.NHK.SLSNB.19.08.04

FAL.NHK.SLS.20.08.04

CHLORAL HYDRATE
IIVS
A1

RF

AA61FJ

104

0.626

0.650

59.25%

2

1

0.9885B1DF

AA61FJ

114

0.686

0.601

3.48%

5

3

0.9882B2DF

AA61FJ

111

0.674

0.513

0.29%

5

3

0.9904B3DF

AA61FJ

111

0.672

0.517

6.49%

3

3

0.9917

AA61KB-A1

RF

AA61KB

NA

NA

0.268

59.01%

1

0NA

AA61KB-B1

DF

AA61KB

170

1.027

0.553

2.62%

3

5

0.9314

AA61KB-B2

DF

AA61KB

148

0.892

0.825

2.87%

4

4

0.9619

AA61KB-B3

DF

AA61KB103

0.62153

0.394

3.13%

4

4

0.9671

FAL.NHK.LK.A1.25.03.04

RF

AA61LK

103

0.620

0.412

65.79%

2

1

0.3337

FAL.NHK.LK.B1.25.04.04

DF

AA61LK

NA

NA

0.039

12.80%

2

1

NA

FAL.NHK.LK.B2.28.04.04

DF

AA61LK

142

0.860

0.825

0.16%

3

5

0.9864

FAL.NHK.LK.B2.11.06.04(should be B3)

DF

AA61LK

135

0.816

0.797

3.73%

3

3

0.9586

FAL.NHK.LK.B4.23.06.04

DF

AA61LK

215

1.299

0.970

1.58%

3

3

0.9863

FAL.NHK.LK.B5.25.06.04

DF

AA61LK

119

0.722

0.927

2.14%

3

3

0.9801

5000, 2273, 1033, 470,
213, 97.0, 44.1, 20.0
5000, 2273, 1033, 470,
213, 97.0, 44.1, 20.0
5000, 2273, 1033, 470,
213, 97.0, 44.1, 20.0

range finder; %VC
difference >0volatility problem; VC1 OD
values much lower than
VC2; VC1 removed from
subsequent analysis by SD

SLS-A2-N040320B

SLS-B1-N040423A
used plate sealer

SLS-B2-N040424A
SLS-B3-N040506A

ECBC
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
500, 340, 231, 157, 107,
72.8, 49.6, 33.7
500, 340, 231, 157, 107,
72.8, 49.6, 33.7
500, 340, 231, 157, 107,
72.8, 49.6, 33.7

10

RF

1.47

YES

range finder

probable volatility problem

SLS-P6SLS-P20

1.47

YES

SLS-P22

1.47

YES

SLS-P24

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RF1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

2.15

NO

2.15

YES

2.15

NO

2.15

YES

FAL.NHK.SLS.23.06.04

2.15

YES

FAL.NHK.SLS.25.06.04

FRAME

1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7,
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7,
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7,

range finder; %VC
possible volatility problem
difference > 15
wrong desorb
solution used in
NRU; SD rejects thistest

FAL.NHK.SLS.25.03.03

FAL.NHK.SLS.25.04.04
FAL.NHK.SLS.28.04.03

PC failed

FAL.NHK.SLS.11.06.04

CHLORAMPHENICOL
IIVS
A2

RF

AA61GJ

355

1.099

0.801

5.41%

0

2

0.6374B1

DF

AA61GJ

296

0.916

0.487

7.17%

2

6

0.9691I-70

100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
560, 311, 173, 96, 53.3,
29.6, 16.5, 9.15

10

RF

1.80

YES

range finder

SLS-A2
SLS-B1In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

B2

DF

AA61GJ

351

1.086

0.358

5.44%

1

6

0.9165B3DF

AA61GJ

453

1.402

0.377

0.99%

1

5

0.93

AA61JS-A1

RF

AA61JS

239

0.740

0.706

3.80%

1

7

0.8464

AA61JS-B1

DF

AA61JS

252

0.780

1.175

3.03%

2

5

0.9626

AA61JS-B2

DF

AA61JS

222

0.687

0.975

0.22%

3

5

0.9452

AA61JS-B3

DF

AA61JS

481

1.488

0.767

0.14%

2

6

0.9349FAL.NHK.MU.A1.30.07.03

RF

AA61MU

232

0.718

1.246

1.87%

1

6

0.8736

FAL.NHK.MU.B1.07.08.03

DF

AA61MU

160

0.495

0.187

55.29%

5

2

0.0978

FAL.NHK.MU.B2.15.08.03

DF

AA61MU

873

2.702

0.394

6.64%

1

2

0.6646

FAL.NHK.MU.B3.23.08.03

DF

AA61MU

587

1.816

0.329

2.15%

2

3

0.8892

FAL.NHK.MU.B4.28.08.03

DF

AA61MU

476

1.473

0.472

15.82%

1

5

0.8489FAL.NHK.MU.B5.05.09.03

DF

AA61MU

473

1.464

0.171

10.94%

2

4

0.8686

FAL.NHK.MU.B6.01.10.03

DF

AA61MU

173

0.535

1.304

7.20%

2

6

0.5745

FAL.NHK.MU.B6.15.10.03(should be B7?)DF

AA61MU

625

1.934

0.485

0.38%

2

5

0.9212

FAL.NHK.MU.B7.19.10.03

DF

AA61MU

916

2.835

0.164

2.34%

1

2

0.7152

FAL.NHK.MU.B8.23.10.03

DF

AA61MU

362

1.120

0.249

8.70%

2

5

0.8807

FAL.NHK.MU.B9.24.10.03

DF

AA61MU

194

0.600

0.861

4.38%

3

4

0.8814

A1

RF

AA61MH

298

1.551

0.413

4.09%

2

1

0.9217B1

DF

AA61MH

447

2.325

0.547

4.83%

4

4

0.9681B2

DF

AA61MH

407

2.121

0.562

0.18%

2

4

0.9655B3DF

AA61MH

444

2.309

0.477

2.95%

2

5

0.9609AA61HH-A1

RF

AA61HH

295

1.54

0.511

3.95%

2

1

0.9327

AA61HH-B1

DF

AA61HH

557

2.900

1.160

3.05%

2

6

0.9595

AA61HH-B2

DF

AA61HH

589

3.065

1.191

1.62%

2

6

0.9588

AA61HH-B3

DF

AA61HH

433

2.252

0.740

2.11%

2

6

0.9690RF

AA61RB

406

2.111

1.459

3.77%

2

6

0.9700

Exposure
Concentrations(ug/mL)7
560, 311, 173, 96, 53.3,
29.6, 16.5, 9.15
560, 311, 173, 96, 53.3,
29.6, 16.5, 9.15

Dilution

Acceptable

Factor8

Tests91.80

YES

SLS-B2

1.80

YES

SLS-B3

10

RF

2.15

YES

SLS-P8

2.15

YES

SLS-P10

2.15

YES

SLS-P12

Rationale for
Unacceptability

Notes

PC ID

ECBC
1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
2000, 930.2, 432.7,
201.2, 93.6, 43.5, 20.2,
9.4
2000, 930.2, 432.7,
201.2, 93.6, 43.5, 20.2,
9.4
2000, 930.2, 432.7,
201.2, 93.6, 43.5, 20.2,
9.4

range finder

SLS-P4

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1162, 541, 251,
171, 79.6, 54.1, 25.2
2500, 1163, 541, 252,
117, 54, 25, 12
2500, 1162, 541, 251,
171, 79.6, 54.1, 25.2
2500, 1162, 541, 251,
171, 79.6, 54.1, 25.2
2500, 1162, 541, 251,
171, 79.6, 54.1, 25.2
2500, 1162, 541, 251,
171, 79.6, 54.1, 25.2
2500, 1162, 541, 251,
171, 79.6, 54.1, 25.2
2500, 1162, 541, 251,
171, 79.6, 54.1, 25.2
2500, 1162, 541, 251,
171, 79.6, 54.1, 25.2
2500, 1162, 541, 251,
171, 79.6, 54.1, 25.2

10RF

range finder

FAL.NHK.SLS.30.07.03

2.15

NO

VC difference >15%; low r2

FAL.NHK.SLS.07.08.03

2.15

NO

low r2

FAL.NHK.SLS.15.08.03

2.15

NO

PC failed

FAL.NHK.SLS.230803

2.15

NO

% VC difference >15

FAL.NHK.SLS.280803

2.15

YES

2.15

NO

2.15

YES

FAL.NHK.SLS.050903
low r2

FAL.NHK.SLS.01.10.03
FAL.NHK.SLS.15.10.03

2.15

NO

2.15

YES

low r2

FAL.NHK.SLS.19.10.03
FAL.NHK.SLS.23.10.03

2.15

YES

FAL.NHK.SLS.24.10.03

CITRIC ACID

IIVS

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10000, 4545, 2066, 939,
427, 194, 88.2, 40.1
10000, 4545, 2066, 939,
427, 194, 88.2, 40.1
3000, 1667, 926, 514,
286, 159, 88.2, 49.0

10

RF

2.2

YES

2.2

YES

1.8

YES

range finder

SLS-A1-N040317B
SLS-B1-N040423A
SLS-B2-N040424A
ppt in 1X C1-C2

SLS-B3-N040506A

ECBC
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 680, 463, 315, 214,
146, 99.1, 67.4
1000, 680, 463, 315, 214,
146, 99.1, 67.4
1000, 680, 463, 315, 214,
146, 99.1, 67.4

10

RF

1.47

YES

range finder

SLS-P1SLS-P7

1.47

YES

SLS-P9

1.47

YES

SLS-P11

10

RF

FRAME
FA.NH.HV.A1.11.02.04 (should
be RB)

I-71

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

range finder

pH and color of 2X matches
FAL.NHK.SLS.11.02.04
citric acid for 3T3

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

FAL.NHK.RB.A2.18.02.04

DF

AA61RB

362

1.886

0.210

4.13%

6

0

0.7857

FAL.NHK.RB.B1.26.02.04

DF

AA61RB

348

1.809

0.183

5.10%

3

5

0.9225

FAL.NHK.RB.B2.27.02.04

DF

AA61RB

361

1.881

0.415

5.54%

4

3

0.9577

FAL.NHK.RB.B3.18.03.04

DF

AA61RB

288

1.501

0.361

12.24%

4

3

0.9324

FAL.NHK.RB.B4.19.03.04

DF

AA61RB

251

1.308

0.510

2.65%

4

4

0.9369RF

AA61FL

3.94

0.010

0.705

0.78%

4

3

Exposure
Concentrations(ug/mL)7

10000, 4651, 2163, 1006,
468, 218, 101, 47
10000, 4651, 2163, 1006,
468, 218, 101, 47
10000, 4651, 2163, 1006,
468, 218, 101, 47
5000, 2326, 1082, 503,
234, 109, 50.6, 23.5
5000, 2326, 1082, 503,
234, 109, 50.6, 23.5

Dilution

Acceptable

Factor8

Tests9

2.15

NO

2.15

YES

Rationale for
Unacceptability

PC failed; no points
between 50-100%

Notes

PC ID

this is a definitive test sinceconc. series is different from FAL.NHK.SLS.18.02.04
A1 range finder
FAL.NHK.SLS/MO.26.0
2.03
ppt detected in C1-C3 at
end of test

2.15

YES

2.15

YES

FAL.NHK.SLS.27.02.04
FAL.NHK.SLS.18.03.03

2.15

YES

FAL.NHK.SLS.19.03.03

10RF

range finder

SLS-A2

1.8

NONo points 50-100%;low R2

SLS-B1

1.8

NONo points 50-100%;low R2

SLS-B2

1.8

NONo points 50-100%;low R2

SLS-B3

3.19

NONo points 50-100%;low R2

SLS-B4

1.5

NO

Low R2

SLS-B7

1.8

NO

PC failedslow NHK growth; media
problems

SLS-B8

1.8

NO

PC failed; no points
between 0 - 50%

slow NHK growth; media
problems

SLS-B91.8

NO

PC failed; no points
between 0 - 50%;
low r2; %VC
difference > 15

slow NHK growth; media
problems

SLS-B10

1.8

NO

PC failedslow NHK growth; media
problems

SLS-B11

1.5

YES

morning (a.m.) harvest;
SMT accepts this test

SLS-B12

1.5

YES

afternoon (p.m.) harvest

SLS-B13

1.5

YES

1.5

NO

PC failed

SLS-B1510RF

low r2; couldn't calc.
ICx values; range
range finderfinderSLS-P3

COLCHICINE
IIVS
A2
B1

DF

AA61FL0.00184

0.0000046

0.384

4.49%

8

0

B2

DF

AA61FL

0.000675

0.0000017

0.289

9.86%

8

0

B3

DF

AA61FL

0.0000306

0.0000001

0.335

7.90%

8

0

B4

DF

AA61FL0.02150.0000538

0.2

4.67%

5

0

B7

DF

AA61FL

0.000733

0.0000018

0.624

0.50%

6

2

B8* Hill function
w/unconstrained bottom

DF

AA61FL0.005070.0000127

0.677

4.22%

1

5

B9* Hill function
w/unconstrained bottom

DF

AA61FL0.00506

0.0000127

0.598

3.21%

0

6

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1.0, 0.56, 0.31, 0.17,
0.6346 0.095, 0.053, 0.029,
0.016
1.0, 0.56, 0.31, 0.17,
0.5984 0.095, 0.053, 0.029,
0.016
1.0, 0.56, 0.31, 0.17,
0.3037 0.095, 0.053, 0.029,
0.016
1.0, 0.313, 0.098, 0.031,
0.7647 0.0095, 0.0030, 0.00093,
0.000290.03, 0.02, 0.013, 0.0089,
0.06259 0.0059, 0.0040, 0.0026,
0.00180.1, 0.056, 0.031, 0.017,
0.4741 0.0095, 0.0053,
0.0029,0.0016
0.1, 0.056, 0.031, 0.017,
0.5162 0.0095, 0.0053,
0.0029,0.0016
0.4952

B10* Hill function
w/unconstrained bottom

DF

AA61FL

NA

NA

0.44

22.49%

07

0.6108B11* Hill function
w/unconstrained bottom

DF

AA61FL0.00609

0.0000152

0.436

4.74%

5

1

0.8455B12* Hill function
w/unconstrained bottom

DF

AA61FL0.009270.0000232

0.727

5.52%

3

3

0.7899B13* Hill function
w/unconstrained bottom

DF

AA61FL0.008920.0000223

0.237

1.66%

5

1

0.9513

B14* Hill function
w/unconstrained bottom

DF

AA61FL0.006170.0000154

0.351

8.77%

5

1

0.9223

B15* Hill function
w/unconstrained bottom

DF

AA61FL0.005710.0000143

0.276

4.29%

5

2

0.873

RF

AA61JZ

NA

NA

0.326

23.32%

5

2

0.0097

0.1, 0.056, 0.031, 0.017,
0.0095, 0.0053,
0.0029,0.0016
0.1, 0.056, 0.031, 0.017,
0.0095, 0.0053,
0.0029,0.0016
0.045, 0.030, 0.020,
0.0133, 0.0089, 0.0059,
0.0040, 0.0026
0.045, 0.030, 0.020,
0.0133, 0.0089, 0.0059,
0.0040, 0.0026
0.045, 0.030, 0.020,
0.0133, 0.0089, 0.0059,
0.0040, 0.0026
0.045, 0.030, 0.020,
0.0133, 0.0089, 0.0059,
0.0040, 0.0026

SLS-B14ECBC
AA61JZ-A1

I-72

10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7
10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001

Dilution

Acceptable

Factor8

Tests910NO

no r2 nor ICx values
range finder
could be calculated

SLS-P5

2.15

NO

PC failed

SLS-P11

2.15

NO

PC failed

SLS-P13

2.15

NO

PC failed; low r2

SLS-P13

1.47

NO

low r2SLS-P15

1.47

YES

SLS-P16

1.47

YES

SLS-P18

1.47

YES

SLS-P19

Rationale for
Unacceptability

AA61JZ-A2

RF

AA61JZ

NA

NA

0.202

3.41%

6

2

NA

AA61JZ-B1

DF

AA61JZ

557

1.394

0.770

0.63%

4

4

0.9016

AA61JZ-B2

DF

AA61JZ

817

2.045

0.099

1.01%

3

4

0.9437

AA61JZ-B3

DF

AA61JZ

0.017

0.00004

0.089

9.22%

1

2

0.4165

AA61JZ-B4

DF

AA61JZ

0.012

0.00003

0.089

9.29%

2

3

0.5530AA61JZ-B5

DF

AA61JZ

0.003

0.00001

0.884

5.21%

5

3

0.8528AA61JZ-B6

DF

AA61JZ

0.011

0.00003

0.494

4.09%

3

2

0.7228AA61JZ-B7

DF

AA61JZ

0.009

0.00002

0.687

1.01%

4

3

0.7162

RF

AA61NW

0.198

0.00050

0.305

17.20%

5

3

0.6953

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

RFrange finder

0.6233

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

NO

RF format

NO

RF format; low r2

10000, 4651.2, 2163.3,
1006.2, 468, 217.7,
101.2, 47.1
10000, 4651.2, 2163.3,
1006.2, 468, 217.7,
101.2, 47.1
0.02140, 0.00995,
0.00463, 0.00215, 0.001,
0.00046, 0.00022, 0.0001
0.0200, 0.0136, 0.0093,
0.0063, 0.0043, 0.0029,
0.0020, 0.0014
0.0200, 0.0136, 0.0093,
0.0063, 0.0043, 0.0029,
0.0020, 0.0014
0.0200, 0.0136, 0.0093,
0.0063, 0.0043, 0.0029,
0.0020, 0.0014
0.0200, 0.0136, 0.0093,
0.0063, 0.0043, 0.0029,
0.0020, 0.0014

Notes

PC ID

FRAME
FAL.NHK.NW.A1.010803
FAL.NHK.NW.B1.080803

DF

AA61NW

0.024

0.00006

0.713

705.50%7

1

FAL.NHK.NW.B2.15.08.03

DF

AA61NW

1.00

0.00250

0.510

4.47%

6

1

0.5677

FAL.NHK.NW.B3.19.10.03

DF

AA61NW

0.008

0.00002

0.312

8.59%

4

2

0.8637

FAL.NHK.NW.B4.23.10.03

DF

AA61NW

0.007

0.00002

0.340

0.96%

4

1

0.9166

FAL.NHK.NW.B5.24.10.03

DF

AA61NW

0.008

0.00002

0.974

0.55%

4

4

0.886910000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
0.100, 0.047, 0.022,
0.01006, 0.00468,
0.00218, 0.00101,
0.000470.100, 0.047, 0.022,
0.01006, 0.00468,
0.00218, 0.00101,
0.000470.100, 0.047, 0.022,
0.01006, 0.00468,
0.00218, 0.00101,
0.00047

SD says toxicity biphasic;chemical may be volatile
high background; biphasic
response; determined ICx
values with only 3 points
biphasic response

FAL.NHK.SLS.010803
FAL.NHK.SLS.07.08.03
FAL.NHK.SLS.15.08.03

YES

FAL.NHK.SLS.19.10.03

YES

FAL.NHK.SLS.23.10.03

YES

FAL.NHK.SLS.24.10.03

CUPRIC SULFATE PENTAHYDRATE
IIVS
A1

RF

AA61LA

NA

NA

0.643

3.80%

0

2

NA

100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001

10

RF

B1

DF

AA61LA

213

0.854

0.646

5.61%

3

3

0.9907750, 536, 383, 273, 195,
139, 99.6, 71.1

1.4

YES

B2

DF

AA61LA

199

0.797

0.583

1.02%

3

3

0.9957

1.4

YES

B3

DF

AA61LA

208

0.833

0.675

1.17%

3

3

0.9811

1.4

YES

AA61HX-A1

RF

AA61HX

NA

NA

0.487

1.42%

0

1

NA

AA61HX-B1

DF

AA61HX

195

0.783

0.880

2.81%

6

1

0.9370AA61HX-B2

DF

AA61HX

168

0.672

0.675

3.43%

6

2

0.9871750, 536, 383, 273, 195,
139, 99.6, 71.1
750, 536, 383, 273, 195,
139, 99.6, 71.1

range finder; nopoints between 0 50%

SLS-A2-N040320B
ppt in 2X C1 (homogeneous
blue suspension); ppt in 1X SLS-B12-N041022B
C1-C8
ppt in 2X C1; ppt in 1X C1C8
ppt in 2X C1; ppt in 1X C1C8

SLS-B113-N041029B
SLS-B14-N041030A

ECBC

I-73

100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
500, 413, 342, 282, 233,
193, 159, 132
500, 413, 342, 282, 233,
193, 159, 132

10

RF

1.21

YES

range finder

SLS-P6SLS-P47

1.21

YES

SLS-P48

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC ID

DF

AA61HX

206

0.823

1.320

1.52%

5

3

0.9814500, 413, 342, 282, 233,
193, 159, 132

1.21

YES

FAL.NHK.LP.A1.20.10 .04

RF

AA61LP

8.41

0.034

0.998

4.10%

3

0

0.9793

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10

RF

FAL.NHK.LP.B1.29.10.04

DF

AA61LP

NA

NA

0.545

7.44%

0

1

NA

2.15

NO

FAL.NHK.LP.B2.10.11.04

DF

AA61LP

189

0.756

1.026

0.20%

5

3

0.9474

1.47

YES

FAL.NHK.LP.B3.12.11.04

DF

AA61LP

186

0.746

0.696

6.80%

2

1

0.97942.15

YES

FAL.NHK.SLS.12.11.04

FAL.NHK.LP.B4.17.11.04

DF

AA61LP

209

0.837

0.999

3.03%

2

1

0.98222.15

YES

FAL.NHK.SLS.17.11.04

A1

RF

AA61GL0.05890.0002

0.518

2.80%

5

1

0.9832

10

RF

B1

DF

AA61GL0.07530.0003

0.534

1.79%

4

3

0.9783

2.2

YES

SLS-B4-N040513C

B2

DF

AA61GL0.05660.0002

0.499

1.72%

4

4

0.9931

2.2

YES

SLS-B5-N040514B

B3

DF

AA61GL0.08220.0003

0.712

3.28%

4

2

0.98582.2

YES

SLS-B6-N040716A

AA61KK-A1

RF

AA61KK

0.04410.0002

0.456

2.74%

6

1

0.966010

RF

AA61KK-B1

DF

AA61KK

0.05580.0002

0.737

3.19%

4

4

0.9741

2.15

YES

SLS-P33

AA61KK-B2

DF

AA61KK

0.06340.0002

0.823

3.39%

4

4

0.9764

2.15

YES

SLS-P35

AA61KK-B3

DF

AA61KK

0.04010.0001

0.418

6.74%

5

3

0.9655

2.15

YES

SLS-P36

FAL.NHK.PF.A1.28.07.04

RF

AA61PF0.08730.0003

0.042

0.79%

4

2

0.810610RFrange finder

FAL.NHK.SLS.28.07.04

FAL.NHK.PF.B1.12.08.04

DF

AA61PF

0.432

0.0015

0.862

1.46%

6

2

0.9511

3.16

NO

PC failed

FAL.NHK.SLS.12.08.04

FAL.NHK.PF-NB.B2.25.08.04

DF

AA61PF

0.06750.0002

1.104

1.57%

7

1

0.96903.16

YES

FAL.NHK.SLS.25.08.04

FAL.NHK.PF.B3.20.10 .04

DF

AA61PF

0.22850.0010

1.179

5.59%

5

3

0.9771

2.15

YES

FAL.NHK.SLS.20.10.04

FAL.NHK.PF.B4.29.10.04

DF

AA61PFNA

0.0000

0.507

2.36%

8

0

0.93782.15

NO

no points between
50 - 100%

FAL.NHK.PF.B5.05.11.04

DF

AA61PF

NA

NA

0.475

3.35%

6

0

NA

2.15

NO

no points between
50 - 100%

FAL.NHK.PF.B6.12.11.04

DF

AA61PF

0.06470.0002

0.725

2.10%

4

4

0.9513

2.15

YES

AA61HX-B3

SLS-P50

FRAME

100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
1000, 680, 463, 315, 214,
146, 99.1, 67.4
500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

range finder ; nopoints between 50 - outlier removed by SD
100%
no points between 0 50%
outliers removed by SD

FAL.NHK.SLS.20.10.04
FAL.NHK.SLS.29.10.04
FAL.NHK.SLS.10.11.04

CYCLOHEXIMIDE
IIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1.00, 0.455, 0.207, 0.094,
0.043, 0.019, 0.0088,
0.00401.00, 0.455, 0.207, 0.094,
0.043, 0.019, 0.0088,
0.00401.00, 0.455, 0.207, 0.094,
0.043, 0.019, 0.0088,
0.0040range finder

SLS-A4-N040331N

ECBC
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1.00, 0.465, 0.216, 0.101,
0.047, 0.022, 0.010,
0.0005
1.00, 0.465, 0.216, 0.101,
0.047, 0.022, 0.010,
0.005
1.00, 0.465, 0.216, 0.101,
0.047, 0.022, 0.010,
0.005

range finder

SLS-P17

FRAME

I-74

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100, 31.6, 10.0, 3.17,
1.00, 0.317, 0.100,
0.0318100, 31.6, 10.0, 3.17,
1.00, 0.317, 0.100,
0.031810.0, 4.65, 2.16, 1.01,
0.468, 0.218, 0.101,
0.047
10.0, 4.65, 2.16, 1.01,
0.468, 0.218, 0.101,
0.047
10.0, 4.65, 2.16, 1.01,
0.468, 0.218, 0.101,
0.047
1.00, 0.47, 0.22, 0.10,
0.05, 0.02, 0.010, 0.0047

toxicity curve doesn't go
above 20% viability

FAL.NHK.SLS.29.10.04
FAL.NHK.SLS.05.11.04
FAL.NHK.SLS.12.11.04

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9

10

RF

2.2

YES

2.2

YES

1.8

YES

Rationale for
Unacceptability

Notes

PC ID

DIBUTYL PHTHALATE
IIVS
A1

RF

AA61FD

25.2

0.090

0.684

8.39%

2

1

0.9676B1

DF

AA61FD

23.2

0.083

0.562

2.55%

5

3

0.9704B2DF

AA61FD

22.3

0.080

0.613

1.33%

3

3

0.9866B3DF

AA61FD

20.6

0.074

0.515

7.46%

4

4

0.9634

AA61JX-A1

RF

AA61JX

26.8

0.096

0.892

1.40%

2

2

0.9594

AA61JX-B1

DF

AA61JX

34.0

0.122

0.957

0.03%

3

5

0.9281

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 455, 207, 93.9,
42.7, 19.4, 8.82, 4.01
1000, 455, 207, 93.9,
42.7, 19.4, 8.82, 4.01
200, 111, 61.7, 34.3, 19.1,
10.6, 5.88, 3.27

range finder

ppt in 1X C1-C2; ppt in 2X
C1-C2
ppt in 1X C1-C4; ppt in 2X
C1-C5
ppt in 1X C1-C5; ppt in 2X
C1-C5
ppt in 1X C1-C4; ppt in 2X
C1-C4

SLS-A3-N040331A
SLS-B1-N040423A
SLS-B2-N040424A
SLS-B3-N040506A

ECBC

AA61JX-B2
AA61JX-B3

DF

AA61JX

19.6

0.071

0.698

0.13%

3

5

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
200, 93.0, 43.3, 20.1, 9.4,
4.4, 2.0, 0.9

10

RF

2.15

YES

0.9518200, 93.0, 43.3, 20.1, 9.4,
4.4, 2.0, 0.9

2.15

YES

200, 93.0, 43.3, 20.1, 9.4,
4.4, 2.0, 0.9

2.15

YES

DF

AA61JX

31.2

0.112

1.251

5.20%

3

4

0.9461

FAL.NHK.MK.A1.14.05.04

RF

AA61MK

152

0.546

0.692

8.77%

1

1

0.7744

FAL.NHK.MK.B1.19.08.04 nb

DF

AA61MK

NA

NA

0.342

2.58%

8

0

0.0000FAL.NHK.MK-RB.B2.25.08.04

DF

AA61MK

17.5

0.063

0.972

4.85%

4

4

0.9053

FAL.NHK.MK.B3.07.10.04

DF

AA61MK

39.7

0.143

0.602

7.72%

4

4

0.9531

FAL.NHK.MK.B4.20.10 .04

DF

AA61MK

84.9

0.305

1.289

5.24%

3

3

0.9716

A1

RF

AA61NP

12.6

0.057

0.702

59.99%

2

1

0.9650B1DFAA61NP

12.1

0.055

0.599

10.60%

5

3

0.9934B2DF

AA61NP

11.9

0.054

0.627

7.89%

4

3

0.9912B3DF

AA61NP

12.7

0.057

0.581

1.03%

4

2

0.9802

AA61PZ-A1

RF

AA61PZ

NA

NA

0.532

72.53%

1

2

NA

AA61PZ-B1(sealer)DF

AA61PZ

8.44

0.038

0.631

6.94%

4

4

0.9304

AA61PZ-B2 (sealer)DF

AA61PZ

10.9

0.049

0.860

3.50%

3

5

0.9861

AA61PZ-B3 (sealer)DF

AA61PZ

6.35

0.029

0.381

4.51%

4

4

0.9428

FAL.NHK.HS.A1.14.05.04

RF

AA61HS

9.55

0.043

0.391

72.35%

3

00.4969FAL.NHK.HS.B1.25.06.04

DF

AA61HS

13.2

0.060

1.094

9.37%

2

3

0.9630range finder

ppt in 2X C2SLS-P15
SLS-P46

ppt in 2X C2; 1X C1-C3 hassmall chunks-possibly
SLS-P49
chemical crystals
chunks of chemical in 1X C1SLS-P51
C3; ppt in 2X C4

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 1361, 926, 630,
428, 291, 198, 135
500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

10RF

range finder

ppt in 1X C1

1.47

NO

no points between
50 - 100%

ppt in 1X C1-C8

FAL.NHK.SLS.14.05.03

2.15

YES

2.15

YES

ppt in 1X C1-C5

FAL.NHK.SLS.07.10.03

2.15

YES

ppt in 1X C1-C4

FAL.NHK.SLS.20.10.04

10RF

volatility problem; VC1 OD
values much lower than
VC2; VC1 removed from
subsequent analysis by SD

SLS-A3-N040331A

2.2

YES

2.2

YES

used plate sealer

SLS-B2-N040424A

2.2

YES

used plate sealer

SLS-B3-N040506A

FAL.NHK.SLSNB.19.08.04
FAL.NHK.SLSRB.20.08.04

DICHLORVOS
IIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
500, 227,103, 47.0, 21.3,
9.70, 4.41, 2.00
500, 227,103, 47.0, 21.3,
9.70, 4.41, 2.00
200, 90.9, 41.3, 18.8,
8.54, 3.88, 1.76, 0.802

range finder; % VC
difference > 15

SLS-B1-N040423A

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

10

RF

2.15

YES

range finder

SLS-P15
SLS-P32

2.15

YES

SLS-P34

2.15

YES

SLS-P36

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10RF50.0, 23.3, 10.8, 5.03,
2.34, 1.09, 0.506, 0.235

2.15

YES

FRAME

I-75

range finder; %VC
difference > 0; nopoints between 50 100%

volatility problem

FAL.NHK.SLS.14.05.03
FAL.NHK.SLS.25.06.04

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

FAL.NHK.HS.B2.12.08.04

Assay Substance
ID
Type1

DF

AA61HS

IC50(ug/mL)

IC50(mM)

Mean VC

18.9

0.085

0.677

OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3
5.08%

2

2

R2 6

Exposure
Concentrations(ug/mL)7

0.630450.0, 23.3, 10.8, 5.03,
2.34, 1.09, 0.506, 0.235
50.0, 23.3, 10.8, 5.03,
2.34, 1.09, 0.506, 0.235

FAL.NHK.HS.B3.19.08.04 nb

DF

AA61HS

NA

NA

0.510

1.27%

0

7

0.0466

FAL.NHK.HS-RB.B4.25.08.04

DF

AA61HS

15.7

0.071

0.773

1.27%

2

1

0.6376

FAL.NHK.HS.B5.27.08.04

DF

AA61HS

8.35

0.038

0.506

9.96%

2

6

0.8021

50.0, 23.3, 10.8, 5.03,
2.34, 1.09, 0.506, 0.235
50.0, 23.3, 10.8, 5.03,
2.34, 1.09, 0.506, 0.235

Dilution

Acceptable

Factor8

Tests92.15

NO

PC failed

FAL.NHK.SLS.12.08.04

2.15

NO

no toxicity detected; SD
no points between 0 removed column of data;50%
odd toxicity curve

FAL.NHK.SLSNB.19.08.04

2.15

YES

FAL.NHK.SLSRB.20.08.04

2.15

YES

FAL.NHK.SLS.27.08.04

10

RF

1.8

YES

1.8

YES

1.8

YES

Rationale for
Unacceptability

Notes

PC ID

DIETHYL PHTHALATE
IIVS
A1

RF

AA61NX

116

0.523

0.556

0.99%

1

1

0.8983B1DF

AA61NX

192

0.863

0.570

3.77%

3

4

0.9757B2DF

AA61NX

221

0.996

0.505

1.47%

3

3

0.9758B3DF

AA61NX

155

0.695

0.790

6.15%

3

3

0.9904

AA61GA-A1

RF

AA61GA

122

0.551

0.898

5.79%

1

3

0.9642

AA61GA-B1

DF

AA61GA

168

0.757

1.039

5.26%

2

4

0.9636

AA61GA-B2

DF

AA61GA

163

0.732

0.920

1.89%

3

2

0.9498

AA61GA-B3

DF

AA61GA

190

0.854

0.776

1.33%

2

3

0.9633

FAL.NHK.KZ.A1.28.07.04

RF

AA61KZ

124

0.560

0.079

10.77%

1

1

0.6487

FAL.NHK.KZ.B1.11.08.04

DF

AA61KZ

27.7

0.125

0.765

6.15%

1

2

0.9160FAL.NHK.KZ.B2.08.10.04

DF

AA61KZ

147

0.660

0.737

18.98%

2

5

0.9382

FAL.NHK.KZ.B3.22.10.04

DF

AA61KZ

149

0.670

0.731

9.65%

2

4

0.9568

FAL.NHK.KZ.B4.28.10.04

DF

AA61KZ

37.9

0.171

0.650

11.96%

4

4

0.9425

A1

RF

AA61MF

0.000750.0000010

0.695

0.29%

7

0

0.9294

B1

DF

AA61MF

0.003900.0000050

0.575

3.87%

3

1

0.9597B2

DF

AA61MF

0.003740.0000048

0.543

0.21%

3

1

0.9615B3DF

AA61MF0.004310.0000055

0.804

1.90%

2

3

0.98481000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3

range finder

ppt in 2X C1-C2

SLS-A4-N040331N
SLS-B4-N040513C

ppt in 2X C1SLS-B5-N040514B
SLS-B6-N040716A

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
800, 372, 173, 80.5, 37.4,
17.4, 8.1, 3.8
800, 372, 173, 80.5, 37.4,
17.4, 8.1, 3.8
800, 372, 173, 80.5, 37.4,
17.4, 8.1, 3.8

10

RF

2.15

YES

range finder

ppt in 2X C1SLS-P14

2.15

YES

ppt in 2X C1-C2

SLS-P29

2.15

YES

ppt in 2X C1-C2; oily

SLS-P30

SLS-P27

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 930, 433, 201, 94,
44, 20, 9500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35

10

RF

2.15

YES

2.15

NO

2.15

YES

2.15

YES

10

RF

2.2

YES

2.2

YES

2.2

YES

range finder% VC difference >15

FAL.NHK.SLS.28.07.04
ppt in 2X C1-C4 and 1X C1FAL.NHK.SLS.11.08.04
C4
volatility issue; incorrect
solvent listed in Addendum FAL.NHK.SLS.08.10.03
III; SD corrected
FAL.NHK.SLS.22.10.04(MO)
FAL.NHK.SLS.28.10.04

DIGOXIN

IIVS

ECBC

I-76

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
0.020, 0.0091, 0.0041,
0.0019, 0.00085,
0.00039, 0.00018,
0.000080
0.020, 0.0091, 0.0041,
0.0019, 0.00085,
0.00039, 0.00018,
0.000080
0.020, 0.0091, 0.0041,
0.0019, 0.00085,
0.00039, 0.00018,
0.000080

range finder; nopoints between 50 100%

ppt in 1X C1 and 2X C1SLS-A3-N040331A

SLS-B4-N040513C

outlier removed by SD

SLS-B5-N040514B

SLS-B6-N040716A

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

AA61PP-A1

RF

AA61PP

0.008650.0000111

1.002

8.88%

5

0

0.9920AA61PP-B1

DF

AA61PP

0.005180.0000066

0.864

4.37%

4

4

0.9591

AA61PP-B2

DF

AA61PP

0.006150.0000079

0.890

1.28%

4

4

0.9932

AA61PP-B3

DF

AA61PP

0.004810.0000062

0.477

0.96%

5

2

0.9770FAL.NHK.HN.A1.14.05.04

RF

AA61HN

0.000020.0000000

0.756

7.58%

5

0

0.9437

FAL.NHK.HN.B1.25.06.04

DF

AA61HN

0.00006

0.0000001

1.205

0.03%

4

3

0.9543

FAL.NHK.HN.B2.20.08.04

DF

AA61HN

0.00006

0.0000001

0.845

3.03%

4

3

0.9762

FAL.NHK.HN.B3.27.08.04

DF

AA61HN

0.000030.0000000

0.404

5.62%

5

3

0.9091

Exposure
Concentrations(ug/mL)7
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
0.100, 0.0465, 0.0216,
0.0101, 0.0047, 0.0022,
0.0010, 0.0005
0.100, 0.0465, 0.0216,
0.0101, 0.0047, 0.0022,
0.0010, 0.0005
0.100, 0.0465, 0.0216,
0.0101, 0.0047, 0.0022,
0.0010, 0.0005

Dilution

Acceptable

Factor8

Tests9

10

RF

2.15

YES

SLS-P33

2.15

YES

SLS-P35

2.15

YES

SLS-P37

10

RF

2.15

YES

FAL.NHK.SLS.25.06.04

2.15

YES

row C data removed by SD;
FAL.NHK.SLS.20.08.04
most of wells were outliers

2.15

YES

FAL.NHK.SLS.27.08.04

10

RF

1.4

YES

ppt in 1X C1SLS-B8-N040819A

1.4

YES

ppt in 1X C1SLS-B9-N040820A

1.4

YES

10

RF

1.47

YES

SLS-P40

1.47

YES

SLS-P42

1.47

YES

SLS-P44

Rationale for
Unacceptability
range finder; nopoints between 50 100%Notes

ppt in 1X C1 and 2X C1PC ID

SLS-P13

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
0.0010000, 0.0004651,
0.0002163, 0.0001006,
0.0000468, 0.0000218,
0.0000101, 0.0000047
0.0010000, 0.0004651,
0.0002163, 0.0001006,
0.0000468, 0.0000218,
0.0000101, 0.0000047
0.0010000, 0.0004651,
0.0002163, 0.0001006,
0.0000468, 0.0000218,
0.0000101, 0.0000047

range finder

outlier removed by SD; ppt
in 1X C1-C2

FAL.NHK.SLS.14.05.03

DIMETHYLFORMAMIDE
IIVS
A1

RF

AA61FN5750

78.720

0.495

3.49%

1

1

0.8849B1DF

AA61FN

6180

84.544

0.553

1.90%

3

4

0.9725B2DF

AA61FN

6580

89.967

0.543

5.48%

3

3

0.9801B3DF

AA61FN6430

87.919

0.544

0.29%

3

3

0.9823

RF

AA61MW

NA

NA

0.773

5.14%

1

0

NA

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
15000, 10714, 7653,
5466, 3905, 2789, 1992,
1423
15000, 10714, 7653,
5466, 3905, 2789, 1992,
1423
15000, 10714, 7653,
5466, 3905, 2789, 1992,
1423

range finder

SLS-A5-N040401A

SLS-B10-N040903A

ECBC
AA61MW-A1

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
30000, 20408, 13883,
9444, 6425, 4371, 2973,
2023
30000, 20408, 13883,
9444, 6425, 4371, 2973,
2023
30000, 20408, 13883,
9444, 6425, 4371, 2973,
2023

range finder; nopoints between 50 100%

SLS-P19

AA61MW-B1

DF

AA61MW

9350

127.962

0.595

0.67%

2

4

0.9730

AA61MW-B2

DF

AA61MW

9510

130.042

0.722

1.78%

3

4

0.9847

AA61MW-B3

DF

AA61MW

9200

125.916

0.961

1.49%

2

4

0.9788

FAL.NHK.KF.A1.24.09.04

RF

AA61KF

1940

26.551

0.501

2.32%

1

1

0.3487

10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001

10RFrange finder

FAL.NHK.SLS.24.09.03

FAL.NHK.KF.B1.01.10.04

DF

AA61KF

7690

105.216

0.990

2.68%

1

7

0.974115000, 6977, 3245, 1509,
702, 327, 152, 70.6

2.15

NO

PC failed

FAL.NHK.SLS.01.10.04

FAL.NHK.KF.B2.10.11.04

DF

AA61KF7930

108.413

1.031

2.19%

1

4

0.929015000, 6977, 3245, 1509,
702, 327, 152, 70.6

2.15

YES

FRAME

I-77

ppt In 2X C1-C5

FAL.NHK.SLS.10.11.04

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9FAL.NHK.KF.B3.12.11.04

DF

AA61KF

6040

82.6200.668

16.78%

1

2

0.892915000, 6977, 3245, 1509,
702, 327, 152, 70.6

2.15

NO

FAL.NHK.KF.B4.17.11.04

DF

AA61KF

7780

106.435

1.146

1.64%

1

2

0.928115000, 6977, 3245, 1509,
702, 327, 152, 70.6

2.15

YES

FAL.NHK.KF.B5.19.11.04

DF

AA61KF

7740

105.946

0.465

5.14%

1

2

0.851415000, 6977, 3245, 1509,
702, 327, 152, 70.6

2.15

YES

Rationale for
Unacceptability

Notes%VC difference >15 outliers removed bySD

PC ID

FAL.NHK.SLS.12.11.04
FAL.NHK.SLS.17.11.04

outliers removed bySD

FAL.NHK.SLS.19.11.04

DIQUAT DIBROMIDE MONOHYDRATE
IIVS
A1

RF

AA61GN

5.71

0.016

0.711

0.12%

4

2

0.9904

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

B1

DF

AA61GN

4.10

0.011

0.570

1.86%

6

2

0.9823100, 55.6, 30.9, 17.1,
9.53, 5.29, 2.94, 1.63

1.8

YES

SLS-B4-N040513C

B2

DF

AA61GN

3.49

0.010

0.513

5.54%

6

2

0.9793100, 55.6, 30.9, 17.1,
9.53, 5.29, 2.94, 1.63

1.8

YES

SLS-B5-N040514B

B3

DF

AA61GN

3.92

0.011

0.652

0.15%

4

2

0.9871

100, 55.6, 30.9, 17.1,
9.53, 5.29, 2.94, 1.63

1.8

YES

SLS-B6-N040716A

AA61KS-A1

RF

AA61KS

3.04

0.008

0.862

7.32%

4

4

0.973010000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RF

AA61KS-B1

DF

AA61KS

3.62

0.010

0.671

2.01%

5

3

0.9904100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

2.15

YES

SLS-P33

AA61KS-B2

DF

AA61KS

4.40

0.012

0.570

0.19%

5

2

0.9601

100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

2.15

YES

SLS-P34

AA61KS-B3

DF

AA61KS

2.75

0.008

0.361

4.41%

5

3

0.9603100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

2.15

YES

SLS-P36

RF

AA61NV

3.88

0.011

0.640

4.87%

4

1

0.9854

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RF500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

range finder

SLS-A3-N040331A

ECBC
range finder

SLS-P14FRAME
FAL.NHK.NV.A1.14.05.04
FAL.NHK.NV.B1.12.08.04

DF

AA61NV

7.22

0.020

0.899

3.27%

6

2

0.9571

FAL.NHK.NV.B2.19.08.04 rb

DF

AA61NV

43.3

0.119

0.271

2.15%

4

1

0.7846

FAL.NHK.NV.B3.20.08.04

DF

AA61NV

6.09

0.017

0.762

8.68%

6

2

0.9750FAL.NHK.NV-RB.B4.25.08.04

DF

AA61NV

11.9

0.033

0.583

7.52%

5

3

0.9780FAL.NHK.NV.B5.27.08.04

DF

AA61NV

0.812

0.002

0.493

3.41%

7

0

0.8924

FAL.NHK.NV.30.09.04

DF

AA61NV

2.97

0.008

0.677

0.21%

5

3

0.9830FAL.NHK.NV.B7.07.10.04

DF

AA61NV

6.13

0.017

0.665

1.98%

4

4

0.9794RF

AA61FC

140

0.509

0.559

3.49%

1

2

0.5182500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

range finder

FAL.NHK.SLS.14.05.03
row of data removed fromanalysis by the SD due tolow cell growth

2.15

NO

PC failed

FAL.NHK.SLS.12.08.04

2.15

NO

PC failed

2.15

YES

2.15

YES

2.15

NO

2.15

YES

FAL.NHK.SLS.30.09.03

2.15

YES

FAL.NHK.SLS.07.10.03

10RF

FAL.NHK.SLSRB.19.08.04
row C data removed by SD;
FAL.NHK.SLS.20.08.04
several wells were outliers
FAL.NHK.SLSRB.20.08.04

no points between
50 - 100%

FAL.NHK.SLS.27.08.04

DISULFOTON
IIVS
A1

I-78

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

range finder

ppt in 2X C2SLS-A4-N040331N

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

B1

DF

AA61FC

176

0.641

0.619

10.61%

4

4

0.9647B2DF

AA61FC

133

0.486

0.566

5.12%

4

4

0.9650B3DFAA61FC

250

0.911

0.668

3.22%

3

5

0.9138

AA61NY-A1

RF

AA61NY

NA

NA

0.798

10.85%

1

3

NA

AA61NY-B1

DF

AA61NY

139

0.508

0.623

2.86%

2

5

0.8924

Exposure
Concentrations(ug/mL)7

2000, 909, 413, 188,
85.4, 38.8, 17.6, 8.02
2000, 909, 413, 188,
85.4, 38.8, 17.6, 8.02
2000, 909, 413, 188,
85.4, 38.8, 17.6, 8.02

Dilution

Acceptable

Factor8

Tests9

2.2

YES

2.2

YES

2.2

YES

Rationale for
Unacceptability

Notes

PC ID

ppt in 1X C1-C5; ppt in 2X
C1-C7; visual observations
SLS-B12-N041022Bof the cells are different fromthe NRU viability results.
ppt in 1X C1-C6; ppt in 2X
C1-C6;
ppt in 1X C1-C5; ppt in 2X
C1-C6;SLS-B113-N041029B
SLS-B14-N041030A

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

10

RF

2.15

YES

range finder

ppt in 2X C2SLS-P39

ppt in 2X C1-C4

SLS-P55

chem. pieces C1-C4 in 96well plate; ppt in 2X C1-C2;
C1 toxicity < C2; curve
SLS-P56
rises; SD originally failed
test; good toxicity curvewhen C1 removed by SD
no PRISM file generated;no points between 0globules of chemical in 1X SLS-P57
50%
C1-C6; ppt in 2X C1-C4
chem. globules in all conc.
in test plate; ppt in 2X C1C5;C1 toxicity< C2 and C3;
curve rises; SD originally
SLS-P58
failed test; good tox. curvewhen C1 and C2 removed
by SD

AA61NY-B2aDF

AA61NY

167

0.610

0.781

1.34%

1

6

0.8173500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

2.15

YES

AA61NY-B3

DF

AA61NY

NA

NA

0.533

0.92%

0

8

NA

300, 204, 139, 94, 64, 44,
30, 201.47

NOAA61NY-B4aDF

AA61NY

113

0.413

0.128

6.62%

1

6

0.7376300, 204, 139, 94, 64, 44,
30, 201.47

YES

FAL.NHK.LC.A1.28.07.04

RF

AA61LC

NA

NA

0.052

15.74%

1

2-0.3837

10RF

FAL.NHK.LC.B1.11.08.04

DF

AA61LC

828

3.017

0.764

7.18%

1

5

0.7436

2.15

YES

FAL.NHK.LC.B2.17.09.04

DF

AA61LC

1670

6.104

0.685

4.15%

0

7

0.8707

2.15

NO

FAL.NHK.LC.B3.08.10.04

DF

AA61LC

586

2.136

0.681

9.54%

2

6

0.88302.15

YES

ppt in 2X C1-C4; 1X C1FAL.NHK.SLS.08.10.03

FAL.NHK.LC.B4.20.10 .04

DF

AA61LC

1010

3.678

1.071

13.87%

2

6

0.93192.15

YES

ppt in 2X C1; ppt in 1X C1C8

FAL.NHK.SLS.20.10.04

A1

RF

AA61HZ

0.817

0.002

0.637

37.84%

2

3

0.9532

10

RF

volatility problem; VC1 OD
values much lower than
VC2; VC1 removed from
subsequent analysis by SD

SLS-A2-N040320B

B1

DF

AA61HZ

2.66

0.007

0.690

3.49%

1

3

0.9857

1.8

YES

ppt in 2X C2SLS-B1-N040423A

B2

DF

AA61HZ

2.10

0.005

0.674

1.76%

3

2

0.99101.8

YES

ppt in 2X C2; ppt in 1X C1SLS-B2-N040424A

B3

DF

AA61HZ

1.80

0.004

0.554

0.89%

3

2

0.9590

1.6

YES

AA61LG-A1

RF

AA61LG

NA

NA

0.612

31.27%

2

1

NA

10

RF

AA61LG-B1(sealer)DF

AA61LG

4.46

0.011

0.935

2.18%

0

5

0.87322.15

NOAA61LG-B2 (sealer)DF

AA61LG

4.09

0.010

1.218

0.21%

2

6

0.9121

1.47

YES

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

range finder; % VC
difference > 15

FAL.NHK.SLS.28.07.04
ppt in C3

no points between 0 - ppt in C1-C4;outliers
50%
removedFAL.NHK.SLS.11.08.04
FAL.NHK.SLS.17.09.04

ENDOSULFAN
IIVS100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
50.0, 27.8, 15.4, 8.57,
4.76, 2.65, 1.47, 0.817
50.0, 27.8, 15.4, 8.57,
4.76, 2.65, 1.47, 0.817
20.0, 12.5, 7.81, 4.88,
3.05, 1.91, 1.19, 0.745

range finder; %VC
difference >0

SLS-B3-N040506A

ECBC

I-79

100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
10.0, 4.65, 2.16, 1.01,
0.47, 0.22, 0.10, 0.05
9.00, 6.12, 4.17, 2.83,
1.93, 1.31, 0.892, 0.607

range finder; % VC
ppt in 2X C1 and C1
difference > 15
no points between 0 50%

SLS-P39
SLS-P46
SLS-P51

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

AA61LG-B3 (sealer)DF

AA61LG

3.00

0.007

0.613

0.94%

3

5

0.9278

AA61LG-B4 (sealer)DF

AA61LG

3.24

0.008

0.631

4.02%

3

4

0.9089RF

AA61PW

1.79

0.004

0.592

24.69%

1

2

0.4155

Exposure
Concentrations(ug/mL)7
9.00, 6.12, 4.17, 2.83,
1.93, 1.31, 0.892, 0.607
9.00, 6.12, 4.17, 2.83,
1.93, 1.31, 0.892, 0.607

Dilution

Acceptable

Factor8

Tests9

1.47

YES

SLS-P52

1.47

YES

SLS-P54

10

RF

Rationale for
Unacceptability

Notes

PC ID

FRAME
FAL.NHK.PW.A1.28.04.04
FAL.NHK.PW.B1.11.06.04

DF

AA61PW

1.05

0.003

0.953

2.52%

5

1

0.6822

FAL.NHK.PW.B2.25.06.04

DF

AA61PW

2.19

0.005

1.109

6.72%

5

3

0.9113

FAL.NHK.PW.B3.17.09.04

DF

AA61PW

1.24

0.003

0.820

0.67%

5

2

0.8280FAL.NHK.PW.B4.07.10.04

DF

AA61PW

0.822

0.002

0.731

4.68%

7

1

0.792910, 1, 0.1, 0.01, 0.001,
0.0001, 0.00001,
0.000001
10, 6.80, 4.63, 3.15, 2.14,
1.46, 0.99, 0.67
10, 6.80, 4.63, 3.15, 2.14,
1.46, 0.99, 0.67
10, 6.80, 4.63, 3.15, 2.14,
1.46, 0.99, 0.67
10, 6.80, 4.63, 3.15, 2.14,
1.46, 0.99, 0.67

1.47

NO

1.47

YES

1.47

YES

1.47

YES

range finder; %VC
difference > 15

possible volatility problem

FAL.NHK.SLS.28.04.03

PC failedincorrect solvent listed;biphasic response

FAL.NHK.SLS.11.06.04
FAL.NHK.SLS.25.06.04

outlier removed by SD

FAL.NHK.SLS.17.09.04
FAL.NHK.SLS.07.10.03

EPINEPHRINE BITARTRATE
IIVS
A1

RF

AA61LT

91.2

0.274

0.637

6.28%

2

1

0.9359B1

DF

AA61LT

61.1

0.183

0.430

3.51%

5

3

0.9623

B2

DF

AA61LT

83.8

0.251

0.562

3.01%

2

3

0.9796

B3

DF

AA61LT

80.0

0.240

0.513

2.26%

2

5

0.9398

AA61HW-A1

RF

AA61HW

73.5

0.220

0.337

4.12%

2

0

0.6969AA61HW-B1

DF

AA61HW

124

0.371

0.897

6.82%

2

2

0.8018

AA61HW-B2

DF

AA61HW

118

0.354

0.959

3.84%

3

3

0.9373

AA61HW-B3

DF

AA61HW

103

0.308

0.692

0.84%

4

2

0.9411

FAL.NHK.RK.A1.26.03.04

RF

AA61RK

93.5

0.281

0.552

10.97%

3

00.7362

FAL.NHK.RK.B1.25.04.04

DF

AA61RK

112

0.337

0.705

1.25%

3

1

FAL.NHK.RK.B2.28.04.04

DF

AA61RK

77.3

0.232

0.887

5.93%

4

FAL.NHK.RK.B3.13.05.04

DF

AA61RK

55.8

0.168

0.606

0.81%

RF

AA61FH

NA

NA

0.628

2.73%

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
200, 143, 102, 72.9, 52.1,
37.2, 26.6, 19.0

10

RF

1.8

YES

range finder
ppt in 1X C1SLS-A2-N040320B
SLS-B1-N040423A

1.8

YES

ppt in 1X C1-C2

SLS-B2-N040424A

1.4

YES

10

RF

1.47

YES

SLS-P26

1.21

YES

SLS-P29

1.21

YES

SLS-P31

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RF

0.84281000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

2.15

YES

two "outliers" in C4 removed
FAL.NHK.SLS.25.04.04
b SD due to low OD

1

0.9755

1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7,

2.15

YES

two "outliers" in C4 removed
FAL.NHK.SLS.28.04.03
by SD; no NR uptake

430.9907

1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

2.15

YES

FAL.NHK.SLS.13.05.04

01

0.429910000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RFrange finder; nopoints between 0 50%

1.8

NO

%VC difference >15

Left VC was removed from
SLS-B8-N040819A
calculations due to volatility

1.8

NO

%VC difference >15

Left VC was removed from
SLS-B9-N040820A
calculations due to volatility

SLS-B3-N040506A

ECBC
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
200, 136, 92.6, 63.0,
42.8, 29.1, 19.8, 13.5
200, 165, 137, 113, 93.3,
77.1, 63.7, 52.7
200, 165, 137, 113, 93.3,
77.1, 63.7, 52.7

range finder

ppt in 2X C1SLS-P6

FRAME
range finder

pts between 50 - 100% but
several above 100% ; ppt in FAL.NHK.SLS.26.03.04
C1

ETHANOL
IIVS
A1
B1

DF

AA61FH

7240

157.2470.461

100.30%3

20.9851B2DF

AA61FH

6430

139.5020.509

100.04%2

2

0.9844I-80

150000, 83333, 46296,
25720, 14289, 7938,
4410, 2450
150000, 83333, 46296,
25720, 14289, 7938,
4410, 2450

SLS-A2-N040320B

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7
150000, 83333, 46296,
25720, 14289, 7938,
4410, 2450
150000, 83333, 46296,
25720, 14289, 7938,
4410, 2450
50000, 31250, 19531,
12207, 7629, 4768, 2980,
1863

Dilution

Acceptable

Factor8

Tests9

1.8

YES

SLS-B11-N040904H

1.8

YES

SLS-B10-N040903A

1.6

YES

SLS-B12-N041022B

10

RF

1.47

YES

SLS-P28

1.47

YES

SLS-P31

1.47

YES

SLS-P34

B3DF

AA61FH

10800

234.197

0.586

1.92%

2

3

0.9760B4DFAA61FH

9250

200.716

0.709

2.59%

1

3

0.9781B5DF

AA61FH

10700

232.050

0.627

1.78%

3

4

0.9858AA61JU-A1

RF

AA61JU

NA

NA

0.436

7.58%

0

1

NA

AA61JU-B1(sealer)DF

AA61JU

7940

172.418

0.701

3.02%

6

1

0.9000AA61JU-B2(sealer)DF

AA61JU8710

189.052

0.741

5.60%

5

3

0.9616

AA61JU-B3(sealer)DF

AA61JU

8220

178.477

0.788

1.41%

3

4

0.9617

RF

AA61PC

11800

256.792

0.646

14.49%

01-0.7906

100000, 1000, 100,10, 1,
0.1, 0.01, 0.001

10

RF

2.15

YES

Rationale for
Unacceptability

Notes

PC ID

ECBC
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
50000, 34014, 23139,
15740, 10708, 7284,
4955, 3371
30000, 20408, 13883,
9444, 6425, 4371, 2973,
2023

range finder

SLS-P5FRAME
FAL.NHK.PC.A1.25.04.04

range finder

FAL.NHK.SLS.25.04.04

FAL.NHK.PC.A2.28.04.04

DF

AA61PC

9640

209.210

0.959

3.42%

2

6

0.942825000, 11628, 5408,
2516, 1170, 544, 253, 118

FAL.NHK.PC.B2.11.06.04

DF

AA61PC

11400

247.504

0.753

2.64%

1

3

0.897225000, 11628, 5408,
2516, 1170, 544, 253, 118

2.15

NO

FAL.NHK.PC.B3.23.06.04

DF

AA61PC

14200

308.022

0.896

9.81%

1

4

0.895825000, 11628, 5408,
2516, 1170, 544, 253, 118

2.15

YES

FAL.NHK.SLS.23.06.04

FAL.NHK.PC.B4.25.06.04

DF

AA61PC

12200

265.816

0.899

4.29%

1

3

0.887525000, 11628, 5408,
2516, 1170, 544, 253, 118

2.15

YES

FAL.NHK.SLS.25.06.04

10

RF

1.78

YES

SLS-B1

1.78

YES

SLS-B2

1.78

YES

SLS-B3

1.78

YES

SLS-B4

FAL.NHK.SLS.28.04.03
PC failedincorrect solvent listed

FAL.NHK.SLS.11.06.04

ETHYLENE GLYCOL
IIVSPreliminaryRF

AA61HR

44900

723.027

0.588

4.11%

0

1

0.6185B1DF

AA61HR

40900

658.615

0.552

1.95%

1

2

0.9752B2DF

AA61HR

32200

518.519

0.734

3.50%

1

3

0.9755B3DF

AA61HR

43200

695.652

0.798

1.30%

1

1

0.9797B4DF

AA61HR

43700

703.704

0.826

4.36%

1

1

0.9780RF

AA61LM

NA

NA

0.788

1.16%

0

0

-0.5039

RF

AA61LM

17700

285.024

1.125

7.69%

0

1

0.9617DF

AA61LM

42100

677.939

1.282

1.23%

2

2

0.9764

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100000, 56250, 31600,
17800, 10000, 5600,
3160, 1770
100000, 56250, 31600,
17800, 10000, 5600,
3160, 1770
100000, 56250, 31600,
17800, 10000, 5600,
3160, 1770
100000, 56250, 31600,
17800, 10000, 5600,
3160, 1770

range finder

Preliminary

ECBC
ECBC-NHK-Ib-01
AA61LM-A1
ECBC-NHK-Ib-02
AA61LM-A2
ECBC-NHK-Ib-03
AA61LM-B1

I-81

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100000, 10000, 1000,
100, 10, 1, 0.1, 0.01
100000, 68000, 46300,
31500, 21400, 14600,
9910, 6740

10RF

range finder

SLS-P2

10NONo points between
10 and 50%

SLS-P3

1.47

YES

SLS-P4

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7
84869.6, 57656.0,
39168.5, 26609.0,
18076.8, 12280.4,
8342.7, 5667.6
100000, 68000, 46300,
31500, 21400, 14600,
9910, 6740
60030, 46200, 35500,
27300, 21000, 16200,
12400, 9570

Dilution

Acceptable

Factor8

Tests9

1.47

YES

1.47

NO

1.3

YES

Rationale for
Unacceptability

Notes

PC ID

ECBC-NHK-Ib-04
AA61LM-B2 (correction rec'd
4/30/03)DF

AA61LM

39000

628.019

1.148

5.83%

1

2

0.9491

ECBC-NHK-Ib-05
AA61LM-B3

DF

AA61LM

44000

708.535

1.119

0.98%

0

2

0.9719ECBC-NHK-Ib-06
AA61LM-B4

DF

AA61LM

32900

529.791

0.910

3.05%

3

3

0.9383A3 1b/NHK/DF1/FAL/PDDF

AA61PD

16.1

0.259

0.047

1.95%

5

1

0.3772100, 68.02, 46.27, 31.47,
21.40, 14.50, 9.90, 6.70

1.47

RF

A4 1b/NHK/DF2/FAL/PDDF

AA61PD

4.17

0.067

0.125

25.74%

4

1

0.1465100, 68.02, 46.27, 31.47,
21.41, 14.56, 9.90, 6.74

1.47

NO

A5 1b/NHK/DF3/FAL/PDDF

AA61PD

NA

NA

0.140

1.78%

6

1

NA

100, 68.02, 46.27, 31.47,
21.41, 14.56, 9.90, 6.74

1.47

NO

A6 1b/NHK/DF4/FAL/PDDF

AA61PD

67.1

1.081

0.920

0.29%

1

0

0.5955100, 68.02, 46.27, 31.47,
21.40, 14.50, 9.90, 6.70

1.47

NO

A10 1b/NHK/DF5/FAL/PDDF

AA61PD

48400

779.3881.203

10.37%

1

6

0.8164

1.47

YES

A11 1b/NHK/DF6/FAL/PDDF

AA61PD

54700

880.837

1.706

4.22%

2

2

0.8960

1.47

YES

A12 1b/NHK/DF7/FAL/PDDF

AA61PD

33200

534.6220.372

17.37%

1

5

0.86781.47

NO

1b/NHK/DF3/FAL/PDDF

AA61PD

46300

745.572

0.773

12.10%

1

5

0.9074

1.47

YES

10

RF

2.2

YES

ppt in 2X C1-C3

SLS-B1-N040423A

2.2

YES

ppt in 2X C1-C3

SLS-B2-N040424A

YES

ppt in 2X C1-C3; ppt in 1X
C1

SLS-B3-N040506A

SLS-P5
No points between
10 and 50%

SLS-P7SLS-P8

FRAME

100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742

R2 < 0.8; PC failed;range finder

NR crystal problems; usedmedium not normally used

A3 1b/NHK/CTR4/FAL/

VC difference >NR crystal problems; usedmedium not normally used

A4 1b/NHK/CTR5/FAL

15%; R2 < 0.8
No R2 or ICx; PC
failedUsed different medium; OD
values of test wells slightly
A5 1b/NHK/CTR6/FAL
higher than bkgd. ODs;
negative values for VC

No point between 50 recalc w/o outlier didn't
improve fit, so outlier was
not removed& 90%; R2 < 0.8

no outliers

A6 1b/NHK/CTR7/FAL
A10
1b/NHK/CTR11/FAL
A11
1b/NHK/CTR12/FAL
A12
1b/NHK/CTR13/FAL/SLS

VC difference > 15%

1b/NHK/CTR14/FAL/SLS

FENPROPATHRIN
IIVS
A1

RF

AA61HY

1.38

0.004

0.552

4.86%

3

1

0.9698B1DF

AA61HY

2.18

0.006

0.580

3.12%

5

3

0.9412B2DF

AA61HY

1.67

0.005

0.600

4.40%

5

2

0.9440B3DFAA61HY

1.62

0.005

0.528

1.77%

5

2

0.9228

AA61LJ-A1

RF

AA61LJ

4.46

0.013

0.569

6.52%

3

3

0.9479AA61LJ-B1

DF

AA61LJ

3.71

0.0106

1.025

3.17%

8

0

0.8224

AA61LJ-B2

DF

AA61LJ

2.94

0.008

1.265

0.48%

5

3

0.9897

AA61LJ-B3

DF

AA61LJ

3.38

0.010

0.779

5.84%

5

3

0.9503

AA61LJ-B4

DF

AA61LJ

4.87

0.014

0.991

1.87%

5

3

0.9448

RF

AA61PT

5.51

0.016

1.226

1.06%

3

5

0.96101000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
75.0, 34.1, 15.5, 7.04,
3.20, 1.46, 0.661, 0.301
75.0, 34.1, 15.5, 7.04,
3.20, 1.46, 0.661, 0.301
75.0, 34.1, 15.5, 7.04,
3.20, 1.46, 0.661, 0.301

2.2

range finder

ppt in 2X C1-C2 and 1X C1SLS-A1-N040317B
C2

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

10RFrange finder

ppt in 2X C1-C2 and 1X C1 SLS-P2

1.47

NO

no points between
50 - 100%

ppt in 2X C1-C5 and 1X C1 SLS-P8

2.15

YES

ppt in 2X C1-C3

2.15

YES

ppt in 2X C1-C3 and 1X C1 SLS-P11

2.15

YES

ppt in 2X C1-C3 and 1X C1 SLS-P23

10

RF

SLS-P10FRAME
FAL.NHK.A1.11/02/04

I-82

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

range finder

C5 outliers removed by SD;
FAL.NHK.SLS.11.02.04
ppt in 2X C1 and 1X C1-C2

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

FAL.NHK.PT.B1.26.02.04

DF

AA61PT

0.012

0.000

0.185

9.24%

8

0

0.4977

FAL.NHK.PT.18.03.04 (B2 notin identifier)DF

AA61PT

2.77

0.008

0.321

1.46%

4

1

0.7108

FAL.NHK.PT.B3.19.03.04

DF

AA61PT

2.37

0.007

0.587

8.52%

5

2

0.9693

FAL.NHK.PT.B4.25.03.04

DF

AA61PT

1.56

0.004

0.693

8.69%

6

2

0.9644

AA61RE

NA

NA

0.542

1.18%

0

1

0.0000

Exposure
Concentrations(ug/mL)7
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
50.0, 23.3, 10.8, 5.03,
2.34, 1.09, 0.51, 0.24
50.0, 23.3, 10.8, 5.03,
2.34, 1.09, 0.51, 0.24
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

Dilution

Acceptable

Factor8

Tests9

2.15

NO

2.15

YES

ppt in 2X C1 and 1X C1FAL.NHK.SLS.18.03.03

2.15

YES

ppt in 2X C1FAL.NHK.SLS.19.03.03

2.15

YES

ppt in 2X C1 and 1X C1-C4 FAL.NHK.SLS.25.03.03

10

RF

1.6

YES

SLS-B12-N041022B

1.4

YES

SLS-B113-N041029B

1.4

YES

SLS-B14-N041030A

Rationale for
Unacceptability
no points between
50-100%

Notes

PC ID

ppt in 2X C1-C5 and 1X C1- FAL.NHK.SLS/MO.26.0
C4
2.03

GIBBERELLIC ACID
IIVS
A1

RF

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
3750, 2344, 1465, 916,
572, 358, 224, 1403750, 2679, 1913, 1367,
976, 697, 498, 356
3750, 2679, 1913, 1367,
976, 697, 498, 356

range finder; nopoints between 0 50%

outlier in C7 removed by SD SLS-A4-N040331N

B1

DF

AA61RE

2820

8.155

0.594

4.88%

1

4

0.9686B2

DF

AA61RE

2920

8.442

0.499

1.94%

1

2

0.9503B3DF

AA61RE

2680

7.735

0.646

1.50%

1

5

0.9492

RF

AA61FR

NA

NA

0.958

1.55%

0

6

NA

2500, 250, 25, 2.5, 0.25,
0.025, 0.0025, 0.00025

10

RF

1.47

YES

ppt in 2X C1-C4 and 1X C1 SLS-P49

ECBC
AA61FR-A1

range finder

SLS-P22

AA61FR-B1

DF

AA61FR

2470

7.136

0.689

0.27%

4

4

0.920910000, 6803, 4628, 3148,
2142, 1457, 991, 674

AA61FR-B2

DF

AA61FR

3270

9.429

1.151

0.64%

3

5

0.9334

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

ppt in 2X C1-C5

AA61FR-B3

DF

AA61FR

2810

8.118

0.643

1.28%

4

4

0.9736

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

ppt in 2X C1-C3 and 1X C1 SLS-P53

FAL.NHK.GY.A1.28.07.04(should be 11.08.04)

RF

AA61GY

NA

NA

0.596

2.46%

0

1

NA

FAL.NHK.GY.B1.08.10.04

DF

AA61GY

3030

8.739

0.629

2.48%

1

7

0.8918

FAL.NHK.GY.B2.20.10 .04

DF

AA61GY

3160

9.130

1.110

2.21%

1

2

0.9820FAL.NHK.GY.B3 .22.10.04

DF

AA61GY

2630

7.594

0.641

8.86%

1

1

0.8601A1

RF

AA61NN

119

0.546

0.579

1.28%

0

1

0.9782B1DF

AA61NN

190

0.873

0.634

3.05%

4

3

0.9710B2DFAA61NN

193

0.889

0.541

0.86%

4

2

0.9455B3DF

AA61NN

144

0.664

0.806

8.24%

4

4

0.9734

AA61FE-A1

RF

AA61FE

171

0.789

0.574

1.65%

1

6

0.9668

AA61FE-B1

DF

AA61FE

114

0.524

0.799

6.19%

3

5

0.9192

AA61FE-B2

DF

AA61FE

236

1.086

0.688

1.79%

2

1

0.9489SLS-P50

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
5000, 2326, 1082, 503,
234, 109, 50.6, 23.6
5000, 2326, 1082, 503,
234, 109, 50.6, 23.6
5000, 2326, 1082, 503,
234, 109, 50.6, 23.6

10

RF

2.15

YES

range finder

FAL.NHK.SLS.11.08.04
FAL.NHK.SLS.08.10.03

2.15

YES

FAL.NHK.SLS.20.10.04

2.15

YES

FAL.NHK.SLS.22.10.04
(MO)

10

RF

1.8
1.8
1.8

GLUTETHIMIDE
IIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3

range finder; nopoints between 0 50%

ppt in 2X C1SLS-A5-N040401A

YES

ppt in 2X C1-C3

SLS-B4-N040513C

YES

ppt in 2X C1-C3

SLS-B5-N040514B

YES

ppt in 1X C1 and 2X C1SLS-B6-N040716A

ECBC

I-83

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7

10

RF

2.15

YES

range finder

SLS-P25
SLS-P40

2.15

YES

SLS-P43

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

AA61FE-B3

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6DF

AA61FE

210

0.966

1.015

6.51%

3

4

0.9724

FAL.NHK.KY.A1.24.09.04

RF

AA61KY

200

0.922

0.492

0.10%

1

1

0.0402

FAL.NHK.KY.B1.01.10.04

DF

AA61KY

222

1.021

1.023

10.48%

5

3

0.8909FAL.NHK.KY.B2.07.10.04

DF

AA61KY

147

0.674

0.668

1.24%

6

2

0.9631

FAL.NHK.KY.B3.05.11.04

DF

AA61KY

195

0.899

0.502

0.78%

3

5

0.9246

FAL.NHK.KY.B4.10.11.04

DF

AA61KY

167

0.771

1.009

9.60%

3

3

0.9317RF

AA61JF

NA

NA

0.446

6.43%

0

2

NA

Exposure
Concentrations(ug/mL)7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7

Dilution

Acceptable

Factor8

Tests9

2.15

YES

Rationale for
Unacceptability

Notes

PC ID

SLS-P45

FRAME
1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
1000, 680, 463, 315, 214,
146, 99.1, 67.4
1000, 680, 463, 315, 214,
146, 99.1, 67.4
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

10RFrange finder

FAL.NHK.SLS.24.09.03

1.47

NO

PC failed

FAL.NHK.SLS.01.10.04

1.47

YES

FAL.NHK.SLS.07.10.03

2.15

YES

FAL.NHK.SLS.05.11.04

2.15

YES

FAL.NHK.SLS.10.11.04

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RF

0.9818

100000, 71429, 51020,
36443, 26031, 18593,
13281, 9486

1.4

YES

SLS-B12-N041022B

1.4

YES

130 ul of 2X doses wereapplied. Final conc. values
adjusted in data sheets by
SD; data from wells G3-G10 SLS-B113-N041029B
removed from EXCEL and
PRISM analyses (by SD)
since they were not dosed

1.4

YES

SLS-B14-N041030A

10RFrange finder

10

RFrange finder

2.15

YES

SLS-P8

1.47

YES

SLS-P14

1.47

YES

SLS-P16

GLYCEROL

IIVS
A1
B1

DF

AA61JF

27500

298.392

0.509

14.14%

3

3

range finder; nopoints between 0 50%

SLS-A1-N040317B

B2

DF

AA61JF

34200

371.354

0.519

9.50%

3

5

0.9761101960, 72829, 52020,
37157, 26541, 18958,
13541, 9672

B3

DF

AA61JF

25400

275.923

0.627

0.03%

3

4

0.9671

100000, 71429, 51020,
36443, 26031, 18593,
13281, 9486

AA61HG-A1

RF

AA61HG

NA

NA

0.612

4.48%

0

7

NA

AA61HG-A2

RF

AA61HG

15600

168.961

0.497

3.56%

1

1

0.8792

AA61HG-B1

DF

AA61HG

51200

555.693

1.001

1.36%

1

3

0.9717

AA61HG-B2

DF

AA61HG

30500

330.969

0.880

0.09%

3

5

0.9505

AA61HG-B3

DF

AA61HG

21100

229.503

0.481

14.05%

5

2

0.9533

FAL.NHK.RA.A1.11/02/04

RF

AA61RA

NA

NA

0.662

0.55%

0

0

NA

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RFrange finder

FAL.NHK.RA.A2.18.02.04

DF

AA61RA

57300

621.996

0.180

11.45%

1

3

0.2547

100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742

1.47

NO

PC failed

FAL.NHK.RA.B1.26.02.04

DF

AA61RA

21800

237.0210.205

15.32%

2

1

0.93892.15

YES

FAL.NHK.SLS/NB.26.02.03

FAL.NHK.RA.B2.18.03.04

DF

AA61RA

8470

92.000

0.438

7.92%

4

4

0.96292.15

YES

FAL.NHK.SLS.18.03.03

FAL.NHK.RA.B3.19.03.04

DF

AA61RA

23800

258.100

0.407

10.70%

2

4

0.94252.15

YES

FAL.NHK.SLS.19.03.03

ECBC
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100000, 10000, 1000,
100, 10, 1, 0.1, 0.01
100000, 46512, 21633,
10062, 4680, 2177, 1012,
471
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742
100000, 68027, 46277,
31481, 21416, 14568,
9911, 6742

no toxicity detected

SLS-P1SLS-P3

FRAME

I-84

100000, 46512, 21633,
10062, 4680, 2177, 1012,
471
100000, 46512, 21633,
10062, 4680, 2177, 1012,
471
100000, 46512, 21633,
10062, 4680, 2177, 1012,
471

FAL.NHK.SLS.11.02.04
this is a definitive test sinceconc. series is different from FAL.NHK.SLS.18.02.04
A1 range finder

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC

2.86

0.008

0.589

OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC ID

HALOPERIDOL
IIVS
A1

RF

AA61LW

2.46%

2

5

0.9764100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001

10

RF

2.2

YES

SLS-B4-N040513C

range finder

SLS-A4-N040331N

B1

DF

AA61LW

4.51

0.012

0.585

0.93%

2

5

0.971550.0, 22.7, 10.3, 4.70,
2.13, 0.970, 0.441, 0.200

B2

DF

AA61LW

3.11

0.008

0.576

4.43%

3

4

0.9736

50.0, 22.7, 10.3, 4.70,
2.13, 0.970, 0.441, 0.200

2.2

YES

SLS-B5-N040514B

B3

DF

AA61LW

2.24

0.006

0.764

4.42%

3

4

0.9571

50.0, 22.7, 10.3, 4.70,
2.13, 0.970, 0.441, 0.200

2.2

YES

SLS-B6-N040716A

AA61JC-A1

RF

AA61JC

4.88

0.013

0.947

6.60%

2

6

0.938310RF

AA61JC-B1

DF

AA61JC

2.70

0.007

0.700

2.99%

4

3

0.9630ECBC100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
80.0, 37.2, 17.3, 8.05,
3.74, 1.74, 0.81, 0.38

range finder

ppt in 1X C1SLS-P19

2.15

YES

SLS-P41

2.15

YES

SLS-P42SLS-P44

AA61JC-B2

DF

AA61JC

3.66

0.010

0.687

7.99%

4

3

0.9516

40.0, 18.6, 8.65, 4.03,
1.87, 0.871, 0.405, 0.188

AA61JC-B3

DF

AA61JC

4.72

0.013

1.060

1.49%

4

4

0.9411

40.0, 18.6, 8.65, 4.03,
1.87, 0.871, 0.405, 0.188

2.15

YES

RF

AA61PM

0.329

0.001

0.803

11.63%

3

30.8526100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001

10

RF

3.15

YES

FAL.NHK.SLS.08.10.03

FRAME
FAL.NHK.PM.A1.11.08.04

range finder

ppt in 1X C1-C2

FAL.NHK.SLS.11.08.04

FAL.NHK.PM.B1.08.10.04

DF

AA61PM

4.52

0.012

0.680

14.55%

2

4

0.9665100, 31.8, 10.1, 3.2, 1.02,
0.322, 0.102, 0.0325

FAL.NHK.PM.B2.22.10.04

DF

AA61PM

4.99

0.013

0.743

2.20%

2

5

0.9658100, 31.8, 10.1, 3.2, 1.02,
0.322, 0.102, 0.0325

3.15

YES

FAL.NHK.SLS.22.10.04(MO)

FAL.NHK.PM.B3.29.10.04

DF

AA61PM

1.64

0.004

0.629

7.30%

5

3

0.9621

100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01,0.47

2.15

YES

FAL.NHK.SLS.29.10.04

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10

RF

2.2

YES

SLS-B1-N040423A

2.2

YES

SLS-B2-N040424A

2.2

YES

SLS-B3-N040506A

10

RF

1.47

YES

HEXACHLOROPHENE
IIVS

A1

RF

AA61JN

0.025

0.00006

0.509

3.75%

2

3

0.9760B1DFAA61JN

0.02230.00005

0.609

3.49%

3

3

0.9868B2DF

AA61JN

0.01860.00005

0.611

0.44%

4

1

0.9891B3DF

AA61JN

0.02270.00006

0.520

1.39%

3

2

0.9885

AA61ND-A1

RF

AA61ND

NA

NA

0.421

16.43%

3

0NA

AA61ND-B1

DF

AA61ND

0.02940.00007

0.684

6.18%

5

3

0.95900.500, 0.227, 0.103,
0.047, 0.021, 0.010,
0.004, 0.002
0.500, 0.227, 0.103,
0.047, 0.021, 0.010,
0.004, 0.002
0.500, 0.227, 0.103,
0.047, 0.021, 0.010,
0.004, 0.002

range finder

Due to high ppt in 2X C1C2and 1X C1-C2; SDremoved these two doses
from Hill function analyses
and set the bottom to 0

SLS-A1-N040317B

ECBC

I-85

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
0.200, 0.136, 0.093,
0.063, 0.043, 0.029,
0.020, 0.013

range finder

ppt in 2X C1-C2 and 1X C1SLS-P4
C2
SLS-P21

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

AA61ND-B2

DF

AA61ND

0.03010.00007

0.891

1.12%

5

3

0.9862

AA61ND-B3

DF

AA61ND

0.02210.00005

0.586

1.63%

2

6

0.9707

FAL.NHK.HB.A2.26.02.03

RF

AA61HB

NA

NA

0.249

7.29%

NA

NA

0.0000FAL.NHK.HB.B1.18.03.04

DF

AA61HB

NA

NA

0.654

5.98%

0

0

-1.2210

FAL.NHK.HB.B2.19.03.04

DF

AA61HB

NA

NA

0.523

6.30%

0

0

-1.2210

FAL.NHK.HB.B2.25.03.04(should be B3)DF

AA61HB

NA

NA

0.544

7.76%

0

0

0.1438

Exposure
Concentrations(ug/mL)7
0.200, 0.136, 0.093,
0.063, 0.043, 0.029,
0.020, 0.013
0.200, 0.136, 0.093,
0.063, 0.043, 0.029,
0.020, 0.013

Dilution

Acceptable

Factor8

Tests9

1.47

YES

SLS-P23

1.47

YES

SLS-P25

NA

RF

3.15

NO

3.15

NO

2.15

NO

Data not analysed; chem.
reacts w/ NR & gives false +curve is going in the
results in columns C1-C4;
FAL.NHK.SLS.25.03.03
wrong direction
cells in first 3-4 col. incorp.
large amount of dye

2.15

NO

Data not analysed; chem.
seems to react w/ NR &curve is going in the gives false + results in col.
FAL.NHK.SLS.26.03.04
wrong direction
C1-C4; cells in first 3-4 col.
Incorp. large amount of dye;
ppt in 1X C1-C2

2.15

YES

FAL.NHK.SLS.25.04.04

2.15

YES

FAL.NHK.SLS.28.04.03

Rationale for
Unacceptability

Notes

PC ID

FRAME

FAL.NHK.HB.B3.26.03.04(should be B4)DF

AA61HB

NA

NA

0.652

15.30%

00-1.2210

FAL.NHK.HB.B4.25.04.04(should be B5)DF

AA61HB

0.05210.00013

0.850

3.86%

4

2

0.9900FAL.NHK.HB.B5.28.04.04(should be B6)DF

AA61HB

0.06190.00015

0.928

2.72%

4

1

0.9862NA

0.010, 0.003, 0.001,
0.00032, 0.00010,
0.0000322, 0.0000102,
0.0000032
0.010, 0.003, 0.001,
0.00032, 0.00010,
0.0000322, 0.0000102,
0.0000032

100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

1.0, 0.465, 0.216, 0.101,
0.046, 0.022, 0.010,
0.005
1.00, 0.465, 0.216, 0.101,
0.047, 0.022, 0.010,
0.005

range finder

SD says ppt binds or reacts
with NR;gives "nonsense"
FAL.NHK.SLS/MO.26.0
data; tox. curve goes wrong 2.03
direction; ppt in 1X C1-C3

SD notes incorrect range
no points between 0- used; considers 100 ug/ml
100%
as start conc. w/ dil. factor
2.15
SD notes incorrect range
no points between 0- used; considers 100 ug/ml
100%
as start conc. w/ dil. factor
2.15

FAL.NHK.SLS.18.03.03

FAL.NHK.SLS.19.03.03

1.00, 0.465, 0.216, 0.101,
0.047, 0.022, 0.010,
0.005

2.15

NO

odd plate; looks as if theno points between
dilutions ran left to right for
50-100%; SD rejects
FAL.NHK.SLS.13.05.04
top three wells & right to left
test
for bottom three.

0.97991.0, 0.47, 0.22, 0.10,
0.05, 0.022, 0.010,
0.00472.15

YES

FAL.NHK.SLS.10.06.04

0.9351

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

1.8

YES

SLS-B1-N040423A

FAL.NHK.HB.13.05.04 (shouldbe B7)DF

AA61HB

NA

NA

0.603

2.36%

4

1

NA

FAL.NHK.HB.B7.10.06.04(should be B8)DF

AA61HB

0.02330.00006

0.922

1.93%

5

3

RF

AA61FW

1360

15.114

0.573

1.92%

1

1

LACTIC ACID
IIVS
A1

range finder

SLS-A2-N040320B

B1

DF

AA61FW

1260

13.976

0.552

3.33%

4

2

0.9915

10000, 5556, 3086, 1715,
953, 529, 294, 163

B2

DF

AA61FW

1210

13.3770.561

10.36%

2

2

0.9868

10000, 5556, 3086, 1715,
953, 529, 294, 163

1.8

YES

SLS-B2-N040424A

B3

DF

AA61FW1470

16.344

0.458

4.02%

4

2

0.9836

5000, 3333, 2222, 1481,
988, 658, 439, 293

1.5

YES

SLS-B3-N040506A

RF

AA61NL1060

11.786

0.411

3.08%

1

1

0.8632

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

ECBC
AA61NL-A1

I-86

range finder

SLS-P6

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC ID

AA61NL-B1

DF

AA61NL

1330

14.770

0.999

0.10%

3

4

0.9731

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

YES

SLS-P26

AA61NL-B2

DF

AA61NL

1310

14.418

0.909

0.66%

3

3

0.9901

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

YES

SLS-P28

AA61NL-B3

DF

AA61NL

1230

13.658

0.824

3.46%

3

5

0.9532

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

YES

SLS-P30

RF

AA61JT

1880

20.863

0.777

7.41%

1

1

0.7636

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

2.15

YES

FAL.NHK.SLS.28.04.03

FRAME
FAL.NHK.JT.A1.25.04.04

range finder

FAL.NHK.SLS.25.04.04

FAL.NHK.JT.B1.28.04.04

DF

AA61JT1350

15.010

0.904

0.04%

3

5

0.9767

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

FAL.NHK.JT.B2.13.05.04

DF

AA61JT

1360

15.079

0.597

1.07%

3

4

0.9702

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

YES

FAL.NHK.SLS.13.05.04

FAL.NHK.JT.B3.10.06.04

DF

AA61JT

1250

13.879

0.670

6.11%

3

1

0.9322

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

YES

FAL.NHK.SLS.10.06.04

RF

AA61PJ

46.8

0.161

0.634

0.78%

1

1

0.7927100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001

10

RF

LINDANE
IIVS
A1

B1

DF

AA61PJ

15.7

0.054

0.547

10.52%

5

2

0.9540200, 111, 61.7, 34.3, 19.1,
10.6, 5.88, 3.27

1.8

YES

B2

DF

AA61PJ

18.0

0.062

0.582

6.00%

4

2

0.9704200, 111, 61.7, 34.3, 19.1,
10.6, 5.88, 3.27

1.8

YES

B3

DF

AA61PJ

13.2

0.045

0.532

6.43%

2

3

0.9626200, 111, 61.7, 34.3, 19.1,
10.6, 5.88, 3.27

1.8

YES

AA61FK-A1

RF

AA61FK

40.6

0.140

0.821

9.29%

2

2

0.8809AA61FK-B1

DF

AA61FK

21.4

0.074

0.550

6.75%

5

2

0.9657

AA61FK-B2

DF

AA61FK

15.5

0.053

0.558

2.09%

5

2

0.8770AA61FK-B3

DF

AA61FK

20.3

0.070

0.619

6.30%

4

4

0.9653

FAL.NHK.KN.A1.14.05.04

RF

AA61KN

61.7

0.212

0.694

7.78%

2

1

0.8847

FAL.NHK.KN.B1.20.08.04

DF

AA61KN

30.8

0.106

0.752

5.39%

6

2

0.9626

FAL.NHK.KN.B2.29.10.04

DF

AA61KN

16.8

0.058

0.450

9.76%

7

1

0.9529FAL.NHK.KN.B3.05.11.04

DF

AA61KN

21.9

0.075

0.453

7.72%

6

2

0.9894

range finder

ppt in 2x C1 and 1X C1
ppt in 1X C1-C3 & 2X C1C3; SD removed top 3
doses from Hill analyses;
ppts and flattening of
response curve wereobserved
ppt in 1X C1-C3 & 2X C1C2; SD removed top 3
doses from Hill analyses;
ppts and flattening of
response curve wereobserved
ppt in 1X C1-C4 & 2X C1C3; SD removed top 3
doses from Hill analyses;
ppts and flattening of
response curve wereobserved

SLS-A3-N040331A

SLS-B8-N040819A

SLS-B9-N040820A

SLS-B10-N040903A

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7

10

RF

2.15

YES

1.47

YES

1.47

YES

range finder

ppt in 2X C1-C2; ppt in 1X C1-C2
SLS-P15
ppt in 2X C1-C3; ppt in 1X
C1-C4;
ppt in 2X C1-C3; ppt in 1X
C1-C4
ppt in 2X C1-C3; ppt in 1X
C1-C4

SLS-P49
SLS-P53
SLS-P55

FRAME

I-87

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 930, 433, 201,93.6, 43.5, 20.3, 9.42
2000, 930, 433, 201,93.6, 43.5, 20.3, 9.42
2000, 930, 433, 201,93.6, 43.5, 20.3, 9.42

10

RF

2.15

YES

2.15

YES

2.15

YES

range finder

ppt in 1X C1
ppt in 2X C1-C3; ppt in 1X
C1-C6
ppt in 2X C1-C4; ppt in 1X
C1-C5
ppt in 1X C1-C5

FAL.NHK.SLS.14.05.03
FAL.NHK.SLS.20.08.04
FAL.NHK.SLS.29.10.04
FAL.NHK.SLS.05.11.04

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC ID

LITHIUM I CARBONATE
IIVS
A2

RF

AA61RN839

11.355

0.736

1.65%

1

0

0.9100B1DF

AA61RN

524

7.092

0.364

1.54%

3

2

0.9453B2

DF

AA61RN

519

7.024

0.26

7.33%

3

2

0.9436B3DF

AA61RN

571

7.728

0.315

8.55%

3

2

0.958

AA61RR-A1

RF

AA61RR767

10.380

0.750

3.35%

1

1

0.8957

AA61RR-B1

DF

AA61RR

308

4.168

0.361

2.25%

6

2

0.9095

AA61RR-B2

DF

AA61RR

541

7.322

1.107

4.03%

4

4

0.9425

AA61RR-B3

DF

AA61RR

384

5.197

0.803

0.21%

5

3

0.9639FAL.NHK.RM.A1.010803

RF

AA61RM

78.5

1.062

0.568

13.97%

250.7509FAL.NHK.RM.B1.080803

DF

AA61RM

378

5.116

0.794

1.03%

2

6

0.8188

FAL.NHK.RM.B2.15.08.03

DF

AA61RM

518

7.010

0.433

6.00%

1

4

0.8092

FAL.NHK.RM.B3.23.08.03

DF

AA61RM

478

6.469

0.614

1.71%

2

4

0.8168

FAL.NHK.RM.B4.05.09.03

DF

AA61RM

303

4.101

0.095

9.10%

2

2

0.5447

FAL.NHK.RM.B5.01.10.03

DF

AA61RM887

12.004

1.302

0.06%

1

3

0.8807

FAL.NHK.RM.B5.15.10.03(should be B6?)DF

AA61RM

471

6.374

0.529

0.71%

2

6

0.2797

FAL.NHK.RM.28.11.03

DF

AA61RM

561

7.592

0.153

3.93%

1

5

0.7316

A1

RF

AA61LS

507

2.322

0.431

13.02%

1

2

0.8210B1

DF

AA61LS

631

2.890

0.650

3.10%

3

4

0.9748B2DF

AA61LS

705

3.228

0.691

2.97%

3

4

0.9666

B3

DF

AA61LS

537

2.460

0.649

2.00%

3

3

0.9670AA61RJ-A1

RF

AA61RJ

324

1.49

0.677

2.99%

1

5

0.9463

AA61RJ-B1

DF

AA61RJ

746

3.419

1.112

0.28%

3

4

0.9663

AA61RJ-B2

DF

AA61RJ

883

4.045

1.180

2.65%

2

6

0.9767

AA61RJ-B3

DF

AA61RJ

653

2.992

0.784

1.54%

3

5

0.9321

RF

AA61HV

982

4.497

1.600

0.24%

1

4

0.80901000, 100, 10, 1,
0.1,0.01, 0.001, 0.0001
2000, 1333, 889, 593,
395, 263, 176, 117
2000, 1333, 889, 593,
395, 263, 176, 117
2000, 1333, 889, 593,
395, 263, 176, 117

10

RF

1.5

YES

range finder

SLS-A2
SLS-B1

1.5

YES

SLS-B2

1.5

YES

SLS-B3ECBC
1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
2000, 1361, 926, 630,
428, 291, 198, 135
2000, 1361, 926, 630,
428, 291, 198, 135
2000, 1361, 926, 630,
428, 291, 198, 135

10

RF

1.47

YES

range finder

range finder

SLS-P2SLS-P5

1.47

YES

SLS-P7

1.47

YES

SLS-P9FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7

10

RF

2.15

YES

range finder

FAL.NHK.SLS.010803

2.15

YES

2.15

NO

PC failed

FAL.NHK.SLS.230803

2.15

NO

low r2

FAL.NHK.SLS.050903

2.15

NO

PC failed

FAL.NHK.SLS.01.10.03

2.15

NO

low r2

2.15

YEShigh background

FAL.NHK.SLS.08.08.03
FAL.NHK.SLS.15.08.03

FAL.NHK.SLS.15.10.03
challenging chemical; SMT
accepts this test

FAL.NHK.SLS.28.11.03

MEPROBAMATE
IIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 1250, 781, 488,
305, 191, 119, 74.5
2000, 1250, 781, 488,
305, 191, 119, 74.5
2000, 1250, 781, 488,
305, 191, 119, 74.5

10

RF

1.6

YES

range finder

SLS-A1-N040317B
SLS-B8-N040819A

1.6

YES

SLS-B9-N040820A

1.6

YES

SLS-B10-N040903A

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 1361, 926, 630,
428, 291, 198, 135
2000, 1361, 926, 630,
428, 291, 198, 135
2000, 1361, 926, 630,
428, 291, 198, 135

10

RF

1.47

YES

range finder

SLS-P2SLS-P8

1.47

YES

SLS-P10

1.47

YES

SLS-P11

10

RF

FRAME
FAL.NHK.HV.A1.11/02/04

I-88

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

range finder

C8 outlier removed by SD

FAL.NHK.SLS.11.02.04

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

FAL.NHK.HV.A2.18/02/04

DF

AA61HV4980

22.801

1.600

0.24%

1

4

0.4736

FAL.NHK.HV.B1.26/02/04

DF

AA61HV

30.8

0.141

0.254

10.02%

6

2

0.9661

FAL.NHK.HV.B2.18/03/04

DF

AA61HV

77.8

0.356

0.378

0.13%

4

4

0.9274

FAL.NHK.HV.B3.25.03.04

DF

AA61HV

379

1.738

0.803

0.65%

2

5

0.7687

3.25

0.012

0.485

7.23%

3

0

0.9831

Exposure
Concentrations(ug/mL)7

5000, 2326, 1082, 503,
234, 109, 50.6, 23.5
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

Dilution

Acceptable

Factor8

Tests9

2.15

NO

2.15

YES

FAL.NHK.SLS/NB.26.02
.03

2.15

YES

FAL.NHK.SLS.18.03.03

2.15

YES

FAL.NHK.SLS.25.03.03

10

RF

1.6

NO

1.6

NO

1.33

NO

1.1

YES

SLS-B6-N040716A

1.1

YES

SLS-B7-N040717B

1.1

YES

SLS-B8-N040819A

Rationale for
Unacceptability

PC failed

Notes

PC ID

this is a definitive test sinceconc.series is different from FAL.NHK.SLS.18.02.04
A1 range finder

MERCURY II CHLORIDE
IIVS
A1

RF

AA61MX

B1

DF

AA61MX

4.54

0.017

0.632

0.90%

4

0

0.9852B2DF

AA61MX

5.17

0.019

0.568

4.76%

0

2

0.9915B3DF

AA61MX

5.10

0.019

0.495

6.71%

0

1

0.9819B4DF

AA61MX

5.26

0.019

0.785

2.29%

2

3

0.9359B5DF

AA61MX

5.44

0.020

0.715

4.31%

1

3

0.9529B6DF

AA61MX

5.35

0.020

0.612

0.00%

2

2

0.9585

AA61KP-A1

RF

AA61KP

2.24

0.008

0.432

8.13%

3

1

0.9582

AA61KP-B1

DF

AA61KP

6.95

0.026

1.076

3.04%

1

1

0.9276

AA61KP-B2

DF

AA61KP

7.87

0.029

1.169

3.40%

2

6

0.9666

AA61KP-B3

DF

AA61KP

5.79

0.021

0.831

1.85%

2

5

0.9856

FAL.NHK.HA.A1.11/02/04

RF

AA61HA

3.56

0.013

1.321

3.96%

3

0

0.9647

FAL.NHK.HA.B1.18.03.04

DF

AA61HA

4.66

0.017

0.486

2.93%

2

3

0.9663

FAL.NHK.HA.B2.19.03.04

DF

AA61HA

4.98

0.018

0.533

9.73%

2

6

0.9174

FAL.NHK.HA.B2.25.03.04(should be B3)DF

AA61HA

6.56

0.024

0.533

4.35%

2

6

0.8230A1

RF

AA61FZ

601

18.763

0.567

1.73%

1

1

0.9073B1DF

AA61FZ

2160

67.345

0.597

1.70%

1

7

0.8425B2DF

AA61FZ1850

57.851

0.546

2.01%

1

4

0.9223B3DF

AA61FZ

2290

71.336

0.790

3.64%

1

3

0.9218B4DF

AA61FZ

NA

NA

0.707

6.86%

0

3

0.90301000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
20.0, 12.5, 7.81, 4.88,
3.05, 1.91, 1.19, 0.745
20.0, 12.5, 7.81, 4.88,
3.05, 1.91, 1.19, 0.745
20.0, 15.0, 11.3, 8.50,
6.39, 4.81, 3.61, 2.72
8.00, 7.27, 6.61, 6.01,
4.46, 4.97, 4.52, 4.11
8.00, 7.27, 6.61, 6.01,
4.46, 4.97, 4.52, 4.11
8.00, 7.27, 6.61, 6.01,
4.46, 4.97, 4.52, 4.11

range finder; nopoints between 50 - ppt in 1X C1
100%
no points between
50 - 100%no points between 0 50%
no points between 0 50%

SLS-A1-N040317B
SLS-B1-N040423A
SLS-B2-N040424A
SLS-B3-N040506A

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 8.26, 6.83, 5.65,
4.67, 3.86, 3.19, 2.63
10.0, 8.26, 6.83, 5.65,
4.67, 3.86, 3.19, 2.63

10

RF

1.47

YES

range finder

ppt in 1X C1SLS-P2SLS-P8

1.21

YES

SLS-P10

1.21

YES

SLS-P11

10

RF

1.47

YES

FAL.NHK.SLS.18.03.03

1.47

YES

FAL.NHK.SLS.19.03.03

1.47

YES

FAL.NHK.SLS.25.03.03

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

range finder

ppt in 1X C1FAL.NHK.SLS.11.02.04

METHANOL
IIVS

ECBC

I-89

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 1250, 781, 488,
305, 191, 119, 74.5
2000, 1250, 781, 488,
305, 191, 119, 74.5
2500, 1563, 977, 610,
381, 238, 149, 93.1
2500, 1563, 977, 610,
381, 238, 149, 93.1

10

RF

1.6

YES

range finder

SLS-A5-N040401A
SLS-B4-N040513C

1.6

YES

SLS-B5-N040514B

1.6

YES

1.6

NO

SLS-B6-N040716A
no points between 0 50%

SLS-B7-N040717B

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9

AA61MJ-A1

RF

AA61MJ

NA

NA

0.909

0.96%

0

8

NA

2500, 250, 25, 2.5, 0.25,
0.025, 0.0025, 0.00025

10

RF

AA61MJ-B1

DF

AA61MJ

NA

NA

0.606

0.30%

0

4

NA

3500, 2381, 1620, 1102,
750, 510, 347, 236

1.47

NO

1.21

NO

1.21

NO

AA61MJ-B2

DF

AA61MJ

NA

NA

0.759

0.65%

0

8

NA

AA61MJ-B3

DF

AA61MJ

NA

NA

0.831

3.88%

0

8

NA

FAL.NHK.RG.A1.24.09.04

RF

AA61RG

635

19.829

0.632

0.55%

1

3

0.6562

FAL.NHK.RG.B1.01.10.04

DF

AA61RG

8610

268.725

1.078

6.69%

0

8

0.4209FAL.NHK.RG.B2.07.10.04

DF

AA61RG

1360

42.297

0.649

3.62%

1

7

0.9324

FAL.NHK.RG.B3.22.10.04

DF

AA61RG

2170

67.812

0.809

0.56%

0

8

0.9463

FAL.NHK.RG.B4.28.10.04

DF

AA61RG

1100

34.301

0.625

8.71%

2

1

0.9422

FAL.NHK.RG.B5.05.11.04

DF

AA61RG

938

29.262

0.467

6.43%

2

6

0.5431A1

RF

AA61HL

143

0.881

0.498

34.80%

1

1

0.9606B1

DF

AA61HL

127

0.785

0.572

1.82%

4

4

0.9551B2DF

AA61HL

128

0.791

0.552

4.42%

4

4

0.9558B3DF

AA61HL

79.6

0.491

0.736

1.75%

5

3

0.9593

AA61NA-A1

RF

AA61NA

225

1.390

0.541

27.12%

1

2

0.8258

AA61NA-B1(sealer)DF

AA61NA

69.7

0.429

0.718

4.19%

5

2

0.8884

AA61NA-B2 (sealer)DF

AA61NA

94.2

0.581

0.680

5.37%

5

3

0.9635

AA61NA-B3 (sealer)DF

AA61NA

119

0.734

0.871

4.38%

5

3

0.9418

FAL.NHK.KL.A1.11.08.04

RF

AA61KL

277

1.706

0.455

16.01%

1

1

0.5525

FAL.NHK.KL.B1.17.09.04

DF

AA61KL

553

3.412

0.487

26.34%

2

5

0.94503500, 2893, 2391, 1976,
1633, 1349, 1115, 922
3500, 2893, 2391, 1976,
1633, 1349, 1115, 922

Rationale for
Unacceptability

Notes

range finder; nopoints between 0 50%

PC ID

SLS-P19

0.02% DMSO in dosing
no points between 0 solutions; highest stock50%
conc. is 700,087 ug/ml
no points between 0 no toxicity
50%
no points between 0 slight toxicity
50%

SLS-P48
SLS-P60
SLS-P61

FRAME
1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
2000, 930, 433, 201,93.6, 43.5, 20.3, 9.42
2000, 930, 433, 201,93.6, 43.5, 20.3, 9.42
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

10RF

range finder

FAL.NHK.SLS.24.09.03

2.15

NO

PC failed; no points
between 50-100%

FAL.NHK.SLS.01.10.04

2.15

YES

2.15

NO

2.15

YES

FAL.NHK.SLS.28.10.04

2.15

YES

FAL.NHK.SLS.05.11.04

10

RF

2.2

YES

2.2

YES

SLS-B5-N040514B

2.2

YES

SLS-B6-N040716A

10

RF

2.15

YES

SLS-P40

2.15

YES

SLS-P42

2.15

YES

SLS-P44

10

RF

2.15

NO

FAL.NHK.SLS.07.10.03
no points between 050%

FAL.NHK.SLS.22.10.04(NB)

NICOTINE
IIVS
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
2000, 909, 413, 188,
85.4, 38.8, 17.6, 8.02
2000, 909, 413, 188,
85.4, 38.8, 17.6, 8.02
2000, 909, 413, 188,
85.4, 38.8, 17.6, 8.02

range finder; % VC
difference > 15

volatility problem; VC1 OD
values much lower than
VC2; VC1 removed from
subsequent analysis by SD

SLS-A4-N040331N

outlier in C6 removed by
SD: used plate sealer

SLS-B4-N040513C

ECBC
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
5000, 2326, 1082, 503,
234, 109, 51, 24
2000, 930, 433, 201,93.6, 43.5, 20.3, 9.42
2000, 930, 433, 201,93.6, 43.5, 20.3, 9.42

range finder; % VC
difference > 15

volatility problem

SLS-P38FRAME

FAL.NHK.KL.B2.30.09.04

DF

AA61KL

80

0.493

0.478

10.61%

2

2

0.4411

FAL.NHK.KL.B3.08.10.04

DF

AA61KL

193

1.191

0.552

19.76%

2

5

0.8957

FAL.NHK.KL.B4 .22.10.04

DF

AA61KL

91

0.561

0.730

2.67%

6

2

0.8631

FAL.NHK.KL.B5.29.10.04

DF

AA61KL

118

0.726

0.455

17.69%

5

3

0.9316

FAL.NHK.KL.B6.05.11.04

DF

AA61KL

224

1.380

0.376

14.23%

3

5

0.8894

I-90

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
5000, 2326, 1082, 503,
234, 109, 50.6, 23.6
5000, 2326, 1082, 503,
234, 109, 50.6, 23.6
5000, 2326, 1082, 503,
234, 109, 50.6, 23.6
5000, 2326, 1082, 503,
234, 109, 50.6, 23.6
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

range finder; % VC
difference > 15
% VC difference >15

FAL.NHK.SLS.11.08.04
outlier removed by SD;
possible volatility problem

FAL.NHK.SLS.17.09.04

2.15

NO

SD rejects curve"roller coaster" curve; somelow concentrations give high FAL.NHK.SLS.30.09.03
toxicity; SD rejects test

2.15

NO

% VC difference >15

volatility issue

2.15

YES

2.15

NO

2.15

YES

FAL.NHK.SLS.08.10.03
FAL.NHK.SLS.22.10.04(NB)% VC difference >15

FAL.NHK.SLS.29.10.04
FAL.NHK.SLS.05.11.04

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

FAL.NHK.KL.B7.12.11.04

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC ID

DF

AA61KL

85.7

0.528

0.727

2.28%

5

3

0.92492500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

2.15

YES

RF

AA61GD

84.5

0.329

0.578

2.76%

3

0

0.9874

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

1.8

YES

SLS-B1-N040423A

1.8

YES

SLS-B2-N040424A

1.8

YES

SLS-B3-N040506A

10

RF

2.15

YES

SLS-P20

2.15

YES

SLS-P22

2.15

YES

SLS-P24

FAL.NHK.SLS.12.11.04

PARAQUAT

IIVS
A1
B1

DF

AA61GD

50.4

0.196

0.564

3.71%

6

2

0.9776B2

DF

AA61GD

59.8

0.233

0.544

0.60%

5

3

0.9719B3DF

AA61GD

50.1

0.194

0.496

3.71%

6

2

0.9679

AA61MP-A1

RF

AA61MP

57.0

0.222

0.407

2.19%

2

2

0.9152

AA61MP-B1

DF

AA61MP

41.4

0.161

0.597

0.17%

5

3

0.9912

AA61MP-B2

DF

AA61MP

50.7

0.197

1.009

3.67%

4

4

0.9822

AA61MP-B3

DF

AA61MP

52.7

0.205

0.528

7.61%

5

3

0.9820FAL.NHK.HP.A1.26.03.04

RF

AA61HP

74.5

0.290

0.562

6.58%

2

1

0.9098

FAL.NHK.HP.B1.25.04.04

DF

AA61HP

57.9

0.225

0.795

3.51%

4

4

0.9828

FAL.NHK.HP.B2.28.04.04

DF

AA61HP

60.1

0.234

0.815

1.88%

8

0

0.9066

FAL.NHK.HP.B3.11.06.04

DF

AA61HP

28.1

0.109

0.790

4.43%

4

4

0.8649FAL.NHK.HP.B4.23.06.04

DF

AA61HP

103

0.399

0.811

17.53%

3

3

0.9562

FAL.NHK.HP.B5.25.06.04

DF

AA61HP

99.8

0.388

0.850

0.84%

3

2

0.9498

FAL.NHK.HP.B6.12.08.04

DF

AA61HP

55.7

0.217

0.880

2.31%

3

5

0.9207

FAL.NHK.HP-RB.B7.25.08.04

DF

AA61HP

132

0.515

0.635

4.72%

2

2

0.8927

A1

RF

AA61PS

95.7

0.329

0.684

5.51%

0

3

0.8685B1

DF

AA61PS

21.2

0.073

0.719

5.83%

6

2

0.9735B2

DF

AA61PS

37.8

0.130

0.656

1.73%

3

3

0.9754B3DF

AA61PS

28.1

0.097

0.752

0.68%

3

4

0.9677

AA61MD-A1

RF

AA61MD

16.0

0.055

0.846

0.38%

2

2

0.9789

AA61MD-B1

DF

AA61MD

25.8

0.088

0.995

5.19%

2

3

0.9372

AA61MD-B2

DF

AA61MD

45.2

0.155

1.228

1.72%

2

6

0.9633

1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3

range finder; nopoints between 50 100%

SLS-A2-N040320B

ECBC
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

range finder

SLS-P5

FRAME
100000, 1000, 100,10, 1,
0.1, 0.01, 0.001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 680, 463, 315, 214,
146, 99.1, 67.4
500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4500, 233, 108, 50.3, 23.4,
10.9, 5.1, 2.4

10

RF

2.15

YES

range finder

FAL.NHK.SLS.26.03.04

1.47

NO

no points between
50 - 100%

FAL.NHK.SLS.28.04.03

2.15

NO

PC failed

FAL.NHK.SLS.11.06.04

2.15

NO

% VC difference >15

FAL.NHK.SLS.23.06.04

2.15

YES

2.15

NO

2.15

YES

10

RF

2.2

YES

1.6

YES

1.6

YES

10

RF

2.15

YES

ppt in 2X C1-C2

SLS-P47

2.15

YES

chunks in1X C1; ppt in 2X
C1-C3

SLS-P51

FAL.NHK.SLS.25.04.04

FAL.NHK.SLS.25.06.04
PC failed

FAL.NHK.SLS.12.08.04
FAL.NHK.SLSRB.20.08.04

PARATHION
IIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 455, 207, 93.9,
42.7, 19.4, 8.82, 4.01
100, 62.5, 39.1, 24.4,
15.3, 9.54, 5.96, 3.73
100, 62.5, 39.1, 24.4,
15.3, 9.54, 5.96, 3.73

range finder; nopoints between 50 100%

SD didn't use data fromhighest dose in Hill
analyses due to the effectsof ppts; ppt in 2X C1-C2 &1X C1-C2
ppt in 2X C1-C6; ppt in 1X
C1-C4
ppt in 2X C1-C3; ppt in 1X
C1
ppt in 2X C1-C3; ppt in 1X
C1-C2

SLS-A3-N040331A

SLS-B12-N041022B
SLS-B113-N041029B
SLS-B14-N041030A

ECBC

I-91

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
200, 93.0, 43.3, 20.1, 9.4,
4.4, 2.0, 0.9

range finder

ppt in 2X C1-C2; ppt in 1X C1SLS-P39

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

AA61MD-B3

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6DFAA61MD

31.1

0.107

0.737

1.12%

3

5

0.9554

FAL.NHK.KE.A1.20.10 .04

DF

AA61KE

87.1

0.299

1.237

0.40%

2

6

0.9819FAL.NHK.KE.B1.29.10.04

DF

AA61KE

33.3

0.114

0.455

24.83%

6

2

0.9604

FAL.NHK.KE.B2.03.11.04

DF

AA61KE

18.9

0.065

0.606

8.86%

6

2

0.9440FAL.NHK.KE.B3.10.11.04

DF

AA61KE

NA

NA

1.144

4.04%

8

0

NA

FAL.NHK.KE.B4.12.11.04

DF

AA61KE

32.1

0.110

0.809

3.24%

6

2

0.9806

FAL.NHK.KE.B5.17.11.04

DF

AA61KE

42.7

0.146

0.855

10.63%

5

3

0.9385

Exposure
Concentrations(ug/mL)7
200, 93.0, 43.3, 20.1, 9.4,
4.4, 2.0, 0.9

Dilution

Acceptable

Factor8

Tests9

2.15

YES

Rationale for
Unacceptability

Notes

PC ID

ppt in 2X C1-C3; ppt in 1X
C1-C2

SLS-P53

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1500, 698, 325, 151,
70.2, 32.7, 15.2, 7.06
1500, 698, 325, 151,
70.2, 32.7, 15.2, 7.06
1500, 1020, 694, 472,
321, 219, 149, 101
1500, 698, 325, 151,
70.2, 32.7, 15.2, 7.06
1500, 698, 325, 151,
70.2, 32.7, 15.2, 7.06

10RFrange finder

ppt in 1X C1FAL.NHK.SLS.20.10.04

2.15

NO

%VC difference >15

ppt in 2X C1-C3; ppt in C1C5; volatility problem

FAL.NHK.SLS.29.10.04

2.15

YES

ppt in 1X C1-C4

FAL.NHK.SLS.03.11.04

1.47

NO

2.15

YES

2.15

YES

no points between
50 - 100%

ppt in 2X C1-C5; ppt in 1X
C1-C7
ppt in 2X C1-C4; ppt in 1X
C1-C3
ppt in 2X C1-C4

FAL.NHK.SLS.10.11.04
FAL.NHK.SLS.12.11.04
FAL.NHK.SLS.17.11.04

PHENOBARBITAL
IIVS
A1

RF

AA61FG

378

1.630

0.575

0.41%

1

1

0.9186B1

DF

AA61FG

458

1.973

0.629

3.11%

3

4

0.9782B2DF

AA61FG

362

1.560

0.655

0.89%

3

4

0.9861B3DF

AA61FG

322

1.387

0.623

0.79%

4

4

0.9867

AA61KV-A1

RF

AA61KV

436

1.875

0.953

0.85%

1

7

0.8831

AA61KV-B1

DF

AA61KV

569

2.450

0.593

0.65%

3

5

0.9763

AA61KV-B2

DF

AA61KV

899

3.873

0.114

1.69%

2

4

0.8199

AA61KV-B3

DF

AA61KV

611

2.631

0.831

1.41%

3

5

0.9887

FAL.NHK.NJ.A1.24.09.04

RF

AA61NJ

253

1.089

0.619

11.58%

1

1

0.7751

FAL.NHK.NJ.B1.08.10.04

DF

AA61NJ

361

1.553

0.654

3.81%

2

6

0.9642

FAL.NHK.NJ.B2.22.10.04

DF

AA61NJ

455

1.959

0.827

4.81%

3

4

0.9826

FAL.NHK.NJ.B3.28.10.04

DF

AA61NJ

264

1.135

0.683

11.67%

3

5

0.9342

A1

RF

AA61PG

34.4

0.366

0.617

98.64%

2

3

0.9801B1DF

AA61PG

79.3

0.842

0.522

2.09%

5

3

0.9749B2

DF

AA61PG

76.6

0.814

0.548

2.89%

3

3

0.9575B3DF

AA61PG

86.5

0.919

0.473

0.39%

4

3

0.9620RF

AA61FV

NA

NA

0.421

99.34%

1

1

NA

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 1111, 617, 343,
191, 106, 58.8, 32.7
2000, 1111, 617, 343,
191, 106, 58.8, 32.7
2000, 1111, 617, 343,
191, 106, 58.8, 32.7

10

RF

1.8

YES

range finder
ppt in 1X C1 and 2X C1SLS-A5-N040401A
SLS-B8-N040819A

1.8

YES

ppt in 2X C1SLS-B9-N040820A

1.8

YES

ppt in 2X C1SLS-B10-N040903A

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
3000, 1395, 649, 302,
140, 65, 30, 14
3000, 1395, 649, 302,
140, 65, 30, 14
3000, 1395, 649, 302,
140, 65, 30, 14

10

RF

2.15

YES

range finder
ppt in 2X C1SLS-P56
SLS-P57

2.15

YES

ppt in 2X C1SLS-P58

2.15

YES

ppt in 2X C1SLS-P59

10

RF

2.15

YES

FAL.NHK.SLS.08.10.03

2.15

YES

FAL.NHK.SLS.22.10.04
(NB)

2.15

YES

FAL.NHK.SLS.28.10.04

10

RF

2.2

YES

2.2

YES

ppt in 1X C1

2.2

YES

used plate sealer; ppt in 1X
SLS-B3-N040506A
C1-C2

10

RF

FRAME
1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
1500, 698, 3.25, 151,
70.2, 32.7, 15.2, 7.06
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

range finder

FAL.NHK.SLS.24.09.03

PHENOL
IIVS
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
2000, 909, 413, 188,
85.4, 38.8, 17.6, 8.02
2000, 909, 413, 188,
85.4, 38.8, 17.6, 8.02
2000, 909, 413, 188,
85.4, 38.8, 17.6, 8.02

range finder; % VC
difference > 15

volatility problem; VC1 OD
values much lower than
VC2; VC1 removed from
subsequent analysis by SD

SLS-A3-N040331A

SLS-B1-N040423A
SLS-B2-N040424A

ECBC
AA61FV-A1

I-92

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

range finder; % VC
difference > 15

volatility problem

SLS-P12In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

AA61FV-B1(sealer)DF

AA61FV

62.8

0.667

0.622

8.17%

4

3

0.9585

AA61FV-B2 (sealer)DF

AA61FV

78.5

0.834

0.668

7.31%

3

4

0.9576

AA61FV-B3 (sealer)DF

AA61FV

36.1

0.383

0.318

2.99%

5

3

0.9402

FAL.NHK.MS.A1.14.05.04

RF

AA61MS

91.0

0.967

0.279

98.26%

3

00.2986

FAL.NHK.MS.B1.12.08.04

DF

AA61MS

381

4.049

0.654

13.72%

1

2

0.8273

Exposure
Concentrations(ug/mL)7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.7

Dilution

Acceptable

Factor8

Tests92.15

YES

SLS-P32

2.15

YES

SLS-P34

2.15

YES

SLS-P3610RFrange finder; %VC
difference > 15; nopts between 50100%

FAL.NHK.SLS.14.05.03

2.15

NO

PC failed

FAL.NHK.SLS.12.08.04

2.15

NO

PC failed; % VC
difference > 15

FAL.NHK.SLSRB.19.08.04

2.15

YES

2.15

YES

Rationale for
Unacceptability

Notes

PC ID

FRAME

FAL.NHK.MS.B2.19.08.04
(RB)

DF

AA61MS

170

1.805

0.168

FAL.NHK.MS-NB.B3.25.08.04

DF

AA61MS

86.7

0.921

FAL.NHK.MS.B4.17.09.04

DF

AA61MS

94.6

1.005

FAL.NHK.MS.B5.30.09.04
FAL.NHK.MS.B6.07.10.04

DF
DF

AA61MS793

8.421

46.79%

3

1

0.4991

1.034

8.73%

4

3

0.98220.760

15.15%

3

4

0.9736

0.589

5.43%

1

0

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

0.8202

1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

2.15

NO

1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

2.15

YES

AA61MS

98.4

1.046

0.650

8.37%

4

3

0.9794

FAL.NHK.SLS.25.08.04

no points between
50 - 100%

outlier removed by SD;
potential volatility problem
SD removed data from C8
due to low OD; "roller
coaster" curve

FAL.NHK.SLS.17.09.04
FAL.NHK.SLS.30.09.03
FAL.NHK.SLS.07.10.03

PHENYLTHIOUREA
IIVS
A1

RF

AA61PV

467

3.066

0.775

1.12%

1

2

0.9466B1

DF

AA61PV

252

1.658

0.643

1.48%

5

3

0.9786B2

DF

AA61PV

352

2.321

0.623

0.41%

4

4

0.9605B3DF

AA61PV

213

1.401

0.654

4.04%

5

3

0.9788

AA61LN-A1

RF

AA61LN

294

1.930

0.995

4.15%

1

7

0.8497

AA61LN-B1

DF

AA61LN

362

2.380

0.577

2.20%

3

2

0.9609AA61LN-B2

DF

AA61LN

306

2.012

0.705

1.12%

3

5

0.9632

AA61LN-B3

DF

AA61LN

422

2.771

0.972

5.43%

3

5

0.9477

FAL.NHK.JB.A1.14.05.04

RF

AA61 JB

555

3.644

0.678

3.82%

1

7

0.9193

FAL.NHK.JB.B1.29.10.04

DF

AA61JB

335

2.201

0.575

8.89%

3

5

0.9804

FAL.NHK.JB.B2.03.11.04

DF

AA61JB

373

2.452

0.526

0.65%

3

5

0.9615

FAL.NHK.JB.B3.05.11.04

DF

AA61JB

495

3.255

0.371

11.87%

3

1

0.8795

A1

RF

AA61NF

136

0.494

0.555

4.16%

1

2

0.9514B1DF

AA61NF

146

0.531

0.647

3.80%

4

4

0.9767

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1389, 772, 429,
238, 132, 73.5, 40.8
2500, 1389, 772, 429,
238, 132, 73.5, 40.8
2500, 1389, 772, 429,
238, 132, 73.5, 40.8

10

RF

1.8

YES

range finder
ppt in 2X C1SLS-A3-N040331A
SLS-B8-N040819A

1.8

YES

ppt in 2X C1SLS-B9-N040820A

1.8

YES

ppt in 2X C1-C2

SLS-B10-N040903A

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 930, 433, 201,93.6, 43.5, 20.3, 9.42
2000, 930, 433, 201,93.6, 43.5, 20.3, 9.42
2000, 930, 433, 201,93.6, 43.5, 20.3, 9.42

10

RF

2.15

YES

2.15

YES

2.15

YES

range finder

SLS-P39
SLS-P41
SLS-P43
ppt in 2X C1SLS-P45

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

10

RF

2.15

YES

2.15

YES

2.15

YES

range finder

FAL.NHK.SLS.14.05.03
ppt in 2X C1FAL.NHK.SLS.29.10.04
FAL.NHK.SLS.03.11.04

ppt in 2X C1FAL.NHK.SLS.05.11.04

PHYSOSTIGMINE
IIVS

I-93

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3

10

RF

1.8

YES

range finder

SLS-A5-N040401A
SLS-B4-N040513C

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

B2

DF

AA61NF

129

0.467

0.596

5.79%

3

3

0.9845B3DF

AA61NF

141

0.511

0.834

1.84%

3

4

0.9527

AA61FT-A1

RF

AA61FT

123

0.447

0.863

2.71%

1

6

0.9452

AA61FT-B1

DF

AA61FT

158

0.575

0.691

3.04%

2

5

0.9669AA61FT-B2

DF

AA61FT

164

0.596

0.674

5.99%

2

3

0.9348

AA61FT-B3

DF

AA61FT

169

0.612

1.001

2.86%

2

6

0.8953

FAL.NHK.GT.A1.24.09.04

RF

AA61GT

153

0.555

0.662

6.01%

1

1

0.6638

FAL.NHK.GT.B1.01.10.04

DF

AA61GT

225

0.819

1.035

7.61%

2

6

0.9354

Exposure
Concentrations(ug/mL)7
1000, 556, 309, 171,95.3, 52.9, 29.4, 16.3
500, 357, 255, 182, 130,93.0, 66.4, 47.4

Dilution

Acceptable

Factor8

Tests9

1.8

YES

SLS-B5-N040514B

1.4

YES

SLS-B6-N040716A

Rationale for
Unacceptability

Notes

PC ID

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
700, 326, 151, 70.4, 32.8,
15.2, 7.09, 3.30
300, 204, 139, 94.4, 64.2,
43.7, 29.7, 20.2
300, 204, 139, 94.4, 64.2,
43.7, 29.7, 20.2

10

RF

2.15

YES

range finder

SLS-P38
SLS-P41

1.47

YES

SLS-P43

1.47

YES

SLS-P45

10RFrange finder

FAL.NHK.SLS.24.09.03

2.15

NO

PC failed

FAL.NHK.SLS.01.10.04

FAL.NHK.SLS.07.10.03

FAL.NHK.SLS.08.10.03

FRAME
1000, 100, 10, 1.0, 0.1,
0.01, 0.001, 0.0001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

FAL.NHK.GT.B2.07.10.04

DF

AA61GT

107

0.387

0.508

1.13%

3

2

0.9741

1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

2.15

NO

wrong solvent used(medium); should be
DMSO; SD will retest

FAL.NHK.GT.B3.08.10.04

DF

AA61GT

157

0.570

0.695

5.70%

3

5

0.9843

1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

2.15

NO

wrong solvent used(medium); should be
DMSO; SD will retest

FAL.NHK.GT.B4.20.10 .04

DF

AA61GT

470

1.706

1.324

1.47%

1

5

0.93822.15

YES

FAL.NHK.GT.B5.22.10.04

DF

AA61GT

0.366

0.001

0.767

7.78%

7

1

0.9929FAL.NHK.GT.B6.28.10.04

DF

AA61GT

167

0.605

0.596

9.68%

3

4

0.97401490

19.987

0.680

4.54

0

1

0.9413

1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 317, 101, 32.0,
10.2, 3.22, 1.02, 0.32
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71

FAL.NHK.SLS.20.10.04
reach 100% cytotoxicityat
C7

FAL.NHK.SLS.22.10.04
(NB)

3.15

YES

2.15

YES

FAL.NHK.SLS.28.10.04

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

SLS-A2

1.5

YES

SLS-B1POTASSIUM I CHLORIDE
IIVS
A2

RF

AA61FF

B1

DF

AA61FF

2040

27.364

0.355

1.41

4

4

0.9755

10000, 6667, 4444, 2963,
1975, 1317, 878, 585

B2

DF

AA61FF

2120

28.437

0.274

8.41

2

4

0.980910000, 6667, 4444, 2963,
1975, 1317, 878, 585

1.5

YES

SLS-B2B3DF

AA61FF

1810

24.279

0.295

8.80

4

3

0.984

10000, 6667, 4444, 2963,
1975, 1317, 878, 585

1.5

YES

SLS-B3

AA61KM-A1

RF

AA61KM1460

19.584

0.687

3.96

1

6

0.8761

AA61KM-B1

DF

AA61KM

2650

35.547

0.949

0.35

3

5

0.9297

AA61KM-B2

DF

AA61KM

2090

28.035

0.960

0.99

3

4

0.9645

AA61KM-B3

DF

AA61KM

2250

30.181

0.797

5.97

3

4

0.9805

AA61KM-B4

DF

AA61KM

2940

39.437

0.666

2.17

3

3

0.9170FAL.NHK.MY.A1.010803

RF

AA61MY

1030

13.816

0.503

3.16

0

6

0.7001

FAL.NHK.MY.B1.080803

DF

AA61MY

1610

21.596

0.625

3.72

3

5

0.8175ECBC
10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001
8000, 5442, 3702, 2518,
1714, 1166, 793, 539
8000, 5442, 3702, 2518,
1714, 1166, 793, 539
8000, 5442, 3702, 2518,
1714, 1166, 793, 539
8000, 5442, 3702, 2518,
1714, 1166, 793, 539

10

RF

1.47

YES

1.47

YES

1.47

NO

1.47

YES

range finder

SLS-P3SLS-P7SLS-P9PC failed

SLS-P11SLS-P19

FRAME

I-94

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
5000, 3401, 2313, 1574,
1070, 728, 496, 337

10

RF

1.47

YES

FAL.NHK.SLS.010803
high background

FAL.NHK.SLS.08.08.03

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

FAL.NHK.MY.B2.15.08.03

DF

AA61MY4760

63.850

0.250

36.21

1

2

0.2925

FAL.NHK.MY.B3.23.08.03

DF

AA61MY

1880

25.218

0.554

7.67

2

6

0.7555

FAL.NHK.MY.B4.28.08.04

DF

AA61MY2860

38.364

0.385

5.19

2

6

0.8496

FAL.NHK.MY.B5.05.09.03
FAL.NHK.MY.B5.15.10.03(should be B6?)DFDF

AA61MY
AA61MY

NA

NA

0.113

NA

NA

NA

Exposure
Concentrations(ug/mL)7
5000, 3401, 2313, 1574,
1070, 728, 496, 337
5000, 3401, 2313, 1574,
1070, 728, 496, 337
5000, 3401, 2313, 1574,
1070, 728, 496, 337

Dilution

Acceptable

Factor8

Tests9

1.47

NO% VC difference>15; low r2

FAL.NHK.SLS.15.08.03

1.47

NO

PC failed

FAL.NHK.SLS.230803

1.47

YES

FAL.NHK.SLS.280803

Rationale for
Unacceptability

Notes

PC ID

NA

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

NO

curve going in wrong
direction; plate reversed 180 FAL.NHK.SLS.050903
degrees when reading?

2.15

YES

FAL.NHK.SLS.15.10.03

10RF

range finder; nopoints between 50 100%; % VC
difference > 15

volatility problem; VC1 OD
values much lower than
VC2; VC1 removed from
subsequent analysis bySD

SLS-A5-N040401A

2.2

NO

no points between 0 - used plate sealer; induced
50%
shift in response

SLS-B4-N040513C

2.2

NO

no points between 0 no toxicity detected
100%

SLS-B5-N040514B

2.2

YES

SLS-B6-N040716A

2.2

YES

SLS-B7-N040717B

2.2

YES

SLS-B8-N040819A

10

RF

range finder; noponts between 50 100%; % VC
difference > 15

SLS-P38

range finder

SLS-P442390

32.059

0.482

3.11

1

6

0.8444

5000, 2326, 1082, 503,
234, 109, 50.6, 23.5

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

POTASSIUM CYANIDE
IIVS
A1

RF

AA61KW

0.00060.00001

0.173

100.39%300.7469B1

DF

AA61KW

NA

NA

0.656

2.12%

0

1

NA

B2

DF

AA61KW

NA

NA

0.541

1.12%

0

0

NA

B3

DF

AA61KW

19.2

0.295

0.670

0.68%

3

3

0.9761B4DF

AA61KW

16.6

0.255

0.613

5.27%

3

3

0.9799B5DF

AA61KW

14.8

0.227

0.584

5.68%

3

3

0.9770

AA61MN-A1

RF

AA61MN

NA

NA

0.017

103.07%

4

0NA

AA61MN-A2 (sealer)RF

AA61MN

15.3

0.235

0.758

2.90%

2

3

0.9585

AA61MN-B1 (sealer)DF

AA61MN

36.1

0.554

0.744

0.85%

3

4

0.9264

AA61MN-B2 (sealer)DF

AA61MN

29.4

0.452

0.939

0.10%

3

5

0.8814

AA61MN-B3 (sealer)DF

AA61MN

22.3

0.342

0.498

4.97%

3

2

0.9697

RF

AA61GP

NA

NA

0.005

87.41%

00-0.0679

0.100, 0.045, 0.021,
0.0094, 0.0043, 0.0019,
0.00088, 0.00040
0.100, 0.045, 0.021,
0.0094, 0.0043, 0.0019,
0.00088, 0.00040
100, 45.5, 20.7, 9.39,
4.27, 1.94, 0.882, 0.401
100, 45.5, 20.7, 9.39,
4.27, 1.94, 0.882, 0.401
100, 45.5, 20.7, 9.39,
4.27, 1.94, 0.882, 0.401

ECBC
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
300, 140, 64.9, 30.2,
14.0, 6.53, 3.04, 1.41
300, 140, 64.9, 30.2,
14.0, 6.53, 3.04, 1.41
300, 140, 64.9, 30.2,
14.0, 6.53, 3.04, 1.41

10

RF

2.15

YES

SLS-P46

2.15

YES

SLS-P50

2.15

YES

SLS-P52

10RF

range finder

FAL.NHK.SLS.24.09.03

2.15

NO

PC failed; no points
between 0-50%

FAL.NHK.SLS.01.10.04

3.15

NO

%VC difference >15 volatility problems

FAL.NHK.SLS.07.10.03

2.15

NO

1.47

NO

1.47

NO

2.15

YES

FAL.NHK.SLS.24.11.04

2.15

YES

FAL.NHK.SLS.26.11.04

FRAME
FAL.NHK.GP.A1.24.09.04
FAL.NHK.GP.B1.01.10.04

DF

AA61GP

4.07

0.062

1.025

7.20%

0

6

0.2038

FAL.NHK.B2.07.10.04

DF

AA61GP

16.4

0.251

0.331

40.76%

6

1

0.8792

FAL.NHK.GP.B3.20.10 .04

DF

AA61GP

NA

NA

1.150

0.46%

0

0

NA

FAL.NHK.GP.B4.11.11.04

DF

AA61GP

NA

NA

0.679

9.53%

6

0

NA

FAL.NHK.GP.B5.17.11.04

DF

AA61GP

71.9

1.105

0.622

22.40%

5

00.9016

FAL.NHK.GP.B6.24.11.04

DF

AA61GP

53.2

0.817

0.906

10.92%

3

4

0.9588

FAL.NHK.GP.B7.26.11.04

DF

AA61GP

11.9

0.182

0.460

1.72%

3

3

0.9363

I-95

10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001
0.100, 0.0465, 0.0216,
0.0101, 0.0047, 0.0022,
0.0010, 0.0005
5000, 1587, 504, 160,
50.8, 16.1, 5.12, 1.62
500, 232, 108, 50.3, 23.9,
10.4, 5.06, 2.35
2000, 1361, 926, 630,
428, 291,198,135
1000, 680, 463, 315, 214,
146, 99.1, 67.4
500, 232, 108, 50.3, 23.9,
10.4, 5.06, 2.35
500, 232, 108, 50.3, 23.9,
10.4, 5.06, 2.35

no points between 0FAL.NHK.SLS.20.10.04
100%
no points between
all concentrations were toxic FAL.NHK.SLS.10.11.04
50-100%no points between
50-100%; %VC
outlier removed bySD
FAL.NHK.SLS.17.11.04
difference >15

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

FAL.NHK.GP.B8.10.12.04

DF

AA61GP

202

3.107

0.993

1.92%

1

7

0.9318500, 232, 108, 50.3, 23.9,
10.4, 5.06, 2.35

2.15

YES

FAL.NHK.GP.B9.10.12.04

DF

AA61GP

31.6

0.484

0.903

1.34%

2

3

0.9469500, 232, 108, 50.3, 23.9,
10.4, 5.06, 2.35

2.15

NO

PC failedrange finder; nopoints between 0 100%

Notes

PC ID

SD has little confidence invalues due to chem.
volatility & interaction withplate
sealerSD
haslittle confidence in

FAL.NHK.SLS(MO).10.1
2.04

values due to chem.
volatility & interaction withplate sealer

FAL.NHK.SLS.10.12.04

PROCAINAMIDE HCL
IIVS
A1

RF

AA61ML

3890

14.314

0.499

3.99%

0

0

0.9391

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

B1

DF

AA61ML

2210

8.143

0.558

0.88%

3

2

0.9836

10000, 7519, 5653, 4251,
3196, 2403, 1807, 1358

1.33

YES

SLS-B4-N040513C

B2

DF

AA61ML

1770

6.498

0.510

6.82%

4

1

0.8603

10000, 7519, 5653, 4251,
3196, 2403, 1807, 1358

1.33

YES

SLS-B5-N040514B

B3

DF

AA61ML

2100

7.740

0.694

1.43%

3

2

0.992010000, 7519, 5653, 4251,
3196, 2403, 1807, 1358

1.33

YES

SLS-B6-N040716A

AA61KC-A1

RF

AA61KC

5120

18.826

0.703

1.72%

0

4

0.943910000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RF

AA61KC-B1

DF

AA61KC

1380

5.091

0.752

4.76%

5

2

0.9773

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

SLS-P32

1.47

YES

SLS-P37

1.47

YES

SLS-P38

10

RF

2.15

YES

FAL.NHK.SLS.11.08.04

1.47

YES

FAL.NHK.SLS.17.09.04

1.47

YES

FAL.NHK.SLS.07.10.03

10RF

range finder

SLS-A210RFrange finder

SLS-A3

1.4

NO

VC difference > 15%

SLS-B1

1.4

YES

1.4

NO

1.4

YES

SLS-A4-N040331N

ECBC

AA61KC-B2

DF

AA61KC

1350

4.963

0.410

2.83%

4

2

0.9664

AA61KC-B3

DF

AA61KC

1710

6.277

0.647

0.26%

2

4

0.9710FAL.NHK.GV.A1.28.07.04

RF

AA61GV

1330

4.884

0.055

6.80%

1

1

0.6423

FAL.NHK.GV.B1.11.08.04

DF

AA61GV

1730

6.365

0.464

0.97%

1

1

0.9180FAL.NHK.GV.B2.17.09.04

DF

AA61GV

2030

7.478

0.775

4.46%

2

1

0.9417

FAL.NHK.GV.B3.07.10.04

DF

AA61GV

1600

5.885

0.613

7.61%

3

3

0.9809A2

RF

AA61GC

28100

467.554

0.731

5.06

0

4

0.6596

A2 with plate coverRF

AA61GC

9820

163.394

0.556

2.40

1

1

0.8691B1DF

AA61GC

15100

251.248

0.296

20.61

2

4

0.8006

B1 with plate coverDF

AA61GC

6610

109.983

0.316

4.51

3

3

0.9817B2

DF

AA61GC

13600

226.290

0.233

23.35

2

4

0.8

B2 with plate coverDF

AA61GC

7570

125.957

0.243

9.58

2

3

0.9695

5000, 3401, 2314, 1574,
1071, 728, 496, 337
5000, 3401, 2314, 1574,
1071, 728, 496, 337

range finder; nopoints between 0 50%

SLS-P18

FRAME
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10000, 4651, 2163, 1006,
468, 218, 101, 47
5000, 3401, 2314, 1574,
1071, 728, 496, 337
5000, 3401, 2314, 1574,
1071, 728, 496, 337

range finder

FAL.NHK.SLS.28.07.04

2-PROPANOL

IIVS

I-96

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
20000, 14286, 10204,
7289, 5206, 3719, 2656,
1897
20000, 14286, 10204,
7289, 5206, 3719, 2656,
1897
20000, 14286, 10204,
7289, 5206, 3719, 2656,
1897
20000, 14286, 10204,
7289, 5206, 3719, 2656,
1897

SLS-B1
VC difference > 15%

SLS-B2
SLS-B2

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7
20000, 14286, 10204,
7289, 5206, 3719, 2656,
1897
20000, 14286, 10204,
7289, 5206, 3719, 2656,
1897

Dilution

Acceptable

Factor8

Tests9

1.4

NO

1.4

YES

Rationale for
Unacceptability

Notes

VC difference >15%; no points 50100%; low R2

PC ID

B3

DF

AA61GC

19200

319.468

0.25

26.08

0

5

0.617

SLS-B3B3 with plate coverDF

AA61GC7080

117.804

0.313

3.69

4

4

0.9821

AA61JL-A1

RF

AA61JL

NA

NA

0.726

0.28

0

5

NA

10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001

10RF

AA61JL-B1

DF

AA61JL

NA

NA

0.457

63.96

6

1

NA

50000, 34014, 23139,
15740, 10707, 7284,
4955, 3370

1.47

NO

AA61JL-B2

DF

AA61JL

NA

NA

0.554

35.73

4

2

NA

50000, 34014, 23139,
15740, 10707, 7284,
4955, 3370

1.47

NO

AA61JL-B3 sealer

DF

AA61JL

4610

76.705

0.646

7.33

3

4

0.92801.47

YES

SLS-P12

AA61JL-B4 sealer

DF

AA61JL5450

90.682

0.480

2.76

2

5

0.8957

1.47

YES

SLS-P18

AA61JL-B5 sealer

DF

AA61JL5730

95.341

0.582

1.85

4

3

0.9429

1.47

YES

SLS-P19

FAL.NHK.NG.A1.30.07.03

RF

AA61NG

NA

NA

1.332

1.06

0

7

0.384910000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RFrange finder; nopoints between 0.1 - Little toxicity
50%

FAL.NHK.NG.B1.07.08.03

DF

AA61NG

1220

20.300

0.400

5.06

3

5

0.1851

10000, 6802, 4628, 3148,
2142, 1457, 991, 674.1

1.47

NO

low r2

FAL.NHK.NG.B2.15.08.03

DF

AA61NG

2390

39.767

0.474

3.95

2

1

0.675610NO

low r2

FAL.NHK.NG.B4.05.09.03(plate sealer)DF

AA61NG

21800

362.729

0.129

15.55

1

3

0.775010NO% VC difference >15

FAL.NHK.NG.B5.15.10.03
plate sealer and mineral oil

DF

AA61NG

7460

124.126

0.624

3.14

1

5

0.6032

10NO

RF format; low r2

FAL.NHK.SLS.15.10.03

1.47

NO

% VC difference >15

FAL.NHK.SLS.19.10.03

1.47

NO

Mineral oil

2.15

NO

low r2

FAL.NHK.SLS.23.10.03

2.15

NO

low r2

FAL.NHK.SLS.23.10.03

2.15

YES

2.15

NO

2.15

YES

SLS-B3

ECBC

20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348

no points between
0.1 - 50%; no r2 nor
ICx values could be
calculated%VC difference >15; no r2 nor ICx
values could be
calculated
PC failed; %VC
difference > 15; no
r2 nor ICx values
could be calculated;

range finder

SLS-P2Volatility of largest conc
contaminated VC & others

SLS-P9Volatility of largest conc
contaminated VC & others

SLS-P11FRAME

FAL.NHK.NG.B6.19.10.03
plate sealer

DF

AA61NG

5850

97.338

0.262

19.17

4

3

0.9245

FAL.NHK.NG.B6.19.10.03
mineral oil

DF

AA61NG

5020

83.527

0.182

3.99

1

4

0.7943DF

AA61NG

2410

40.100

0.236

9.93

4

4

0.6362DF

AA61NG

4710

78.369

0.251

8.11

3

3

0.5306DF

AA61NG

5220

86.855

0.622

0.92

2

3

0.8150DF

AA61NG

4730

78.702

0.709

2.74

2

4

0.7880FAL.NHK.NG.B7.23.10.03
plate sealer
FAL.NHK.NG.B7.23.10.03
mineral oil
FAL.NHK.NG.B8.24.10.03
plate sealer
FAL.NHK.NG.B8.24.10.03
mineral oil

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348
20000, 13605, 9255,
6296, 4283, 2914, 1982,
1348
20000, 9302, 4327, 2012,
936, 435, 202, 94
20000, 9302, 4327, 2012,
936, 435, 202, 94
20000, 9302, 4327, 2012,
936, 435, 202, 94
20000, 9302, 4327, 2012,
936, 435, 202, 94

FAL.NHK.NG.B9.05.11.03ps
plate sealer

DF

AA61NG

4590

76.373

0.561

4.88

2

1

0.8354

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

FAL.NHK.NG.B9.05.11.03 minoil (mineral oil)DF

AA61NG

4480

74.542

0.564

20.01

2

2

0.7822

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

NO

FAL.NHK.NG.B10.07.11.03Ps
plate sealer

DF

AA61NG

3010

50.083

0.243

1.37

3

1

0.725620000, 9302, 4327, 2012,
936, 435, 202, 94

2.15

YES

I-97

FAL.NHK.SLS.30.07.03

SD wonders if chemical is a
FAL.NHK.SLS.07.08.03
mitotic inhibitor
FAL.NHK.SLS.15.08.03
SD provided revised file tocorrect data entry error

experimental

FAL.NHK.SLS.050903

FAL.NHK.SLS.19.10.03

FAL.NHK.SLS.24.10.03
low r2; Mineral oil

experimental

low r2; VC difference
experimental>15%; Mineral oil
challenging chemical; SMT
accepts this test

FAL.NHK.SLS.24.10.03
FAL.NHK.SLS.05.11.03(revised by study
director)
FAL.NHK.SLS.05.11.03(revised by study
director)
FAL.NHK.SLS.07.11.03

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells
FAL.NHK.NG.B10.07.11.03.mo (mineral oil)

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7
20000, 9302, 4327, 2012,
936, 435, 202, 94

Dilution

Acceptable

Factor8

Tests92.15

NO

Mineral oil

RFrange finder

Rationale for
Unacceptability

Notes

experimental

PC ID

DF

AA61NG

2610

43.428

0.270

5.07

2

1

0.8214

FAL.NHK.SLS.07.11.03

PreliminaryRF

AA61GU

23.1

0.078

0.606

4.44%

0

0

0.9617B1DF

AA61GU

29.6

0.100

0.582

4.61%

2

1

0.9576B2

DF

AA61GU

26.9

0.091

0.764

0.61%

2

2

0.9790B3DFAA61GU

25.2

0.085

1.001

0.94%

2

4

0.9652B4DF

AA61GU

32.7

0.111

0.907

4.02%

1

2

0.9864

RF

AA61KH

15.8

0.053

1.006

0.13%

0

2

0.9629DF

AA61KH

33.1

0.112

1.153

0.37%

1

3

0.9724DF

AA61KH

40.1

0.136

1.216

7.40%

2

1

0.9856DF

AA61KH

41.6

0.141

1.153

5.14%

2

1

0.9683A1 1b/NHKRF1b/FAL/NMRF

AA61NM

3.53

0.012

0.149

7.05%

0

3

0.8056100, 20, 4, 0.8, 0.16,
0.032, 0.0064, 0.00128

5

RF

range finder

A1
1b/NHKCTR1/FAL/SLS

A2 1b/NHKRF2/FAL/NMRF

AA61NM

8.66

0.029

0.475

9.32%

1

2

0.8193100, 68.02, 46.27, 31.47,
21.40, 14.50, 9.90, 6.70

1.47

RF

range finder

A2
1b/NHKCTR2/FAL/SLS

A3 1b/NHK/DF2/FAL/NMDF

AA61NM

24.4

0.082

0.042

11.04%

02

0.325730, 20.4, 13.8, 9.4, 6.42,
4.37, 2.97, 2.02

1.47

NO

A4 1b/NHK/DF3/FAL/NMDF

AA61NM

1.22

0.004

0.140

15.20%

04

0.068030, 20.4, 13.8, 9.4, 6.42,
4.37, 2.97, 2.02

1.47

NO

A5 1b/NHK/DF4/FAL/NMDF

AA61NM

NA

NA

0.008

9.78%

0

0

NC

30, 20.4, 13.8, 9.4, 6.42,
4.37, 2.97, 2.02

1.47

NO

A6 1b/NHK/DF5/FAL/NM
recalculated w/o outliersDF

AA61NM

54.0

0.183

1.686

2.60%

0

8

0.718630, 20.4, 13.8, 9.4, 6.42,
4.37, 2.97, 2.02

1.47

NO

A8 1b/NHK/DF7/FAL/NMDF

AA61NM

NA

NA

1.045

2.91%

0

5

NC

50, 34.01, 23.13, 15.74,
10.70, 7.28, 4.95,3.36

1.47

NO

A9 1b/NHK/DF8/FAL/NMDF

AA61NM

3.21

0.011

1.026

25.70%

04

0.147650, 34.01, 23.13, 15.74,
10.70, 7.28, 4.95,3.36

1.47

NO

PROPRANOLOL
IIVS

ECBC
ECBC-NHK-Ib-01
AA61KH-A1
ECBC-NHK-Ib-02
AA61KH-B1
ECBC-NHK-Ib-03
AA61KH-B2
ECBC-NHK-Ib-04
AA61KH-B3

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 56.3, 31.6, 17.8,
10.0, 5.6, 3.2, 1.8100, 56.3, 31.6, 17.8,
10.0, 5.6, 3.2, 1.8100, 56.3, 31.6, 17.8,
10.0, 5.6, 3.2, 1.8100, 56.3, 31.6, 17.8,
10.0, 5.6, 3.2, 1.8
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.91, 6.74

Preliminary

YES

SLS-B1YES

SLS-B2YES

SLS-B3YES

SLS-B4RF

range finder

SLS-P1YESSLS-P3YES

SLS-P4YES

SLS-P5

FRAME

15%; no point
between 10 & 50%;
R2 < 0.8; PC failed

A10 1b/NHK/DF9/FAL/NMDF

AA61NM

42.8

0.145

0.954

2.32%

1

3

0.5573350, 238.1, 162.0, 110.2,
75.0, 51.0, 34.7, 23.6

1.47

NO

A11 1b/NHK/DF10/FAL/NMDF

AA61NM

46.5

0.157

1.280

0.27%

1

2

0.8686350, 238.1, 162.0, 110.2,
75.0, 51.0, 34.7, 23.6

1.47

YES

A12 1b/NHK/DG11/FAL/NMDF

AA61NM

26.0

0.088

0.539

6.14%

3

0

0.8391350, 238.1, 162.0, 110.2,
75.0, 51.0, 34.7, 23.6

1.47

NO

I-98

No point between 10 NR crystal problems; usedmedium not normally used;
A3 1b/NHK/CTR4/FAL/& 50%; R2 < 0.8; PC removing outlier doesn't
failedsignificantly improve R2
No point between 10
NR crystal problems; used
& 50% viability; R2 < medium not normally used A4 1b/NHK/CTR5/FAL
0.8
NR crystal problems; usedmedium not normally used;
No points between
OD values of test wells no2
A5 1b/NHK/CTR6/FAL
10 & 90%; no R or different than theICx; PC failed
background ODs; negativevalues for VC
No point between 10 removed two outliers; didn't
A6 1b/NHK/CTR7/FAL
reach IC50
& 50%; R2 < 0.8
PRISM couldn't docalculations;didn'treach
No point between 10
IC50; recalc w/o outliers
A8 1b/NHK/CTR9/FAL& 50%; no R2 or ICx didn't improve curve fit, so
they
have
not
been
removedVC difference >R2 < 0.8

U-shaped dose-response

A9 1b/NHK/CTR10/FAL

no outliers; nonmonotonic
response

A10
1b/NHK/CTR11/FAL

removed 3 outliers
No point between 50
& 90%

A11
1b/NHK/CTR12/FAL
A12
1b/NHK/CTR13/FAL/SLSIn Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC ID

1b/NHK/DF12/FAL/NMDF

AA61NM

43.4

0.147

0.650

5.04%

1

2

0.9265350, 238.1, 162.0, 110.2,
75.0, 51.0, 34.7, 23.6

1.47

YES

1b/NHK/CTR14/FAL/SLS

1b/NHK/DF13/FAL/NMDF

AA61NM

41.5

0.140

0.897

2.57%

2

2

0.9555350, 238.1, 162.0, 110.2,
75.0, 51.0, 34.7, 23.6

1.47

YES

1b/NHK/CTR15/FAL/SLS

PROPYLPARABEN
IIVS
A1

RF

AA61PX

15.0

0.083

0.719

1.51%

2

2

0.9878B1

DF

AA61PX

13.4

0.075

0.631

1.14%

5

3

0.9849B2

DF

AA61PX

15.2

0.085

0.664

3.40%

5

3

0.9935B3DF

AA61PX

12.9

0.072

0.512

1.92%

4

3

0.9841

AA61PK-A1

RF

AA61PK

14.8

0.082

0.534

9.07%

2

1

0.8856

AA61PK-B1

DF

AA61PK

20.7

0.115

0.960

0.09%

4

4

0.9856

AA61PK-B2

DF

AA61PK

15.9

0.088

1.059

0.57%

4

4

0.9647

AA61PK-B3

DF

AA61PK

17.7

0.098

0.760

0.66%

4

4

0.9877

RF

AA61HT

23.4

0.130

0.486

8.29%

2

2

0.7353

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
200, 111, 61.7, 34.3, 19.1,
10.6, 5.88, 3.27
200, 111, 61.7, 34.3, 19.1,
10.6, 5.88, 3.27
200, 111, 61.7, 34.3, 19.1,
10.6, 5.88, 3.27

10

RF

1.8

YES

range finder

SLS-A2-N040320B
SLS-B1-N040423A

1.8

YES

SLS-B2-N040424A

1.8

YES

SLS-B3-N040506A

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
300, 140, 64.9, 30.2,
14.0, 6.53, 3.04, 1.41
300, 140, 64.9, 30.2,
14.0, 6.53, 3.04, 1.41
300, 140, 64.9, 30.2,
14.0, 6.53, 3.04, 1.41

10

RF

2.15

YES

range finder

ppt in 2X C1 and 1X C1SLS-P5SLS-P27

2.15

YES

SLS-P29

2.15

YES

SLS-P30

10RFrange finder

FAL.NHK.SLS.26.03.04

10RF

range finder; wrong
desorb solution used same application date andin NRU; SD rejects PC as HT A1
test

FAL.NHK.SLS.26.03.04

2.15

YES

2.15

NO

PC failed

2.15

NO

% VC difference >
15

2.15

YES

FAL.NHK.SLS.25.06.04

2.15

YES

FAL.NHK.SLS.20.08.04

FRAME
FAL.NHK.HT.A1.26.03.04

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

FAL.NHK.HT.A2.25.04.04

RF

AA61HT

NA

NA

0.729

50.05%

2

2

NA

FAL.NHK.HT.B1.28.04.04

DF

AA61HT

20.4

0.113

1.018

5.66%

2

3

0.9749FAL.NHK.HT.B2.11.06.04

DF

AA61HT

10.7

0.060

0.892

2.02%

4

4

0.9211

FAL.NHK.HT.B3.23.06.04

DF

AA61HT

NA

NA

0.521

99.17%NA

NA

NA

FAL.NHK.HT.B4.25.06.04

DF

AA61HT

15.3

0.085

1.063

4.00%

3

5

0.9548

FAL.NHK.HT.B5.20.08.04

DF

AA61HT

20.0

0.11072

0.906

0.85%

2

2

0.9443

AA61MV

0.581

0.004

0.393

15.03%

2

1

0.9631

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

2.2

100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47
100, 46.5, 21.6, 10.1,
4.68, 2.18, 1.01, 0.47

FAL.NHK.SLS.28.04.03
FAL.NHK.SLS.11.06.04
no cells in VC2; no PRISM
file

FAL.NHK.SLS.23.06.04

SODIUM ARSENITE
IIVS
A1

RF

volatile effects in VC1 andVC2SLS-A5-N040401A

YES

used plate sealer

SLS-B4-N040513C

range finder

B1

DF

AA61MV

0.440

0.003

0.590

11.98%

3

1

0.9426

30.0, 13.6, 6.20, 2.82,
1.28, 0.582, 0.265, 0.120

B2

DF

AA61MV

0.546

0.004

0.580

1.54%

4

1

0.972430.0, 13.6, 6.20, 2.82,
1.28, 0.582, 0.265, 0.120

2.2

YES

plate sealer used

SLS-B5-N040514B

B3

DF

AA61MV

0.424

0.003

0.666

3.98%

3

2

0.9931

30.0, 13.6, 6.20, 2.82,
1.28, 0.582, 0.265, 0.120

2.2

YES

plate sealer used

SLS-B6-N040716A

RF

AA61KA

0.506

0.004

0.850

0.23%

3

2

0.9923

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

ECBC
AA61KA-A1

I-99

range finder

SLS-P18

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

AA61KA-B1

DF

AA61KA

1.05

0.008

0.822

1.69%

3

3

0.9450AA61KA-B2

DF

AA61KA

0.764

0.006

1.005

1.85%

4

4

0.9892

AA61KA-B3

DF

AA61KA

0.555

0.004

0.801

0.43%

4

4

0.9804

FAL.NHK.GS.A1.24.09.04

RF

AA61GS

0.056

0.0004

0.652

2.90%

1

3

0.9075

FAL.NHK.GS.B1.01.10.04

DF

AA61GS

1.07

0.008

0.961

3.18%

2

4

0.9814

FAL.NHK.GS.B2.07.10.04

DF

AA61GS

0.275

0.002

0.516

3.33%

5

3

0.9843

FAL.NHK.GS.B3 .22.10.04

DF

AA61GS

0.545

0.004

0.712

5.53%

4

1

0.9815

FAL.NHK.GS.B4 .28.10.04

DF

AA61GS

0.187

0.001

0.759

3.27%

6

2

0.9854

Exposure
Concentrations(ug/mL)7
8.00, 3.72, 1.73, 0.805,
0.374, 0.174, 0.081,
0.038
8.00, 3.72, 1.73, 0.805,
0.374, 0.174, 0.081,
0.038
8.00, 3.72, 1.73, 0.805,
0.374, 0.174, 0.081,
0.038

Dilution

Acceptable

Factor8

Tests92.15

YES

SLS-P26

2.15

YES

SLS-P28

2.15

YES

SLS-P30

Rationale for
Unacceptability

Notes

PC ID

FRAME
10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001
10.0, 4.65, 2.16, 1.01,
0.47, 0.22, 0.10, 0.05
10.0, 4.65, 2.16, 1.01,
0.47, 0.22, 0.10, 0.05
10.0, 4.65, 2.16, 1.01,
0.47, 0.22, 0.10, 0.05
10.0, 4.65, 2.16, 1.01,
0.47, 0.22, 0.10, 0.05

10RFrange finder

FAL.NHK.SLS.24.09.03

2.15

NO

PC failed

FAL.NHK.SLS.01.10.04

2.15

YES

FAL.NHK.SLS.07.10.03

2.15

YES

FAL.NHK.SLS.22.10.04
(MO)

2.15

YES

FAL.NHK.SLS.28.10.04

SODIUM CHLORIDE
IIVS
A1

RF

AA61PE2100

35.999

0.630

2.05%

1

1

0.9570B1DF

AA61PE

NA

NA

0.549

1.11%

0

0

NA

B2

DF

AA61PE

NA

NA

0.518

0.68%

0

2

NA

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 625, 391, 244, 153,
95.4, 59.6, 37.3
1000, 625, 391, 244, 153,
95.4, 59.6, 37.3

10

RF

1.6

NO

1.6

NO

1.4

YES

range finder

SLS-A3-N040331A

no points between 0 100%no points between 0 toxicity not detected
100%

SLS-B4-N040513C
SLS-B5-N040514B

B3

DF

AA61PE

3170

54.236

0.707

4.08%

3

4

0.9471

10000, 7143, 5102, 3644,
2603, 1859, 1328, 949

B4DF

AA61PE

3470

59.332

0.599

10.23%

3

5

0.9518

10000, 7143, 5102, 3644,
2603, 1859, 1328, 949

1.4

YES

SLS-B7-N040717B

B5

DF

AA61PE

3770

64.460

0.550

2.04%

2

3

0.928010000, 7143, 5102, 3644,
2603, 1859, 1328, 949

1.4

YES

SLS-B8-N040819A

AA61JW-A1

RF

AA61JW

2250

38.485

0.817

2.63%

1

5

0.9346

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RF

AA61JW-B1

DF

AA61JW

3730

63.869

0.949

2.37%

3

5

0.9583

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

SLS-P26

AA61JW-B2

DF

AA61JW

3740

64.016

0.999

4.56%

3

4

0.955910000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

SLS-P28

AA61JW-B3

DF

AA61JW

3280

56.142

0.746

0.28%

3

5

0.9504

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

SLS-P30

FAL.NHK.FM.A1.14.05.04

RF

AA61FM2330

39.837

0.715

0.68%

1

4

0.9613

FAL.NHK.FM.B1.25.06.04

DF

AA61FM

366

6.256

0.954

1.08%

1

4

0.9769FAL.NHK.FM.B2.12.08.04

DF

AA61FM

NA

NA

0.658

6.32%

0

0

NA

FAL.NHK.FM.B3.19.08.04 nb

DF

AA61FM

NA

NA

0.397

0.95%

0

1

NA

FAL.NHK.FM.B4.30.09.04

DF

AA61FM

NA

NA

0.558

4.48%

0

4

0.7866outlier removed by SD

SLS-B6-N040716A

ECBC
range finder

SLS-P12

FRAME

I-100

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 680, 463, 315, 214,
146, 99.1, 67.4
1000, 680, 463, 315, 214,
146, 99.1, 67.4
2500, 930, 433, 201,93.6, 43.5, 20.3, 9.42

10

RF

2.15

YES

1.47

NO

1.47

NO

2.15

NO

range finder

FAL.NHK.SLS.14.05.03
FAL.NHK.SLS.25.06.04

PC failed; no points
between 0 - 100%no points between 0 no toxicity detected50%
toxicity curve begins to riseno points between 0 at high concentrations;50%
maybe affecting NRU

FAL.NHK.SLS.12.08.04
FAL.NHK.SLSNB.19.08.04
FAL.NHK.SLS.30.09.03

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

FAL.NHK.FM.B5.05.11.04

DF

AA61FM

268

4.584

0.455

0.60%

1

6

0.8717

FAL.NHK.FM.B3. 12.11.04(should be B6)

DF

AA61FM

NA

NA

0.694

14.43%03

NA

FAL.NHK.FM.B7.17.11.04

DF

AA61FM

NA

NA

0.919

5.26%

0

8

NA

FAL.NHK.FM.B8.26.11.04

DF

AA61FM

2720

Exposure
Concentrations(ug/mL)7
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
1000, 465, 216, 101,
46.8, 21.8, 10.1, 4.71
2000, 1527, 1165, 890,
679, 518, 396, 302

46.590

0.636

2.88%

2

6

0.9214

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

0.001

0.545

2.40%

2

2

0.9955

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

Dilution

Acceptable

Factor8

Tests9

2.15

YES

2.15

NO

1.31

NO

2.15

YES

10

RF

1.8

YES

SLS-B4-N040513C

1.8

YES

SLS-B5-N040514B

1.8

YES

SLS-B6-N040716A

10

RF

1.47

YES

SLS-P26

1.47

YES

SLS-P28

1.47

YES

SLS-P30

10

RF

3.16

YES

3.16

NO

3.16

YES

3.16

YES

FAL.NHK.SLS.12.11.04

3.16

YES

FAL.NHK.SLS.24.11.04

Rationale for
Unacceptability

Notes

PC ID

FAL.NHK.SLS.05.11.04
no points between 0 ppt in 1X C1-C4
50%
no points between 0 50%

FAL.NHK.SLS.12.11.04
FAL.NHK.SLS.17.11.04
FAL.NHK.SLS.26.11.04

SODIUM DICHROMATE DIHYDRATE
IIVS
A1

RF

AA61FP

0.390

B1

DF

AA61FP

0.527

0.002

0.587

1.15%

3

4

0.9863B2DF

AA61FP

0.511

0.002

0.522

0.67%

4

4

0.9863B3DF

AA61FP

0.691

0.002

0.711

0.67%

4

4

0.9841

RF

AA61NT

0.284

0.0010

0.542

1.94%

4

3

0.98195.00, 2.78, 1.54, 0.857,
0.476, 0.265, 0.147,
0.082
5.00, 2.78, 1.54, 0.857,
0.476, 0.265, 0.147,
0.082
5.00, 2.78, 1.54, 0.857,
0.476, 0.265, 0.147,
0.082

range finder

SLS-A4-N040331N

ECBC
AA61NT-A1
AA61NT-B1

DF

AA61NT

0.781

0.003

0.837

1.68%

1

7

0.8935

AA61NT-B2

DF

AA61NT

0.899

0.003

0.915

2.34%

2

6

0.9495

AA61NT-B3

DF

AA61NT

0.673

0.002

0.762

1.72%

3

5

0.9680RF

AA61HK

0.112

0.000

0.059

15.81%

5

3

0.746010000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1.00, 0.680, 0.463, 0.315,
0.214, 0.146, 0.099,
0.067
2.00, 1.361, 0.926, 0.630,
0.428, 0.291, 0.198,
0.135
2.00, 1.361, 0.926, 0.630,
0.428, 0.291, 0.198,
0.135

range finder

SLS-P16FRAME
FAL.NHK.HK.A1.28.07.04
FAL.NHK.HK.A1.28.07.04(should be 11.08.04)

RF

AA61HK

0.770

0.003

0.623

6.22%

1

1

0.9797

FAL.NHK.HK-NB.B2.25.08.04

DF

AA61HK

48.8

0.164

0.877

4.03%

1

4

0.9276

FAL.NHK.HK.B3.03.11.04

DF

AA61HK

0.512

0.002

0.518

1.50%

1

3

0.9921

FAL.NHK.HK.B3.12.11.04(should be B4)DF

AA61HK

0.882

0.003

0.792

0.95%

5

3

0.9919FAL.NHK.HK.B4.24.11.04(should be B5)DF

AA61HK

1.24

0.004

1.060

0.46%

1

2

0.9962

I-101

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
100, 31.6, 10.0, 3.17,
1.00, 0.317, 0.100,
0.0318100, 31.6, 10.0, 3.17,
1.00, 0.317, 0.100,
0.0318100, 31.6, 10.0, 3.17,
1.00, 0.317, 0.100,
0.0318100, 31.6, 10.0, 3.17,
1.00, 0.317, 0.100,
0.0318100, 31.6, 10.0, 3.17,
1.00, 0.317, 0.100,
0.0318range finder; % VC
difference > 15

FAL.NHK.SLS.28.07.04
FAL.NHK.SLS.11.08.04

SD rejects

FAL.NHK.SLS.25.08.04
solvent listed as DMSO-should be medium; SD
confirmed medium wasused

FAL.NHK.SLS.03.11.04

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

Notes

PC ID

SODIUM I FLUORIDE
IIVS
A2

RF

AA61HF

50.2

1.196

0.624

2.61%

2

1

0.9754B1

DF

AA61HF

50.1

1.193

0.355

6.81%

5

1

0.9643B2DF

AA61HF

51.9

1.236

0.275

12.46%

5

2

0.9713B3DF

AA61HF

49.1

1.169

0.321

2.29%

5

3

0.9679B6DF

AA61HF

63.8

1.519

0.56

6.98%

4

4

0.9088

AA61MG-A1

RF

AA61MG

35.2

0.838

0.673

0.47%

2

3

0.9552

AA61MG-B1

DF

AA61MG

55.0

1.310

0.359

0.67%

3

5

0.9146

AA61MG-B2

DF

AA61MG

41.3

0.984

0.855

2.57%

4

4

0.9376

AA61MG-B3

DF

AA61MG

49.8

1.186

0.942

1.56%

4

4

0.9160FAL.NHK.RH.A1.010803

RF

AA61RH

3.94

0.094

1.113

4.56%

3

4

0.9474

FAL.NHK.RH.B1.080803

DF

AA61RH

28.6

0.681

0.762

0.08

1

5

0.9046

FAL.NHK.RH.B2.15.08.03

DF

AA61RH

45.2

1.076

0.549

0.03

4

3

0.9257

FAL.NHK.RH.B3.01.10.03

DF

AA61RH

51.2

1.219

1.140

0.01

4

4

0.9761

FAL.NHK.RH.B3.15.10.03(should be B4?)DF

AA61RH

45.3

1.079

0.531

0.01

4

3

0.97711250

16.796

0.439

6.83%

0

2

0.9817

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
300, 188, 117, 73.2, 45.8,
28.6, 17.9, 11.2
300, 188, 117, 73.2, 45.8,
28.6, 17.9, 11.2
300, 188, 117, 73.2, 45.8,
28.6, 17.9, 11.2
150, 115, 88.8, 68.3, 52.5,
40.4, 31.1, 23.9

10

RF

1.6

YES

range finder

SLS-A2
SLS-B1

1.6

YES

SLS-B2

1.6

YES

SLS-B3

1.46

YES

SLS-B7

ECBC
10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001
1000, 300, 100, 30, 10, 3,
1, 0.3150, 102.5, 69.4, 47.2,
32.1, 21.8, 14.9, 10.1
150, 102.5, 69.4, 47.2,
32.1, 21.8, 14.9, 10.1

10

RF

3.33

YES

range finder

range finder

SLS-P2SLS-P5

1.47

YES

SLS-P7

1.47

YES

SLS-P9

FRAME
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35
500, 233, 108, 50.3, 23.4,
10.9, 5.06, 2.35

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RFrange finder

10

YES

range finder format

2.15

YES

FAL.NHK.SLS.010803
high background

FAL.NHK.SLS.08.08.03
FAL.NHK.SLS.15.08.03

2.15

NO

2.15

YES

PC failed

PC fails

FAL.NHK.SLS.01.10.03

10

RF

1.8

YES

SLS-B1-N040423A

1.8

YES

SLS-B2-N040424A

1.4

YES

SLS-B3-N040506A

FAL.NHK.SLS.15.10.03

SODIUM HYPOCHLORITE
IIVS
A1

RF

AA61RD

10000, 5556, 3086,
1715,953, 529, 294, 163
10000, 5556, 3086,
1715,953, 529, 294, 163
4000, 2857, 2041, 1458,
1041, 744, 531, 379

range finder; nopoints between 0 50%

SLS-A1-N040317B

B1

DF

AA61RD

1620

21.787

0.530

4.61%

4

2

0.9847B2

DF

AA61RD

1460

19.642

0.571

5.89%

2

1

0.9828B3DF

AA61RD1820

24.389

0.515

7.20%

3

3

0.9820RF

AA61HE1030

13.874

0.465

7.39%

0

1

0.8508

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RF

0.930910000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

YES

SLS-P7

1.47

YES

SLS-P9

1.47

YES

SLS-P12ECBC
AA61HE-A1
AA61HE-B1

DF

AA61HE

1960

26.375

0.975

3.79%

2

3

5000, 3401, 2313, 1574,
1071, 728, 496, 337
5000, 3401, 2313, 1574,
1071, 728, 496, 337

AA61HE-B2

DF

AA61HE

2390

32.151

1.161

1.44%

2

5

0.9791

AA61HE-B3

DF

AA61HE

1240

16.718

0.725

0.10%

4

3

0.9857

FAL.NHK.LU.A1.13.02.03

RF

AA61LU

955

12.829

0.077

1.41%

1

0

0.0662

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

FAL.NHK.LU.A2.20.02.03

DF

AA61LU

738

9.913

0.204

12.54%

6

1

0.9071

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

range finder

SLS-P3

FRAME

I-102

range finder

rejected by SD due tobacterial contam. in some of FAL.NHK.SLS.13.02.03
the plates in this test series
this is a definitive test sinceconc. series is different from FAL.NHK.SLS.20.02.03
A1 RF

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7
2500, 1701, 1157, 787,
535, 364, 248, 169

Dilution

Acceptable

Factor8

Tests91.47

NO

no points between 0- used wrong solvent; should
100%; wrong solvent be medium instead of
FAL.NHK.SLS.27.02.03
used
DMSO

1.47

YES

FAL.NHK.SLS.19.03.03

1.47

YES

FAL.NHK.SLS.25.03.03

FAL.NHK.LU.B1.27.02.04

DF

AA61LU

NA

NA

0.492

9.65%

0

0

NA

FAL.NHK.LU.B2.19.03.04

DF

AA61LU1120

15.073

0.437

3.51%

2

6

0.9027

FAL.NHK.LU.B3.25.03.04

DF

AA61LU1870

25.130

0.628

1.58%

1

2

0.7836

AA61GX

NA

NA

0.503

2.45%

0

2

NA

100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001

10RF

0.9647500, 357, 255, 182, 130,93.0, 66.4, 47.4

1.4

YES

2500, 1701, 1157, 787,
535, 364, 248, 169
2500, 1701, 1157, 787,
535, 364, 248, 169

Rationale for
Unacceptability

Notes

PC ID

SODIUM OXALATE
IIVS
A1
B1

RF
DF

AA61GX

252

1.879

0.631

2.24%

2

6

B2

DF

AA61GX

428

3.191

0.565

1.71%

1

5

0.8879510, 364, 260, 186, 133,
94.8, 67.7, 48.4

1.4

YES

B3

DF

AA61GX

400

2.985

0.669

2.53%

1

7

0.8426500, 357, 255, 182, 130,93.0, 66.4, 47.4

1.4

YES

AA61LZ-A1

RF

AA61LZ

230

1.717

0.621

2.94%

2

6

0.9507

AA61LZ-B1

DF

AA61LZ

312

2.328

0.636

0.73%

3

5

0.8613

AA61LZ-B2

DF

AA61LZ

337

2.517

0.709

1.12%

2

6

0.9490AA61LZ-B3

DF

AA61LZ

417

3.111

0.928

5.95%

1

5

0.9635

FAL.NHK.RC.A1.24.09.04

RF

AA61RC

687

5.127

0.404

1.28%

2

0

0.6286

FAL.NHK.RC.B1.29.10.04

DF

AA61RC

134

1.002

0.598

5.63%

5

3

0.8555

FAL.NHK.RC.B2.03.11.04

DF

AA61RC

422

3.147

0.465

1.00%

1

7

0.7013

FAL.NHK.RC.B3.10.11.04

DF

AA61RC

384

2.863

1.082

0.92%

5

1

0.9714

FAL.NHK.RC.B4.17.11.04

DF

AA61RC

460

3.435

1.002

2.39%

2

5

0.9280range finder; nopoints between 0 50%

SLS-A5-N040401A
SLS-B12-N041022B
130 ul of 2X doses wereapplied. Final conc. values
adjusted in data sheets
bySD

SLS-B113-N041029B
SLS-B14-N041030A

ECBC
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
1000, 680, 463, 315, 214,
146, 99.1, 67.4
600, 408, 278, 189, 128,
87.4, 59.5, 40.5
600, 408, 278, 189, 128,
87.4, 59.5, 40.5

10

RF

1.47

YES

range finder
ppt in 1X C1-C3

SLS-P20
SLS-P40

1.47

YES

ppt in 1X C1-C2

SLS-P42

1.47

YES

ppt in 1X C1SLS-P44

ppt in 1X C1-C2

FAL.NHK.SLS.24.09.03

ppt In 1X C1-C5

FAL.NHK.SLS.29.10.04

FRAME
10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001
1000, 680, 463, 315, 214,
146, 99.1, 67.4
500, 340, 231, 157, 107,
72.8, 49.6, 33.7
2000, 1361, 926, 630,
428, 291,198,135
1000, 680, 463, 315, 214,
146, 99.1, 67.4

10

RF

1.47

YES

1.47

YES

1.47

YES

1.47

YES

range finder

FAL.NHK.SLS.03.11.04
ppt In 1X C1-C5

FAL.NHK.SLS.10.11.04
FAL.NHK.SLS.17.11.04

SODIUM SELENATE
IIVS
A2

RF

AA61FS

7.44

0.039

0.646

4.12

4

1

0.9744B1DF

AA61FS

11.0

0.058

0.366

1.07

7

1

0.9841B2DF

AA61FS

10.5

0.056

0.29

12.33

4

1

0.9854B3DF

AA61FS

8.49

0.045

0.339

3.42

4

2

0.9763

AA61LF-A1

RF

AA61LF

7.91

0.042

0.605

6.62

3

2

0.9431

AA61LF-B1

DF

AA61LF

7.99

0.042

0.361

5.82

7

1

0.9236

AA61LF-B3

DF

AA61LF

7.95

0.042

0.890

1.82

4

3

0.9492

AA61LF-B4

DF

AA61LF

4.85

0.026

0.836

5.88

4

3

0.9845

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
556, 309, 172, 95.3, 53.0,
29.4, 16.3, 9.07
556, 309, 172, 95.3, 53.0,
29.4, 16.3, 9.08
100, 55.6, 30.9, 17.1, 9.5,
5.3, 2.94, 1.63

10

RF

1.8

YES

range finder

SLS-A2
SLS-B1

1.8

YES

SLS-B2

1.8

YES

SLS-B3

ECBC

I-103

10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001
100, 68, 46.3, 31.5, 21.4,
14.6, 9.9, 6.7
100, 46.5, 21.6, 10.1, 4.7,
2.2, 1.0, 0.47
100, 46.5, 21.6, 10.1, 4.7,
2.2, 1.0, 0.47

10

RF

1.47

YES

2.15

YES

2.15

NO

range finder

range finder

SLS-P1SLS-P5SLS-P9PC failed

SLS-P11

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

AA61LF-B5

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6DFAA61LF

6.48

0.034

0.647

1.62

4

2

0.8997

FAL.NHK.NS.A1.010803

RF

AA61NS

10.4

0.055

0.360

5.76

2

3

0.9256

FAL.NHK.NS.A2.080803

RF

AA61NS

14.6

0.077

0.716

5.02

6

0

0.9642

FAL.NHK.NS.B2.15.08.03(should be B1)

DF

AA61NS

12.2

0.065

0.551

5.35

4

4

0.9509FAL.NHK.NS.B2.230803

DF

AA61NS

9.34

0.049

0.490

0.47

5

3

0.9542

FAL.NHK.NS.B3.28.08.06

DF

AA61NS

34.0

0.180

0.398

3.79

1

6

0.6981

FAL.NHK.NS.B4.05.09.03

DF

AA61NS

9.14

0.048

0.207

7.21

6

2

0.9566

FAL.NHK.NS.B5.01.10.03

DF

AA61NS

7.75

0.041

1.124

6.36

6

2

Exposure
Concentrations(ug/mL)7
100, 46.5, 21.6, 10.1, 4.7,
2.2, 1.0, 0.47

Dilution

Acceptable

Factor8

Tests9

2.15

YES

Rationale for
Unacceptability

Notes

PC ID

SLS-P19FRAME

FAL.NHK.NS.B5.15.10.03(should be B6?)

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
250, 170, 116, 78.7, 53.6,
36.4, 24.8, 16.9
50, 34.01, 23.14, 15.74,
10.71, 7.28, 4.96, 3.37
50, 34.01, 23.14, 15.74,
10.71, 7.28, 4.96, 3.37
50, 34.01, 23.14, 15.74,
10.71, 7.28, 4.96, 3.37

10RFrange finder

1.47

RF

range finder

1.47

YES

1.47

NO

PC failed

FAL.NHK.SLS.230803

1.47

NO

low r2

FAL.NHK.SLS.280803

75, 51.02, 34.71, 23.61,
16.06, 10.93, 7.433, 5.06

1.47

YES

0.914775, 51.02, 34.71, 23.61,
16.06, 10.93, 7.433, 5.06

1.47

NO

50, 34.01, 23.14, 15.74,
10.71, 7.28, 4.96, 3.37

1.47

YES

DF

AA61NS

27.0

0.143

0.565

1.67

2

4

0.9272

A1

RF

AA61JY

67.1

0.201

0.490

3.17%

1

1

0.8475B1DF

AA61JY

59.0

0.176

0.606

1.54%

2

6

0.9699B2DF

AA61JY

52.7

0.158

0.598

3.50%

2

6

0.9122B3DF

AA61JY

53.5

0.160

0.616

2.26%

2

6

0.9020RF

AA61NR

183

0.548

0.882

6.19%

1

6

0.8663

FAL.NHK.SLS.010803
high background

FAL.NHK.SLS.08.08.03

this is the first definitive test FAL.NHK.SLS.15.08.03

FAL.NHK.SLS.050903
PC failed

FAL.NHK.SLS.01.10.03
FAL.NHK.SLS.15.10.03

STRYCHNINE

IIVS100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49

10RF

ppt in 2X C1SLS-A5-N040401A

1.4

YES

range finder

ppt in 2X C1SLS-B12-N041022B

1.4

YES

ppt in 2X C1SLS-B14-N041030A

1.4

YES

ppt in 2X C1SLS-B15-N041110A

10

RF

1.47

YES

ppt in 2X C1-C8

SLS-P47

1.47

YES

ppt in 2X C1-C3

SLS-P50

1.47

YES

ppt in 2X C1-C5

SLS-P52

1.47

YES

ppt in 2X C1SLS-P54ECBC
AA61NR-A1
AA61NR-B1

DF

AA61NR

66.5

0.199

0.878

3.32%

5

3

0.8150AA61NR-B2

DF

AA61NR

214

0.641

1.230

1.92%

2

6

0.9262

AA61NR-B3

DF

AA61NR

72.3

0.216

0.593

3.86%

5

3

0.9316

AA61NR-B4

DF

AA61NR

48.1

0.144

0.676

2.33%

6

2

0.9227

FAL.NHK.FY.A1.24.09.04

RF

AA61FY

87.7

0.262

0.520

1.43%

1

0

-0.0136

FAL.NHK.FY.B1.01.10.04

DF

AA61FY

60.3

0.180

0.965

14.61%

1

2

0.6474

FAL.NHK.FY.B2.08.10.04

DF

AA61FY

83.9

0.251

0.595

2.95%

2

3

0.9088

FAL.NHK.FY.B3.29.10.04

DF

AA61FY

29.9

0.089

0.585

9.13%

4

3

0.9623

FAL.NHK.FY.B4.05.11.04

DF

AA61FY

43.8

0.131

0.475

5.37%

4

3

0.9636

AA61KJ

0.09820.00020.448

10.68%

4

00.9741500, 50.0, 5.0, 0.50, 0.05,
0.005, 0.0005, 0.00005
400, 272, 185, 126, 85.7,
58.3, 39.6, 27.0
400, 272, 185, 126, 85.7,
58.3, 39.6, 27.0
400, 272, 185, 126, 85.7,
58.3, 39.6, 27.0
400, 272, 185, 126, 85.7,
58.3, 39.6, 27.0

range finder

SLS-P39

FRAME
100, 10, 1.0, 0.1, 0.01,
0.001, 0.0001, 0.00001
125, 58.1, 27.0, 12.6,
5.85, 2.72, 1.27, 0.59
250, 116, 54.1, 25.2, 11.7,
5.44, 2.53, 1.18
500, 232, 108, 50.3, 23.9,
10.4, 5.06, 2.35
500, 232, 108, 50.3, 23.9,
10.4, 5.06, 2.35

10RFrange finder

FAL.NHK.SLS.24.09.03

2.15

NO

PC failed

FAL.NHK.SLS.01.10.04

2.15

YES

2.15

YES

2.15

YES

10

RF

FAL.NHK.SLS.08.10.03
FAL.NHK.SLS.29.10.04
outlier removed by SD

FAL.NHK.SLS.05.11.04

THALLIUM I SULFATE
IIVS
A1

RF

I-104

100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001

range finder; nopoints between 50 100%

SLS-A1-N040317B

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

B1

DF

AA61KJ

0.137

0.0003

0.574

0.51%

4

3

0.9864

B2

DF

AA61KJ

0.141

0.0003

0.553

1.22%

4

2

0.9838

Exposure
Concentrations(ug/mL)7
1.00, 0.556, 0.309, 0.171,
0.095, 0.053, 0.029,
0.016
1.00, 0.556, 0.309, 0.171,
0.095, 0.053, 0.029,
0.016

Dilution

Acceptable

Factor8

Tests9

1.8

YES

1.8

YES

Rationale for
Unacceptability

Notes

PC ID

SLS-B1-N040423A
SLS-B2-N040424A
Mimimal to no NRU in C1C4 although visualobservatios appeared aslevel 2.

SLS-B3-N040506A

ppt in 2X C1SLS-P38DF

AA61KJ

0.104

0.0002

0.471

0.27%

4

3

0.9906

1.00, 0.556, 0.309, 0.171,
0.095, 0.053, 0.029,
0.016

1.8

YES

AA61PB-A1

RF

AA61PB

NA

NA

0.610

3.77%

6

1

NA

500, 50.0, 5.00, 0.5, 0.05,
0.005, 0.0005, 0.00005

10RF

range finder

AA61PB-B1

DF

AA61PB

NA

NA

0.975

2.18%

0

8

NA

0.005, 0.00233, 0.00108,
0.0005, 0.00023, 0.00011,
0.00005, 0.00002

2.15

NO

no points between 0 50%

AA61PB-B2

DF

AA61PB

0.313

0.0006

1.127

6.67%

2

6

0.8224

2.15

YES

AA61PB-B3

DF

AA61PB

0.132

0.0003

0.635

0.47%

4

4

0.9863

2.15

YES

AA61PB-B4

DF

AA61PB

0.149

0.0003

0.727

1.40%

4

4

0.97722.15

YES

FAL.NHK.GB.A1.13.02.03

RF

AA61GB

0.07080.0001

0.203

6.82%

3

3

0.6722

10RF

FAL.NHK.GB.B1.18.03.04

DF

AA61GB

0.167

0.00030.449

10.16%

3

2

0.96292.15

YES

FAL.NHK.SLS.18.03.03

FAL.NHK.GB.B2.19.03.04

DF

AA61GB

0.175

0.0003

0.448

0.84%

3

5

0.97142.15

YES

FAL.NHK.SLS.19.03.03

FAL.NHK.GB.B3.25.03.04

DF

AA61GB

0.118

0.0002

0.736

5.85%

4

3

0.92442.15

YES

FAL.NHK.SLS.25.03.03

661

4.043

0.513

1.38%

2

1

0.9403

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10

RF

1.8

B3

ECBC

1.00, 0.465, 0.216, 0.101,
0.047, 0.022, 0.010,
0.005
2.00, 0.930, 0.433, 0.201,
0.094, 0.044, 0.020,
0.009
2.00, 0.930, 0.433, 0.201,
0.094, 0.044, 0.020,
0.009

SLS-P46

SLS-P50
ppt in 2X C1SLS-P52
SLS-P54

FRAME
500, 50, 5, 0.5, 0.05,
0.005, 0.0005, 0.00005
1.0, 0.47, 0.22, 0.10,
0.05, 0.022, 0.010,
0.0047
1.0, 0.47, 0.22, 0.10,
0.05, 0.022, 0.010,
0.0047
1.0, 0.47, 0.22, 0.10,
0.05, 0.022, 0.010,
0.0047

range finder

rejected by SD due tobacterial contam. in some of FAL.NHK.SLS.13.02.03
the plates in this test series

TRICHLOROACETIC ACID
IIVS
A1

RF

AA61MR

range finder

ppt in 1X C1SLS-A4-N040331N

YES

ppt in 1X C1-C2

SLS-B4-N040513C

B1

DF

AA61MR

423

2.587

0.572

0.22%

5

2

0.9761

10000, 5556, 3086, 1715,
953, 529, 294, 163

B2

DF

AA61MR

423

2.587

0.665

0.91%

4

2

0.9853

10000, 5556, 3086, 1715,
953, 529, 294, 163

1.8

YES

ppt in 1X C1-C2

SLS-B5-N040514B

B3

DF

AA61MR

335

2.050

0.672

8.28%

3

2

0.9732

10000, 5556, 3086, 1715,
953, 529, 294, 163

1.8

YES

ppt in 1X C1-C2

SLS-B6-N040716A

AA61KT-A1

RF

AA61KT

348

2.132

0.561

3.44%

2

4

0.956010

RF

AA61KT-B1

DF

AA61KT

400

2.448

0.789

0.01%

4

3

0.9754

2.15

YES

SLS-P33

AA61KT-B2

DF

AA61KT

366

2.243

0.666

4.87%

4

4

0.9886

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
7000, 3256, 1514, 704,
328, 152, 70.9, 33.0
7000, 3256, 1514, 704,
328, 152, 70.9, 33.0

2.15

YES

SLS-P35

ECBC

I-105

range finder

SLS-P17

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

AA61KT-B3

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6DF

AA61KT

277

1.693

0.500

0.20%

4

4

0.9697

FAL.NHK.GH.A1.28.07.04

RF

AA61GH

627

3.835

0.053

4.54%

2

1

0.8134

FAL.NHK.GH.B1.11.08.04

DF

AA61GH

649

3.970

0.507

12.88%

4

4

0.8715

FAL.NHK.GH.B2.27.08.04

DF

AA61GH

370

2.263

0.439

1.88%

4

4

0.8671

FAL.NHK.GH.B3.17.09.04

DF

AA61GH

604

3.696

0.711

5.96%

4

4

0.9901

Exposure
Concentrations(ug/mL)7
7000, 3256, 1514, 704,
328, 152, 70.9, 33.0

Dilution

Acceptable

Factor8

Tests9

2.15

YES

Rationale for
Unacceptability

Notes

ppt in 1X C1PC ID

SLS-P37

FRAME
10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001
10000, 4651, 2163, 1006,
468, 218, 101, 47
10000, 4651, 2163, 1006,
468, 218, 101, 47
10000, 4651, 2163, 1006,
468, 218, 101, 47

10

RF

2.15

YES

2.15

YES

2.15

YES

range finder

FAL.NHK.SLS.28.07.04
FAL.NHK.SLS.11.08.04
FAL.NHK.SLS.27.08.04
outlier removed by SD

FAL.NHK.SLS.17.09.04

1,1,1-TRICHLOROETHANE
IIVS
A1

RF

AA61KG

NA

NA

0.516

5.11%

0

1

NA

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RFrange finder; nopoints between 0 50%

SLS-A5-N040401A

B1

DF

AA61KG

NA

NA

0.573

1.92%

0

5

-3.2450

10000, 7143, 5102, 3644,
2603, 1859, 1328, 949

1.4

NO

no points between 0 ppt in 1X C1
50%

SLS-B113-N041029B

B2

DF

AA61KG

NA

NA

0.677

2.29%

0

3

0.713012500, 8929, 6378, 4555,
3254, 2324, 1660, 1186

1.4

NO

no points between 0 ppt in 1X C1-C3
50%

SLS-B14-N041030A

B3DF

AA61KG

9400

70.439

0.598

4.99%

0

2

0.882812500, 8929, 6378, 4555,
3254, 2324, 1660, 1186

1.4

NO

ppt in 1X C1-C3; ppt in 2X
C1-C4; test article wasno points between 0 - noted to form droplets and
SLS-B15-N041110A
50%
adhere to the dilution vesel;maximum plausible dose
was tested.

RF

AA61JV

5300

39.702

0.614

8.77%

1

7

0.8101

10000, 1000, 100, 10, 1,
0.1, 0.01, 0.001

10RFrange finder

0.9418

10000, 6803, 4628, 3148,
2142, 1457, 991, 674

1.47

YES

ppt in 2X C1-C8

SLS-P46

1.21

YES

ppt in 2X C1; 1X C1 haslarge globules of chemical;
outlier removed by SD

SLS-P48

1.21

YES

ppt in 2X C1-C4; 1X C1 has
SLS-P51
large globules of chemical;

ECBC
AA61JV-A1(sealer)
AA61JV-B1(sealer)DF

AA61JV7530

56.469

0.920

1.02%

1

6

AA61JV-B2 (sealer)DF

AA61JV8710

65.285

0.674

2.11%

1

6

0.9422

10000, 8264, 6830, 5645,
4665, 3855, 3186, 2633

AA61JV-B3 (sealer)DF

AA61JV

8170

61.208

1.119

2.10%

1

7

0.853010000, 8264, 6830, 5645,
4665, 3855, 3186, 2633

FAL.NHK.PN.A1.24.09.04

RF

AA61PN

NA

NA

0.472

8.81%

0

2

NA

FAL.NHK.PN.B1.29.10.04

DF

AA61PN

NA

NA

0.543

4.83%

0

0

0.9623SLS-P20

FRAME
10000, 1000, 100, 10,
1.0, 0.1, 0.01, 0.001
2500, 1701, 1157, 787,
535, 364, 248, 169

10RF

range finder

FAL.NHK.SLS.24.09.03

1.47

NO

no points between 0100%

FAL.NHK.SLS.29.10.04
FAL.NHK.SLS.19.11.04

FAL.NHK.PN.B2.19.11.04

DF

AA61PN

NA

NA

0.417

4.54%

0

1

NA

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

NO

no points between 050%

FAL.NHK.PN.B3.24.11.04

DF

AA61PN

NA

NA

1.211

2.37%

0

6

NA

10000, 4651, 2163, 1006,
468, 218, 101, 47.1

2.15

NO

odd curve; two columns of
no points between 0data removed by SD (wells
50%
not seeded with cells?)

FAL.NHK.SLS.24.11.04

1.64

0.008

0.690

3.71%

1

2

0.9531

10, 1, 0.1, 0.01, 0.001,
0.0001, 0.00001,
0.000001

10RF

range finder

SLS-A2-N040320B

TRIETHYLENEMELAMINE
IIVS
A1

RF

AA61MT

I-106

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

B1

DF

AA61MT

1.66

0.008

0.543

8.55%

3

5

0.9632B2DF

AA61MT

2.12

0.010

0.572

4.28%

3

3

0.9763B3DF

AA61MT

2.62

0.013

0.544

3.49%

2

4

0.9730AA61GE-A1

RF

AA61GE

0.791

0.004

0.881

0.27%

0

7

0.9461

AA61GE-B1

DF

AA61GE

1.33

0.007

0.642

6.27%

2

6

0.8577

AA61GE-B2

DF

AA61GE

2.77

0.014

0.979

1.34%

1

6

0.9306

AA61GE-B3

DF

AA61GE

0.964

0.005

0.561

1.05%

2

6

0.9283

FAL.NHK.LB.A1.26.03.04

RF

AA61LB

1.13

0.006

0.805

2.56%

1

1

0.8822

FAL.NHK.LB.B1.25.04.04

DF

AA61LB

2.37

0.012

0.846

8.90%

1

3

0.9664

FAL.NHK.LB.B2.28.04.04

DF

AA61LB

2.22

0.011

0.851

4.98%

3

4

0.8151

FAL.NHK.LB.B3.11.06.04

DF

AA61LB

2.18

0.011

0.975

1.63%

3

4

0.9221

FAL.NHK.LB.B4.25.06.04

DF

AA61LB

1.49

0.007

1.155

0.33%

1

6

0.8420

Exposure
Concentrations(ug/mL)7
10.0, 5.56, 3.09, 1.71,
0.953, 0.529, 0.294,
0.163
10.0, 5.56, 3.09, 1.71,
0.953, 0.529, 0.294,
0.163
10.0, 5.56, 3.09, 1.71,
0.953, 0.529, 0.294,
0.163

Dilution

Acceptable

Factor8

Tests9

1.8

YES

SLS-B1-N040423A

1.8

YES

SLS-B2-N040424A

1.8

YES

SLS-B3-N040506A

10

RF

2.15

YES

SLS-P21

2.15

YES

SLS-P23

2.15

YES

SLS-P25

10

RF

2.15

YES

1.47

YES

1.47

NO

2.15

YES

10

RF

range finder

SLS-A2-N040320B

1.8

NO

no points between
50 - 100%

SLS-B1-N040423A

1.8

NO

no points between
50 - 100%

SLS-B2-N040424A

1.5

YES

SLS-B3-N040506A

1.5

YES

SLS-B8-N040819A

1.5

YES

SLS-B9-N040820A

10

RF

Rationale for
Unacceptability

Notes

PC ID

ECBC
10, 1, 0.1, 0.01, 0.001,
0.0001, 0.00001,
0.000001
5.00, 2.33, 1.08, 0.503,
0.234, 0.109, 0.051,
0.024
5.00, 2.33, 1.08, 0.503,
0.234, 0.109, 0.051,
0.024
5.00, 2.33, 1.08, 0.503,
0.234, 0.109, 0.051,
0.024

range finder; nopoints between 0 50%

ppt in 2X C1SLS-P13FRAME
10, 1, 0.1, 0.01, 0.001,
0.0001, 0.00001,
0.000001
10.0, 4.65, 2.16, 1.01,
0.468, 0.218, 0.101,
0.047
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 4.65, 2.16, 1.01,
0.468, 0.218, 0.101,
0.047

range finder

FAL.NHK.SLS.26.03.04
FAL.NHK.SLS.25.04.04
FAL.NHK.SLS.28.04.03

PC failed

FAL.NHK.SLS.11.06.04
FAL.NHK.SLS.25.06.04

TRIPHENYLTIN HYDROXIDE
IIVS
A1

RF

AA61JR

0.013

0.00004

0.729

1.45%

2

1

0.9887B1DF

AA61JR

0.015

0.00004

0.602

4.32%

2

0

0.9758B2DF

AA61JR

0.015

0.00004

0.630

3.36%

2

0

0.9907B3DF

AA61JR

0.012

0.00003

0.485

9.45%

3

2

0.9779B4DF

AA61JR

0.012

0.00003

0.658

0.37%

4

3

0.9917B5DF

AA61JR

0.014

0.00004

0.610

0.07%

3

4

0.9907

RF

AA61LL

0.015

0.00004

0.542

3.67%

0

2

0.988010, 1, 0.1, 0.01, 0.001,
0.0001, 0.00001,
0.000001
1.00, 0.556, 0.309, 0.171,
0.095, 0.053, 0.029,
0.016
1.00, 0.556, 0.309, 0.171,
0.095, 0.053, 0.029,
0.016
0.067, 0.045, 0.030,
0.020, 0.0132, 0.0088,
0.0059, 0.0039
0.067, 0.045, 0.030,
0.020, 0.013, 0.0088,
0.0059, 0.0039
0.067, 0.045, 0.030,
0.020, 0.013, 0.0088,
0.0059, 0.0039

ECBC
AA61LL-A1

I-107

10, 1, 0.1, 0.01, 0.001,
0.0001, 0.00001,
0.000001

range finder

SLS-P5

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

Exposure
Concentrations(ug/mL)7
0.080, 0.054, 0.037,
0.025, 0.017, 0.012,
0.008, 0.005
0.080, 0.054, 0.037,
0.025, 0.017, 0.012,
0.008, 0.005
0.080, 0.054, 0.037,
0.025, 0.017, 0.012,
0.008, 0.005

Dilution

Acceptable

Factor8

Tests9

1.47

YES

SLS-P22

1.47

YES

SLS-P25

1.47

YES

SLS-P27

Rationale for
Unacceptability

Notes

PC ID

AA61LL-B1

DF

AA61LL

0.021

0.00006

1.065

0.78%

4

4

0.9633

AA61LL-B2

DF

AA61LL

0.015

0.00004

0.599

0.01%

4

3

0.9832

AA61LL-B3

DF

AA61LL

0.029

0.00008

0.987

5.68%

3

4

0.9754

FAL.NHK.GG.A1.26.03.04

RF

AA61GG

0.010

0.00003

0.616

6.20%

2

0

0.8151

10.0, 1.0, 0.1, 0.01,
0.001, 0.0001, 0.00001,
0.000001

10RF

range finder

ppt in 1X C1FAL.NHK.SLS.26.03.04

FAL.NHK.GG.A2.25.04.04

DF

AA61GG

NA

NA

0.052

12.10%

2

6

NA

0.1, 0.0465, 0.0216,
0.0101, 0.0047, 0.0022,
0.0010, 0.0005

2.15

NO

wrong desorb
solution used in
ppt in 1X C1NRU; SD rejects thistest

FAL.NHK.SLS.25.04.04

FAL.NHK.GG.B1.28.04.04

DF

AA61GG

0.002

0.00001

0.877

1.40%

5

2

0.98842.15

YES

FAL.NHK.SLS.28.04.03

FAL.NHK.GG.B2.13.05.04

DF

AA61GG

0.003

0.00001

0.701

2.72%

2

3

0.9701

2.15

YES

FAL.NHK.SLS.13.05.04

FAL.NHK.GG.B3.10.06.04

DF

AA61GG

0.015

0.00004

0.894

5.53%

3

2

0.9727

1.47

YES

FAL.NHK.SLS.10.06.04

A1

RF

AA61MZ

710

4.921

0.730

0.79%

1

2

0.9232B1

DF

AA61MZ

394

2.735

0.633

8.35%

4

4

0.9086B2

DF

AA61MZ

512

3.548

0.676

4.33%

3

5

0.9566B3DF

AA61MZ

383

2.655

0.657

7.25%

3

4

0.9436

AA61JJ-A1

RF

AA61JJ

406

2.812

0.953

4.71%

1

1

0.9319AA61JJ-B1

DF

AA61JJ

575

3.991

0.920

0.13%

2

4

0.9458AA61JJ-B2

DF

AA61JJ

484

3.358

0.963

0.38%

2

4

0.9533

AA61JJ-B3

DF

AA61JJ

344

2.383

0.717

0.17%

2

6

0.9570FAL.NHK.GK.A1.25.03.04

RF

AA61GK

NA

NA

0.666

0.25%

0

0

NA

FAL.NHK.GK.B1.25.04.04

DF

AA61GK

757

5.248

0.874

6.22%

3

5

0.8798

FAL.NHK.GK.B2.28.04.04

DF

AA61GK

828

5.742

0.735

2.30%

3

5

0.8571

FAL.NHK.GK.B2.13.05.04(should be B3)DF

AA61GK

522

3.623

0.778

1.46%

2

3

0.9880RF

AA61NH

78.3

0.160

0.566

5.81%

1

0

0.8763FRAME

0.100, 0.047, 0.022,
0.010, 0.005, 0.002,
0.001, 0.0005
0.1, 0.0465, 0.0216,
0.0101, 0.0047, 0.0022,
0.0010, 0.0005
0.100, 0. 68, 0.0463,
0.0315, 0.0214, 0.0146,
0.0099, 0.0067

VALPROIC ACID
IIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2500, 1563, 977, 610,
381, 238, 149, 93.1
2500, 1563, 977, 610,
381, 238, 149, 93.1
2500, 1563, 977, 610,
381, 238, 149, 93.1

10

RF

1.6

YES

range finder

SLS-A2-N040320B
SLS-B8-N040819A

1.6

YES

SLS-B9-N040820A

1.6

YES

SLS-B10-N040903A

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1861, 865, 403, 187,
87.1, 40.5, 18.8, 8.8
2000, 930, 433, 201,93.6, 43.5, 20.2, 9.4
2000, 930, 433, 201,93.6, 43.5, 20.2, 9.4

10

RF

2.15

YES

range finder

SLS-P15

2.15

YES

ppt in 2X C1-C2; oily

SLS-P29

2.15

YES

ppt in 2X C1; oily

SLS-P30

SLS-P27

FRAME
2000, 200, 20, 2, 0.2,
0.02, 0.002, 0.0002
2500, 1701, 1157, 787,
535, 364, 248, 169
2500, 1701, 1157,
787,535, 364, 248, 169
2500, 1163, 541, 252,
117, 54.4, 25.3, 11.8

10

RF

1.47

YES

1.47

YES

2.15

YES

10

RF

range finder

FAL.NHK.SLS.25.03.03
FAL.NHK.SLS.25.04.04
ppt in 2X C1FAL.NHK.SLS.28.04.03
FAL.NHK.SLS.13.05.04

VERAPAMIL HCL
IIVS
A1

I-108

100, 10, 1, 0.1, 0.01,
0.001, 0.0001, 0.00001

range finder; nopoints between 50 100%

SD chose to use bottom = 0
SLS-A4-N040331N
instead of bottom > 0;

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3

R2 6

B1

DF

AA61NH

67.5

0.137

0.656

5.17%

4

4

0.9864B2DF

AA61NH

71.0

0.144

0.669

0.10%

4

3

0.9788B3DF

AA61NH

60.1

0.122

0.577

7.59%

3

4

0.9794

AA61LY-A1

RF

AA61LY

64.6

0.131

0.423

5.73%

2

3

0.9492

AA61LY-B1

DF

AA61LY

65.3

0.133

0.821

0.23%

4

4

0.9735

AA61LY-B2

DF

AA61LY

71.0

0.144

0.861

1.55%

4

4

0.9820AA61LY-B3

DF

AA61LY

45.2

0.092

0.455

1.81%

3

4

0.9523

FAL.NHK.MC.A1.28.07.04

RF

AA61MC

81.1

0.165

0.070

23.68%

2

1

0.6033

FAL.NHK.MC.B1.20.08.04

DF

AA61MC

73.3

0.149

0.892

3.87%

1

4

0.9216

FAL.NHK.MC.B2.08.10.04

DF

AA61MC

50.0

0.102

0.728

0.31%

3

3

0.9778

FAL.NHK.MC.B3.20.10 .04

DF

AA61MC

115

0.233

1.206

5.67%

1

2

0.9892

A1

RF

AA61MA

871

8.203

0.746

0.09%

1

0

0.8848B1DF

AA61MA

374

3.524

0.700

5.04%

3

2

0.7194

Exposure
Concentrations(ug/mL)7
200, 143, 102, 72.9, 52.1,
37.2, 26.6, 19.0
200, 143, 102, 72.9, 52.1,
37.2, 26.6, 19.0
200, 143, 102, 72.9, 52.1,
37.2, 26.6, 19.0

Dilution

Acceptable

Factor8

Tests91.4

YES

SLS-B8-N040819A

1.4

YES

SLS-B9-N040820A

1.4

YES

SLS-B10-N040903A

Rationale for
Unacceptability

Notes

PC ID

ECBC
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
800, 372, 173, 80.5, 37.4,
17.4, 8.1, 3.8
800, 372, 173, 80.5, 37.4,
17.4, 8.1, 3.8
800, 372, 173, 80.5, 37.4,
17.4, 8.1, 3.8

10

RF

2.15

YES

range finder

ppt in 2X C1SLS-P17

2.15

YES

ppt in 2X C1SLS-P35

2.15

YES

ppt in 2X C1SLS-P37

10

RF

2.15

YES

ppt in 2X C1-C3; outliers
removed by SD

FAL.NHK.SLS.20.08.04

2.15

YES

ppt in 2X C1FAL.NHK.SLS.08.10.03

2.15

YES

SLS-P33

FRAME
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
1500, 698, 325, 151,
70.2, 32.7, 15.2, 7.06
1500, 698, 325, 151,
70.2, 32.7, 15.2, 7.06
1500, 698, 325, 151,
70.2, 32.7, 15.2, 7.06

range finder; % VC
difference > 15

FAL.NHK.SLS.28.07.04

FAL.NHK.SLS.20.10.04

XYLENE
IIVS
1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001
2000, 1429, 1020, 729,
521, 372, 266, 190

10

RF

1.4

YES

range finderwell-to-well variability in 3
lowest doses observed

SLS-A3-N040331A
SLS-B8-N040819A

SLS-B9-N040820A

B2

DF

AA61MA

700

6.592

0.660

6.57%

2

3

0.77392000, 1429, 1020, 729,
521, 372, 266, 190

1.4

YES

ppt in 2X C1-C3; variability
in 4 highest doses
observed; top 2 doses notincluded in the Hill analysis

B3

DF

AA61MA

385

3.631

0.629

2.40%

2

2

0.8182

2000, 1429, 1020, 729,
521, 372, 266, 190

1.4

YES

ppt in 2X C1-C4; variabilityin 7 highest doses
observed;Top dose not
SLS-B10-N040903A
included in Hill analysis (SDdecision)RF

AA61GM

164

1.545

1.075

3.37%

0

5

0.9337

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10

RF

1.47

NO

1.47

NO

1.21

NO

ECBC
AA61GM-A1

800, 544, 370, 252, 171,
117, 79.3, 53.9
2000, 1361, 926, 630,
428, 291, 198, 135
4000, 3306, 2732, 2258,
1866, 1542, 1275, 1053

AA61GM-B1

DF

AA61GM

NA

NA

1.106

0.20%

0

8

NA

AA61GM-B2

DF

AA61GM

NA

NA

0.675

0.96%

0

5

NA

AA61GM-B3

DF

AA61GM

NA

NA

0.699

4.39%

0

4

NA

RF

AA61JG

NA

NA

0.725

2.43%

0

0

NA

1000, 100, 10, 1, 0.1,
0.01, 0.001, 0.0001

10RF

0.3835

2500, 1701, 1157, 787,
535, 364, 248, 169

1.47

NO

range finder; nopoints between 0 ppt in 2X C1
50%
no points between 0 50%
no points between 0 ppt in 2X C1-C5
50%
no points between 0 - ppt in 2X C1-C8; no toxicity
50%
detected

SLS-P13SLS-P47
SLS-P49
SLS-P53

FRAME
FAL.NHK.JG.A1.14.05.04
FAL.NHK.JG.B1.08.10.04

DF

AA61JG

NA

NA

0.834

13.03%

0

7

I-109

range finder; nopoints between 0 100%no points between 0 50%

FAL.NHK.SLS.14.05.03
FAL.NHK.SLS.08.10.03

In Vitro Cytotoxicity Test Methods BRD Appendix I2

November 2006

NHK NRU Reference Substance Data

Experiment ID
NHK Cells

Assay Substance
ID
Type1

IC50
(ug/mL)

IC50(mM)

Mean VC
OD

2

Difference
Number of Number ofof right/left
PointsPointsVC from
0 - 50 %4 50 - 100 %5
mean VC3R2 6

FAL.NHK.JG.B2.22.10.04

DF

AA61JG

3130

29.444

0.798

7.28%

0

6

0.6066

FAL.NHK.JG.B3.28.10.04

DF

AA61JG

NA

NA

0.559

1.04%

0

0

NA

Exposure
Concentrations(ug/mL)7
2500, 1701, 1157, 787,
535, 364, 248, 169
2500, 1701, 1157, 787,
535, 364, 248, 169

Dilution

Acceptable

Factor8

Tests9

1.47

NO

1.47

NO

Rationale for
Unacceptability
no points between 0 50%
no points between 0 100%

Notes

PC ID

FAL.NHK.SLS.22.10.04(NB)
FAL.NHK.SLS.28.10.04

Abbreviations: ppt=Precipitate; SD=Study Director; RF=Range Finder; DF=Definitive Test; PC=Positive Control; C1 - C8=Concentration series applied to the the cells. C1 is the highest concentration and C8 is lowest; NA=Not Available; RC=Registry of Cytotoxicity; 2X=Two times the concentration applied to the cells; VC=Vehicle Control;
R2=Coefficient of Determination; OD=Optical Density; ID=Identification. Substance ID was the code assigned by the chemical distributor (BioReliance Corp.). Experiment ID and PC ID are test identification numbers assigned by the cytotoxicity testing laboratory.
1

Range finder or definitive test
Mean OD value for all VC wells in test plate
Difference of right and left VC column of wells in the test plate4% Viability values between 0 and 50% viability; test acceptance criterion. Phase Ib used the range of 10 -50%.
5% Viability values between 50 and 100% viability; test acceptance criterion. Phase Ib used the range of 50 - 90%.
6
Calculated value from the Prism® software
7
Reference substance concentrations applied to the cells
8
Step-wise dilution factor used to determine reference substance exposure concentrations
9
Determination for whether test meets or doesn’t meet test acceptance criteria; not applied to RF tests
Shaded boxes identify values that do not meet the specific test acceptance criteria
2
3

I-110

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

Appendix I3

3T3 NRU Positive Control (SLS) Data

I-111

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

[This Page Intentionally Left Blank]

I-112

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

SLS-B1

45.2

0.157

13-Aug-02

0.187

17.06%

1

1

0.8361

SLS-B2

40.4

0.140

27-Aug-02

0.385

3.88%

3

4

0.7841

SLS-B3

38.6

0.134

27-Aug-02

0.410

0.04%

1

5

0.8376

SLS-B4

33.3

0.116

28-Aug-02

0.288

15.91%

1

2

0.9378SLS-B5

26.6

0.092

28-Aug-02

0.233

4.43%

2

4

0.8086

39.5

0.137

4-Sep-02

0.255

7.59%

1

2

0.9621

39.1

0.136

4-Sep-02

0.330

3.18%

1

2

0.9749

36.5

0.126

4-Sep-02

0.508

3.64%

1

3

0.9639

33.1

0.115

4-Sep-02

0.457

1.39%

1

4

0.9678SLS-B11

42.9

0.149

9-Sep-02

0.349

6.33%

1

2

0.9332

SLS-B12

35.3

0.123

10-Sep-02

0.326

5.41%

1

3

0.9211

SLS-B13

33.0

0.114

10-Sep-02

0.414

6.50%

1

4

0.8802

37.6

0.130

11-Sep-02

0.347

1.97%

1

3

0.9241

42.8

0.148

11-Sep-02

0.303

3.16%

1

1

0.8408

34.8

0.121

11-Sep-02

0.345

3.43%

1

2

0.9770SLS-B17 (33 ug/ml
NR)

34.3

0.119

11-Sep-02

0.389

17.94%

04

0.8377

SLS-B18

39.2

0.136

17-Sep-02

0.430

7.88%

1

2

0.9472

SLS-B19

44.7

0.155

17-Sep-02

0.422

13.89%

1

1

0.9389SLS-B20

34.8

0.121

17-Sep-02

0.445

4.12%

1

3

0.9364SLS-B21

38.6

0.134

17-Sep-02

0.402

1.66%

1

3

0.8969SLS-B22

43.5

0.151

18-Sep-02

0.394

2.94%

1

1

0.9271

Experiment ID1
3T3 Cells

Mean VC
2

OD

Difference ofright/left VC
from mean
VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

5

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9Rationale for
Unacceptability

1.47

NO

VC difference > 15%.

1.47

NO

Inadequate curve fit.

1.47

YES

1.47

NO

VC difference > 15%.

1.47

NO

Inadequate curve fit.

1.47

NO

1.47

NO

1.47

NO

1.47

NO

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

NO

1.47

YES

1.47

NO

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

ECBC
Phase Ia

SLS-B6 (25 ug/ml
NR 1 hr)SLS-B7 (50 ug/ml
NR 1 hr)SLS-B8 (25 ug/ml
NR 3 hr)SLS-B9 (50 ug/ml
NR 3 hr)SLS-B14 (33 ug/ml
NR)
SLS-B15 (33 ug/ml
NR)
SLS-B16 (33 ug/ml
NR)

I-113

100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7

R&D: 3 replicate
ODs/concentration
R&D: 3 replicate
ODs/concentration
R&D: 3 replicate
ODs/concentration
R&D: 3 replicate
ODs/concentration

Inadequate curve fit.

VC difference > 15%. No
points between 10 &50%.

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

SLS-B23

39.7

0.138

SLS-B24

45.6

SLS-B25

OD

Difference ofright/left VC
from mean

18-Sep-02

0.423

1.71%

1

2

0.92530.158

18-Sep-02

0.283

10.48%

020.8502

44.6

0.155

18-Sep-02

0.311

13.03%

1

00.8784

34.0

0.118

22-Jan-03

0.300

2.23%

1

3

0.9245

31.3

0.109

22-Jan-03

0.214

2.18%

1

4

0.8744

13.2

0.046

29-Jan-03

0.270

23.27%

230.8703

56.1

0.195

4-Feb-03

0.438

7.34%

1

2

0.8206

43.0

0.149

25-Feb-03

0.750

3.31%

1

1

0.9827

40.8

0.141

26-Feb-03

0.443

6.47%

1

1

0.9702

44.9

0.156

4-Mar-03

0.450

3.57%

1

1

0.9403ECBC-3T3-Ib-07
SLS-P12

37.3

0.129

11-Mar-03

0.568

10.54%

1

4

0.9314

ECBC-3T3-Ib-08
SLS-P13

47.2

0.164

18-Mar-03

0.517

6.58%

1

1

0.9566SLS-P1

41.4

0.144

17-Jun-03

0.409

4.01%

3

3

0.9561SLS-P2

36.1

0.125

17-Jun-03

0.452

16.14%

3

4

0.9411SLS-P3

44.5

0.154

24-Jun-03

0.427

8.32%

3

3

0.9434SLS-P4

39.5

0.137

24-Jun-03

0.460

0.14%

3

4

0.9202SLS-P5

44.2

0.153

1-Jul-03

0.619

2.60%

3

4

0.9365SLS-P6

37.8

0.131

1-Jul-03

0.563

3.20%

2

4

0.9361SLS-P7

42.1

0.146

8-Jul-03

0.485

5.48%

1

5

0.9162SLS-P8

41.5

0.144

8-Jul-03

0.630

4.97%

2

4

0.9461SLS-P9

40.3

0.140

15-Jul-03

0.450

6.36%

1

5

0.9250Experiment ID1
3T3 Cells

Mean VC
2

VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

5

R2 6

Phase Ib
ECBC-3T3-Ib-01
SLS-P1
ECBC-3T3-Ib-01
SLS-P2ECBC-3T3-Ib-02
SLS-P3ECBC-3T3-Ib-03
SLS-P4
ECBC-3T3-Ib-04
SLS-P5ECBC-3T3-Ib-05
SLS-P7
ECBC-3T3-Ib-06
SLS-P9Exposure
Concentrations

Dilution

Acceptable

Factor8

Tests9

1.47

YES

1.47

NO

1.47

NO

1.47

YES

1.47

YES

1.47

NO

VC difference > 15%;
IC50 out of range

1.47

NO

IC50 out of range

1.47

YES

1.47

YES

1.47

YES

80.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

80.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

1.21

YES

1.21

NO

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

(ug/mL)7

100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7

Rationale for
Unacceptability

No points between 10 &50%.
No points between 50 &90%.

Phase II

I-114

80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

% VC difference > 15

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

SLS-P10

35.2

0.122

SLS-P11

38.7

SLS-P12OD

Difference ofright/left VC
from mean

15-Jul-03

0.629

4.12%

3

3

0.97510.134

22-Jul-03

0.488

3.70%

2

4

0.9769

39.1

0.136

22-Jul-03

0.554

1.92%

3

4

0.9760SLS-P13

41.6

0.144

29-Jul-03

0.700

0.18%

3

4

0.9440SLS-P14

40.7

0.141

29-Jul-03

0.730

3.11%

3

4

0.9663SLS-P15

43.2

0.150

5-Aug-03

0.649

0.59%

2

4

0.9591SLS-P16

44.1

0.153

6-Aug-03

0.276

3.23%

4

4

0.9790SLS-P17

37.3

0.129

31-Aug-03

0.710

5.38%

2

4

0.9482SLS-P18 sealer

32.4

0.112

31-Aug-03

0.545

4.39%

3

3

0.8897SLS-P19

41.4

0.144

1-Sep-03

0.613

2.00%

3

3

0.9625SLS-P20

38.4

0.133

9-Sep-03

0.350

0.88%

3

4

0.9350SLS-P21

43.0

0.149

23-Sep-03

0.650

3.04%

2

4

0.9637SLS-P22

41.2

0.143

29-Oct-03

0.406

1.21%

3

4

0.9289SLS-P23

41.8

0.145

4-Nov-03

0.378

8.20%

4

4

0.9577SLS-P24

44.7

0.155

5-Nov-03

0.333

3.43%

4

3

0.9518SLS-P1

37.5

0.130

13-Jan-04

0.355

3.82%

3

3

0.8860SLS-P2

34.9

0.121

13-Jan-04

0.442

8.96%

3

3

SLS-P3

40.8

0.142

21-Jan-04

0.461

4.62%

2

SLS-P4

29.4

0.102

21-Jan-04

0.511

3.62%

SLS-P5

43.7

0.151

27-Jan-04

0.299

2.09%

Experiment ID1
3T3 Cells

Mean VC
2

VC

3

Number of
PointsNumber of
PointsExposure
Concentrations

Dilution

Acceptable

Factor8

Tests9

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

NO

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

80.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.964180.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

3

0.975180.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

2

3

0.967280.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

NO

3

4

0.976680.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0 - 50 %4

50 - 100 %

5

R2 6

Phase III

I-115

(ug/mL)7

80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1
80, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

Rationale for
Unacceptability

R&D

IC50 out of range

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

SLS-P6

42.8

0.148

27-Jan-04

0.384

1.89%

2

3

0.9558SLS-P7

43.1

0.149

3-Feb-04

0.378

6.60%

4

4

SLS-P8

38.5

0.134

3-Feb-04

0.379

7.38%

2

SLS-P9

38.5

0.134

10-Feb-04

0.375

8.36%

SLS-P10

35.9

0.124

10-Feb-04

0.374

SLS-P11

40.5

0.140

24-Feb-04

SLS-P12

37.3

0.129

SLS-P13

39.3

SLS-P14

Mean VC

Difference ofright/left VC
from mean

Number of
PointsNumber of
PointsExposure
Concentrations

Dilution

Acceptable

Factor8

Tests980.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.977980.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

4

0.966280.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

3

4

0.931580.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

3.25%

3

4

0.964080.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.297

2.83%

3

4

0.955480.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

24-Feb-04

0.334

0.02%

2

3

0.966580.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.136

25-Feb-04

0.385

0.30%

3

4

0.962480.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

37.9

0.132

25-Feb-04

0.422

5.43%

4

4

0.956180.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P15

44.7

0.155

2-Mar-04

0.526

3.85%

2

5

0.984080.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P16

41.9

0.145

2-Mar-04

0.605

0.29%

2

4

0.973980.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P17

38.9

0.135

3-Mar-04

0.453

7.56%

3

4

0.949680.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P18

35.5

0.123

3-Mar-04

0.522

0.59%

3

3

0.940480.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P19

41.3

0.143

9-Mar-04

0.539

7.29%

3

4

0.958680.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P20

37.7

0.131

9-Mar-04

0.535

0.73%

2

4

0.973180.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P21

42.7

0.148

16-Mar-04

0.563

0.59%

2

3

0.984980.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

Experiment ID1
3T3 Cells

2

OD

VC

3

0 - 50 %4

50 - 100 %

I-116

5

R2 6

(ug/mL)7

Rationale for
Unacceptability

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

SLS-P22

38.9

0.135

16-Mar-04

0.548

0.03%

3

4

0.9759SLS-P23

43.4

0.150

23-Mar-04

0.632

3.43%

3

4

SLS-P24

42.1

0.146

23-Mar-04

0.707

2.19%

2

SLS-P25

52.7

0.183

30-Mar-04

0.667

2.75%

SLS-P26

43.0

0.149

30-Mar-04

0.623

SLS-P27

45.9

0.159

6-Apr-04

SLS-P28

43.9

0.152

SLS-P29

46.3

SLS-P30

Mean VC

Difference ofright/left VC
from mean

Number of
PointsNumber of
PointsExposure
Concentrations

Dilution

Acceptable

Factor8

Tests980.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.971480.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

4

0.985880.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

2

5

0.966180.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

NO

0.88%

3

3

0.955680.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.521

2.17%

2

4

0.976680.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

6-Apr-04

0.614

1.41%

3

4

0.978580.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.161

13-Apr-04

0.477

4.37%

3

5

0.957980.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

43.1

0.149

13-Apr-04

0.609

1.67%

1

5

0.942080.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P31

44.1

0.153

20-Apr-04

0.473

5.99%

1

5

0.945680.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P32

39.4

0.136

20-Apr-04

0.481

2.79%

3

4

0.976280.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P33

44.8

0.155

27-Apr-04

0.434

8.49%

2

4

0.954880.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P34

42.1

0.146

27-Apr-04

0.448

8.96%

3

4

0.962480.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P35

49.3

0.171

4-May-04

0.611

1.23%

3

4

0.982880.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P36

42.4

0.147

4-May-04

0.680

4.09%

2

4

0.962680.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P37

44.8

0.155

11-May-04

0.588

2.31%

2

5

0.971380.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

Experiment ID1
3T3 Cells

2

OD

VC

3

0 - 50 %4

50 - 100 %

I-117

5

R2 6

(ug/mL)7

Rationale for
Unacceptability

IC50 out of range

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

SLS-P38

43.2

0.150

11-May-04

0.682

3.69%

3

4

0.9645SLS-P39

37.8

0.131

18-May-04

0.418

7.64%

3

4

SLS-P40

37.0

0.128

18-May-04

0.408

1.70%

2

SLS-P41

45.0

0.156

25-May-04

0.506

2.77%

SLS-P42

42.1

0.146

25-May-04

0.575

SLS-P43

42.8

0.148

15-Jun-04

SLS-P44

42.2

0.146

SLS-P45

45.9

SLS-P46

Mean VC

Difference ofright/left VC
from mean

Number of
PointsNumber of
PointsExposure
Concentrations

Dilution

Acceptable

Factor8

Tests980.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.957880.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

4

0.954180.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

2

5

0.977280.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

1.65%

2

4

0.973380.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.698

6.20%

3

4

0.968980.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

15-Jun-04

0.695

8.92%

4

4

0.964880.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.159

22-Jun-04

0.561

1.81%

3

5

0.971880.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

46.1

0.160

22-Jun-04

0.650

1.33%

2

5

0.977280.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P47

40.2

0.139

29-Jun-04

0.421

8.18%

4

4

0.960380.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P48

37.6

0.130

29-Jun-04

0.468

10.36%

3

4

0.951280.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P49

40.2

0.139

13-Jul-04

0.325

12.65%

4

4

0.952480.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P50NANA

20-Jul-04

0.414

4.06%

1

1

NA

80.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

NO

IC50 out of range

SLS-P51NANA

20-Jul-04

0.414

16.20%

15NA80.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

NO

IC50 out of range; % VC
difference > 15;

SLS-P52NANA

27-Jul-04

0.471

14.02%

31

NA80.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

NO

IC50 out of range

SLS-P53NANA

27-Jul-04

0.555

8.43%

5

1

NA

80.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

NO

IC50 out of range

Experiment ID1
3T3 Cells

2

OD

VC

3

0 - 50 %4

50 - 100 %

I-118

5

R2 6

(ug/mL)7

Rationale for
Unacceptability

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

SLS-P54

44.1

0.153

10-Aug-04

0.797

1.55%

3

5

0.9653SLS-P55

45.1

0.156

10-Aug-04

0.658

5.46%

3

4

SLS-P56NANA

17-Aug-04

0.372

34.25%

2

SLS-P57

40.4

0.140

17-Aug-04

0.523

6.59%

SLS-P58

47.1

0.163

24-Aug-04

0.477

SLS-P59

40.6

0.141

24-Aug-04

SLS-P60

53.7

0.186

SLS-P61

60.1

SLS-P62

Mean VC

Difference ofright/left VC
from mean

Number of
PointsNumber of
PointsExposure
Concentrations

Dilution

Acceptable

Factor8

Tests980.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.957080.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

5

NA

80.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

NO

4

4

0.957980.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

4.19%

2

5

0.921580.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.462

7.30%

4

4

0.958980.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

31-Aug-04

0.754

3.56%

2

6

0.845780.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

NO

IC50 out of range

0.208

31-Aug-04

0.726

3.36%

2

6

0.920380.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

NO

IC50 out of range

43.4

0.150

14-Sep-04

0.635

5.64%

2

5

0.900680.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P63

41.4

0.144

14-Sep-04

0.625

6.52%

2

5

0.961480.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P64

37.4

0.130

28-Sep-04

0.473

6.10%

3

4

0.940080.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P65

38.8

0.135

28-Sep-04

0.394

4.91%

3

4

0.968180.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P66

37.0

0.128

5-Oct-04

0.520

3.86%

2

4

0.949580.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P67

33.4

0.116

5-Oct-04

0.554

4.23%

3

3

0.960380.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P68

42.7

0.148

19-Oct-04

0.472

0.62%

2

5

0.963280.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

SLS-P69

43.6

0.151

19-Oct-04

0.349

0.38%

1

5

0.965980.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

Experiment ID1
3T3 Cells

2

OD

VC

3

0 - 50 %4

50 - 100 %

I-119

5

R2 6

(ug/mL)7

Rationale for
Unacceptability

PC failed; % VC
difference > 15

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

SLS-P70

39.7

0.138

26-Oct-04

0.468

3.33%

3

4

0.9687SLS-P71

44.9

0.156

27-Oct-04

0.504

3.38%

2

3

SLS-P72

45.8

0.159

2-Nov-04

0.517

1.76%

3

SLS-P73

45.7

0.158

2-Nov-04

0.517

0.08%

SLS-P74

46.6

0.161

16-Nov-04

0.510

B1(1a/3T3/DF1/FA
L/SLS)

53.9

0.187

3-Sep-02

B2(1a/3T3/DF2/FA
L/SLS)

NA

NA

50.8

Experiment ID1
3T3 Cells

Mean VC

Difference ofright/left VC
from mean

Number of
PointsNumber of
PointsExposure
Concentrations

Dilution

Acceptable

Factor8

Tests980.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.941680.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

5

0.940580.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

2

5

0.968580.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.42%

2

5

0.946180.0, 66.1, 54.6, 45.2,
37.3, 30.8, 25.5, 21.1

1.21

YES

0.402

11.18%

01

0.9577400, 200, 100, 50, 25,
12.5, 6.3, 3.1

2.00

NO

3-Sep-02

0.419

15.17%

1

1

0.7691400, 200, 100, 50, 25,
12.5, 6.3, 3.1

2.00

NO

0.176

3-Sep-02

0.420

3.73%

0

1

0.9583

2.00

NO

44.4

0.154

3-Sep-02

0.490

2.60%

1

1

0.9800

2.00

NO

51.0

0.177

3-Sep-02

0.503

8.01%

0

1

0.9812

2.00

NO

49.8

0.173

3-Sep-02

0.441

6.29%

1

0

0.9517

2.00

NO

54.2

0.188

4-Sep-02

0.408

5.64%

0

1

0.8134

2.00

NO

50.2

0.174

4-Sep-02

0.337

34.90%

01

0.8010

2.00

NO

52.1

0.181

4-Sep-02

0.484

0.79%

0

1

0.9657

2.00

NO

52.5

0.182

4-Sep-02

0.459

7.20%

0

1

0.9389

2.00

NO

46.4

0.161

4-Sep-02

0.509

6.94%

0

3

0.9422

2.00

NO

23.0

0.080

18-Sep-02

0.900

3.51%

1

3

0.8277

1.47

NO

Inadequate curve fit.

46.7

0.162

18-Sep-02

0.547

7.61%

1

0

0.9736

1.47

NONo point between 50 &90% viability

2

OD

VC

3

0 - 50 %4

50 - 100 %

5

R2 6

(ug/mL)7

Rationale for
Unacceptability

FAL
Phase Ia

B3(1a/3T3/DF3/FA
L/SLS)
B4(1a/3T3/DF4/FA
L/SLS)
B5(1a/3T3/DF5/FA
L/SLS)
B6(1a/3T3/DF6/FA
L/SLS)
B7(1a/3T3/DF7/FA
L/SLS)
B8(1a/3T3/DF8/FA
L/SLS)
B9(1a/3T3/DF9/FA
L/SLS)
B10(1a/3T3/DF10/
FAL/SLS)
B11(1a/3T3/DF11/
FAL/SLS)
1a/3T3/DF14/FAL/
SLS
1a/3T3/DF15/FAL/
SLS

I-120

400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
150, 102, 69.4, 47.2,
32.1, 21.9, 14.9, 10.1
150, 102, 69.4, 47.2,
32.1, 21.9, 14.9, 10.1

No point between 10 &50% viability
Bad values for 6.3 ug/mL
wells. VC difference >15%.
No point between 10 &50% viability
No point between 10 &50% viability
No point between 50 &90% viability.
No point between 10 &50% viability
VC difference > 15%. No
point between 10 & 50%
viability
No point between 10 &50% viability
No point between 10 &50% viability
No point between 10 &50% viability

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
Experiment ID1
3T3 Cells

IC50(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

1a/3T3/DF16/FAL/
SLS

42.4

0.147

18-Sep-02

0.590

21.70%

1

00.9833

46.6

0.161

18-Sep-02

0.442

4.00%

1

0

0.8646

22.6

0.078

18-Sep-02

0.920

4.36%

2

3

0.8319

23.1

0.080

18-Sep-02

0.936

4.30%

1

3

0.8350

48.0

0.166

22-Oct-02

0.488

9.05%

0

1

0.9570

50.7

0.176

22-Oct-02

0.579

10.46%

03

0.8773

42.0

0.146

23-Oct-02

0.768

6.31%

1

3

0.9433

46.8

0.162

23-Oct-02

0.795

2.60%

0

4

0.9321

49.0

0.170

23-Oct-02

0.784

0.24%

0

1

0.9725

48.9

0.169

30-Oct-02

0.676

2.03%

1

2

0.9532

48.0

0.166

30-Oct-02

0.636

4.77%

1

2

0.9788

48.7

0.169

30-Oct-02

0.684

2.23%

1

2

0.9811

53.0

0.184

30-Oct-02

0.545

4.83%

1

1

0.8486

50.8

0.176

31-Oct-02

0.660

1.09%

1

3

0.9261

51.4

0.178

31-Oct-02

0.612

9.54%

1

4

0.9057

51.3

0.178

31-Oct-02

0.630

0.19%

1

2

0.9749

52.5

0.182

31-Oct-02

0.669

6.97%

1

1

0.9879

47.1

0.163

5-Nov-02

0.581

3.57%

1

3

0.9757

46.8

0.162

5-Nov-02

0.564

11.34%

1

3

0.9468

36.6

0.127

6-Nov-02

0.649

6.40%

1

3

0.8929

44.8

0.155

6-Nov-02

0.605

1.06%

2

3

0.9258

40.7

0.141

12-Nov-02

0.618

0.88%

1

3

0.9756

42.3

0.147

12-Nov-02

0.665

0.86%

1

3

0.9599

1a/3T3/DF17/FAL/
SLS
1a/3T3/DF18/FAL/
SLS
1a/3T3/DF19/FAL/
SLS
1a/3T3/DF28/FAL/
SLS
1a/3T3/DF29/FAL/
SLS
1a/3T3/DF30/FAL/
SLS
1a/3T3/DF31/FAL/
SLS
1a/3T3/DF32/FAL/
SLS
1a3T3DF33FALSLS
1a3T3DF34FALSLS
1a3T3DF35FALSLS
1a3T3DF36FALSLS
1a3T3DF37FALSLS
1a3T3DF38FALSLS+
1a3T3DF39FALSLS
1a3T3DF40FALSLS
1a3T3DF41FALSLS+
1a3T3DF42FALSLS
1a3T3DF43FALSLS
1a3T3DF44FALSLS+
1a3T3DF45FALSLS
1a3T3DF46FALSLS

Mean VC
2

OD

Difference ofright/left VC
from mean
VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

I-121

5

R2 6

Exposure
Concentrations(ug/mL)7

150, 102, 69.4, 47.2,
32.1, 21.9, 14.9, 10.1
150, 102, 69.4, 47.2,
32.1, 21.9, 14.9, 10.1
150, 102, 69.4, 47.2,
32.1, 21.9, 14.9, 10.1
150, 102, 69.4, 47.2,
32.1, 21.9, 14.9, 10.1
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 68.0, 46.3, 31.5,
21.4, 14.6, 9.9, 6.7
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 76.9, 59.2, 45.5,
35, 26.9, 20.7, 15.9
100, 76.9, 59.2, 45.5,
35, 26.9, 20.7, 15.9
100, 76.9, 59.2, 45.5,
35, 26.9, 20.7, 15.9
100, 76.9, 59.2, 45.5,
35, 26.9, 20.7, 15.9
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3

Dilution

Acceptable

Factor8

Tests9

1.47

NO

1.47

NO

1.47

NO

Inadequate curve fit.

1.47

NO

Inadequate curve fit.

1.47

NO

1.47

NO

1.47

YES

1.47

NO

1.47

NO

1.21

YES

1.21

YES

1.30

YES

1.30

NO

1.30

YES

1.30

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

Rationale for
Unacceptability
VC difference > 15%. No
point between 50 & 90%
viability.
No point between 50 &90% viability

No point between 10 &50% viability
No point between 10 &50% viability
No point between 10 &50% viability
No point between 10 &50% viability

Inadequate curve fit.

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

42.1

0.146

37.9

OD

Difference ofright/left VC
from mean

12-Nov-02

0.674

3.71%

1

2

0.98110.131

13-Nov-02

0.531

15.94%

2

3

0.8139

38.7

0.134

13-Nov-02

0.561

14.96%

1

3

0.8648

40.6

0.141

13-Nov-02

0.533

11.42%

2

3

0.9179

40.3

0.140

20-Nov-02

0.689

0.29%

1

3

0.9478

42.5

0.147

20-Nov-02

0.780

1.37%

1

3

0.9682

39.9

0.138

20-Nov-02

0.692

7.30%

2

3

0.9403

1b3T3CRT1FALSLS

34.4

0.119

4-Dec-02

0.618

16.76%

3

2

0.84791b3T3CTR2FALSLS

48.8

0.169

10-Dec-02

0.545

6.73%

1

2

0.94091b3T3CTRFALSLS

24.5

0.085

17-Dec-02

0.453

1.97%

1

0

0.8653

43.5

0.151

7-Jan-03

0.597

2.23%

1

2

0.9631

50.9

0.176

8-Jan-03

0.271

14.37%

1

1

0.9136

43.2

0.150

14-Jan-03

0.625

3.68%

1

3

0.9163

32.4

0.112

14-Jan-03

0.417

5.55%

1

2

0.9377

1b3T3CRTFALSL
S

70.1

0.243

15-Jan-03

0.432

2.31%

1

2

0.9000

1b3T3CRTFALSL
S

35.3

0.122

21-Jan-03

0.651

1.86%

1

2

0.9727

Experiment ID1
3T3 Cells
1a3T3DF47FALSLS
1a3T3DF48FALSLS
1a3T3DF49FALSLS
1a3T3DF50FALSLS
1a3T3DF51FALSLS
1a3T3DF52FALSLS
1a3T3DF53FALSLS

Mean VC
2

VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

5

R2 6

Phase Ib

1b3T3CTRFALSLS
1b3T3CTRFALSLS
1b3T3CRTFALSLS
1b3T3CRT2FALSLS

1b3T3CRTFALSL
S

38.1

0.132

28-Jan-03

0.181

17.95%

1

00.9716

1b3T3CRTFALSL
S

58.7

0.204

29-Jan-03

0.646

8.07%

0

2

0.9573

1b3T3CRTFALSL
S

44.3

0.154

4-Feb-03

0.662

0.79%

1

1

0.9848

I-122

Exposure
Concentrations(ug/mL)7

100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3

100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
82.6, 67.7, 56.0, 42.29,
38.25, 31.61,
26.13,21.59
100.00, 82.64, 68.30,
56.45, 46.65, 38.55,
31.86, 26.33
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 68.02, 46.28,
31.48, 21.42, 14.57,
9.91, 6.74
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3

Dilution

Acceptable

Factor8

Tests9

1.21

YES

1.21

NO

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

NO

1.21

YES

1.21

NO

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

1.21

Rationale for
Unacceptability

VC difference > 15%.

VC difference > 15%

IC50 out of range; no points
between 50 & 90% viability

NR crystals in plate;
stopped after 1 h

IC50 out of rangeNO

NR crystals in plate;
stopped after 1 h; VC
difference > 15%; nopoint between 50 & 90%
viability

NO

No point between 10 &50% viability

YES

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
Experiment ID1
3T3 Cells

IC50(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

1b3T3CRTFALSL
S

36.8

0.128

5-Feb-03

0.566

1.65%

1

1

0.9867

1b3T3CRTFALSL
S

48.0

0.166

26-Feb-03

0.310

15.17%

1

2

0.9457A1SLS190603

49.1

0.170

17-Jun-03

1.031

2.49%

2

5

0.7802

54.6

0.189

18-Jun-03

0.684

6.26%

4

3

0.9851

50.8

0.176

24-Jun-03

0.483

3.45%

3

4

0.9788

50.7

0.176

25-Jun-03

0.564

0.19%

2

2

0.9878

57.5

0.199

1-Jul-03

0.516

7.13%

1

4

0.9913

55.8

0.193

2-Jul-03

0.562

4.86%

4

3

0.9788

52.5

0.182

8-Jul-03

0.640

0.86%

2

3

0.9794

50.6

0.175

9-Jul-03

0.533

2.92%

2

3

0.9869

50.2

0.174

15-Jul-03

0.708

0.81%

2

3

0.9905

43.2

0.150

16-Jul-03

0.502

5.68%

2

3

0.9763

47.6

0.165

23-Jul-03

0.435

5.81%

1

2

0.9633

30.5

0.106

5-Aug-03

0.725

0.11%

7

1

0.9204

36.2

0.126

6-Aug-03

0.463

1.17%

5

3

0.7811

39.4

0.137

10-Sep-03

0.768

4.53%

3

4

0.8322

45.2

0.157

16-Sep-03

0.401

0.69%

4

3

0.9582

45.0

0.156

17-Sep-03

0.377

0.62%

1

2

0.9790

35.7

0.124

23-Sep-03

0.379

4.55%

3

2

0.9738

51.2

0.178

1-Oct-03

0.596

5.23%

2

4

0.9344

37.5

0.130

15-Oct-03

0.398

9.90%

3

2

0.9763

49.8

0.173

28-Oct-03

0.310

12.63%

4

1

0.9702

Mean VC
2

OD

Difference ofright/left VC
from mean
VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

5

R2 6

Phase II

FAL.3T3.SLS2.A1.
200603
FAL.3T3.SLS.A2.2
6.06.03
FAL.3T3.SLS.A2.2
7.06.03
FAL.3T3.SLS.B1.0
3.07.03
FAL.3T3.SLS.04.0
7.03
FAL.3T3.SLS.10.0
7.03
FAL.3T3.SLS.11.0
7.03
FAL.3T3.SLS.17.0
7.03
FAL.3T3.SLS.18.0
7.03
FAL.3T3.SLS.25.0
7.03
FAL.3T3.SLS.0708
03
FAL.3T3.SLS.0808
03
FAL.3T3.SLS.1209
03
FAL.3T3.SLS.1809
03
FAL.3T3.SLS.1909
03
FAL.3T3.SLS.2509
03
FAL.3T3.SLS.0310
03
FAL.3T3.SLS.1710
03
FAL.3T3.SLS.3010
03

I-123

Exposure
Concentrations

Dilution

Acceptable

Factor8

Tests9100, 82.645, 68.301,
56.447, 46.651,
38.554, 31.863, 26.333

1.21

YES

100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3

1.21

YES

1.21

NO

1.21

YES

1.21

YES

1.21

YES

1.21

YES

IC50 out of range

1.21

NO

IC50 out of range

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

NO

IC50 out of range

1.21

NO

low r2

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

(ug/mL)7

100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4

Rationale for
Unacceptability

r2 too low

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
Experiment ID1
3T3 Cells

IC50(ug/mL)

IC50(mM)

FAL.3T3.SLS.3010
03 (should be311003)

Reference
Substance
Application
Date

39.6

0.137

29-Oct-03

0.313

8.62%

3

3

0.9886

55.0

0.191

6-Jan-04

0.615

0.20%

4

4

0.9771

53.3

0.185

7-Jan-04

0.592

7.04%

4

4

0.9727

67.0

0.232

13-Jan-04

0.841

1.98%

2

6

0.8901

30.4

0.105

14-Jan-04

1.161

0.39%

6

2

0.8932

35.7

0.124

20-Jan-04

0.382

7.11%

3

2

0.9685

30.8

0.107

21-Jan-04

0.792

2.31%

2

2

0.9194

41.4

0.144

27-Jan-04

0.467

0.43%

5

3

0.9671

44.3

0.153

28-Jan-04

0.453

1.44%

4

4

0.9721

26.9

0.093

3-Feb-04

0.417

2.14%

4

0

0.9317

38.8

0.135

4-Feb-04

0.427

4.23%

5

3

0.9136

47.9

0.166

23-Feb-04

0.637

2.29%

3

4

0.9829

49.8

0.173

15-Mar-04

0.356

5.91%

4

3

0.9831

44.0

0.152

30-Mar-04

0.404

1.46%

2

2

0.9593

42.3

0.147

27-Apr-04

0.310

2.34%

3

5

0.9881

31.3

0.108

28-Apr-04

0.249

4.22%

6

1

0.9874

40.7

0.141

4-May-04

0.320

9.70%

2

3

0.9897

40.2

0.139

5-May-04

0.313

0.03%

3

3

0.9865

45.2

0.157

18-May-04

0.422

3.24%

2

3

0.9797

32.7

0.114

19-May-04

0.337

0.94%

2

2

0.9720

44.2

0.153

25-May-04

0.406

5.89%

3

3

0.9466

40.6

0.141

22-Jun-04

0.434

3.69%

4

3

0.9826

Mean VC
2

OD

Difference ofright/left VC
from mean
VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

5

R2 6

Exposure
Concentrations(ug/mL)7

100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4

Dilution

Acceptable

Factor8

Tests9

1.21

YES

1.21

YES

1.21

YES

1.21

NO

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

NO

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

Rationale for
Unacceptability

Phase IIIFAL.3T3.SLS.0801
04
FAL.3T3.SLS.09/0
1/04
FAL.3T3.SLS.15/0
1/04
FAL.3T3.SLS.16/0
1/04
FAL.3T3.SLS.22/0
1/04
FAL3T3.23-01-04
FAL3T3.SLS.2901-04
FAL.3T3.SLS.29/0
1/04
FAL.3T3.SLS.5/02/
04
FAL.3T3.SLS.06/0
2/04
FAL3T3.SLS.25.02.04
FAL.3T3.SLS.17/0
3/04
FAL.3T3.SLS.01/0
4/04
FAL.3T3.SLS.29/0
4/04
FAL.3T3.SLS.30/0
4/04
FAL.3T3.SLS.06/0
5/04
FAL.3T3.SLS.07/0
5/04
FAL.3T3.SLS.20/0
5/04
FAL.3T3.SLS.21.0
5.04
FAL.3T3.SLS.27/0
5/04
FAL.3T3.SLS.24.0
6.04

I-124

100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.5, 56.5,
46.7, 38.6, 31.9, 26.4
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3

IC50 out of range

recalculated values:
IC50 out of range; nopoints between 50-100

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

39.7

0.138

40.3

OD

Difference ofright/left VC
from mean

6-Jul-04

0.324

7.16%

2

3

0.96590.140

7-Jul-04

0.408

2.92%

2

3

0.9765

35.6

0.124

14-Jul-04

0.402

5.43%

2

2

0.9676

40.3

0.140

15-Sep-04

0.411

1.89%

3

3

0.979640.7

0.14126

21-Sep-04

0.333

2.60%

2

3

0.971842.9

0.14860

12-Oct-04

0.320

5.42%

3

2

0.990139.9

0.13836

2-Nov-04

0.259

2.51%

4

3

0.9816B1

NANA

24-Aug-02

0.306

17.18%

1

00.5129

B2

53.7

0.186

24-Aug-02

0.280

38.89%

1

00.3966

B3

34.7

0.120

25-Aug-02

0.452

1.92%

0

1

0.9877

B4

34.2

0.119

25-Aug-02

0.428

4.07%

0

3

0.9664

B5

35.9

0.125

26-Aug-02

0.409

3.71%

0

1

0.9872

B6

39.0

0.135

26-Aug-02

0.382

0.09%

0

0

0.9649

B7

35.7

0.124

27-Aug-02

0.302

2.98%

0

2

0.9773

B8

36.1

0.125

27-Aug-02

0.299

6.86%

0

1

0.9792

B9

41.5

0.144

29-Aug-02

0.342

6.02%

1

1

0.9831

B10

45.1

0.156

29-Aug-02

0.358

1.51%

1

1

0.9664

B11

43.8

0.152

30-Aug-02

0.366

4.26%

1

0

0.9936

B12

44.6

0.155

30-Aug-02

0.359

0.95%

1

1

0.9864

B13

44.5

0.154

4-Sep-02

0.538

0.37%

1

1

0.9799Experiment ID1
3T3 Cells
FAL.3T3.SLS.08.0
7.04
FAL.3T3.SLS.09.0
7.04
FAL.3T3.SLS.16.0
7.04
FAL.3T3.SLS.17.0
9.04
FAL.3T3.SLS.23.0
9.04
FAL.3T3.SLS.14.1
0.04
FAL.3T3.SLS.04.1
1.04

Mean VC
2

VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

5

R2 6

Exposure
Concentrations

Dilution

Acceptable

Factor8

Tests9

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

1.21

YES

100, 56.2, 31.6, 17.8,
10, 5.63, 3.17, 1.78

1.78

NO

100, 56.2, 31.6, 17.8,
10, 5.63, 3.17, 1.78

1.78

NO

1.78

NO

1.78

NO

1.78

NO

1.78

NO

1.78

NO

1.78

NO

1.47

YES

1.47

YES

1.47

NO

1.47

YES

1.47

YES

(ug/mL)7

100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3
100, 82.6, 68.3, 56.4,
46.7, 38.6, 31.9, 26.3

Rationale for
Unacceptability

IIVS
Phase Ia

I-125

100, 56.2, 31.6, 17.8,
10, 5.63, 3.17, 1.78
100, 56.2, 31.6, 17.8,
10, 5.63, 3.17, 1.78
100, 56.2, 31.6, 17.8,
10, 5.63, 3.17, 1.78
100, 56.2, 31.6, 17.8,
10, 5.63, 3.17, 1.78
100, 56.2, 31.6, 17.8,
10, 5.63, 3.17, 1.78
100, 56.2, 31.6, 17.8,
10, 5.63, 3.17, 1.78
100, 68.1, 46.4, 31.6,
21.5, 14.7, 10, 6.8100, 68.1, 46.4, 31.6,
21.5, 14.7, 10, 6.8100, 68.1, 46.4, 31.6,
21.5, 14.7, 10, 6.8100, 68.1, 46.4, 31.6,
21.5, 14.7, 10, 6.8100, 68.1, 46.4, 31.6,
21.5, 14.7, 10, 6.8

VC difference > 15%. No
points between 50 &90% viability.
VC difference > 15%. No
points between 50 &90% viability.
No points between 10 &50% viability.
No points between 10 &50% viability.
No points between 10 &50% viability.
No points between 10 &90% viability.
No points between 10 &50% viability.
No points between 10 &50% viability.

No points between 50 &90% viability.

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

B14

43.9

0.152

B15

37.8

B16

OD

Difference ofright/left VC
from mean

4-Sep-02

0.491

6.43%

1

1

0.98690.131

5-Sep-02

0.357

9.90%

1

1

0.9906

40.4

0.140

5-Sep-02

0.336

10.55%

1

1

0.9832

B17

39.7

0.138

6-Sep-02

0.464

2.31%

1

2

0.9780

B18

38.1

0.132

6-Sep-02

0.426

11.25%

1

1

0.9910

B19

36.7

0.127

7-Sep-02

0.378

4.90%

1

1

0.9928

B20

36.5

0.127

7-Sep-02

0.354

12.49%

1

1

0.9954

B21

46.7

0.162

8-Sep-02

0.453

0.44%

0

2

0.9800

B22

41.8

0.145

8-Sep-02

0.439

0.63%

1

1

0.9802A1 Preliminary

41.1

0.143

15-Jan-03

0.389

8.42%

1

1

0.9890

B1

43.5

0.151

22-Jan-03

0.569

6.41%

1

1

0.9822

B2

44.8

0.155

29-Jan-03

0.514

2.88%

1

1

0.9830

B3

38.5

0.133

5-Feb-03

0.519

1.00%

1

1

0.9854

B4

49.4

0.171

12-Feb-03

0.548

10.23%

02

0.9770

B5

41.9

0.145

26-Feb-03

0.507

5.41%

1

1

0.9747

A1

41.3

0.143

23-Jul-03

0.546

3.97%

1

3

0.9902

B1

39.6

0.137

28-Jul-03

0.375

1.11%

1

5

0.9559

B2

38.8

0.135

29-Jul-03

0.529

5.36%

2

5

0.9711

B3

30.0

0.104

30-Jul-03

0.527

1.74%

1

4

0.9854

B4

42.6

0.148

13-Aug-03

0.483

7.35%

1

5

0.9891

B5

39.1

0.136

16-Sep-03

0.510

6.44%

3

5

0.9568

B6

38.2

0.132

23-Sep-03

0.433

2.75%

1

5

0.9668Experiment ID1
3T3 Cells

Mean VC
2

VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

5

R2 6

Phase Ib

Phase II

I-126

Exposure
Concentrations

Dilution

Acceptable

Factor8

Tests9

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

NO

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

100, 68.1, 46.4, 31.6,
21.5, 14.7, 10.0, 6.81

1.47

NO

100, 68.1, 46.4, 31.6,
21.5, 14.7, 10.0, 6.81

1.47

YES

1.50

YES

1.40

YES

1.40

YES

1.40

NO

1.40

YES

1.40

YES

1.40

YES

(ug/mL)7

100, 68.1, 46.4, 31.6,
21.5, 14.7, 10, 6.8100, 68.1, 46.4, 31.6,
21.5, 14.7, 10, 6.8100, 68.1, 46.4, 31.6,
21.5, 14.7, 10, 6.8100, 68.1, 46.4, 31.6,
21.5, 14.7, 10, 6.8100, 68.1, 46.4, 31.6,
21.5, 14.7, 10, 6.8100, 68.1, 46.4, 31.6,
21.5, 14.7, 10, 6.8100, 68.1, 46.4, 31.6,
21.5, 14.7, 10, 6.8100, 68.1, 46.4, 31.6,
21.5, 14.7, 10, 6.8100, 68.1, 46.4, 31.6,
21.5, 14.7, 10, 6.8100, 68.1, 46.4, 31.6,
21.5, 14.7, 10.0, 6.81
100, 68.1, 46.4, 31.6,
21.5, 14.7, 10.0, 6.81
100, 68.1, 46.4, 31.6,
21.5, 14.7, 10.0, 6.81
100, 68.1, 46.4, 31.6,
21.5, 14.7, 10.0, 6.81

100, 66.7, 44.4, 29.6,
19.8, 13.2, 8.78, 5.85
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.5100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.5100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.5100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.5100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.5100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.5

Rationale for
Unacceptability

No points between 10 &50% viability.

No points between 10
and 50%; IC50 out of
range

IC50 out of range

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

B7

38.9

0.135

B8

45.2

A1

OD

Difference ofright/left VC
from mean

24-Sep-03

0.479

2.49%

1

5

0.97100.157

1-Oct-03

0.547

3.52%

1

5

0.9798

42.1

0.146

3-Feb-04

0.429

3.86%

2

5

0.9691

A2

42.4

0.147

10-Feb-04

0.494

0.10%

2

4

0.9874

A3

41.0

0.142

17-Feb-04

0.458

1.06%

1

4

0.9858

A4

37.2

0.129

9-Mar-04

0.417

7.26%

1

4

0.9893

A5

33.0

0.114

23-Mar-04

0.346

1.01%

2

3

0.9758

B1

45.9

0.159

26-Jul-04

0.399

0.81%

1

5

0.9709

B2

44.5

0.154

27-Jul-04

0.379

5.70%

3

4

B3

40.1

0.139

28-Jul-04

0.344

14.50%

2

B4

42.2

0.146

23-Aug-04

0.493

3.37%

B5

47.2

0.164

24-Aug-04

0.485

B6

46.1

0.160

28-Sep-04

B7

40.7

0.141

B8

41.2

B9

Mean VC

Number of
PointsNumber of
PointsExposure
Concentrations

Dilution

Acceptable

Factor8

Tests9

1.40

YES

1.40

YES

100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49,

1.40

YES

100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49,

1.40

YES

1.40

YES

1.40

YES

1.40

YES

100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49,

1.40

YES

0.9828100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49,

1.40

YES

5

0.9364100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49,

1.40

YES

1

3

0.9874100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49,

1.40

YES

7.64%

2

2

0.9864

1.40

YES

0.462

1.12%

1

4

0.9824

1.40

YES

1-Oct-04

0.372

10.21%

1

5

0.9808

1.40

YES

0.143

4-Oct-04

0.427

0.90%

1

4

0.9826

1.40

YES

43.4

0.150

12-Oct-04

0.413

4.72%

1

5

0.9758

1.40

YES

B10

43.7

0.151

13-Oct-04

0.465

2.54%

2

5

0.9833

1.40

YES

B11

42.3

0.147

2-Nov-04

0.398

4.84%

1

3

0.9920

1.40

YES

B12

32.5

0.113

9-Nov-04

0.355

1.15%

1

3

0.9888

1.40

YES

B13

41.6

0.144

10-Nov-04

0.362

5.53%

1

4

0.9831

1.40

YES

Experiment ID1
3T3 Cells

2

VC

3

0 - 50 %4

50 - 100 %

5

R2 6

Phase III

I-127

(ug/mL)7

100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.5100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.5100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49

100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49,
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49

Rationale for
Unacceptability

In Vitro Cytotoxicity Test Methods BRD Appendix I3

November 2006

3T3 NRU Positive Control (SLS) Data
IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

B14

21.4

0.074

B15

43.5

B16

OD

Difference ofright/left VC
from mean

16-Nov-04

0.445

4.98%

3

3

0.95680.151

8-Dec-04

0.442

2.26%

1

3

0.9932

37.2

0.129

14-Dec-04

0.436

5.18%

1

5

0.9757

B17

43.2

0.150

15-Dec-04

0.373

3.10%

1

3

0.9869

B18

41.0

0.142

19-Jan-05

0.385

1.43%

1

3

0.9739Experiment ID1
3T3 Cells

Mean VC
2

VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

5

R2 6

Exposure
Concentrations(ug/mL)7

100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49
100, 71.4, 51.0, 36.4,
26.0, 18.6, 13.3, 9.49,

Dilution

Acceptable

Factor8

Tests9

1.40

NO

1.40

YES

1.40

YES

1.40

YES

1.40

YES

Rationale for
Unacceptability
IC50 out of range

Abbreviations: NR=Neutral red; R&D=Research and development; PC=Positive control; C1 - C8=Concentration series applied to the the cells. C1 is the highest concentration and C8 is lowest; NA=Not Available; RC=Registry of Cytotoxicity;
PC test ID
Mean OD value for all VC wells in test plate3
Difference of right and left VC column of wells in the test plate4% Viability values between 0 and 50% viability; test acceptance criterion; Phases Ia and Ib = number of points between 10 - 50%
5% Viability values between 50 and 100% viability; test acceptance criterion; Phases Ia and Ib = number of points between 50 - 90%
6
Calculated value from the Prism® software
7
Reference substance concentrations applied to the cells
8
Step-wise dilution factor
9
Determination whether test meets or doesn’t meet test acceptance criteria
Shaded boxes identify values that do not meet the specific test acceptance criteria
1
2

Phase
Ib (3T3)
II (3T3)
III (3T3)

Acceptance Limits for PC IC50
ECBC (ug/mL)
FAL (ug/mL)
28.8 – 47.7
25.2 – 59.5
26.4 – 56.3
31.5 – 54.9
30.8 – 51.6
27.2 – 64.7

IIVS (ug/mL)
34.5 – 47.3
33.6 – 50.6
31.8 – 49.3

I-128

In Vitro Cytotoxicity Test Methods BRD Appendix I4Appendix I4

NHK NRU Positive Control (SLS) Data

I-129

November 2006

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

[This Page Intentionally Left Blank]

I-130

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

NHK NRU Positive Control (SLS) Data

IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

SLS-B1

5.47

0.019

12-Aug-02

0.559

13.30%

1

00.9772

SLS-B2

5.92

0.021

12-Aug-02

0.782

3.07%

1

0

SLS-B3

3.40

0.012

12-Sep-02

0.285

21.73%

3

SLS-B4

3.91

0.014

12-Sep-02

0.369

3.41%

SLS-B5

7.02

0.024

9-Sep-02

2.277

SLS-B6

4.77

0.017

9-Sep-02

1.898

SLS-B7)

4.90

0.017

9-Sep-02

SLS-B8

5.61

0.019

SLS-B96.65

SLS-B10

3.71

SLS-B11

Mean VC

Difference ofright/left VC
from mean

Number of
PointsNumber of
PointsExposure
Concentrations

Dilution

Acceptable

Factor8

Tests9100, 68, 46.3, 31.5, 21.4,
14.6, 9.9, 6.7

1.47

NO

0.9717100, 68, 46.3, 31.5, 21.4,
14.6, 9.9, 6.7

1.47

NO

0

0.818250, 34, 23.2, 15.8, 10.7,
7.3, 5, 3.4

1.47

NO

3

0

0.8615

1.47

NO

5.94%

1

4

0.9229

1.47

YES

5.47%

2

4

0.8750

1.47

YES

2.301

2.51%

2

3

0.9331

1.47

YES

9-Sep-02

2.312

4.42%

2

4

0.9273

1.47

NO

0.023

10-Sep-02

1.181

6.10%

1

5

0.8680

1.47

YES

0.013

10-Sep-02

1.007

7.50%

4

2

0.9338

1.47

YES

3.84

0.013

9-Sep-02

1.531

11.76%

3

3

0.9413

1.47

YES

SLS-B12 (no re-feed)

4.10

0.014

16-Sep-02

0.763

7.92%

2

3

0.9683

1.47

YES

SLS-B13 (re-feed)

2.78

0.010

16-Sep-02

0.404

10.90%

3

2

0.9131

1.47

YES

2.82

0.010

16-Sep-02

0.924

0.12%

3

2

0.9583

1.47

YES

3.42

0.012

16-Sep-02

0.271

2.12%

3

2

0.8829

1.47

YES

SLS-B16 (no re-feed)

2.71

0.009

23-Sep-02

0.313

9.38%

2

2

0.9026

1.47

YES

SLS-B17 (re-feed)+

3.13

0.011

23-Sep-02

0.078

14.92%

2

2

0.7987

1.47

NO

Inadequate curve fit.

SLS-B18 (no re-feed)

3.19

0.011

23-Sep-02

0.258

19.12%

3

2

0.8196

1.47

NO

VC difference > 15%.

SLS-B19 (re-feed)

3.19

0.011

23-Sep-02

0.079

4.56%

2

3

0.69301.47

NO

Inadequate curve fit.

SLS-B20

3.48

0.012

9-Oct-02

0.892

1.31%

2

3

0.9455

1.47

YES

SLS-B21

3.17

0.011

9-Oct-02

0.863

0.47%

3

2

0.9539

1.47

YES

Experiment ID1
NHK Cells

2

OD

VC

3

0 - 50 %4

50 - 100 %

5

R2 6

(ug/mL)7

Rationale for
Unacceptability

ECBC
Phase Ia

SLS-B14 (no re-feed)
SLS-B15 (re-feed)

+

I-131

50, 34, 23.2, 15.8, 10.7,
7.3, 5, 3.4
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6
23.2, 15.8, 10.7, 7.3, 5,
3.4, 2.3, 1.6

26% highest viability. No
points between 50 & 90%
viability.
32% highest viability. No
points between 50 & 90%
viability.
VC difference > 15%. No
points between 50 & 90%
viability.
No points between 50 &90% viability.

< 8 of 12 vehicle control
replicates.

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

NHK NRU Positive Control (SLS) Data

IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

3.98

0.014

23-Jan-03

0.861

0.42%

1

4

0.9559

4.57

0.016

23-Jan-03

0.788

2.50%

2

4

0.9326

2.20

0.008

28-Jan-03

1.023

6.41%

2

2

0.9391

3.16

0.011

3-Feb-03

1.135

1.67%

2

3

0.9623

3.76

0.013

10-Feb-03

1.267

0.53%

2

2

0.9559

3.75

0.013

24-Feb-03

1.154

1.28%

2

3

0.9757

3.92

0.014

24-Feb-03

1.135

4.94%

1

4

0.9316

3.05

0.011

17-Mar-03

0.964

7.32%

2

3

0.9603SLS-P1

2.78

0.010

16-Jun-03

0.610

5.82%

4

2

0.9491SLS-P2

2.76

0.010

16-Jun-03

0.671

11.64%

6

2

0.9346SLS-P3

2.38

0.008

23-Jun-03

0.583

2.99%

6

2

0.9074SLS-P4

2.46

0.009

23-Jun-03

0.607

0.81%

3

2

0.9167SLS-P5

1.96

0.007

30-Jun-03

0.380

4.50%

7

1

0.8647SLS-P7

2.38

0.008

7-Jul-03

1.023

4.31%

6

2

0.8829SLS-P8

2.34

0.008

7-Jul-03

0.967

1.28%

6

2

0.9475SLS-P9

2.76

0.010

14-Jul-03

1.054

5.19%

6

2

0.8590SLS-P10

2.53

0.009

14-Jul-03

0.950

3.83%

6

2

0.9316SLS-P11

6.64

0.023

21-Jul-03

0.823

4.52%

3

4

0.9677SLS-P12

5.75

0.020

21-Jul-03

0.748

1.27%

3

5

0.9376SLS-P13

7.88

0.027

28-Jul-03

0.088

4.75%

3

1

0.7990SLS-P15

3.00

0.010

25-Aug-03

0.139

7.92%

4

3

0.8397SLS-P16

3.55

0.012

31-Aug-03

0.660

0.75%

4

4

0.8686SLS-P17 sealer

3.64

0.013

31-Aug-03

0.642

4.51%

4

4

0.9055SLS-P18

3.50

0.012

1-Sep-03

0.471

7.27%

4

3

0.9184

Experiment ID1
NHK Cells

Phase Ib
ECBC-NHK-Ib-01
SLS-P2ECBC-NHK-Ib-01
SLS-P1
ECBC-NHK-Ib-02
SLS-P3ECBC-NHK-Ib-03
SLS-P4
ECBC-NHK-Ib-04
SLS-P5ECBC-NHK-Ib-05
SLS-P7
ECBC-NHK-Ib-05
SLS-P6
ECBC-NHK-Ib-06
SLS-P8Mean VC
2

OD

Difference ofright/left VC
from mean
VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

5

R2 6

Exposure
Concentrations(ug/mL)7

20, 13.6, 9.25, 6.28, 4.27,
2.90, 1.97, 1.34
20, 13.6, 9.24, 6.28, 4.26,
2.90, 1.97, 1.34
20, 13.6, 9.24, 6.28, 4.26,
2.90, 1.97, 1.34
20, 13.6, 9.24, 6.28, 4.26,
2.90, 1.97, 1.34
20, 13.6, 9.24, 6.28, 4.26,
2.90, 1.97, 1.34
20, 13.6, 9.24, 6.28, 4.26,
2.90, 1.97, 1.34
20, 13.6, 9.24, 6.28, 4.26,
2.90, 1.97, 1.34
20, 13.6, 9.24, 6.28, 4.26,
2.90, 1.97, 1.34

Dilution

Acceptable

Factor8

Tests9

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

NO

1.47

YES

1.47

NO

1.47

YES

1.47

YES

1.47

NO

1.47

YES

Rationale for
Unacceptability

Phase II

I-132

20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3

IC50 out of range

IC50 out of range

R&D experiment

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

NHK NRU Positive Control (SLS) Data

IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

SLS-P19

2.68

0.009

SLS-P20

3.14

SLS-P1OD

Difference ofright/left VC
from mean

2-Sep-03

0.761

0.66%

6

2

0.9106

0.011

2-Sep-03

0.761

6.29%

4

4

0.8461

2.71

0.009

14-Jan-04

0.602

1.54%

6

2

0.9562SLS-P2

2.41

0.008

14-Jan-04

0.593

2.01%

5

2

0.9500SLS-P3

2.75

0.010

4-Feb-04

0.514

2.25%

5

3

0.9521SLS-P4

3.48

0.012

4-Feb-04

0.545

2.19%

5

3

0.9372SLS-P5

2.87

0.010

9-Feb-04

0.400

20.23%

6

20.9787SLS-P6

2.95

0.010

9-Feb-04

0.582

1.37%

5

3

0.9743SLS-P7

4.26

0.015

22-Mar-04

1.064

1.54%

4

4

0.9309SLS-P8

4.65

0.016

22-Mar-04

1.026

2.48%

4

4

0.9055SLS-P9

5.62

0.019

29-Mar-04

1.172

6.87%

3

5

0.9149SLS-P10

5.19

0.018

29-Mar-04

1.211

2.79%

3

5

0.8495SLS-P11

3.27

0.011

5-Apr-04

0.760

3.46%

5

3

0.9345SLS-P12

3.07

0.011

12-Apr-04

0.781

2.78%

5

3

0.9583SLS-P13

2.64

0.009

12-Apr-04

0.847

1.72%

6

2

0.9227SLS-P14

3.09

0.011

19-Apr-04

0.911

3.10%

5

3

0.9541SLS-P15

2.39

0.008

19-Apr-04

0.840

2.00%

5

2

0.9495SLS-P16

2.57

0.009

26-Apr-04

0.594

0.48%

6

2

0.9722SLS-P17

2.59

0.009

26-Apr-04

0.507

1.33%

6

2

0.9605SLS-P18

2.36

0.008

3-May-04

0.667

2.30%

4

3

0.9382SLS-P19

3.28

0.011

3-May-04

0.786

0.06%

5

3

0.9557SLS-P20

2.10

0.007

10-May-04

0.684

2.79%

6

2

0.9517SLS-P21

2.71

0.009

10-May-04

0.591

0.47%

5

2

0.9609SLS-P22

3.62

0.013

24-May-04

0.967

0.75%

4

4

0.9317SLS-P23

3.57

0.012

24-May-04

0.944

1.32%

4

4

0.9164

Experiment ID1
NHK Cells

Mean VC
2

VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

5

R2 6

Exposure
Concentrations(ug/mL)7
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3
20.0, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.3

Dilution

Acceptable

Factor8

Tests9

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

Rationale for
Unacceptability

Phase III

I-133

20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35

1.47

NO

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

% VC difference >15

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

NHK NRU Positive Control (SLS) Data

IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

SLS-P24

1.78

0.006

SLS-P25

2.37

SLS-P26

3.46

SLS-P27

OD

Difference ofright/left VC
from mean

14-Jun-04

0.623

4.06%

6

1

0.9431

0.008

14-Jun-04

0.523

5.18%

6

2

0.93030.012

21-Jun-04

0.901

0.40%

4

4

0.8960

3.41

0.012

21-Jun-04

1.021

0.50%

4

4

0.9365SLS-P28

2.45

0.008

28-Jun-04

0.946

1.45%

6

2

0.9476SLS-P29

2.34

0.008

28-Jun-04

0.918

3.97%

6

2

0.9517SLS-P30

2.65

0.009

6-Jul-04

0.784

0.62%

5

3

0.9483SLS-P31

2.85

0.010

6-Jul-04

0.673

0.82%

4

3

0.9655SLS-P32

2.53

0.009

12-Jul-04

0.626

2.25%

6

2

0.9348SLS-P33

2.28

0.008

12-Jul-04

0.756

2.45%

6

2

0.9521SLS-P34

2.58

0.009

19-Jul-04

0.759

0.59%

5

2

0.9536SLS-P35

2.71

0.009

19-Jul-04

0.781

1.21%

5

3

0.9599SLS-P36

2.72

0.009

26-Jul-04

0.373

0.31%

4

3

0.9411SLS-P37

2.50

0.009

26-Jul-04

0.427

1.21%

6

2

0.9482SLS-P38

3.26

0.011

2-Aug-04

0.628

12.01%

3

4

0.8904SLS-P39

2.59

0.009

2-Aug-04

0.839

3.43%

5

3

0.9302SLS-P40

2.74

0.010

9-Aug-04

0.632

3.96%

5

3

0.9279SLS-P41

2.90

0.010

9-Aug-04

0.663

2.35%

5

3

0.9480SLS-P42

2.94

0.010

16-Aug-04

0.697

0.23%

5

2

0.9599SLS-P43

3.04

0.011

16-Aug-04

0.751

0.50%

5

3

0.9240SLS-P44

2.46

0.009

23-Aug-04

0.908

2.01%

6

2

0.9487SLS-P45

3.38

0.012

23-Aug-04

0.926

1.47%

5

3

0.9464SLS-P46

4.04

0.014

30-Aug-04

0.936

2.46%

4

4

0.9318SLS-P47

4.58

0.016

30-Aug-04

0.943

1.02%

4

4

0.8656SLS-P48

2.64

0.009

7-Sep-04

0.721

6.39%

5

3

0.9543SLS-P49

1.99

0.007

7-Sep-04

0.641

0.69%

4

2

0.9585Experiment ID1
NHK Cells

Mean VC
2

VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

I-134

5

R2 6

Exposure
Concentrations(ug/mL)7
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35

Dilution

Acceptable

Factor8

Tests9

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

Rationale for
Unacceptability

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

NHK NRU Positive Control (SLS) Data

IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

SLS-P50

2.99

0.010

SLS-P51

3.72

SLS-P52

2.70

SLS-P53

OD

Difference ofright/left VC
from mean

13-Sep-04

1.123

3.25%

5

3

0.89080.013

13-Sep-04

1.042

0.19%

4

4

0.92170.009

27-Sep-04

0.529

1.54%

6

2

0.9508

2.76

0.010

27-Sep-04

0.604

1.75%

4

2

0.9270SLS-P54

3.45

0.012

4-Oct-04

0.745

0.79%

4

4

0.9265SLS-P55

3.12

0.011

4-Oct-04

0.639

5.10%

3

3

0.9318SLS-P56

3.77

0.013

18-Oct-04

0.826

1.61%

5

3

0.9471SLS-P57

3.02

0.010

25-Oct-04

0.612

1.55%

4

3

0.9690SLS-P58

2.83

0.010

26-Oct-04

0.155

8.34%

3

3

0.9318

8.13

0.028

9-Sep-02

1.333

6.67%

1

2

0.9823

7.63

0.026

9-Sep-02

1.294

6.43%

1

2

0.9889

8.06

0.028

9-Sep-02

1.289

6.39%

1

2

0.9839

4.62

0.016

9-Sep-02

1.169

13.44%

1

1

0.9683

5.23

0.018

9-Sep-02

1.089

9.96%

1

1

0.9645

5.19

0.018

9-Sep-02

1.184

9.32%

1

1

0.9253

6.72

0.023

11-Sep-02

0.333

0.73%

2

2

0.8307

7.79

0.027

11-Sep-02

1.000

11.26%

1

1

0.9666

7.63

0.026

11-Sep-02

1.076

8.62%

1

2

0.9339

5.30

0.018

11-Sep-02

1.698

7.44%

1

1

0.9810

7.70

0.027

23-Sep-02

1.534

4.79%

1

5

0.93283 (no re-feed)

8.66

0.030

23-Sep-02

1.559

0.38%

1

5

0.92022 (re-feed)

6.84

0.024

23-Sep-02

1.485

1.38%

1

3

0.9695Experiment ID1
NHK Cells

Mean VC
2

VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

5

R2 6

Exposure
Concentrations(ug/mL)7
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35
20.0, 13.61, 9.26, 6.30,
4.28, 2.91, 1.98, 1.35

Dilution

Acceptable

Factor8

Tests9

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

2.00

YES

2.00

YES

2.00

YES

2.00

YES

2.00

YES

2.00

YES

2.00

NO

2.00

YES

2.00

YES

2.00

YES

1.47

NO

1.47

NO

1.47

NO

Rationale for
Unacceptability

FAL
Phase Ia
B1(1a/NHK/DF4/F
AL/SLS)B2(1a/NHK/DF5/F
AL/SLS)B3(1a/NHK/DF6/F
AL/SLS)+B4(1a/NHK/DF7/F
AL/SLS)B5(1a/NHK/DF8/F
AL/SLS)
B6(1a/NHK/DF12/
FAL/SLS)
B7(1a/NHK/DF14/
FAL/SLS)
B8(1a/NHK/DF15/
FAL/SLS)
B9(1a/NHK/DF16/
FAL/SLS)
B10(1a/NHK/DF17/FAL/SLS)+
1 (no re-feed)

I-135

400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
400, 200, 100, 50, 25,
12.5, 6.3, 3.1
20, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.4
20, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.4
20, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.4

Inadequate curve fit.

405 nm OD subtracted
from 540 nm OD
405 nm OD subtracted
from 540 nm OD
405 nm OD subtracted
from 540 nm OD

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

NHK NRU Positive Control (SLS) Data

IC50
(ug/mL)

IC50(mM)

Reference
Substance
ApplicationDate

4 (re-feed)

5.60

0.019

5 (no re-feed)

8.26

6 (no re-feed)

Experiment ID1
NHK Cells

1a/NHK/DF23/FAL/SLS
1a/NHK/DF24/FAL/SLS
1a/NHK/DF25/FAL/SLS
1a/NHK/DF26/FAL/SLS
1a/NHK/DF27/FAL/SLS
1a/NHK/DF28/FAL/SLS
1a/NHK/DF29/FAL/SLS
1a/NHK/DF30/FAL/SLS
1a/NHK/DF31/FAL/SLS+
1a/NHK/DF32/FAL/SLS
1a/NHK/DF33/FAL
/SLS+

OD

Difference ofright/left VC
from mean

23-Sep-02

1.301

14.78%

1

4

0.8851

0.029

25-Sep-02

1.122

9.11%

2

2

0.8930

11.75

0.041

25-Sep-02

0.633

16.43%

2

4

0.6280

3.33

0.012

22-Oct-02

0.246

8.25%

2

0

0.9216

4.63

0.016

23-Oct-02

0.493

3.46%

2

1

0.9721

3.22

0.011

23-Oct-02

0.393

41.08%

300.8731

4.45

0.015

23-Oct-02

0.505

20.88%

2

1

0.9385

4.41

0.015

23-Oct-02

0.484

7.93%

2

1

0.9076

6.66

0.023

24-Oct-02

0.693

1.54%

1

2

0.8672

5.57

0.019

24-Oct-02

0.545

9.79%

1

1

0.9244

14.43

0.050

19-Nov-02

1.094

2.67%

1

6

0.6304

13.38

0.046

19-Nov-02

1.354

3.71%

2

6

0.6670

13.37

0.046

19-Nov-02

0.890

3.18%

2

5

0.6136

11.89

0.041

19-Nov-02

0.766

7.34%

3

3

0.8476

3.74

0.013

11-Dec-02

0.164

7.05%

1

1

0.9725

6.46

0.022

13-Dec-02

0.743

9.94%

1

5

4.88

0.017

14-Jan-03

0.086

3.20%

2

4

Mean VC
2

VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %5

Acceptable

Factor8

Tests9

1.47

NO

1.47

NO

1.47

NO

1.47

NO

1.47

YES

1.47

NO

VC difference > 15%. No
point between 50 & 90%
viability.

1.47

NO

VC difference > 15%.

1.47

YES

1.47

YES

1.47

YES

1.30

NO

Inadequate curve fit.

1.30

NO

Inadequate curve fit.

1.30

NO

Inadequate curve fit.

1.30

YES

15, 11.54, 8.88, 6.83,
5.25, 4.04, 3.11, 2.39

1.30

YES

0.801715, 11.54, 8.88, 6.83,
5.25, 4.04, 3.11, 2.39

1.30

YES

0.752615, 11.54, 8.88, 6.83,
5.25, 4.04, 3.11, 2.39

1.30

NO

Phase Ib
A1
1b/NHKCTR1/FAL/
SLS
A2
1b/NHKCTR2/FAL/
SLS
A3
1b/NHK/CTR4/FAL/ recalculated w/ooutlier

I-136

Exposure
Concentrations

Dilution

R2 6(ug/mL)7

20, 13.6, 9.3, 6.3, 4.3,
2.9, 2.0, 1.4
25, 17, 11.6, 7.87, 5.35,
3.64, 2.48, 1.69
25, 17, 11.6, 7.87, 5.35,
3.64, 2.48, 1.69
50, 34, 23, 15.7, 10.7,
7.3, 4.9, 3.4
50, 34, 23, 15.7, 10.7,
7.3, 4.9, 3.4
50, 34, 23, 15.7, 10.7,
7.3, 4.9, 3.4
50, 34, 23, 15.7, 10.7,
7.3, 4.9, 3.4
50, 34, 23, 15.7, 10.7,
7.3, 4.9, 3.4
50, 34, 23, 15.7, 10.7,
7.3, 4.9, 3.4
50, 34, 23, 15.7, 10.7,
7.3, 4.9, 3.4
30.0, 23.08, 17.75,
13.65, 10.50, 8.08,
6.22, 4.78
30.0, 23.08, 17.75,
13.65, 10.50, 8.08,
6.22, 4.78
30.0, 23.08, 17.75,
13.65, 10.50, 8.08,
6.22, 4.78
30.0, 23.08, 17.75,
13.65, 10.50, 8.08,
6.22, 4.78

Rationale for
Unacceptability
405 nm OD subtracted
from 540 nm OD
405 nm OD subtracted
from 540 nm OD
405 nm OD subtracted
from 540 nm OD. VC
difference > 15%.
No point between 50 &90% viability

R2 < 0.8

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

NHK NRU Positive Control (SLS) Data

Experiment ID1
NHK Cells

IC50(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

A4
1b/NHK/CTR5/FAL

3.12

0.011

15-Jan-03

0.146

3.42%

2

1

0.8444A5
1b/NHK/CTR6/FAL

NC#VALUE!

17-Jan-03

0.003

286.96%1

0

Mean VC
2

OD

Difference ofright/left VC
from mean
VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %5

Exposure
Concentrations

Dilution

Acceptable

Factor8

Tests915, 11.54, 8.88, 6.83,
5.25, 4.04, 3.11, 2.39

1.30

YES

NC

15, 11.54, 8.88, 6.83,
5.25, 4.04, 3.11, 2.39

1.30

NO

1.30

YES

R2 6

(ug/mL)7

7.80

0.027

27-Jan-03

1.210

2.15%

2

2

0.962615, 11.54, 8.88, 6.83,
5.25, 4.04, 3.11, 2.39

A8
1b/NHK/CTR9/FAL

5.48

0.019

3-Feb-03

0.935

12.58%

1

4

0.936215, 11.54, 8.88, 6.83,
5.25, 4.04, 3.11, 2.39

1.30

YES

4.12

0.014

4-Feb-03

0.648

23.68%

2

4

0.716015, 11.54, 8.88, 6.83,
5.25, 4.04, 3.11, 2.39

1.30

NO

3.92

0.014

19-Mar-03

1.068

6.94%

2

3

0.886815, 11.54, 8.88, 6.83,
5.25, 4.04, 3.11, 2.39

1.30

YES

5.08

0.018

20-Mar-03

1.542

0.79%

3

3

0.879215, 11.54, 8.88, 6.83,
5.25, 4.04, 3.11, 2.39

1.30

YES

3.14

0.011

23-Mar-03

0.403

13.53%

3

1

0.872015, 11.54, 8.88, 6.83,
5.25, 4.04, 3.11, 2.39

1.30

YES

3.32

0.012

24-Mar-03

0.831

3.67%

1

2

0.9652

1.30

YES

2.91

0.010

2-May-03

0.973

0.92%

2

2

0.9586

1.47

YES

4.52

0.016

2-May-03

0.843

5.43%

2

2

0.9229

1.47

YES

3.10

0.011

7-Jul-03

1.114

4.61%

3

4

0.9350

1.47

YES

1.34

0.005

30-Jul-03

0.609

2.17%

3

2

0.9358

1.47

YES

1.40

0.005

5-Aug-03

0.526

4.20%

4

2

0.9077

1.47

YES

1.74

0.006

6-Aug-03

0.810

2.34%

4

3

0.9517

1.47

YES

2.75

0.010

11-Aug-03

0.639

0.03%

4

4

0.3154

1.47

NO

3.56

0.012

13-Aug-03

0.462

6.70%

3

5

0.8954

1.47

YES

3.03

0.011

21-Aug-03

0.401

0.35%

4

2

0.7230

1.47

NO

3.45

0.012

26-Aug-03

0.454

2.31%

2

3

0.9372

1.47

YES

Phase II

FAL.NHK.SLS.30.
07.03
FAL.NHK.SLS.010
803
FAL.NHK.SLS.07.
08.03
FAL.NHK.SLS.08.
08.03
FAL.NHK.SLS.13.
08.03
FAL.NHK.SLS.15.
08.03
FAL.NHK.SLS.230
803
FAL.NHK.SLS.280
803

I-137

15, 11.54, 8.88, 6.83,
5.25, 4.04, 3.11, 2.39
15, 10.2, 6.94, 4.72,
3.21, 2.19, 1.49, 1.01
10, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
12.0, 8.2, 5.6, 3.2, 2.6,
1.8, 1.2, 0.8
12.0, 8.2, 5.6, 3.2, 2.6,
1.8, 1.2, 0.8
12.0, 8.2, 5.6, 3.2, 2.6,
1.8, 1.2, 0.8
12.0, 8.2, 5.6, 3.2, 2.6,
1.8, 1.2, 0.8
10, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10, 6.8, 4.6, 3.14, 2.14,
1.5, 0.9, 0.68
10, 6.8, 4.6, 3.14, 2.14,
1.5, 0.9, 0.68

VC difference > 15%; nopoint between 50 & 90%;
no R2 or ICx

A6
1b/NHK/CTR7/FAL

A9
1b/NHK/CTR10/FA
L
A10
1b/NHK/CTR11/FA
L
A11
1b/NHK/CTR12/FA
L
A12
1b/NHK/CTR13/FA
L/SLS
1b/NHK/CTR14/FA
L/SLS
1b/NHK/CTR15/FA
L/SLS
1b/NHK/DF1/FAL/
SLS

Rationale for
Unacceptability

VC difference > 15%; R2< 0.8

low r2

low r2

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

NHK NRU Positive Control (SLS) Data

Experiment ID1
NHK Cells
FAL.NHK.SLS.050
903
FAL.NHK.SLS.01.
10.03
FAL.NHK.SLS.01.
10.03
FAL.NHK.SLS.15.
10.03
FAL.NHK.SLS.19.
10.03
FAL.NHK.SLS.23.
10.03
FAL.NHK.SLS.24.
10.03
FAL.NHK.SLS.05.1
1.03
FAL.NHK.SLS.07.1
1.03
FAL.NHK.SLS.28.1
1.03

IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

3.20

0.011

4.59

OD

Difference ofright/left VC
from mean

3-Sep-03

0.110

8.54%

2

3

0.91580.016

29-Sep-03

1.292

1.62%

2

6

0.9168

5.50

0.019

29-Sep-03

0.895

20.89%

2

5

0.9276

2.90

0.010

13-Oct-03

0.547

4.65%

3

5

0.8927

3.85

0.013

17-Oct-03

0.340

2.89%

3

5

0.9637

4.90

0.017

21-Oct-03

0.279

8.61%

3

2

0.7996

2.96

0.010

22-Oct-03

0.932

1.31%

3

5

0.9119

3.69

0.013

3-Nov-03

0.515

1.10%

3

5

0.8516

3.95

0.014

5-Nov-03

0.351

4.18%

3

3

0.9316

3.46

0.012

26-Nov-03

0.174

6.01%

3

5

0.9543

5.28

0.018

9-Feb-04

1.131

1.33%

2

6

0.9062

4.83

0.017

9-Feb-04

1.131

1.33%

2

6

0.8318

3.63

0.013

11-Feb-04

0.106

6.36%

4

4

0.7409

6.22

0.022

16-Feb-04

0.155

6.02%

2

2

0.4330

2.24

0.008

18-Feb-04

0.254

1.35%

4

4

0.9233

3.25

0.011

24-Feb-04

0.292

4.37%

4

4

0.9347

4.04

0.014

24-Feb-04

0.280

4.67%

3

3

0.9265

2.78

0.010

25-Feb-04

0.472

3.50%

3

5

0.9173

4.48

0.016

16-Mar-04

0.424

2.34%

3

5

0.8934

2.76

0.010

17-Mar-04

0.555

1.67%

3

5

0.8882

2.93

0.010

23-Mar-04

0.584

8.67%

4

4

0.9493

3.96

0.014

24-Mar-04

0.593

3.86%

3

5

0.9244

3.06

0.011

26-Apr-04

0.762

0.95%

3

5

0.9561

Mean VC
2

VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

5

R2 6

Phase III

FAL.NHK.SLS.11.0
2.04
FAL.NHK.SLS.11.0
2.04
FAL.NHK.SLS.13.
02.03
FAL.NHK.SLS.18.
02.04
FAL.NHK.SLS.20.
02.03
FAL.NHK.SLS/NB.
26.02.03
FAL.NHK.SLS/MO.26.02.03
FAL.NHK.SLS.27.
02.03
FAL.NHK.SLS.18.
03.03
FAL.NHK.SLS.19.
03.03
FAL.NHK.SLS.25.
03.03
FAL.NHK.SLS.26.
03.04
FAL.NHK.SLS.28.
04.03

I-138

Exposure
Concentrations(ug/mL)7

10, 6.8, 4.6, 3.14, 2.14,
1.5, 0.9, 0.68
10, 6.8, 4.6, 3.14, 2.14,
1.5, 0.9, 0.68
10, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10, 6.8, 4.6, 3.14, 2.14,
1.5, 0.9, 0.68
10, 6.8, 4.6, 3.14, 2.14,
1.5, 0.9, 0.68
10, 6.8, 4.6, 3.14, 2.14,
1.5, 0.9, 0.68
10, 6.8, 4.6, 3.14, 2.14,
1.5, 0.9, 0.68
10, 6.8, 4.6, 3.14, 2.14,
1.5, 0.9, 0.68
10, 6.8, 4.6, 3.14, 2.14,
1.5, 0.9, 0.68
10, 6.8, 4.6, 3.14, 2.14,
1.5, 0.9, 0.68
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67
10.0, 6.8, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

Dilution

Acceptable

Factor8

Tests9

1.47

YES

1.47

YES

1.47

NO

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

NO

bottom not set to 0

1.47

NO

r2 < 0.85

1.47

NO

r2 < 0.85

1.47

NO

r2 < 0.85; IC50 out of
range

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

Rationale for
Unacceptability% VC difference >15

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

NHK NRU Positive Control (SLS) Data

Experiment ID1
NHK Cells

IC50(ug/mL)

IC50(mM)

FAL.NHK.SLS.13.
05.04

Reference
Substance
Application
Date

2.79

0.010

FAL.NHK.SLS.14.
05.03

3.80

OD

Difference ofright/left VC
from mean

11-May-04

0.612

0.80%

4

4

0.97820.013

12-May-04

0.594

7.47%

3

3

FAL.NHK.SLS.25.06.042.62

0.009

23-Jun-04

1.347

0.43%

4

FAL.NHK.SLS.28.
07.04

NA

NA

26-Jul-04

0.073

22.93%

FAL.NHK.SLS.11.0
8.04

3.77

0.013

9-Aug-04

0.512

FAL.NHK.SLS.12.
08.04

5.86

0.020

10-Aug-04

FAL.NHK.SLSRB.19.08.04

4.49

0.016

FAL.NHK.SLSNB.19.08.04

1.85

FAL.NHK.SLS.20.
08.04

Mean VC

Number of
PointsNumber of
PointsExposure
Concentrations

Dilution

Acceptable

Factor8

Tests910, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

0.9301

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

4

0.873010.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

2

5

0.7622

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

NO

4.88%

3

5

0.847010.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

0.701

8.17%

2

1

0.9776

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

NO

IC50 out of range

17-Aug-04

0.337

0.10%

3

1

0.7397

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

NO

r2 too low

0.006

17-Aug-04

0.537

10.04%

3

4

0.858910.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

3.70

0.013

18-Aug-04

0.738

8.90%

3

5

0.975010.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

FAL.NHK.SLS.25.
08.04

3.56

0.012

23-Aug-04

0.991

2.23%

2

6

0.8697

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

FAL.NHK.SLSRB.20.08.04(should be
25.08.04)

5.20

0.018

23-Aug-04

0.645

2.80%

2

1

0.8472

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

FAL.NHK.SLS.27.
08.04

3.00

0.010

23-Aug-04

0.546

7.84%

3

5

0.8783

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

FAL.NHK.SLS.17.
09.04

3.30

0.011

15-Sep-04

0.803

1.34%

3

5

0.940810.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

FAL.NHK.SLS.30.
09.03

2.78

0.010

28-Sep-04

0.562

3.86%

3

4

0.95595000, 2326, 1082, 503,
234, 109, 50.6, 23.6

1.47

YES

FAL.NHK.SLS.01.
10.04

8.25

0.029

29-Sep-04

1.103

3.49%

1

7

0.966910.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

NO

FAL.NHK.SLS.07.
10.03

2.23

0.008

5-Oct-04

0.602

6.09%

4

4

0.948810.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

2

VC

3

0 - 50 %4

50 - 100 %

I-139

5

R2 6

(ug/mL)7

Rationale for
Unacceptability% VC differnece > 15; r2
too low

IC50 out of range

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

NHK NRU Positive Control (SLS) Data

Experiment ID1
NHK Cells

IC50(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

FAL.NHK.SLS.08.
10.03

2.91

0.010

6-Oct-04

0.827

4.33%

3

5

0.9222

FAL.NHK.SLS.20.
10.04

4.95

0.017

18-Oct-04

1.231

5.58%

2

6

FAL.NHK.SLS.22.
10.04 (NB)

3.62

0.013

20-Oct-04

0.675

0.86%

3

FAL.NHK.SLS.28.
10.04

3.39

0.012

26-Oct-04

0.641

7.85%

FAL.NHK.SLS.29.
10.04

2.33

0.008

27-Oct-04

0.502

FAL.NHK.SLS.03.1
1.04

3.19

0.011

1-Nov-04

FAL.NHK.SLS.05.1
1.04

2.16

0.007

FAL.NHK.SLS.10.1
1.04

4.07

FAL.NHK.SLS.12.1
1.04

Mean VC

Difference ofright/left VC
from mean

Number of
PointsNumber of
PointsExposure
Concentrations

Dilution

Acceptable

Factor8

Tests910.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

0.909910.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

5

0.9405

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

3

5

0.9366

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

1.46%

4

4

0.9531

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

0.447

8.60%

3

5

0.9331

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

3-Nov-04

0.538

0.62%

4

4

0.9467

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

0.014

8-Nov-04

1.011

0.89%

2

6

0.921010.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

3.76

0.013

10-Nov-04

0.742

3.04%

2

6

0.9085

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

FAL.NHK.SLS.17.1
1.04

4.04

0.014

15-Nov-04

1.050

1.74%

2

6

0.8732

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

FAL.NHK.SLS.19.1
1.04

3.91

0.014

17-Nov-04

0.509

4.62%

3

3

0.9793

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

FAL.NHK.SLS.24.1
1.04

4.09

0.014

22-Nov-04

1.124

2.91%

2

6

0.8654

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

FAL.NHK.SLS.26.1
1.04

3.00

0.010

24-Nov-04

0.620

1.45%

3

5

0.9524

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

FAL.NHK.SLS(MO).10.12.04

6.02

0.021

8-Dec-04

1.017

1.35%

2

6

0.8137

10.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

YES

FAL.NHK.SLS.10.
12.04

4.18

0.014

8-Dec-04

0.928

0.25%

3

5

0.917010.0, 6.80, 4.63, 3.15,
2.14, 1.46, 0.99, 0.67

1.47

NO

2

OD

VC

3

0 - 50 %4

50 - 100 %

I-140

5

R2 6

(ug/mL)7

Rationale for
Unacceptability

IC50 out of range; low r2

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

NHK NRU Positive Control (SLS) Data

IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

B1

3.70

0.013

19-Aug-02

0.785

11.83%

1

5

0.8579

B2

2.93

0.010

19-Aug-02

0.778

5.60%

1

6

0.8406

B3

59.28

0.206

24-Aug-02

1.883

3.30%

1

6

0.0862

B4

10.06

0.035

24-Aug-02

1.680

8.59%

0

2

0.6253

B5

3.72

0.013

25-Aug-02

1.129

7.89%

1

5

0.9213

B6

3.88

0.013

25-Aug-02

1.130

5.10%

1

5

0.8956

B7

3.57

0.012

26-Aug-02

1.083

7.51%

1

6

0.8251

B8

3.30

0.011

26-Aug-02

0.867

11.48%

3

5

0.8592

B9

3.85

0.013

27-Aug-02

0.985

10.80%

2

5

0.8840

B10

3.72

0.013

27-Aug-02

1.026

2.70%

1

6

0.8212

B11

4.92

0.017

4-Sep-02

1.240

0.59%

1

5

0.8987

B12

4.13

0.014

4-Sep-02

1.218

4.81%

1

6

0.8888

B13

4.02

0.014

5-Sep-02

1.082

0.78%

1

6

0.8669

B14

4.18

0.014

5-Sep-02

1.111

3.22%

1

6

0.8742

B15

4.36

0.015

6-Sep-02

0.693

12.53%

1

6

0.8170

B16

5.07

0.018

6-Sep-02

0.747

12.82%

2

6

0.7516

B17

3.70

0.013

7-Sep-02

0.550

3.51%

1

5

0.8953

B18

3.50

0.012

7-Sep-02

0.558

9.32%

1

6

0.8518

B19

3.45

0.012

8-Sep-02

0.658

10.32%

1

6

0.8785

B20

3.03

0.011

8-Sep-02

0.682

5.43%

2

5

0.9061

B23 (no re-feed)

3.54

0.012

21-Sep-02

1.084

4.29%

2

4

0.9573

B23 (re-feed)

3.46

0.012

21-Sep-02

0.824

4.80%

2

3

0.9531

Experiment ID1
NHK Cells

Mean VC
2

OD

Difference ofright/left VC
from mean
VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

5

R2 6

Exposure
Concentrations(ug/mL)7

Dilution

Acceptable

Factor8

Tests9

1.79

YES

1.79

YES

1.47

NO

1.47

NO

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

NO

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

Rationale for
Unacceptability

IIVS
Phase Ia

I-141

10, 5.6, 3.2, 1.8, 1.0,
0.6, 0.3, 0.2
10, 5.6, 3.2, 1.8, 1.0,
0.6, 0.3, 0.2
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7

Major precipitation
problems
Major precipitation
problems. No points
between 10 & 50%.

Inadequate curve fit.

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

NHK NRU Positive Control (SLS) Data

Experiment ID1
NHK Cells

IC50(ug/mL)

IC50(mM)

Reference
Substance
ApplicationDate

B24 (no re-feed)

3.89

0.013

B24 (re-feed)

3.72

B25 (no re-feed)

OD

Difference ofright/left VC
from mean

21-Sep-02

1.120

0.13%

1

5

0.9361

0.013

21-Sep-02

0.784

2.36%

2

4

0.9265

3.92

0.014

22-Sep-02

1.078

1.34%

1

5

0.9426

B25 (re-feed)

4.19

0.015

22-Sep-02

0.938

2.24%

2

5

0.9540B26 (no re-feed)

3.44

0.012

22-Sep-02

1.037

7.19%

2

3

0.9495

B26 (re-feed)

3.64

0.013

22-Sep-02

0.775

4.29%

2

4

0.9491

B27 (no re-feed)

2.87

0.010

23-Sep-02

1.050

1.79%

2

5

0.8907

B27 ( re-feed)

2.68

0.009

23-Sep-02

0.841

2.77%

2

5

0.9212

B28 (no re-feed)

3.30

0.011

23-Sep-02

1.029

0.04%

2

5

0.9088B28 (re-feed)

2.78

0.010

23-Sep-02

0.819

3.87%

3

4

0.9476Preliminary

2.78

0.010

4-Jan-03

0.631

3.03%

3

3

0.9588

B1

2.98

0.010

17-Jan-03

0.518

0.50%

2

5

0.9403

B2

3.31

0.011

18-Jan-03

0.726

9.52%

2

3

0.9621

B3

3.00

0.010

31-Jan-03

0.845

3.64%

2

4

0.9420

B4

3.64

0.013

1-Feb-03

0.781

1.49%

2

4

0.9550

A2

3.11

0.011

9-Aug-03

0.682

5.04%

3

4

0.9538

B1

3.24

0.011

16-Aug-03

0.351

7.73%

3

3

0.9661

B2

4.42

0.015

17-Aug-03

0.26

3.34%

2

4

0.9394

B3

4.10

0.014

18-Aug-03

0.284

4.05%

3

2

0.9569

B4

4.52

0.016

25-Aug-03

0.201

2.12%

2

4

0.9434

B7

3.98

0.014

29-Aug-03

0.605

7.45%

2

4

0.945

B8

6.56

0.023

13-Sep-03

0.512

9.47%

1

4

0.8297

Mean VC
2

VC

3

Number of
Points
0 - 50 %4

Number of
Points50 - 100 %

5

R2 6

Phase Ib

Phase II

I-142

Exposure
Concentrations(ug/mL)7

10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.5, 1.0, 0.7
10, 6.8, 4.6, 3.2, 2.2,
1.47, 1.0, 0.68
10, 6.8, 4.6, 3.2, 2.2,
1.47, 1.0, 0.68
10, 6.8, 4.6, 3.2, 2.2,
1.47, 1.0, 0.68
10, 6.8, 4.6, 3.2, 2.2,
1.47, 1.0, 0.68
10, 6.8, 4.6, 3.2, 2.2,
1.47, 1.0, 0.68
10, 6.7, 4.4, 3.0, 2.0,
1.3, 0.88, 0.59
10, 6.7, 4.4, 3.0, 2.0,
1.3, 0.88, 0.59
10, 6.7, 4.4, 3.0, 2.0,
1.3, 0.88, 0.59
10, 6.7, 4.4, 3.0, 2.0,
1.3, 0.88, 0.59
10, 6.7, 4.4, 3.0, 2.0,
1.3, 0.88, 0.59
10, 6.7, 4.4, 3.0, 2.0,
1.3, 0.88, 0.59
10, 6.7, 4.4, 3.0, 2.0,
1.3, 0.88, 0.59

Dilution

Acceptable

Factor8

Tests9

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.47

YES

1.50

YES

1.50

YES

1.50

YES

1.50

YES

1.50

YES

1.50

YES

1.50

NO

Rationale for
Unacceptability

IC50 out of range

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

NHK NRU Positive Control (SLS) Data

IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

B9

5.85

0.020

B10

5.25

B11

OD

Difference ofright/left VC
from mean

14-Sep-03

0.551

4.08%

2

3

0.90420.018

15-Sep-03

0.475

1.75%

2

3

0.8811

6.15

0.021

16-Sep-03

0.38

1.21%

1

3

0.7715

B12

4.27

0.015

29-Sep-03

0.642

4.75%

2

5

0.924

B13

4.27

0.015

29-Sep-03

0.242

1.41%

2

4

0.928

B14

3.98

0.014

30-Sep-03

0.317

1.85%

2

5

0.9696

B15

6.36

0.022

1-Oct-03

0.294

0.97%

2

2

0.8797

A1

2.88

0.010

15-Mar-04

0.474

1.95%

3

5

0.9576

A2

3.42

0.012

18-Mar-04

0.581

5.05%

2

6

A3

3.90

0.014

29-Mar-04

0.610

0.07%

3

A4

2.67

0.009

29-Mar-04

0.509

3.50%

A5

2.65

0.009

30-Mar-04

0.533

B1

2.84

0.010

21-Apr-04

B2

3.38

0.012

B3

2.79

B4
B5

Experiment ID1
NHK Cells

Mean VC

Number of
PointsNumber of
PointsExposure
Concentrations

Dilution

Acceptable

Factor8

Tests9

1.50

NOIC50 out of range

1.50

NO

IC50 out of range

1.50

NO

IC50 out of range; low r2

1.50

YES

1.50

YES

1.50

YES

1.50

NO

10.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

0.9176

10.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

5

0.8815

10.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

3

5

0.962910.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

5.08%

3

5

0.9534

10.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

0.621

3.08%

4

4

0.9377

10.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

22-Apr-04

0.526

2.69%

3

5

0.956810.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

0.010

4-May-04

0.531

6.18%

3

5

0.946910.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

4.20

0.015

11-May-04

0.528

11.31%

2

6

0.8904

10.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

3.51

0.012

12-May-04

0.537

7.15%

2

6

0.914910.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

2

VC

3

0 - 50 %4

50 - 100 %

5

R2 6

Phase III

I-143

(ug/mL)7

10, 6.7, 4.4, 3.0, 2.0,
1.3, 0.88, 0.59
10, 6.7, 4.4, 3.0, 2.0,
1.3, 0.88, 0.59
10, 6.7, 4.4, 3.0, 2.0,
1.3, 0.88, 0.59
10, 6.7, 4.4, 3.0, 2.0,
1.3, 0.88, 0.59
10, 6.7, 4.4, 3.0, 2.0,
1.3, 0.88, 0.59
10, 6.7, 4.4, 3.0, 2.0,
1.3, 0.88, 0.59
10, 6.7, 4.4, 3.0, 2.0,
1.3, 0.88, 0.59

Rationale for
Unacceptability

IC50 out of range

In Vitro Cytotoxicity Test Methods BRD Appendix I4

November 2006

NHK NRU Positive Control (SLS) Data

IC50
(ug/mL)

IC50(mM)

Reference
Substance
Application
Date

B6

2.72

0.009

14-Jul-04

0.629

6.79%

3

5

0.9380

B7

2.58

0.009

15-Jul-04

0.611

0.67%

3

5

B8

2.95

0.010

17-Aug-04

0.587

10.35%

3

B9

3.08

0.011

18-Aug-04

0.554

1.95%

B10

4.14

0.014

1-Sep-04

0.597

B11

3.55

0.012

2-Sep-04

B12

2.93

0.010

B13

2.50

B14
B15

Experiment ID1
NHK Cells

Mean VC

Difference ofright/left VC
from mean

Number of
PointsNumber of
PointsExposure
Concentrations

Dilution

Acceptable

Factor8

Tests910.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

0.9646

10.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

4

0.9304

10.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

3

4

0.960910.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

6.80%

2

6

0.944810.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

0.669

1.77%

2

6

0.943810.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

20-Oct-04

0.599

3.40%

3

5

0.9561

10.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

0.009

27-Oct-04

0.629

3.01%

3

5

0.9645

10.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

3.10

0.011

28-Oct-04

0.702

3.78%

3

5

0.9615

10.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

2.51

0.009

8-Nov-04

0.623

2.50%

4

4

0.9151

10.0, 6.67, 4.44, 2.96,
1.98, 1.32, 0.88, 0.59

1.50

YES

2

OD

VC

3

0 - 50 %4

50 - 100 %

5

R2 6

(ug/mL)7

Rationale for
Unacceptability

Abbreviations: NR=Neutral red; R&D=Research and development; PC=Positive control; C1 - C8=Concentration series applied to the the cells. C1 is the highest concentration and C8 is lowest; NA=Not Available; RC=Registry of Cytotoxicity;
PC test ID
Mean OD value for all VC wells in test plate3
Difference of right and left VC column of wells in the test plate4% Viability values between 0 and 50% viability; test acceptance criterion; Phases Ia and Ib = number of points between 10 - 50%
5% Viability values between 50 and 100% viability; test acceptance criterion; Phases Ia and Ib = number of points between 50 - 90%
6
Calculated value from the Prism® software
7
Reference substance concentrations applied to the cells
8
Step-wise dilution factor
9
Determination whether test meets or doesn’t meet test acceptance criteria
Shaded boxes identify values that do not meet the specific test acceptance criteria
1
2

Phase
Ib (NHK)
II (NHK)
III (NHK)

Acceptance Limits for PC IC50
ECBC (ug/mL)
FAL (ug/mL)
1.40 – 6.67
1.34 – 13.6
1.22 – 6.10
0 – 11.1
0.07 – 7.11
0.57 – 5.82

IIVS (ug/mL)
2.57 – 4.79
2.10 – 5.04
1.94 – 5.61

I-144

In Vitro Cytotoxicity Test Methods BRD Appendix J

November 2006

Appendix J

LD50 and Toxicity Category Predictions

J1

3T3 NRU Predictions: RC Millimole Regression ........................ J-5

J2

NHK NRU Predictions: RC Millimole Regression.................... J-11

J3

3T3 NRU Predictions: RC Rat-Only Millimole Regression ..... J-17

J4

NHK NRU Predictions: RC Rat-Only Millimole Regression ... J-21

J5

3T3 NRU Predictions: RC Rat-Only Weight Regression.......... J-25

J6

NHK NRU Predictions: RC Rat-Only Weight Regression ....... J-29

J7

Comparison of Millimole Regression with Weight
Regression Regarding Prediction of Toxicity (LD50) for Low
or High Molecular Weight Chemicals........................................ J-34

J-1

In Vitro Cytotoxicity Test Methods BRD Appendix J

November 2006

[This Page Intentionally Left Blank]

J-2

In Vitro Cytotoxicity Test Methods BRD Appendix J

November 2006

Appendix J
The data presented in this appendix support the analyses in Section 6. For the analysis in
Appendices J1 through J6, the IC50 values for each reference substance are the geometricmean of the geometric mean IC50 values obtained for each laboratory. IC50 data for the samereference substances were used with each regression/test method evaluated. Sixty-sevenchemicals were evaluated for the 3T3 NRU test method and 68 chemicals were evaluated for
the NHK NRU test method. Of the original 72 chemicals tested, epinephrine bitartrate,colchicine, and propylparaben were excluded due to the lack of rat oral reference LD50 data.
Carbon tetrachloride and methanol were excluded from the 3T3 NRU evaluations because nolaboratory attained sufficient toxicity in any experiment for the calculation of an IC50. Carbon
tetrachloride was also excluded from the NHK NRU evaluations because no laboratory
attained sufficient toxicity in any test for the calculation of an IC50.
RC Millimole Regression: Appendices J1 (3T3 NRU) and J2 (NHK NRU)
Log LD50 (mmol/kg) = 0.435 log IC50 (mM) + 0.625
Appendices J1 and J2 support the analysis of outlier substances presented in Section 6.2.
Predicted LD50 values in mmol/kg and mg/kg (conversion from the mmol/kg values) for eachreference substance were determined for each test method using the respective IC50 values in
the RC millimole regression. Epinephrine bitartrate, colchicine, and propylparaben were
included in this analysis for a more complete comparison with the results of the RC. The
predicted log LD50 value was subtracted from the observed log LD50 value (initial values inTable 3-2 from the RC, HSDB, or RTECS® were converted to mmol/kg) and the difference(positive or negative) was compared to the RC criterion for outliers (0.699). Reference
substances with absolute values greater than 0.699 were identified as positive or negative
outliers to the RC millimole regression. The observed LD50 value (mg/kg) was used to assign
each reference chemical to an observed toxicity category (GHS acute oral classification [UN2005]). The predicted LD50 value (mg/kg) was used to determine the reference substance’s
predicted toxicity category.

J-3

In Vitro Cytotoxicity Test Methods BRD Appendix J

November 2006

RC Rat-Only Millimole Regression: Appendices J3 (3T3 NRU) and J4 (NHK NRU)
Log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621
Appendices J3 and J4 support the accuracy analyses for GHS acute oral toxicity category
predictions presented in Section 6.4.2. As described in Section 6.3.1, the RC rat-only
millimole regression was calculated using the RC IC50 and LD50 values for the 282 chemicals
that had rat oral LD50 values. The observed LD50 values, which were the reference LD50
values (mg/kg) from Table 4-2, were used to assign each reference substance to an observed
toxicity category (GHS acute oral classification [UN 2005]). The predicted LD50 value(mg/kg) was used to determine the reference substance’s predicted toxicity category.
RC Rat-Only Weight Regression: Appendices J5 (3T3 NRU) and J6 (NHK NRU)
Log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024
Appendices J5 and J6 support the accuracy analyses for GHS acute oral toxicity category
predictions presented in Section 6.4.2. As described in Section 6.3.2, the RC rat-only weight
regression was calculated using the RC IC50 and LD50 data for the 282 chemicals that had rat
oral LD50 values. The regression data were converted into weight units (i.e., LD50 values as
mg/kg and IC50 values as µg/mL). The observed LD50 values, which were the reference LD50
values (mg/kg) from Table 4-2, were used to assign each reference substance to an observed
toxicity category (GHS acute oral classification [UN 2005]). Predicted LD50 values in mg/kg
for each reference substance were determined for each NRU test method using the respective
NRU IC50 values in the RC rat-only weight regression. The predicted LD50 value (mg/kg)
was used to determine the reference substance’s predicted toxicity category.
Comparison of RC Rat-Only Millimole Regression and the RC Rat-Only Weight
Regression for the Prediction of LD50 for Low or High Molecular Weight Substances
Appendix J7 supports Section 6.6.2, which compares the under- and over-prediction of
acute oral toxicity (i.e., using LD50 values) for low and high molecular weight substances for
the RC rat-only millimole regression and the RC rat-only weight regression. The analysis
uses the RC IC50 and LD50 values for the 282 RC substances with rat oral LD50 data, which
are provided in Appendix K-3.

J-4

In Vitro Cytotoxicity Test Methods BRD Appendix J1

November 2006

Appendix J1

3T3 NRU Predictions: RC Millimole Regression

J-5

In Vitro Cytotoxicity Test Methods BRD Appendix J1

November 2006

[This Page Intentionally Left Blank]

J-6

In Vitro Cytotoxicity Methods BRD Appendix J1

November 2006

3T3 NRU Predictions: RC Millimole Regression

RC Millimole Regression: Log LD50 (mmol/kg) = 0.435 log IC50 (mM) + 0.625

Reference Substance1

Log Observed
LD50(mmol/kg)

Observed
LD50
Toxicity

Observed
LD50(mg/kg)2

Category(mg/kg)Log Predicted
LD50
3(mmol/kg)

Predicted
LD50

Predicted
LD50
Toxicity

4

2(mg/kg)Category(mg/kg)

3T3
Log IC50
5(mM)

Log Observed
LD50 - Log

3T3
IC50
5

(ug/mL)

Predicted LD50

Outlier7(mmol/kg)6

1,1,1-Trichloroethane

1.888

10298

> 5000

1.576

5022

> 5000

2.186

20453

0.312

2-Propanol

1.988

5843

> 5000

1.392

1483

300-2000

1.764

3489

0.595

5-Aminosalicylic Acid

1.704

7749

> 5000

1.076

1824

300-2000

1.037

1667

0.628

Acetaminophen

1.201

2404

2000-5000

0.407

385.9

300-2000-0.501

47.7

0.795

Acetonitrile

1.966

3798

2000-5000

1.620

1711

300-2000

2.287

7951

0.346

Acetylsalicylic Acid

0.744

1000

300-2000

0.875

1351

300-2000

0.574

676

-0.131

Aminopterin-2.1673<5-1.48015

5-50-4.8390.006-0.687

Amitriptyline HCl

0.061

361

300-2000-0.092254

50-300-1.648

7.05

0.153

Arsenictrioxide-1.00020

5-50-0.236115

50-300-1.9802.07-0.764Atropine Sulfate-0.036639

300-2000

0.207

1119

300-2000-0.96176.0-0.243

Boric Acid

1.634

2660

2000-5000

1.267

1143.6300-2000

1.476

1850

0.367

Busulfan-2.090

2

<5

0.407

629

300-2000-0.50177.7-2.497

Cadmium chloride-0.31988

50-300-0.48460

50-300-2.549

0.518

0.165

Caffeine-0.005192

50-3000.579

737

300-2000-0.105153-0.584

Carbamazepine

0.918

1957

300-2000

0.468

695

300-2000-0.360103

0.450

Chloral Hydrate

0.462

479

300-2000

0.644

729

300-2000

0.044

183

-0.182Chloramphenicol

1.021

3393

2000-5000

0.453

918

300-2000-0.395130

0.568

Citric Acid

1.194

3000

2000-5000

0.886

1477.5300-2000

0.600

765

0.308

Colchicine
Cupric Sulfate Pentahydrate

-1.82

6

-1.144

28.7

0.034

-0.680

300

0.268

462

5-50
300-2000-4.0660.080

5-50
50-300-0.82237.6-0.188Cycloheximide-2.1482<5-0.75749.3

5-50-3.1770.187-1.391

Dibutyl Phthalate

1.635

11998

> 50000.274

523

300-2000-0.807

43.4

1.361

Positive

Dichlorvos (DDVP)-1.114

17

5-50

0.149

311

300-2000-1.09517.7-1.262

Negative

Diethyl Phthalate

1.588

8602

> 50000.487

683

300-2000-0.316107

1.100

Positive

Digoxin-1.637

18

5-50

0.519

2580

2000-5000-0.244445-2.156

Negative

Dimethylformamide

1.583

2800

2000-5000

1.432

1974

300-2000

1.854

5224

0.152

Diquat Dibromide Monohydrate-0.173243

50-300-0.094291

50-300-1.6548.04-0.079Disulfoton-2.137

2

<5

0.696

1363

300-2000

0.163

400

-2.833

Negative

Endosulfan-1.35418

5-50-0.175272

50-300-1.8405.88-1.179Negative

J-7

Positive

Negative

Negative

Negative

In Vitro Cytotoxicity Methods BRD Appendix J1

November 2006

3T3 NRU Predictions: RC Millimole Regression

Reference Substance1

Log Observed
LD50(mmol/kg)

Observed
LD50
Toxicity

Observed
LD50(mg/kg)2

Category(mg/kg)Log Predicted
LD50
3(mmol/kg)

Predicted
LD50

Predicted
LD50
Toxicity

4

2(mg/kg)Category(mg/kg)

3T3
Log IC50
5(mM)

Log Observed
LD50 - Log

3T3
IC50
5

(ug/mL)

Predicted LD50

Outlier7(mmol/kg)6

Epinephrine bitartrate
Ethanol

-1.92

4

<5

0.298

662

300-2000-0.75259.0-2.219Negative

2.483

14008

> 5000

1.561

1675

300-2000

2.151

6523

0.922

Positive

Ethyleneglycol

2.140

8567

> 5000

1.754

3522

2000-5000

2.595

24436

0.386

Fenpropathrin-1.288

18

5-50

0.114

454

300-2000-1.17523.3-1.402Gibberellic Acid

1.260

6305

> 5000

1.214

5664

> 5000

1.353

7810

0.047

Glutethimide

0.441

600

300-2000

0.590

846

300-2000-0.079181-0.149Glycerol

2.139

12691

> 5000

1.679

4394

2000-5000

2.422

24345

0.461

Haloperidol-0.46812850-300-0.153264

50-300-1.7886.13-0.315Hexachlorophene-0.82461

50-300-0.239235

50-300-1.987

4.19-0.585Lactic Acid

1.617

3730

2000-5000

1.290

1757

300-2000

1.529

3044

0.327

Lindane-0.58576

50-3000.444

808

300-2000-0.416112-1.029Lithium carbonate

1.206

1187

300-2000

1.008

753

300-2000

0.881

562

0.198

Meprobamate

0.561

794

300-2000

0.778

1309

300-2000

0.351

490

-0.217

Mercury Chloride-2.4341<5-0.16618550-300-1.8194.12-2.268

Negative

Nicotine-0.511

50

5-50

0.776

969

300-2000

0.347

361

-1.287

Negative

Paraquat-0.5098050-3000.144

358.14300-2000-1.10620.1-0.652Parathion-2.161

2

<5

0.237

503

300-2000-0.89137.4-2.398

Negative

Phenobarbital-0.154163

50-300

0.800

1465

300-2000

0.402

586

-0.954

Negative

Phenol0.643

414

300-2000

0.559

341

300-2000-0.152

66.3

0.085

Phenylthiourea-1.705

3

<5

0.501

482

300-2000-0.28579.0-2.206

Negative

Physostigmine-1.787

5

<5

0.183

420

300-2000-1.015

26.6-1.970

Negative

Potassium cyanide-0.824

10

5-50

0.506

209

50-300-0.27434.6-1.330

Negative

Potassium chloride

1.543

2602

2000-5000

1.355

1689

300-2000

1.678

3555

0.188

Procainamide HCl

0.856

1950

300-2000

0.716

1414

300-2000

0.210

441

0.140

Propranolol

0.201

470

300-2000

0.050

332

300-2000-1.321

14.1

0.151

Propylparaben

1.550

6326

> 50000.260

328300-2000-0.840

26.1

1.290

Sodium Arsenite-0.50141

5-50-0.34758

50-300-2.2340.759-0.154

Sodium Chloride

1.710

2998

2000-5000

1.456

1669

300-2000

1.910

4746

0.254

Sodium Dichromate Dihydrate-0.71957

50-300-0.55284

50-300-2.7060.587-0.167

Sodium Hypochlorite

2.078

8910

> 50001.123

989

300-2000

1.145

1040

0.955

J-8

Negative

Negative

Positive

Positive

In Vitro Cytotoxicity Methods BRD Appendix J1

November 2006

3T3 NRU Predictions: RC Millimole Regression

Reference Substance1

Log Observed
LD50(mmol/kg)

Observed
LD50
Toxicity

Observed
LD50(mg/kg)2

Category(mg/kg)Log Predicted
LD50
3(mmol/kg)

Predicted
LD50

Predicted
LD50
Toxicity

4

2(mg/kg)Category(mg/kg)

3T3
Log IC50
5(mM)

Log Observed
LD50 - Log

3T3
IC50
5

(ug/mL)

Predicted LD50

Outlier7(mmol/kg)6

Sodium Oxalate

0.063

155

50-300

0.383

323.4

300-2000-0.55737.142

Sodium fluoride

0.632

180

50-3000.742

23250-300

0.269

78.0

-0.319-0.110Sodium selenate-2.072

2

<5

0.271

352.7

300-2000-0.81429.023-2.343

Negative

Strychnine-2.144

2

<5

0.483

1017

300-2000-0.326158-2.627

NegativeThallium Sulfate-1.24129

5-50-0.231296

50-300-1.9685.43-1.009Negative

Trichloroacetic Acid

1.486

4999

2000-5000

0.948

1449

300-2000

0.742

902

0.538

Triethylenemelamine-2.3101<5-0.62648

5-50-2.8750.272-1.684Triphenyltin Hydroxide-0.92144

5-50-1.25820

5-50-4.329

0.017

0.337

Valproic Acid

1.009

1471

300-2000

0.955

1299

300-2000

0.758

826

0.054

Verapamil HCl-0.65810850-3000.126656300-2000-1.14834.9-0.783

Xylene

1.607

4300

2000-5000

0.987

1030

300-2000

0.832

721

0.621

Negative

Negative

Abbreviations: 3T3=Neutral red uptake with mouse fibroblast 3T3 cell line
1

Carbon tetrachloride and methanol were excluded because IC50 values could not be determined. Initial LD50 from Table 3-2 converted to mmol/kg. Initial LD50 values came largely from the RC (1983/84 RTECS®)

for RC susbtances and from the current Hazardous Substances Data Bank (HSDB) or RTECS® and electronic database searches for non-RC substances.
2

Globally Harmonized System (GHS) hazard classification (UN 2005):
Abbreviation
Category
Oral LD50 Limits<5

1

LD50 ≤5 mg/kg

5-50

2

5 < LD50 ≤50 mg/kg

50-3003

50 < LD50 ≤300 mg/kg

300-2000

4300 < LD50 ≤2000 mg/kg

2000-5000

52000 < LD50 ≤5000 mg/kg>5000

Unclassified

LD50 >5000 mg/kg

3

LD50 determined using NRU IC50 in RC millimole regression: Log LD50 (mmol/kg) = 0.435 log IC50 (mM) + 0.6254

Predicted LD50 in mg/kg (converted from results of RC millimole regression)

5

Combined 3T3 IC50 values from three laboratories

6

Calculation to determine outliers to the RC millimole regression line7

Log observed LD50 - log predicted LD50 > 0.699 (or log 5) identifies a chemical as an “outlier”; negative=predicted value below prediction interval of RC millimole regression line; positive=predicted value above
prediction interval of RC millimole regression line (Halle 1998, 2003)

J-9

In Vitro Cytotoxicity Test Methods BRD Appendix J1

November 2006

[This Page Intentionally Left Blank]

J-10

In Vitro Cytotoxicity Test Methods BRD Appendix J2

November 2006

Appendix J2

NHK NRU Predictions: RC Millimole Regression

J-11

In Vitro Cytotoxicity Test Methods BRD Appendix J2

November 2006

[This Page Intentionally Left Blank]

J-12

In Vitro Cytotoxicity Test Methods BRD Appendix J2

November 2006

NHK NRU Predictions: RC Millimole Regression

RC Millimole Regression: Log LD50 (mmol/kg) = 0.435 log IC50 (mM) + 0.625

Reference Substance1

Log Observed
LD50
2(mmol/kg)
1,1,1-Trichloroethane
2-Propanol
5-Aminosalicylic Acid
Acetaminophen
Acetonitrile
Acetylsalicylic Acid
Aminopterin
Amitriptyline HCl
Arsenictrioxide
Atropine Sulfate
Boric Acid
BusulfanCadmium chloride
Caffeine
Carbamazepine
Chloral Hydrate
Chloramphenicol
Citric Acid
Colchicine
Cupric Sulfate Pentahydrate
Cycloheximide
Dibutyl Phthalate
Dichlorvos (DDVP)
Diethyl Phthalate
DigoxinDimethylformamide
Diquat Dibromide Monohydrate
Disulfoton
Endosulfan
Epinephrine bitartrate

1.888
1.988
1.704
1.201
1.966
0.744
-2.1670.061-1.000-0.0361.634-2.090-0.319-0.005
0.918
0.462
1.021
1.194
-1.82
0.080
-2.1481.635-1.1141.588-1.6371.583-0.173-2.137-1.354-1.92

Observed
LD50

Observed
LD50
Toxicity

3

4(mg/kg)

10298
5843
7749
2404
3798
1000
3
361
20
639
2660
2
88
192
1957
479
3393
3000
6.00
300
2
11998
17
8602
18
2800
243
2
18
4

Category(mg/kg)> 5000> 5000> 50002000-5000
2000-5000
300-2000<5300-2000
5-50
300-2000
2000-5000<550-30050-300300-2000
300-2000
2000-5000
2000-5000
5-50
50-300<5> 50005-50> 50005-50
2000-5000
50-300<55-50<5Log Predicted
LD50
5(mmol/kg)

1.401
1.473
0.401
0.858
1.654
0.854
0.702
-0.047-0.011
0.221
0.988
0.635
-0.249
0.850
0.428
0.584
0.637
0.769
-1.45
0.580
-0.934
0.196
0.053
0.509
-1.9371.506-0.211
0.622-0.3680.372

J-13

Predicted
LD50

Predicted
LD50
Toxicity

6

4(mg/kg)

3361
1788
385
1089
1853
1287
2218
282
193
1155
601
1064
103
1374
633
635
1402
1128
14.0
949
33
437
250
718
9
2345
223
1149
175
785

Category(mg/kg)2000-5000
300-2000
300-2000
300-2000
300-2000
300-2000
2000-5000
50-30050-300300-2000
300-2000
300-2000
50-300300-2000
300-2000
300-2000
300-2000
300-2000
5-50
300-2000
5-50
300-2000
50-300300-2000
5-50
2000-5000
50-300300-2000
50-300300-2000NHK
Log IC50
7

(mM)

1.784
1.951
-0.516
0.535
2.367
0.526
0.177
-1.545-1.461-0.9290.833
0.024-2.0090.516-0.453-0.0940.028
0.331-4.780-0.104-3.584-0.987-1.315-0.266-5.8892.026-1.922-0.007-2.282-0.581Log Observed
LD50 - Log

NHK
IC50
8

(ug/mL)

4709
2635
154
934
3065
1099
1557
-13
-2
255
593
676
-26
1167
271
371
894
867
0
550
-31
85
13
366
-17
3533
-47
714
-64
87

Predicted LD50

Outlier10(mmol/kg)9
0.486
0.514
1.304
0.344
0.312
-0.110-2.8690.107-0.989-0.2570.646-2.726-0.070-0.8550.490-0.1220.384
0.425-0.373-0.500-1.2141.439-1.167
1.079
0.299
0.077
0.038
-2.759-0.987-2.293

Positive

Negative
Negative

Negative
Negative

Negative
Positive
Negative
Positive

Negative
Negative

In Vitro Cytotoxicity Test Methods BRD Appendix J2

November 2006

NHK NRU Predictions: RC Millimole Regression

Reference Substance1

Ethanol
Ethyleneglycol
Fenpropathrin
Gibberellic Acid
Glutethimide
GlycerolHaloperidol
Hexachlorophene
Lactic Acid
Lindane
Lithium carbonate
Meprobamate
Mercury Chloride
MethanolNicotine
Paraquat
Parathion
Phenobarbital
PhenolPhenylthiourea
Physostigmine
Potassium Cyanide
Potassium chloride
Procainamide HCl
Propranolol
Propylparaben
Sodium Arsenite
Sodium Chloride
Sodium Dichromate Dihydrate
Sodium Hypochlorite
Sodium Oxalate

Log Observed
LD50

Observed
LD50(mmol/kg)2(mg/kg)3

2.483
2.140
-1.288
1.260
0.441
2.139
-0.468-0.8241.617-0.5851.206
0.561-2.4342.609-0.511-0.509-2.161-0.1540.643-1.705-1.787-0.824
1.543
0.856
0.201
1.550
-0.5011.710-0.719
2.078
0.063

14008
8567
18
6305
600
12691
128
61
3730
76
1187
794
1
13012
50
80
2
163
414
3
5
10
2602
1950
470
6326
41
2998
57
8910
155

Observed
LD50
Toxicity
Category4(mg/kg)> 5000> 50005-50> 5000300-2000> 500050-30050-3002000-5000
50-300300-2000
300-2000<5> 50005-5050-300<550-300300-2000<5<5
5-50
2000-5000
300-2000
300-2000> 50005-50
2000-5000
50-300> 500050-300

Log Predicted
LD50

Predicted
LD50(mmol/kg)5(mg/kg)6

1.642
1.857
-0.314
1.024
0.583
1.682
-0.266-1.180
1.130
0.107
0.974
0.718
-0.102
1.355
0.546
0.355
0.197
0.749
0.582
0.775
0.411
0.472
1.268
0.976
0.228
0.175
-0.4341.292-0.516
1.193
0.801

J-14

2019
4462
170
3657
831
4424
204
27
1215
372
695
1140
215
726
570
582
459
1303
360
906
709
193
1381
2571
500
269
48
1145
91
1160
847

Predicted
LD50
Toxicity
Category4(mg/kg)2000-5000
2000-5000
50-3002000-5000
300-2000
2000-5000
50-3005-50
300-2000
300-2000
300-2000
300-2000
50-300300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
50-300300-2000
2000-5000
300-2000
50-3005-50
300-2000
50-300300-2000
300-2000NHK
Log IC50

NHK
IC50

(mM)7

(ug/mL)8

2.337
2.831
-2.1580.916-0.0982.429-2.049-4.1491.161-1.1910.801
0.213-1.6711.679-0.182-0.621-0.9830.285-0.0980.344-0.493-0.3521.477
0.807-0.912-1.040-2.4351.534-2.622
1.305
0.404

3315
8285
-53
3743
484
7440
-54
-32
1373
40
677
818
-22
984
311
207
90
976
209
702
291
91
1750
2509
114
16.6
-21
1480
-47
1384
678

Log Observed
LD50 - Log
Predicted LD50

Outlier10(mmol/kg)9
0.841
0.283
-0.9740.237-0.1410.458-0.2020.356
0.487-0.6920.232-0.157-2.332
1.253-1.057-0.864-2.358-0.9030.061-2.480-2.197-1.2960.275-0.120-0.0271.375-0.0660.418-0.2040.885-0.737

Positive
Negative

Negative
Positive
Negative
Negative
Negative
Negative
Negative
Negative
Negative

Positive

Positive
Negative

In Vitro Cytotoxicity Test Methods BRD Appendix J2

November 2006

NHK NRU Predictions: RC Millimole Regression

Reference Substance1

Sodium fluoride
Sodium selenate
Strychnine
Thallium Sulfate
Trichloroacetic Acid
Triethylenemelamine
Triphenyltin Hydroxide
Valproic Acid
Verapamil HCl
XyleneLog Observed
LD50

Observed
LD50(mmol/kg)2(mg/kg)3

0.632-2.072-2.144-1.241
1.486-2.310-0.9211.009-0.6581.607

Observed
LD50
Toxicity
Category4(mg/kg)

180
2
2
29
4999
1
44
1471
108
4300

50-300<5<5
5-50
2000-5000<55-50
300-2000
50-3002000-5000

Log Predicted
LD50

Predicted
LD50(mmol/kg)5(mg/kg)6

0.654
0.074
0.302
-0.9070.800-0.263-1.360
0.864
0.247
0.904

189
224
670
62
1032
111
16
1055
868
852

Predicted
LD50
Toxicity
Category4(mg/kg)50-30050-300300-2000
50-300300-2000
50-3005-50
300-2000
300-2000
300-2000

Log Observed
LD50 - Log

NHK
Log IC50

NHK
IC50

(mM)7

(ug/mL)80.066-1.267-0.743-3.5220.403-2.042-4.5620.550-0.869
0.642

124
17
202
-57
826
-29
-22
912
214
770

Predicted LD50

Outlier10(mmol/kg)9-0.021-2.146-2.446-0.3330.685-2.0470.438
0.144-0.905
0.703

Negative
Negative

Negative

Negative
Positive

Abbreviations: NHK=Neutral red uptake with normal human epidermal keratinocytes.
1

Carbon tetrachloride and methanol were excluded because IC50 values could not be determined. Initial LD50 from Table 3-2 converted to mmol/kg. Initial LD50 values came largely from the RC (1983/84 RTECS®)

for RC susbtances and from the current Hazardous Substances Data Bank (HSDB) or RTECS® and electronic database searches for non-RC substances.
2

Globally Harmonized System (GHS) hazard classification (UN 2005):
Abbreviation
Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg<52
5 < LD50 ≤50 mg/kg
5-50
350 < LD50 ≤300 mg/kg
50-3004300 < LD50 ≤2000 mg/kg
300-2000
52000 < LD50 ≤5000 mg/kg
2000-5000
Unclassified
LD50 >5000 mg/kg>5000
3
LD50 determined using NRU IC50 in RC millimole regression: Log LD50 (mmol/kg) = 0.435 log IC50 (mM) + 0.625.
4
Predicted LD50 in mg/kg (converted from results of RC millimole regression).
5
Combined NHK IC50 values from three laboratories.
6
Calculation to determine outliers to the RC millimole regression line.
7

Log observed LD50 - log predicted LD50 > 0.699 (or log 5) identifies a chemical as an “outlier”; negative=predicted value below prediction interval of RC millimole regression line; positive=predicted value above
prediction interval of RC millimole regression line (Halle 1998, 2003).

J-15

In Vitro Cytotoxicity Test Methods BRD Appendix J2

November 2006

[This Page Intentionally Left Blank

J-16

In Vitro Cytotoxicity Test Methods BRD Appendix J3

November 2006

Appendix J3

3T3 NRU Predictions: RC Rat-Only Millimole Regression

J-17

In Vitro Cytotoxicity Test Methods BRD Appendix J3

November 2006

[This Page Intentionally Left Blank]

J-18

In Vitro Cytotoxicity Methods BRD Appendix J3

November 2006

3T3 NRU Predictions: RC Rat-Only Millimole Regression
Log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621

1

Reference Substance

Log
Reference
LD50(mmol/kg)

Reference
LD50(mg/kg)2

Observed
Toxicity
Category3(mg/kg)Log
Predicted
LD50(mmol/kg)4

Predicted
LD50(mg/kg)5Predicted
Toxicity
Category3(mg/kg)

3T3
Log
IC50
(mM)6

1.957
1.929
1.350
1.155
1.942
0.922
-1.7990.046-0.8970.071
1.744-1.308-0.132
0.203
1.075
0.586
1.033
1.489

12078
5105
3428
2162
3595
1506
7
349
25
819
3426
12
135
310
2807
638
3490
5929

>5000
>5000
2000-5000
2000-5000
2000-5000
300-2000
5-50
300-2000
5-50
300-2000
2000-5000
5-50
50-300300-2000
2000-5000
300-2000
2000-5000
>5000

1.580
1.395
1.076
0.401
1.625
0.873
-1.504-0.103-0.248
0.199
1.269
0.401
-0.498
0.575
0.463
0.640
0.448
0.884

5078
1494
1825
381
1731
1346
14
248
112
1099
1149
620
58
730
686
723
906
1472

>5000
300-2000
300-2000
300-2000
300-2000
300-2000
5-50
50-30050-300300-2000
300-2000
300-2000
50-300300-2000
300-2000
300-2000
300-2000
300-2000

2.186
1.764
1.037
-0.501
2.287
0.574-4.839-1.648-1.980-0.9611.476-0.501-2.549-0.105-0.3600.044-0.395
0.600

0.279

475

300-2000

0.260

455

300-2000-0.822-2.1481.504-0.5761.622-1.441
1.861

2
8892
59
9311
28
5305

<5
>5000
50-300
>5000
5-50
>5000

-0.774
0.267
0.140
0.482
0.514
1.435

47
514
305
674
2550
1990

5-50
300-2000
300-2000
300-2000
2000-5000
300-2000-3.177-0.807-1.095-0.316-0.2441.854-0.355160

50-300-0.105284

50-300-1.654-1.739-1.1652.391
2.062-0.664
1.241
0.441
2.332
-0.057-0.6961.606-0.4640.902
0.803-0.830-0.367

5
28
11324
7161
76
6039
600
19770
330
82
3635
100
590
1387
40
70

<5
5-50
>5000
>5000
50-300>5000
300-2000>5000
300-2000
50-3002000-5000
50-300300-2000
300-2000
5-50
50-3000.693-0.187
1.565
1.760
0.105
1.215
0.586
1.684
-0.164-0.251
1.292
0.438
1.008
0.775
-0.177
0.774

1352
265
1693
3574
445
5683
838
4452
258
228
1765
798
752
1301
180
963

300-2000
50-300300-2000
2000-5000
300-2000>5000
300-2000
2000-5000
50-30050-300300-2000
300-2000
300-2000
300-2000
50-300300-2000

0.163-1.8402.151
2.595-1.1751.353-0.0792.422-1.788-1.9871.529-0.4160.881
0.351-1.8190.347

2

1,1,1-Trichloroethane
2-Propanol
5-Aminosalicylic Acid
Acetaminophen
Acetonitrile
Acetylsalicylic Acid
Aminopterin
Amitriptyline HCl
Arsenictrioxide
Atropine Sulfate
Boric Acid
BusulfanCadmium chloride
Caffeine
Carbamazepine
Chloral Hydrate
Chloramphenicol
Citric Acid
Cupric Sulfate
Pentahydrate
Cycloheximide
Dibutyl Phthalate
Dichlorvos (DDVP)
Diethyl Phthalate
DigoxinDimethylformamide
Diquat Dibromide
Monohydrate
Disulfoton
Endosulfan
Ethanol
Ethylene glycol
Fenpropathrin
Gibberellic Acid
Glutethimide
GlycerolHaloperidol
Hexachlorophene
Lactic Acid
Lindane
Lithium carbonate
Meprobamate
Mercury Chloride
Nicotine

J-19

In Vitro Cytotoxicity Methods BRD Appendix J3

November 2006

3T3 NRU Predictions: RC Rat-Only Millimole Regression
Log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621

1

Reference Substance

Log
Reference
LD50(mmol/kg)

Reference
LD50(mg/kg)2

Observed
Toxicity
Category3(mg/kg)Log
Predicted
LD50(mmol/kg)4

Predicted
LD50(mg/kg)5Predicted
Toxicity
Category3(mg/kg)

3T3
Log
IC50
(mM)6

-0.443-1.679-0.0160.765-1.705-1.741-0.956
1.575
0.856
0.197
-0.474
1.841

93
6
224
548
3
5
7
2802
1950
466
44
4050

50-3005-5050-300300-2000<5<5
5-50
2000-5000
300-2000
300-2000
5-50
2000-5000

0.135
0.230
0.798
0.554
0.496
0.175
0.501
1.358
0.713
0.041
-0.360
1.459

351
494
1457
337
477
412
206
1699
1404
325
57
1683

300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
50-300300-2000
300-2000
300-2000
50-300300-2000-1.106-0.891
0.402-0.152-0.285-1.015-0.2741.678
0.210-1.321-2.2341.910-0.77150

50-300-0.56781

50-300-2.706

2.142
0.674
0.480
-1.799-1.725-1.3051.505-1.708-0.0470.839-0.646
1.643

10328
633
127
3
6
25
5229
4
329
996
111
4665

>5000
300-2000
50-300
<5
5-50
5-50
>5000
<5
300-2000
300-2000
50-3002000-5000

1.124
0.376
0.739
0.264
0.478
-0.2430.947-0.641-1.279
0.954
0.117
0.986

990
319
230
347
1005
288
1445
47
19
1296
643
1028

300-2000
300-2000
50-300300-2000
300-2000
50-300300-2000
5-50
5-50
300-2000
300-2000
300-2000

1.145-0.5570.269-0.814-0.326-1.9680.742-2.875-4.3290.758-1.1480.832

2Paraquat
Parathion
Phenobarbital
PhenolPhenylthiourea
Physostigmine
Potassium Cyanide
Potassium chloride
Procainamide HCl
Propranolol
Sodium Arsenite
Sodium Chloride
Sodium Dichromate
Dihydrate
Sodium Hypochlorite
Sodium Oxalate
Sodium fluoride
Sodium selenate
Strychnine
Thallium Sulfate
Trichloroacetic Acid
Triethylenemelamine
Triphenyltin Hydroxide
Valproic Acid
Verapamil HCl
Xylene1

Three chemicals were excluded because no rat oral LD50 was identified: epinephrine bitartrate, colchicine, and propylparaben. Carbon
tetrachloride and methanol were excluded because IC50 values could not be determined.
2
Reference LD50 in mmol/kg from Table 4-2. Reference rat oral LD50 values were developed from rat acute oral LD50 studies located using
literature searches, secondary references, and electronic database searches.
3
Globally Harmonized System (GHS) hazard classification (UN 2005):
Category
Oral LD50 Limits
Abbreviation<5
1
LD50 ≤5 mg/kg
5-50
25 < LD50 ≤50 mg/kg
50-3003
50 < LD50 ≤300 mg/kg
300-2000
4300 < LD50 ≤2000 mg/kg
2000-5000
52000 < LD50 ≤5000 mg/kg>5000
Unclassified LD50 >5000 mg/kg
4

LD50 determined using NRU IC50 in RC rat-only millimole regression: Log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
LD50 in mg/kg (converted from results of RC rat-only millimole regression).
6
Combined 3T3 IC50 values from three laboratories.
5

J-20

In Vitro Cytotoxicity Test Methods BRD Appendix J4

November 2006

Appendix J4

NHK NRU Predictions: RC Rat-Only Millimole Regression

J-21

In Vitro Cytotoxicity Test Methods BRD Appendix J4

November 2006

[This Page Intentionally Left Blank]

J-22

In Vitro Cytotoxicity Methods BRD Appendix J4

November 2006

NHK NRU Predictions: RC Rat-Only Millimole Regression
Log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621
1

Reference Substance

1,1,1-Trichloroethane
2-Propanol
5-Aminosalicylic Acid
Acetaminophen
Acetonitrile
Acetylsalicylic Acid
Aminopterin
Amitriptyline HCl
Arsenictrioxide
Atropine Sulfate
Boric Acid
BusulfanCadmium chloride
Caffeine
Carbamazepine
Chloral Hydrate
Chloramphenicol
Citric Acid
Cupric Sulfate
Pentahydrate
Cycloheximide
Dibutyl Phthalate
Dichlorvos (DDVP)
Diethyl Phthalate
DigoxinDimethylformamide
Diquat Dibromide
Monohydrate
Disulfoton
Endosulfan
Ethanol
Ethyleneglycol
Fenpropathrin
Gibberellic Acid
Glutethimide
GlycerolHaloperidol
Hexachlorophene
Lactic Acid
Lindane
Lithium carbonate
Meprobamate
Mercury Chloride
MethanolNicotine

Log
Reference
LD50(mmol/kg)2
1.957
1.929
1.350
1.155
1.942
0.922
-1.7990.046-0.8970.071
1.744-1.308-0.132
0.203
1.075
0.586
1.033
1.489

12078
5105
3428
2162
3595
1506
7
349
25
819
3426
12
135
310
2807
638
3490
5929

Observed
Toxicity
Category3(mg/kg)>5000>5000
2000-5000
2000-5000
2000-5000
300-2000
5-50
300-2000
5-50
300-2000
2000-5000
5-50
50-300300-2000
2000-5000
300-2000
2000-5000
>5000

Log
Predicted
LD50(mmol/kg)4
3.478
3.411
2.645
3.034
3.505
3.059
3.073
2.378
2.335
2.736
3.000
2.922
2.119
3.067
2.738
2.814
2.968
2.997

3009
2579
442
1081
3196
1145
1184
239
216
544
1001
836
131
1168
547
652
929
992

Predicted
Toxicity
Category3(mg/kg)2000-5000
300-2000
300-2000
300-2000
300-2000
300-2000
5-50
50-30050-300300-2000
300-2000
300-2000
50-300300-2000
300-2000
300-2000
300-2000
300-2000

NHK
Log
IC50
(mM)6
1.784
1.951
-0.516
0.535
2.367
0.526
0.177
-1.545-1.461-0.9290.833
0.024-2.0090.516-0.453-0.094
0.028
0.331

0.279

475

300-2000

2.877

754

300-2000-0.104-2.1481.504-0.5761.622-1.441
1.861

2
8892
59
9311
28
5305

<5
>5000
50-300
>5000
5-50
>5000

1.602
2.566
2.407
2.798
0.909
3.471

40
368
255
628
8
2958

5-50
300-2000
50-300300-2000
5-50
2000-5000-3.584-0.987-1.315-0.266-5.8892.026-0.355160

50-3002.261

18250-300-1.922-1.739-1.1652.391
2.062-0.664
1.241
0.441
2.332
-0.057-0.6961.606-0.4640.902
0.803-0.8302.434-0.367

5
28
11324
7161
76
6039
600
19770
330
82
3635
100
590
1387
40
8710
70

<5
5-50
>5000
>5000
50-300>5000
300-2000>5000
300-2000
50-3002000-5000
50-300300-2000
300-20005-50>5000
50-300

2.928
2.146
3.512
3.744
2.167
3.310
2.857
3.658
2.220
1.451
3.183
2.497
3.017
2.973
2.308
3.209
2.778

848
140
3253
5549
147
2040
720
4553
166
28
1524
314
1040
941
203
1616
600

300-2000
50-3002000-5000
2000-5000
50-3002000-5000
300-2000
2000-5000
50-3005-50
300-2000
300-2000
300-2000
300-2000
50-300300-2000
300-2000-0.007-2.2822.337
2.831-2.1580.916-0.0982.429-2.049-4.1491.161-1.1910.801
0.213-1.6711.679-0.182

Reference
LD50(mg/kg)2

J-23

Predicted
LD50(mg/kg)5In Vitro Cytotoxicity Methods BRD Appendix J4

November 2006

NHK NRU Predictions: RC Rat-Only Millimole Regression
Log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621
1

Reference Substance

Paraquat
Parathion
Phenobarbital
PhenolPhenylthiourea
Physostigmine
Potassium Cyanide
Potassium chloride
Procainamide HCl
Propranolol
Sodium Arsenite
Sodium Chloride
Sodium Dichromate
Dihydrate
Sodium Hypochlorite
Sodium Oxalate
Sodium fluoride
Sodium selenate
Strychnine
Thallium Sulfate
Trichloroacetic Acid
Triethylenemelamine
Triphenyltin Hydroxide
Valproic Acid
Verapamil HCl
XyleneLog
Reference
LD50(mmol/kg)2-0.443-1.679-0.0160.765-1.705-1.741-0.956
1.575
0.856
0.197
-0.474
1.841

93
6
224
548
3
5
7
2802
1950
466
44
4050

Observed
Toxicity
Category3(mg/kg)50-3005-5050-300300-2000<5<5
5-50
2000-5000
300-2000
300-2000
5-50
2000-5000

Log
Predicted
LD50(mmol/kg)4
2.690
2.575
3.010
2.722
2.964
2.748
2.568
3.270
3.230
2.604
1.904
3.252

-0.77150

50-300

2.142
0.674
0.480
-1.799-1.725-1.3051.505-1.708-0.0470.839-0.646
1.643

10328
633
127
3
6
25
5229
4
329
996
111
4665

>5000
300-2000
50-300
<5
5-50
5-50
>5000
<5
300-2000
300-2000
50-3002000-5000

Reference
LD50(mg/kg)2

489
376
1024
527
920
560
370
1862
1697
402
80
1786

Predicted
Toxicity
Category3(mg/kg)300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
50-300300-2000
2000-5000
300-2000
5-50
300-2000

NHK
Log
IC50
(mM)6
-0.621-0.9830.285-0.0980.344-0.493-0.3521.477
0.807-0.912-2.435
1.534

1.969

93

50-300-2.622

3.206
2.965
2.652
2.399
2.687
1.719
2.997
2.124
1.281
3.032
2.702
3.016

1606
923
449
251
486
52
994
133
19
1076
503
1039

300-2000
300-2000
50-30050-300300-2000
50-300300-2000
50-3005-50
300-2000
300-2000
300-2000

1.305
0.404
0.066
-1.267-0.743-3.5220.403-2.042-4.5620.550-0.8690.642

Predicted
LD50(mg/kg)5

1

Three chemicals were excluded because no rat oral LD50 was identified: epinephrine bitartrate, colchicine, and propylparaben. Carbon
tetrachloride was excluded because IC50 values could not be determined.
2
Reference LD50 in mmol/kg from Table 4-2. Reference rat oral LD50 values were developed from rat acute oral LD50 studies located using
literature searches, secondary references, and electronic database searches.
3
Globally Harmonized System (GHS) hazard classification (UN 2005):
Category
Oral LD50 Limits
Abbreviation<5
1
LD50 ≤5 mg/kg
5-50
25 < LD50 ≤50 mg/kg
50-3003
50 < LD50 ≤300 mg/kg
300-2000
4300 < LD50 ≤2000 mg/kg
2000-5000
52000 < LD50 ≤5000 mg/kg>5000
Unclassified LD50 >5000 mg/kg
4

LD50 determined using NRU IC50 in RC rat-only millimole regression: Log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621
LD50 in mg/kg (converted from results of RC rat-only millimole regression)
6
Combined NHK IC50 values from three laboratories
5J-24

In Vitro Cytotoxicity Test Methods BRD Appendix J5

November 2006

Appendix J5

3T3 NRU Predictions: RC Rat-Only Weight Regression

J-25

In Vitro Cytotoxicity Test Methods BRD Appendix J5

November 2006

[This Page Intentionally Left Blank]

J-26

In Vitro Cytotoxicity Methods BRD Appendix J5

November 2006

3T3 NRU Predictions: RC Rat-Only Weight Regression
Log LD50 (mg/kg) = 0.372 log IC50 (ug/mL) + 2.024
1Reference Substance

1,1,1-Trichloroethane
2-Propanol
5-Aminosalicylic Acid
Acetaminophen
Acetonitrile
Acetylsalicylic Acid
Aminopterin
Amitriptyline HCl
Arsenictrioxide
Atropine Sulfate
Boric Acid
BusulfanCadmium chloride
Caffeine
Carbamazepine
Chloral Hydrate
Chloramphenicol
Citric Acid
Cupric Sulfate
Pentahydrate
Cycloheximide
Dibutyl Phthalate
Dichlorvos (DDVP)
Diethyl Phthalate
DigoxinDimethylformamide
Diquat Dibromide
Monohydrate
Disulfoton
Endosulfan
Ethanol
Ethyleneglycol
Fenpropathrin
Gibberellic Acid
Glutethimide
GlycerolHaloperidol
Hexachlorophene
Lactic Acid
Lindane
Lithium carbonate
Meprobamate
Mercury Chloride
Nicotine

Log
Reference
LD50(mg/kg)2
4.082
3.708
3.535
3.335
3.556
3.178
0.845
2.543
1.400
2.913
3.535
1.084
2.131
2.491
3.448
2.805
3.543
3.773

12078
5105
3428
2162
3595
1506
7
349
25
819
3426
12
135
310
2807
638
3490
5929

Observed
Toxicity
Category3(mg/kg)>5000>5000
2000-5000
2000-5000
2000-5000
300-2000
5-50
300-2000
5-50
300-2000
2000-5000
5-50
50-300300-2000
2000-5000
300-2000
2000-5000
>5000

Log
Predicted
LD50(mg/kg)4
3.628
3.342
3.223
2.648
3.475
3.077
1.207
2.340
2.142
2.724
3.239
2.727
1.918
2.836
2.773
2.866
2.811
3.097

5078
1494
1825
381
1731
1346
14
248
112
1099
1149
620
58
730
686
723
906
1472

Predicted
Toxicity
Category3(mg/kg)>5000
300-2000
300-2000
300-2000
300-2000
300-2000
5-50
50-30050-300300-2000
300-2000
300-2000
50-300300-2000
300-2000
300-2000
300-2000
300-2000

2.677

475

300-2000

2.610

455

300-2000

1.576

0.301
3.949
1.769
3.969
1.451
3.725

2
8892
59
9311
28
5305

<5
>5000
50-300
>5000
5-50
>5000

1.753
2.633
2.489
2.779
3.009
3.407

47
514
305
674
2550
1990

5-50
300-2000
300-2000
300-2000
2000-5000
300-2000-0.727
1.637
1.249
2.031
2.649
3.718

2.204

160

50-3002.361

284

50-300

0.905

0.699
1.444
4.054
3.855
1.879
3.781
2.778
4.296
2.519
1.914
3.561
2.000
2.771
3.142
1.604
1.843

5
28
11324
7161
76
6039
600
19770
330
82
3635
100
590
1387
40
70

<5
5-50
>5000
>5000
50-300>5000
300-2000>5000
300-2000
50-3002000-5000
50-300300-2000
300-2000
5-50
50-300

2.992
2.310
3.443
3.656
2.533
3.472
2.864
3.656
2.317
2.256
3.320
2.786
3.047
3.025
2.253
2.975

1352
265
1693
3574
445
5683
838
4452
258
228
1765
798
752
1301
180
963

300-2000
50-300300-2000
2000-5000
300-2000>5000
300-2000
2000-5000
50-30050-300300-2000
300-2000
300-2000
300-2000
50-300300-2000

2.602
0.770
3.814
4.388
1.368
3.893
2.258
4.386
0.787
0.623
3.483
2.047
2.749
2.690
0.615
2.557

Reference
LD50(mg/kg)2

J-27

Predicted
LD50(mg/kg)5

3T3
Log IC50(ug/mL)6
4.311
3.543
3.222
1.678
3.900
2.830
-2.195
0.848
0.316
1.881
3.267
1.890
-0.286
2.183
2.014
2.263
2.115
2.884

In Vitro Cytotoxicity Methods BRD Appendix J5

November 2006

3T3 NRU Predictions: RC Rat-Only Weight Regression
Log LD50 (mg/kg) = 0.372 log IC50 (ug/mL) + 2.024
1Reference Substance
Paraquat
Parathion
Phenobarbital
PhenolPhenylthiourea
Physostigmine
Potassium Cyanide
Potassium chloride
Procainamide HCl
Propranolol
Sodium Arsenite
Sodium Chloride
Sodium Dichromate
Dihydrate
Sodium Hypochlorite
Sodium Oxalate
Sodium fluoride
Sodium selenate
Strychnine
Thallium Sulfate
Trichloroacetic Acid
Triethylenemelamine
Triphenyltin
Hydroxide
Valproic Acid
Verapamil HCl
XyleneLog
Reference
LD50(mg/kg)2
1.967
0.785
2.350
2.739
0.477
0.699
0.857
3.447
3.290
2.668
1.639
3.607

93
6
224
548
3
5
7
2802
1950
466
44
4050

Observed
Toxicity
Category3(mg/kg)50-3005-5050-300300-2000<5<5
5-50
2000-5000
300-2000
300-2000
5-50
2000-5000

Log
Predicted
LD50(mg/kg)4
2.509
2.609
3.054
2.702
2.730
2.554
2.597
3.345
3.008
2.452
1.979
3.392

351
494
1457
337
477
412
206
1699
1404
325
57
1683

Predicted
Toxicity
Category3(mg/kg)300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
50-300300-2000
300-2000
300-2000
50-300300-2000

1.703

50

50-3001.938

8150-300-0.232

4.014
2.801
2.103
0.477
0.799
1.398
3.718
0.602

10328
633
127
3
6
25
5229
4

>5000
300-2000
50-300
<5
5-50
5-50
>5000
<5

3.146
2.608
2.728
2.568
2.842
2.297
3.123
1.814

990
319
230
347
1005
288
1445
47

300-2000
300-2000
50-300300-2000
300-2000
50-300300-2000
5-50

3.017
1.570
1.892
1.463
2.198
0.735
2.955
-0.5652.517

329

300-2000

1.368

19

5-50

-1.764

2.998
2.045
3.669

996
111
4665

300-2000
50-3002000-5000

3.109
2.598
3.087

1296
643
1028

300-2000
300-2000
300-2000

2.917
1.543
2.858

Reference
LD50(mg/kg)2

Predicted
LD50(mg/kg)51

3T3
Log IC50(ug/mL)6
1.304
1.573
2.768
1.822
1.898
1.425
1.540
3.551
2.644
1.150
-0.120
3.676

Three chemicals were excluded because no rat oral LD50 was identified: epinephrine bitartrate, colchicine, and propylparaben. Carbon
tetrachloride and methanol were excluded because IC50 values could not be determined.
2
Reference LD50 in mmol/kg from Table 4-2. Reference rat oral LD50 values were developed from rat acute oral LD50 studies located using
literature searches, secondary references, and electronic database searches.
3
Globally Harmonized System (GHS) hazard classification (UN 2005):
Category
Oral LD50 Limits
Abbreviation<5
1
LD50 ≤5 mg/kg
5-50
25 < LD50 ≤50 mg/kg
50-3003
50 < LD50 ≤300 mg/kg
300-2000
4300 < LD50 ≤2000 mg/kg
2000-5000
52000 < LD50 ≤5000 mg/kg>5000
Unclassified LD50 >5000 mg/kg
4

LD50 determined using NRU IC50 in RC rat-only weight regression: Log LD50 (mg/kg) = 0.372 log IC50 (ug/mL) + 2.024
LD50 in mg/kg (converted from results of RC rat-only weight regression)
6
Combined 3T3 IC50 values from three laboratories
5J-28

In Vitro Cytotoxicity Test Methods BRD Appendix J6

November 2006

Appendix J6

NHK NRU Predictions: RC Rat-Only Weight Regression

J-29

In Vitro Cytotoxicity Test Methods BRD Appendix J6

November 2006

[This Page Intentionally Left Blank]

J-30

In Vitro Cytotoxicity Methods BRD Appendix J6

November 2006

NHK NRU Predictions: RC Rat-Only Weight Regression
Log LD50 (mg/kg) = 0.372 log IC50 (ug/mL) + 2.024
1Reference Substance

1,1,1-Trichloroethane
2-Propanol
5-Aminosalicylic Acid
Acetaminophen
Acetonitrile
Acetylsalicylic Acid
Aminopterin
Amitriptyline HCl
Arsenictrioxide
Atropine Sulfate
Boric Acid
BusulfanCadmium chloride
Caffeine
Carbamazepine
Chloral Hydrate
Chloramphenicol
Citric Acid
Cupric Sulfate
Pentahydrate
Cycloheximide
Dibutyl Phthalate
Dichlorvos (DDVP)
Diethyl Phthalate
DigoxinDimethylformamide
Diquat Dibromide
Monohydrate
Disulfoton
Endosulfan
Ethanol
Ethyleneglycol
Fenpropathrin
Gibberellic Acid
Glutethimide
GlycerolHaloperidol
Hexachlorophene
Lactic Acid
Lindane
Lithium carbonate
Meprobamate
Mercury Chloride
MethanolNicotine

Log
Reference
LD50(mg/kg)2
1.957
1.929
1.350
1.155
1.942
0.922
-1.7990.046-0.8970.071
1.744-1.308-0.132
0.203
1.075
0.586
1.033
1.489

12078
5105
3428
2162
3595
1506
7
349
25
819
3426
12
135
310
2807
638
3490
5929

Observed
Toxicity
Category3(mg/kg)>5000>5000
2000-5000
2000-5000
2000-5000
300-2000
5-50
300-2000
5-50
300-2000
2000-5000
5-50
50-300300-2000
2000-5000
300-2000
2000-5000
>5000

Log
Predicted
LD50(mg/kg)4
3.478
3.411
2.645
3.034
3.505
3.059
3.073
2.378
2.335
2.736
3.000
2.922
2.119
3.067
2.738
2.814
2.968
2.997

3009
2579
442
1081
3196
1145
1184
239
216
544
1001
836
131
1168
547
652
929
992

Predicted
Toxicity
Category3(mg/kg)2000-5000
300-2000
300-2000
300-2000
300-2000
300-2000
2000-5000
50-30050-300300-2000
300-2000
300-2000
50-300300-2000
300-2000
300-2000
300-2000
300-2000

0.279

475

300-2000

2.877

754

300-2000

2.293-2.1481.504-0.5761.622-1.441
1.861

2
8892
59
9311
28
5305

<5
>5000
50-300
>5000
5-50
>5000

1.602
2.566
2.407
2.798
0.909
3.471

40
368
255
628
8
2958

5-50
300-2000
50-300300-2000
5-50
2000-5000-1.134
1.458
1.029
2.081
-2.9963.890-0.355160

50-3002.261

18250-3000.637-1.739-1.1652.391
2.062-0.664
1.241
0.441
2.332
-0.057-0.6961.606-0.4640.902
0.803-0.8302.434-0.367

5
28
11324
7161
76
6039
600
19770
330
82
3635
100
590
1387
40
8710
70

<5
5-50
>5000
>5000
50-300>5000
300-2000>5000
300-2000
50-3002000-5000
50-300300-2000
300-20005-50>5000
50-300

2.928
2.146
3.512
3.744
2.167
3.310
2.857
3.658
2.220
1.451
3.183
2.497
3.017
2.973
2.308
3.209
2.778

848
140
3253
5549
147
2040
720
4553
166
28
1524
314
1040
941
203
1616
600

300-2000
50-3002000-5000
2000-5000
50-3002000-5000
300-2000
2000-5000
50-3005-50
300-2000
300-2000
300-2000
300-2000
50-300300-2000
300-2000

2.431
0.328
4.001
4.624
0.385
3.456
2.239
4.393
0.526
-1.540
3.115
1.272
2.670
2.552
0.763
3.184
2.028

Reference
LD50(mg/kg)2

J-31

Predicted
LD50(mg/kg)5NHK
Log IC50(ug/mL)6
3.910
3.730
1.669
2.714
3.980
2.782
2.821
0.952
0.835
1.913
2.625
2.415
0.255
2.805
1.920
2.125
2.538
2.614

In Vitro Cytotoxicity Methods BRD Appendix J6

November 2006

NHK NRU Predictions: RC Rat-Only Weight Regression
Log LD50 (mg/kg) = 0.372 log IC50 (ug/mL) + 2.024
1Reference Substance
Paraquat
Parathion
Phenobarbital
PhenolPhenylthiourea
Physostigmine
Potassium Cyanide
Potassium chloride
Procainamide HCl
Propranolol
Sodium Arsenite
Sodium Chloride
Sodium Dichromate
Dihydrate
Sodium Hypochlorite
Sodium Oxalate
Sodium fluoride
Sodium selenate
Strychnine
Thallium Sulfate
Trichloroacetic Acid
Triethylenemelamine
Triphenyltin
Hydroxide
Valproic Acid
Verapamil HCl
XyleneLog
Reference
LD50(mg/kg)2-0.443-1.679-0.0160.765-1.705-1.741-0.956
1.575
0.856
0.197
-0.474
1.841

93
6
224
548
3
5
7
2802
1950
466
44
4050

Observed
Toxicity
Category3(mg/kg)50-3005-5050-300300-2000<5<5
5-50
2000-5000
300-2000
300-2000
5-50
2000-5000

Log
Predicted
LD50(mg/kg)4
2.690
2.575
3.010
2.722
2.964
2.748
2.568
3.270
3.230
2.604
1.904
3.252

489
376
1024
527
920
560
370
1862
1697
402
80
1786

Predicted
Toxicity
Category3(mg/kg)300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
50-300300-2000
2000-5000
300-2000
5-50
300-2000-0.77150

50-3001.969

9350-300-0.148

2.142
0.674
0.480
-1.799-1.725-1.3051.505-1.708

10328
633
127
3
6
25
5229
4

>5000
300-2000
50-300
<5
5-50
5-50
>5000
<5

3.206
2.965
2.652
2.399
2.687
1.719
2.997
2.124

1606
923
449
251
486
52
994
133

300-2000
300-2000
50-30050-300300-2000
50-300300-2000
50-300

3.177
2.531
1.689
1.009
1.781
-0.8192.616
0.268-0.047329

300-2000

1.281

19

5-50

-1.9980.839-0.646
1.643

996
111
4665

300-2000
50-3002000-5000

3.032
2.702
3.016

1076
503
1039

300-2000
300-2000
300-2000

2.709
1.823
2.668

Reference
LD50(mg/kg)2

Predicted
LD50(mg/kg)51

NHK
Log IC50(ug/mL)6
1.790
1.481
2.651
1.875
2.527
1.947
1.462
3.350
3.241
1.559
-0.322
3.300

Three chemicals were excluded because no rat oral LD50 was identified: epinephrine bitartrate, colchicine, and propylparaben. Carbon
tetrachloride was excluded because IC50 values could not be determined.
2
Reference LD50 in mmol/kg from Table 4-2. Reference rat oral LD50 values were developed from rat acute oral LD50 studies located using
literature searches, secondary references, and electronic database searches.
3
Globally Harmonized System (GHS) hazard classification (UN 2005):
Category
Oral LD50 Limits
Abbreviation<5
1
LD50 ≤5 mg/kg
5-50
25 < LD50 ≤50 mg/kg
50-3003
50 < LD50 ≤300 mg/kg
300-2000
4300 < LD50 ≤2000 mg/kg
2000-5000
52000 < LD50 ≤5000 mg/kg>5000
Unclassified LD50 >5000 mg/kg
4

LD50 determined using NRU IC50 in RC rat-only weight regression: Log LD50 (mg/kg) = 0.372 log IC50 (ug/mL) + 2.024
LD50 in mg/kg (converted from results of RC rat-only weight regression)
6
Combined NHK IC50 values from three laboratories
5J-32

In Vitro Cytotoxicity Test Methods BRD Appendix J7

November 2006

Appendix J7Comparison of Millimole Regression with Weight
Regression Regarding Prediction of Toxicity (LD50) for Low
or High Molecular Weight Chemicals

J-33

In Vitro Cytotoxicity Test Methods BRD Appendix J7

November 2006

[This Page Intentionally Left Blank]

J-34

In Vitro Cytotoxicity Test Methods BRD Appendix J7

J.7

November 2006

The Prediction of Toxicity for High and Low Molecular Weight Substances
Using Millimole vs. Weight-Based Regressions

The ICCVAM Acute Toxicity Working Group expressed some concern that the RC rat-only
weight regression may less accurately (than the RC rat-only millimole regression) predict the
toxicity of low molecular weight substances and high molecular weight substances. Using the
RC IC50 and LD50 values for the 282 RC substances with rat oral LD50 data, analyses were
performed to• Determine the difference in the over and under-prediction rates of acute oral
toxicity (i.e., LD50) from IC50 values for low molecular weight substances(i.e., molecular weight ≤100 g/mole) vs. substances with higher molecularweights• Determine the difference in the over and under-prediction rates of acute oral
toxicity from IC50 values for high molecular weight substances (i.e., molecularweight ≥400 g/mole) vs. substances with lower molecular weights• Compare the RC rat-only millimole regression with the RC rat-only weight
regression with respect to the over and under-prediction rates of the toxicity of
low and high molecular weight substances
J.7.1
Methods
The data used for to evaluate the over- and under-prediction rates of toxicity of low or highmolecular weight chemicals were the RC data rather than the NICEATM/ECVAM validation
study data because the RC contains data for many more substances. The RC IC50 and LD50
values for the 282 RC substances with rat oral LD50 data were used since substances with rat
data are the focus of the BRD with respect to the prediction of oral LD50 (and starting dose
for acute oral toxicity testing) from IC50 (see Appendix K-3 for the data used). Over- or
under-prediction of toxicity was determined by subtracting the predicted LD50 in mg/kg (i.e.,
the rat oral LD50 calculated using the RC IC50 in the regression equation) from the observed
LD50 in mg/kg (i.e., the in vivo rat oral LD50 from the RC that was used to develop the
regression). Negative values indicated that toxicity was underpredicted by the regression(i.e., predicted LD50 was greater than observed LD50) and positive values indicated that
toxicity was overpredicted by the regression (i.e., predicted LD50 was less than observedLD50). This analysis assumed that the regressions either underpredicted or overpredicted the
toxicity of all of the substances evaluated. In other words, there was a difference between the
LD50 predicted by the regression and the in vivo LD50 used to calculate the regression even if
it was a tiny fraction (i.e., no substances fit the regression exactly).
The proportion of low or high molecular weight chemicals that were under- and overpredicted in terms of acute oral toxicity (i.e., predicted LD50 values were higher or lower thanreported in vivo LD50 values, respectively) using a millimole regression were calculated.
These proportions were compared with those for chemicals that did not have low or highmolecular weights. The same calculations were then performed for a weight-based
regression. The proportions of under- and over-prediction of the toxicity for the millimole
and weight-based regressions were compared to determine whether the weight regression
increased the proportion of low molecular weight chemicals for which toxicity was
underpredicted or the proportion of high molecular weight chemicals for which toxicity was
overpredicted.

J-35

In Vitro Cytotoxicity Test Methods BRD Appendix J7

November 2006

The millimole regression used was the RC rat-only millimole regression. The RC rat-onlyregression in millimole units, was calculated using the IC50 and oral LD50 data from the 282
RC chemicals with rat oral LD50 values and is strikingly similar in slope and intercept to theoriginal RC millimole regression, which was based on 347 chemicals (282 chemicals with rat
LD50 data and 65 chemicals with mouse LD50 data) (see Table J7-1). The weight-based
regression used was the RC rat-only weight regression calculated using the IC50 and oral
LD50 values from the 282 RC chemicals with rat oral LD50 values (see Table J7-1).
Table J7-1

IC50-LD50 Linear Regressions

Moniker
RC millimole regression
RC rat-only millimole
regression
RC rat-only weight regression

Data Used
347 RC substances with oral rat and mouse
LD50 data – millimole units1
282 RC substances with rat oral LD50 data
– millimole units1
282 RC substances with rat oral LD50 data
– weight units2

SlopeIntercept

R2

0.435

0.625

0.452

0.439

0.621

0.452

0.372

2.024

0.325

2

Abbreviations: RC=Registry of Cytotoxicity; R =coefficient of determination
1
IC50 in mM; LD50 in mmol/kg.
2
IC50 in µg/mL; LD50 in mg/kg.

J.7.2
Results
Figures J7-1 and J7-2 show either the low molecular weight or high molecular weightchemicals plotted with either the RC rat-only millimole regression or the RC rat-only weight
regression. Since LD50 is inversely related to toxicity, low LD50 values indicate high toxicity
and high LD50 values indicate low toxicity. The regression lines show the predicted LD50 foreach IC50. The regression lines underpredict the toxicity of chemicals that are plotted below
the lines (i.e., predicted LD50 > in vivo LD50 and predicted toxicity < in vivo toxicity). The
regression lines overpredict the toxicity of chemicals that are plotted above the lines (i.e.,
predicted LD50 < in vivo LD50 and predicted toxicity > in vivo toxicity).
Of the 282 RC substances with rat oral LD50 values, there were 51 substances with molecularweights ≤100 g/mole and 231 substances with molecular weights >100 g/mole. Figure J7-1
shows the 51 low molecular weight chemicals (i.e., with molecular weight ≤100 g/mole)graphed with both the RC rat-only millimole regression (Figure J7-1a) and the RC rat-only
weight regression (Figure J7-1b). The RC rat-only millimole regression underestimated the
toxicity of 20/51 (39%) substances and overestimated the toxicity of 31/51 (61%) substances(see Table J7-2). The RC rat-only weight regression underestimated the toxicity of 24/51(47%) substances and overestimated the toxicity of 27/51 (53%) substances. Fisher’s exact
test indicated that there was no difference between the millimole and weight regressions forthe under and over-prediction rates of toxicity for the 51 low molecular weight substances(two-tailed p=0.549) (see Table J7-3).

J-36

In Vitro Cytotoxicity Test Methods BRD Appendix J7

Figure J7-1

November 2006

Rat-only Regressions Graphed with 51 Chemicals with Molecular Weight≤100 g/mole

a.
1000

log LD50 = 0.439 log IC50 + 0.621
R2 = 0.452; n=282

100

10

1

0.1

0.01

0.001
0.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

1000

10000

100000IC50(mM)b.
100000log LD50 = 0.372 log IC50 + 2.024
R2 = 0.325; n=282

10000

1000

100

10

1

0.1
0.001

0.01

0.1

1

10

100

IC50 (µg/mL)

Figure J7-1a shows the RC rat-only millimole regression. Toxicity is underpredicted (i.e., predicted LD50 > invivo LD50) for 20/51 (39%) chemicals. Toxicity is overpredicted (i.e., predicted LD50 < in vivo LD50) for 31/51(61%) chemicals. Figure J7-1b shows the RC rat-only weight regression. Toxicity is underpredicted (i.e.,
predicted LD50 > in vivo LD50) for 24/51 (47%) chemicals. Toxicity is overpredicted (i.e., predicted LD50 < invivo LD50) for 27/51 (53%) chemicals.

J-37

In Vitro Cytotoxicity Test Methods BRD Appendix J7Table J7-2

Regression
RC Rat-only
WeightRC Rat-only
Millimole

November 2006

Over- and Under Prediction of Toxicity for Low and High Molecular
Weight Chemicals Using RC Rat-only Weight and Millimole Regressions
Toxicity
Toxicity
Underpredicted
Overpredicted
51 Chemicals with Molecular Weight≤100 g/mole
24/51 (47%)

27/51 (53%)

101/231 (44%)

130/231 (57%)

20/51 (39%)

31/51 (61%)

108/231 (47%)

123/231 (53%)

20 Chemicals with Molecular Weight≥400 g/mole
RC Rat-only
WeightRC Rat-only
Millimole

Table J7-3

Toxicity
Toxicity
Underpredicted
Overpredicted
231 Chemicals with Molecular Weight>100 g/mole

262 Chemicals with Molecular Weight<400 g/mole

4/20 (20%)

16/20 (80%)

121/262 (46%)

141/262 (54%)

7/20 (35%)

13/20 (65%)

121/262 (46%)

141/262 (54%)

Over- and Under Prediction of Toxicity for Low and High Molecular
Weight Substances Using RC Rat-Only Weight and Millimole
Regressions
Comparison

RC rat-only millimole vs. RC rat-only weight
regression
RC rat-only millimole vs. RC rat-only weight
regression
51 Low molecular weight (≤100 g/mole)substances vs. 231 other substances (>100g/mole)51 Low molecular weight (≤100 g/mole)substances vs. 231 other substances (>100g/mole)RC rat-only millimole vs. RC rat-only weight
regression
RC rat-only millimole vs. RC rat-only weight
regression
20 High molecular weight substances (≥400
g/mole) vs. 262 other substances (<400 g/mole)
20 High molecular weight substances (≥400
g/mole) vs. 262 other substances (<400 g/mole)For

Fisher’s Exact
Test1

Under- and over-prediction of toxicity
for 51 substances with molecularweight ≤100 g/mole
Under- and over-prediction of toxicity
for 231 substances with molecularweight >100 g/mole

0.549
0.575

RC rat-only millimole regression

0.355

RC rat-only weight regression

0.756

Under- and over-prediction of toxicity
for 20 substances with molecularweight ≥400 g/mole
Under- and over-prediction of toxicity
for 262 substances with molecularweight <400 g/mole

0.480
NT

RC rat-only millimole regression

0.362

RC rat-only weight regression

0.033

Abbreviations: RC=Registry of Cytotoxicity; NT=Not tested since the proportions were the same.
Toxicity was underpredicted for 121/262 (46%) substances and overpredicted for 141/262 (54%) substances.
1
P-values.

J-38

In Vitro Cytotoxicity Test Methods BRD Appendix J7

November 2006

For the 231 substances with molecular weights >100 g/mole, the RC rat-only millimole
regression underestimated the toxicity of 108/231 (47%) substances and overestimated the
toxicity of 123/231 (53%) substances (see Table J7-2). The RC rat-only weight regression
underestimated the toxicity of 101/231 (44%) substances and overestimated the toxicity of
130/231 (57%) substances. Fisher’s exact test indicated that there was no difference between
the millimole and weight regressions for the under- and over-prediction rates for the 231
substances with molecular weight >100 g/mole (two-tailed p=0.575; see Table J7-3).
Additionally, Fisher’s exact test also showed that there was no difference in the under- andover-prediction rates for the 51 substances with molecular weight ≤100 g/mole compared tothe under- and over-prediction of the toxicity of the 231 substances with molecular weight>100 g/mole (two-tailed p=0.756 for the RC rat-only weight regression and two-tailed
p=0.355 for the RC rat-only millimole regression).
Of the 282 RC substances with rat oral LD50 values, there were 20 substances with molecularweights ≥400 g/mole and 262 substances with molecular weights <400 g/mole (see Table
J7-2). Figure J7-2 shows the 20 chemicals with molecular weights ≥400 g/mole plotted with
the RC rat-only milllimole regression (Figure J7-2a) and the RC rat-only weight regression(Figure J7-2b). The RC rat-only millimole regression underestimated the toxicity of 7/20
(35%) substances and overestimated the toxicity of 13/20 (65%) substances (see Table J7-2).
The RC rat-only weight regression underestimated the toxicity of 4/20 (20%) substances andoverestimated the toxicity of 16/20 (80%) substances. Fisher’s exact test indicated that there
was no difference between the millimole and weight regressions for the under- and overprediction of toxicity for the 20 high molecular weight substances (two-tailed p=0.480; see
Table J7-3).
For the remaining 262 substances with molecular weights <400 g/mole, the RC rat-only
millimole and the RC rat-only weight regressions both underestimated the toxicity of
121/262 (46%) substances and overestimated toxicity of 141/262 (54%) substances (seeTable J7-2). Thus, there was no difference in the two regressions in the rates of under- andover-estimation of toxicity for the 262 substances with molecular weights <400 g/mole.
Fisher’s exact test also showed that there was no difference in the rates for under- and overprediction of the toxicity of substances with high molecular weight (≥400 g/mole) comparedwith the under- and over-prediction of the toxicity of substances with lower molecular weight
for the RC rat-only millimole regression (two-tailed p=0.362; see Table J7-3). For the RC
rat-only weight regression, however, there was a significant difference in the under- andover-prediction rates for substances with high molecular weight (>400 g/mole) comparedwith the under- and over-prediction rates for substances with lower molecular weight (twotailed p =0.033). Thus, the weight-based regression overestimated the toxicity of the highmolecular weight substances (compared with substances with lower molecular weight) while
the millimole regression did not.

J-39

In Vitro Cytotoxicity Test Methods BRD Appendix J7

November 2006

Figure J7-2 Rat-only Regressions Graphed with 20 Chemicals with Molecular Weight≥400 g/molea.
1000

log LD50 = 0.439 log IC50 + 0.621
R2= 0.452; n=282

100

10

1

0.1

0.01

0.001
0.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

10000

100000IC50(mM)b.
100000log LD50 = 0.372 log IC50 + 2.024
R2 = 0.325; n=282

10000

1000

100

10

1

0.1
0.001

0.01

0.1

1

10

100

1000

IC50 (µg/mL)

Figure J7-2a shows the RC rat-only millimole regression. Toxicity is underpredicted (i.e., predicted LD50 > invivo LD50) for 7/20 (35%) chemicals. Toxicity is overpredicted (i.e., predicted LD50 < in vivo LD50) for 13/20(65%) chemicals. Figure J7-2b shows the RC rat-only weight regression. Toxicity is underpredicted (i.e.,
predicted LD50 > in vivo LD50) for 4/20 (20%) chemicals. Toxicity is overpredicted (i.e., predicted LD50 < invivo LD50) for 16/20 (80%) chemicals.

J-40

In Vitro Cytotoxicity Test Methods BRD Appendix K

November 2006

Appendix K

IC50 and LD50 Data for Regressions
K1

IC50 and LD50 Values Used for Laboratory-SpecificRegressions .......................................................................................K-3

K2

IC50 and LD50 Values Used for Combined-Laboratory
Regressions .....................................................................................K-17

K3

RC IC50 and LD50 Values for RC Substances with Rat Oral
LD50 Data ........................................................................................K-23

K4

Individual Laboratory LD50 Predictions: RC Rat-Only
Millimole Regression......................................................................K-33

K-1

In Vitro Cytotoxicity Test Methods BRD Appendix K

November 2006

[This Page Intentionally Left Blank]

K-2

In Vitro Cytotoxicity Test Methods BRD Appendix K1

November 2006

Appendix K1

IC50 and LD50 Values Used for Laboratory-Specific Regressions

K-3

In Vitro Cytotoxicity Test Methods BRD Appendix K1

November 2006

[This Page Intentionally Left Blank]

K-4

In Vitro Cytotoxicity Test Methods BRD K1

November 2006

IC50 and LD50 Values Used for Laboratory-Specific Regressions

NRU Test
Method
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3

Substance
1,1,1-Trichloroethane
1,1,1-Trichloroethane
1,1,1-Trichloroethane
2-Propanol
2-Propanol
2-Propanol
5-Aminosalicylic acid
5-Aminosalicylic acid
5-Aminosalicylic acidAcetaminophen
Acetaminophen
Acetaminophen
Acetonitrile
Acetonitrile
Acetonitrile
Acetylsalicylic acid
Acetylsalicylic acid
Acetylsalicylic acid
Aminopterin
Aminopterin
Aminopterin
Amitriptyline HCl
Amitriptyline HCl
Amitriptyline HCl
Arsenic III trioxide
Arsenic III trioxide
Arsenic III trioxide
Atropine sulfate
Atropine sulfate
Atropine sulfate
Boric acid
Boric acid
Boric acid
BusulfanBusulfanBusulfanCadmium II chloride
Cadmium II chloride
Cadmium II chlorideLab

Log IC50 (mM)1

ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS

2.489
2.200
1.868
1.637
1.820
1.835
0.979
1.127
1.005
-0.577-0.375-0.553
2.195
2.312
2.355
0.553
0.827
0.344
-4.926-4.612-4.980-1.724-1.611-1.609-1.937-2.278-1.724-1.151-0.734-0.998
1.370
1.804
1.254
-0.8270.075-0.751-2.585-2.675-2.387

Log Reference

IC50 (mM)1

2

LD50 (mmol/kg)
1.957
1.957
1.957
1.929
1.929
1.929
1.350
1.350
1.350
1.155
1.155
1.155
1.942
1.942
1.942
0.922
0.922
0.922
-1.799-1.799-1.799
0.046
0.046
0.046
-0.897-0.897-0.897
0.071
0.071
0.071
1.744
1.744
1.744
-1.308-1.308-1.308-0.132-0.132-0.132308.185
158.51273.75843.32866.01968.3409.529
13.387
10.116
0.265
0.421
0.280
156.682204.968226.301
3.572
6.708
2.208
0.000012
0.000024
0.000010
0.019
0.024
0.025
0.012
0.005
0.019
0.071
0.184
0.100
23.43263.74817.939
0.149
1.187
0.177
0.00260.00210.0041K-5(mmol/kg)

Molecular
Weight(g/mole)IC50 (µg/mL)1

90.53490.53490.53484.92884.92884.92822.39122.39122.39114.29914.29914.29987.57687.57687.576
8.357
8.357
8.357
0.016
0.016
0.016
1.112
1.112
1.112
0.127
0.127
0.127
1.179
1.179
1.179
55.41055.41055.410
0.049
0.049
0.049
0.738
0.738
0.738

133.410133.410133.41060.11060.11060.110153.100
153.100
153.100
151.200
151.200
151.200
41.05041.05041.050180.200
180.200
180.200
440.470
440.470
440.470
313.900
313.900
313.900
197.840
197.840
197.840694.800
694.800
694.800
61.83061.83061.830246.310246.310246.310183.300
183.300
183.300

41114.974
21147.061
9840.111
2604.458
3968.400
4107.888
1458.814
2049.588
1548.817
40.08763.72842.3646431.812
8413.951
9289.664
643.675
1208.741
397.8020.0052
0.011
0.005
5.920
7.681
7.719
2.285
1.042
3.731
49.128128.135
69.8231448.772
3941.547
1109.175
36.700292.41543.685
0.477
0.387
0.752

Reference LD50
2Reference LD50
(mg/kg)2
12078
12078
12078
5105
5105
5105
3428
3428
3428
2162
2162
2162
3595
3595
3595
1506
1506
1506
7.00
7.00
7.00
349
349
349
25.1
25.1
25.1
819
819
819
3426
3426
3426
12.1
12.1
12.1
135
135
135

In Vitro Cytotoxicity Test Methods BRD K1

November 2006

IC50 and LD50 Values Used for Laboratory-Specific Regressions

NRU Test
Method
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3

SubstanceLab

Log IC50 (mM)1

Caffeine
Caffeine
Caffeine
Carbamazepine
Carbamazepine
Carbamazepine
Chloral hydrate
Chloral hydrate
Chloral hydrate
Chloramphenicol
Chloramphenicol
Chloramphenicol
Citric acid
Citric acid
Citric acidColchicine
Colchicine
Colchicine
Cupric sulfate pentahydrate
Cupric sulfate pentahydrate
Cupric sulfate pentahydrate
Cycloheximide
Cycloheximide
Cycloheximide
Dibutyl phthalate
Dibutyl phthalate
Dibutyl phthalate
Dichlorvos
Dichlorvos
Dichlorvos
Diethyl phthalate
Diethyl phthalate
Diethyl phthalate
DigoxinDigoxinDigoxinDimethylformamide
Dimethylformamide
Dimethylformamide

ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS

-0.165-0.143-0.007-0.457-0.209-0.412-0.0410.162
0.011-0.776-0.088-0.321
0.378
0.774
0.648
-4.292-3.671-4.158-0.480-0.333-1.653-3.384-2.726-3.420-1.079-0.214-1.129-1.373-0.829-1.084-0.430-0.191-0.328-0.3730.039-0.397
1.862
1.874
1.826

Log Reference

IC50 (mM)1

2

LD50 (mmol/kg)
0.203
0.203
0.203
1.075
1.075
1.075
0.586
0.586
0.586
1.033
1.033
1.033
1.489
1.489
1.489
-1.425-1.425-1.425
0.279
0.279
0.279
-2.148-2.148-2.148
1.504
1.504
1.504
-0.576-0.576-0.576
1.622
1.622
1.622
-1.441-1.441-1.441
1.861
1.861
1.861

0.684
0.720
0.984
0.349
0.617
0.387
0.910
1.454
1.025
0.168
0.817
0.478
2.389
5.943
4.444
0.000051
0.000213
0.000070
0.331
0.465
0.022
0.000410.00188
0.00038
0.083
0.611
0.074
0.042
0.148
0.082
0.372
0.644
0.470
0.424
1.093
0.401
72.84874.77467.001K-6(mmol/kg)

Molecular
Weight(g/mole)IC50 (µg/mL)1

1.596
1.596
1.596
11.87911.87911.879
3.857
3.857
3.857
10.80010.80010.80030.86430.86430.864
0.038
0.038
0.038
1.902
1.902
1.902
0.007
0.007
0.007
31.95131.95131.951
0.266
0.266
0.266
41.90441.90441.904
0.036
0.036
0.036
72.57272.57272.572

194.200
194.200
194.200
236.300
236.300
236.300
165.400
165.400
165.400
323.150323.150323.150
192.100
192.100
192.100
399.480
399.480
399.480249.700
249.700
249.700
281.400
281.400
281.400
278.300
278.300
278.300
220.980220.980220.980
222.200
222.200
222.200
780.900
780.900
780.900
73.10073.10073.100132.841
139.744
191.132
82.414145.88191.411150.545240.436
169.56454.147264.039154.402458.846
1141.563
853.755
0.020
0.085
0.028
82.667116.078
5.556
0.116
0.529
0.107
23.227169.92220.6709.358
32.75918.22582.604143.109104.472
330.877853.755
312.9685325.168
5465.962
4897.788

Reference LD50
2Reference LD50
(mg/kg)2
310
310
310
2807
2807
2807
638
638
638
3490
3490
3490
5929
5929
5929
15.0
15.0
15.0
475
475
475
2.0
2.0
2.0
8892
8892
8892
58.7
58.7
58.7
9311
9311
9311
28.3
28.3
28.3
5305
5305
5305

In Vitro Cytotoxicity Test Methods BRD K1

November 2006

IC50 and LD50 Values Used for Laboratory-Specific Regressions

NRU Test
Method
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3

SubstanceLab

Diquat dibromide monohydrate
ECBC
Diquat dibromide monohydrate
FAL
Diquat dibromide monohydrate
IIVS
Disulfoton
ECBC
Disulfoton
FAL
Disulfoton
IIVS
Endosulfan
ECBC
Endosulfan
FAL
Endosulfan
IIVS
Epinephrine bitartrate
ECBC
Epinephrine bitartrate
FAL
Epinephrine bitartrate
IIVS
Ethanol
ECBC
Ethanol
FAL
Ethanol
IIVS
Ethylene glycol
ECBC
Ethylene glycol
FAL
Ethylene glycol
IIVS
Fenpropathrin
ECBC
Fenpropathrin
FAL
Fenpropathrin
IIVS
Gibberellic acid
ECBC
Gibberellic acid
IIVS
Glutethimide
ECBC
Glutethimide
FAL
Glutethimide
IIVS
Glycerol
ECBC
Glycerol
FAL
Glycerol
IIVS
Haloperidol
ECBC
Haloperidol
FAL
Haloperidol
IIVS
Hexachlorophene
ECBC
Hexachlorophene
FAL
Hexachlorophene
IIVS
Lactic acid
ECBC
Lactic acid
FAL
Lactic acid
IIVS
LindaneECBC

Log IC50 (mM)1-1.978-1.146-1.837-0.3611.611-0.760-1.933-1.511-2.076-0.813-0.723-0.721
2.051
2.259
2.143
2.469
2.698
2.618
-1.191-1.012-1.3221.363
1.343-0.1150.117-0.240
2.334
2.477
2.455
-1.851-1.673-1.840-1.939-1.896-2.126
1.513
1.584
1.490
-0.496

Log Reference

IC50 (mM)1

2

LD50 (mmol/kg)-0.355-0.355-0.355-1.739-1.739-1.739-1.165-1.165-1.165-1.921-1.921-1.921
2.391
2.391
2.391
2.062
2.062
2.062
-0.664-0.664-0.664
1.241
1.241
0.441
0.441
0.441
2.332
2.332
2.332
-0.057-0.057-0.057-0.696-0.696-0.696
1.606
1.606
1.606
-0.464

0.011
0.071
0.015
0.435
40.796
0.174
0.012
0.031
0.008
0.154
0.189
0.190
112.439181.516139.075
294.295
499.068
415.216
0.064
0.097
0.048
23.07422.035
0.767
1.308
0.576
215.835
299.942285.400
0.014
0.021
0.014
0.012
0.013
0.007
32.56238.37430.8820.319

K-7(mmol/kg)

Molecular
Weight(g/mole)IC50 (µg/mL)1

0.442
0.442
0.442
0.018
0.018
0.018
0.068
0.068
0.068
0.012
0.012
0.012
245.800
245.800
245.800
115.351
115.351
115.351
0.217
0.217
0.217
17.43617.436
2.761
2.761
2.761
214.681214.681214.681
0.878
0.878
0.878
0.202
0.202
0.202
40.35340.35340.3530.344

362.100
362.100
362.100
274.420274.420274.420406.910406.910406.910333.300
333.300
333.300
46.07046.07046.07062.08062.08062.080349.430
349.430
349.430
346.380
346.380217.300
217.300
217.300
92.09092.09092.090375.900
375.900
375.900
406.910406.910406.91090.08090.08090.080290.800

3.811
25.882
5.268
119.40211195.195
47.7284.751
12.5543.416
51.28663.14463.3385180.043
8362.446
6407.176
18269.837
30982.150
25776.589
22.49133.98216.6477992.206
7632.497
166.725
284.228125.098
19876.198
27621.673
26282.499
5.297
7.977
5.440
4.684
5.172
3.046
2933.144
3456.740
2781.848
92.754

Reference LD50
2Reference LD50
(mg/kg)2
160
160
160
5.0
5.0
5.0
27.8
27.8
27.8
4.0
4.0
4.0
11324
11324
11324
7161
7161
7161
75.7
75.7
75.7
6039
6039
600
600
600
19770
19770
19770
330
330
330
82.0
82.0
82.0
3635
3635
3635
100

In Vitro Cytotoxicity Test Methods BRD K1

November 2006

IC50 and LD50 Values Used for Laboratory-Specific Regressions

NRU Test
Method
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3

SubstanceLab

Log IC50 (mM)1

LindaneLindane
Lithium I carbonate
Meprobamate
Meprobamate
Meprobamate
Mercury II chloride
Mercury II chloride
Mercury II chloride
Nicotine
Nicotine
Nicotine
Paraquat
Paraquat
Paraquat
Parathion
Parathion
Parathion
Phenobarbital
Phenobarbital
Phenobarbital
PhenolPhenolPhenolPhenylthiourea
Phenylthiourea
Phenylthiourea
Physostigmine
Physostigmine
Physostigmine
Potassium cyanide
Potassium cyanide
Potassium cyanide
Potassium I chloride
Potassium I chloride
Potassium I chloride
Procainamide HCl
Procainamide HCl
Procainamide HCl

FAL
IIVS
ECBC
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS

-0.067-0.685
0.881
0.207
0.600
0.247
-1.897-1.670-1.889
0.216
0.386
0.441
-1.103-1.109-1.107-1.147-0.398-1.128
0.429
0.473
0.303
-0.2800.036-0.211-0.7950.183-0.242-1.038-0.863-1.145-0.6370.353-0.538
1.650
1.690
1.695
0.168
0.200
0.261

Log Reference

IC50 (mM)1

2

LD50 (mmol/kg)-0.464-0.464
0.902
0.803
0.803
0.803
-0.830-0.830-0.830-0.367-0.367-0.367-0.443-0.443-0.443-1.679-1.679-1.679-0.016-0.016-0.016
0.908
0.908
0.908
-1.705-1.705-1.705-1.741-1.741-1.741-0.956-0.956-0.956
1.575
1.575
1.575
0.856
0.856
0.856

0.856
0.207
7.601
1.609
3.981
1.766
0.013
0.021
0.013
1.643
2.430
2.760
0.079
0.078
0.078
0.071
0.400
0.074
2.688
2.975
2.011
0.524
1.086
0.615
0.160
1.523
0.573
0.092
0.137
0.072
0.231
2.254
0.289
44.66748.97249.557
1.473
1.585
1.826

K-8

(mmol/kg)

Molecular
Weight(g/mole)IC50 (µg/mL)1

0.344
0.344
7.985
6.353
6.353
6.353
0.148
0.148
0.148
0.430
0.430
0.430
0.360
0.360
0.360
0.021
0.021
0.021
0.965
0.965
0.965
8.097
8.097
8.097
0.020
0.020
0.020
0.018
0.018
0.018
0.111
0.111
0.111
37.58637.58637.586
7.175
7.175
7.175

290.800
290.800
73.890218.300
218.300
218.300
271.500
271.500
271.500
162.200
162.200
162.200
257.200
257.200
257.200
291.300
291.300
291.300
232.230232.230232.230
94.11094.11094.110152.200
152.200
152.200
275.400
275.400
275.400
65.12065.12065.12074.55074.55074.550271.790271.790271.790249.02960.090561.606
351.291868.960
385.478
3.446
5.801
3.505
266.481394.155447.71320.30819.991
20.11620.750116.413
21.695624.214690.770466.928
49.355102.172
57.85424.389231.73987.16325.23537.78619.70215.031146.780
18.8513329.953
3650.893
3694.501
400.252
430.857
496.211

Reference LD50
2Reference LD50
(mg/kg)2
100
100
590
1387
1387
1387
40.2
40.2
40.2
69.7
69.7
69.7
92.7
92.7
92.7
6.1
6.1
6.1
224.0
224.0
224.0
762.0
762.0
762.0
3.0
3.0
3.0
5.0
5.0
5.0
7.2
7.2
7.2
2802.02802.02802.01950.01950.01950.0In Vitro Cytotoxicity Test Methods BRD K1

November 2006

IC50 and LD50 Values Used for Laboratory-Specific Regressions

NRU Test
Method
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3

SubstanceLab

Propranolol
ECBC
Propranolol
FAL
Propranolol
IIVS
Propylparaben
ECBC
Propylparaben
FAL
Propylparaben
IIVS
Sodium arsenite
ECBC
Sodium arsenite
FAL
Sodium arsenite
IIVS
Sodium chloride
ECBC
Sodium chloride
FAL
Sodium chloride
IIVS
Sodium dichromate dihydrate ECBC
Sodium dichromate dihydrate FAL
Sodium dichromate dihydrate IIVS
Sodium hypochlorite
ECBC
Sodium hypochlorite
FAL
Sodium hypochlorite
IIVS
Sodium oxalate
ECBC
Sodium oxalate
FAL
Sodium oxalate
IIVS
Sodium I fluoride
ECBC
Sodium I fluoride
FAL
Sodium I fluoride
IIVS
Sodium selenate
ECBC
Sodium selenate
FAL
Sodium selenate
IIVS
Strychnine
ECBC
Strychnine
FAL
Strychnine
IIVS
Thallium II sulfate
ECBC
Thallium II sulfate
FAL
Thallium II sulfate
IIVS
Trichloroacetic acid
ECBC
Trichloroacetic acid
FAL
Trichloroacetic acid
IIVS
Triethylenemelamine
ECBC
Triethylenemelamine
FAL
Triethylenemelamine
IIVS

Log IC50 (mM)1-1.354-1.378-1.232-0.940-0.553-1.026-2.419-1.998-2.284
1.913
1.896
1.920
-2.697-2.680-2.740
1.041
0.996
1.399
-0.535-0.649-0.488
0.163
0.354
0.290
-1.176-0.548-0.7170.059-0.434-0.603-2.263-1.726-1.916
0.666
0.872
0.687
-3.378-2.153-3.095

Log Reference

IC50 (mM)1

2

LD50 (mmol/kg)
0.197
0.197
0.197
1.546
1.546
1.546
-0.474-0.474-0.474
1.841
1.841
1.841
-0.771-0.771-0.771
2.142
2.142
2.142
0.674
0.674
0.674
0.480
0.480
0.480
-1.799-1.799-1.799-1.725-1.725-1.725-1.305-1.305-1.305
1.282
1.282
1.282
-1.708-1.708-1.708

0.044
0.042
0.059
0.115
0.280
0.094
0.003810.01005
0.0052081.90178.621
83.1680.00201
0.00209
0.00182
10.9959.898
25.058
0.292
0.224
0.325
1.456
2.260
1.950
0.067
0.283
0.192
1.146
0.368
0.249
0.005
0.019
0.012
4.639
7.443
4.869
0.000419
0.007030.000804K-9(mmol/kg)

Molecular
Weight(g/mole)IC50 (µg/mL)1

1.575
1.575
1.575
35.13935.13935.139
0.336
0.336
0.336
69.30269.30269.302
0.169
0.169
0.169
138.737138.737138.737
4.724
4.724
4.724
3.020
3.020
3.020
0.016
0.016
0.016
0.019
0.019
0.019
0.050
0.050
0.050
19.13719.13719.137
0.020
0.020
0.020

295.840295.840295.840
180.200
180.200
180.200
129.900
129.900
129.900
58.44058.44058.440298.000
298.000
298.000
74.44074.44074.440134.000
134.000
134.000
41.99041.99041.990188.940
188.940
188.940
334.400
334.400
334.400
504.800
504.800
504.800
163.400
163.400
163.400
204.230204.230204.230

13.08912.37717.35220.68650.46616.982
0.495
1.305
0.676
4786.301
4594.624
4860.340
0.599
0.622
0.542
818.465736.772
1865.306
39.11430.06143.57661.13694.91181.86012.59453.48736.291
383.119123.12183.432
2.756
9.483
6.124
757.9961216.186
795.548
0.086
1.436
0.164

Reference LD50
2Reference LD50
(mg/kg)2
466.0
466.0
466.0
6332.06332.06332.0
43.6
43.6
43.6
4050.04050.04050.0
50.5
50.5
50.5
10327.6
10327.6
10327.6
633.0
633.0
633.0
126.8
126.8
126.8
3.0
3.0
3.0
6.3
6.3
6.3
25.0
25.0
25.0
3127.03127.03127.04.0
4.0
4.0

In Vitro Cytotoxicity Test Methods BRD K1

November 2006

IC50 and LD50 Values Used for Laboratory-Specific Regressions

NRU Test
Method
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3

Substance
Triphenyltin hydroxide
Triphenyltin hydroxide
Triphenyltin hydroxide
Valproic acid
Valproic acid
Valproic acid
Verapamil HCl
Verapamil HCl
Verapamil HCl
XyleneLab

Log IC50 (mM)1

ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
IIVS

-4.161-4.366-4.459
0.577
1.097
0.600
-1.188-1.153-1.1030.832

Log Reference2

LD50 (mmol/kg)

IC50 (mM)1-0.047-0.047-0.047
0.839
0.839
0.839
-0.646-0.646-0.6461.643

0.000069
0.000043
0.000035
3.776
12.494
3.981
0.065
0.070
0.079
6.787

K-10

(mmol/kg)

Molecular
Weight(g/mole)IC50 (µg/mL)1

0.896
0.896
0.896
6.907
6.907
6.907
0.226
0.226
0.226
43.939367.020367.020367.020144.200
144.200
144.200
491.080491.080491.080
106.170

0.025
0.016
0.013
544.503
1801.634
574.11631.84234.51438.726720.554

Reference LD50
2Reference LD50
(mg/kg)2
329.0
329.0
329.0
996.0
996.0
996.0
111.0
111.0
111.0
4665.0In Vitro Cytotoxicity Test Methods BRD K1

November 2006

IC50 and LD50 Values Used for Laboratory-Specific Regressions

NRU Test
Method
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK

Substance
1,1,1-Trichloroethane
2-Propanol
2-Propanol
2-Propanol
5-Aminosalicylic acid
5-Aminosalicylic acid
5-Aminosalicylic acidAcetaminophen
Acetaminophen
Acetaminophen
Acetonitrile
Acetonitrile
Acetonitrile
Acetylsalicylic acid
Acetylsalicylic acid
Acetylsalicylic acid
Aminopterin
Aminopterin
Aminopterin
Amitriptyline HCl
Amitriptyline HCl
Amitriptyline HCl
Arsenic III trioxide
Arsenic III trioxide
Arsenic III trioxide
Atropine sulfate
Atropine sulfate
Atropine sulfate
Boric acid
Boric acid
Boric acid
BusulfanBusulfanBusulfanCadmium II chloride
Cadmium II chloride
Cadmium II chloride
Caffeine
CaffeineLab

Log IC50 (mM)1

ECBC
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL

1.784
1.940
1.840
2.071
-0.717-0.330-0.501
0.564
0.466
0.574
2.357
2.388
2.354
0.544
0.583
0.452
0.299
0.091
0.141
-1.480-1.696-1.458-1.426-1.968-0.991-0.912-0.943-0.932
0.839
0.786
0.875
0.003
-0.0330.102-1.948-2.083-1.9950.609
0.469

Log Reference2

LD50 (mmol/kg)

IC50 (mM)1

1.957
1.929
1.929
1.929
1.350
1.350
1.350
1.155
1.155
1.155
1.942
1.942
1.942
0.922
0.922
0.922
-1.799-1.799-1.799
0.046
0.046
0.046
-0.897-0.897-0.897
0.071
0.071
0.071
1.744
1.744
1.744
-1.308-1.308-1.308-0.132-0.132-0.1320.203
0.203

60.88187.191
69.247117.715
0.192
0.468
0.316
3.663
2.925
3.754
227.608244.542226.128
3.501
3.827
2.831
1.990
1.234
1.383
0.033
0.020
0.035
0.038
0.011
0.102
0.122
0.114
0.117
6.899
6.111
7.501
1.006
0.926
1.265
0.011
0.008
0.010
4.062
2.947

K-11

(mmol/kg)

Molecular
Weight(g/mole)IC50 (µg/mL)1

90.53484.92884.92884.92822.39122.39122.39114.29914.29914.29987.57687.57687.576
8.357
8.357
8.357
0.016
0.016
0.016
1.112
1.112
1.112
0.127
0.127
0.127
1.179
1.179
1.179
55.41055.41055.410
0.049
0.049
0.049
0.738
0.738
0.738
1.596
1.596

133.41060.11060.11060.110153.100
153.100
153.100
151.200
151.200
151.200
41.05041.05041.050180.200
180.200
180.200
440.470
440.470
440.470
313.900
313.900
313.900
197.840
197.840
197.840694.800
694.800
694.800
61.83061.83061.830246.310246.310246.310183.300
183.300
183.300
194.200
194.200

8122.069
5241.038
4162.458
7075.821
29.39971.66948.343553.775442.249567.5459343.293
10038.450
9282.536
630.957
689.710510.113876.710543.604608.951
10.402
6.328
10.9237.425
2.132
20.21685.04979.18981.345426.580
377.862463.803247.742228.034
311.650
2.066
1.514
1.856
788.860
572.357

Reference LD50
2Reference LD50
(mg/kg)2
12078.1
5105.05105.05105.03428.03428.03428.02162.02162.02162.03595.03595.03595.01506.01506.01506.0
7.0
7.0
7.0
349.0
349.0
349.0
25.1
25.1
25.1
819.0
819.0
819.0
3426.03426.03426.0
12.1
12.1
12.1
135.2
135.2
135.2
310.0
310.0

In Vitro Cytotoxicity Test Methods BRD K1

November 2006

IC50 and LD50 Values Used for Laboratory-Specific Regressions

NRU Test
Method
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK

SubstanceLab

Caffeine
IIVS
Carbamazepine
ECBC
Carbamazepine
FAL
Carbamazepine
IIVS
Chloral hydrate
ECBC
Chloral hydrate
FAL
Chloral hydrate
IIVS
Chloramphenicol
ECBC
Chloramphenicol
FAL
Chloramphenicol
IIVS
Citric acid
ECBC
Citric acid
FAL
Citric acid
IIVS
Colchicine
ECBC
Colchicine
FAL
Colchicine
IIVS
Cupric sulfate pentahydrate ECBC
Cupric sulfate pentahydrate FAL
Cupric sulfate pentahydrate IIVS
Cycloheximide
ECBC
Cycloheximide
FAL
Cycloheximide
IIVS
Dibutyl phthalate
ECBC
Dibutyl phthalate
FAL
Dibutyl phthalate
IIVS
Dichlorvos
ECBC
Dichlorvos
FAL
Dichlorvos
IIVS
Diethyl phthalate
ECBC
Diethyl phthalate
FAL
Diethyl phthalate
IIVS
Digoxin
ECBC
Digoxin
FAL
Digoxin
IIVS
Dimethylformamide
ECBC
Dimethylformamide
FAL
Dimethylformamide
IIVS
Diquat dibromide monohydrate
ECBC
Diquat dibromide monohydrate
FAL

Log IC50 (mM)1
0.471-0.555-0.235-0.569-0.082-0.031-0.169-0.033
0.070
0.048
0.434
0.206
0.352
-4.918-4.720-4.699-0.121-0.109-0.082-3.732-3.418-3.601-1.005-0.854-1.102-1.423-1.265-1.258-0.108-0.615-0.074-5.164-7.209-5.293
2.107
2.029
1.942
-2.012-1.779Log Reference2

LD50 (mmol/kg)
0.203
1.075
1.075
1.075
0.586
0.586
0.586
1.033
1.033
1.033
1.489
1.489
1.489
-1.425-1.425-1.425
0.279
0.279
0.279
-2.148-2.148-2.148
1.504
1.504
1.504
-0.576-0.576-0.576
1.622
1.622
1.622
-1.441-1.441-1.441
1.861
1.861
1.861
-0.355-0.355

IC50 (mM)1
2.956
0.278
0.582
0.270
0.829
0.931
0.677
0.927
1.175
1.117
2.715
1.608
2.250
0.0000121
0.0000190
0.0000200
0.757
0.778
0.828
0.000185
0.000382
0.000251
0.099
0.140
0.079
0.038
0.054
0.055
0.779
0.243
0.843
0.00000685
0.00000006
0.00000509
127.962106.926
87.448
0.010
0.017

K-12

(mmol/kg)

Molecular
Weight(g/mole)IC50 (µg/mL)1

1.596
11.87911.87911.879
3.857
3.857
3.857
10.80010.80010.80030.86430.86430.864
0.038
0.038
0.038
1.902
1.902
1.902
0.007
0.007
0.007
31.95131.95131.951
0.266
0.266
0.266
41.90441.90441.904
0.036
0.036
0.036
72.57272.57272.5720.442
0.442

194.200
236.300
236.300
236.300
165.400
165.400
165.400
323.150323.150323.150
192.100
192.100
192.100
399.480
399.480
399.480249.700
249.700
249.700
281.400
281.400
281.400
278.300
278.300
278.300
220.980220.980220.980
222.200
222.200
222.200
780.900
780.900
780.900
73.10073.10073.100362.100
362.100

574.11665.766
137.61563.728137.088
153.934
112.030
299.663379.588361.049521.595
308.852
432.182
0.005
0.008
0.008
188.944194.387
206.697
0.052
0.108
0.071
27.52138.96422.0128.348
11.991
12.199173.11453.910187.2120.00530.000048
0.00409354.057
7816.278
6392.440
3.525
6.028

Reference LD50
2Reference LD50
(mg/kg)2
310.0
2807.02807.02807.0
638.0
638.0
638.0
3490.03490.03490.05929.05929.05929.0
15.0
15.0
15.0
475.0
475.0
475.0
2.0
2.0
2.0
8892.08892.08892.0
58.7
58.7
58.7
9311.09311.09311.0
28.3
28.3
28.3
5305.05305.05305.0160.0
160.0

In Vitro Cytotoxicity Test Methods BRD K1

November 2006

IC50 and LD50 Values Used for Laboratory-Specific Regressions

NRU Test
Method
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK

SubstanceLab

Diquat dibromide monohydrate
IIVS
Disulfoton
ECBC
Disulfoton
FAL
Disulfoton
IIVS
Endosulfan
ECBC
Endosulfan
FAL
Endosulfan
IIVS
Epinephrine bitartrate
ECBC
Epinephrine bitartrate
FAL
Epinephrine bitartrate
IIVS
Ethanol
ECBC
Ethanol
FAL
Ethanol
IIVS
Ethylene glycol
ECBC
Ethylene glycol
FAL
Ethylene glycol
IIVS
Fenpropathrin
ECBC
Fenpropathrin
FAL
Fenpropathrin
IIVS
Gibberellic acid
ECBC
Gibberellic acid
FAL
Gibberellic acid
IIVS
Glutethimide
ECBC
Glutethimide
FAL
Glutethimide
IIVS
Glycerol
ECBC
Glycerol
FAL
Glycerol
IIVS
Haloperidol
ECBC
Haloperidol
FAL
Haloperidol
IIVS
Hexachlorophene
ECBC
Hexachlorophene
FAL
Hexachlorophene
IIVS
Lactic acid
ECBC
Lactic acid
FAL
Lactic acid
IIVS
Lindane
ECBC
LindaneFAL

Log IC50 (mM)1-1.976-0.2980.458-0.182-2.077-2.493-2.276-0.464-0.628-0.652
2.255
2.411
2.346
2.785
2.903
2.806
-1.982-2.207-2.287
0.912
0.927
0.909
-0.087-0.110-0.096
2.542
2.250
2.495
-2.019-2.053-2.076-4.179-3.984-4.285
1.155
1.166
1.162
-1.188-1.113

Log Reference2

LD50 (mmol/kg)

IC50 (mM)1-0.355-1.739-1.739-1.739-1.165-1.165-1.165-1.921-1.921-1.921
2.391
2.391
2.391
2.062
2.062
2.062
-0.664-0.664-0.664
1.241
1.241
1.241
0.441
0.441
0.441
2.332
2.332
2.332
-0.057-0.057-0.057-0.696-0.696-0.696
1.606
1.606
1.606
-0.464-0.464

0.011
0.504
2.872
0.657
0.00840.00320.0053
0.343
0.236
0.223
179.852
257.780
221.776609.021800.306
639.7410.01040.00620.0052
8.174
8.461
8.106
0.819
0.776
0.802
348.180
177.877312.695
0.00958
0.00886
0.008400.000066
0.0001040.000052
14.27414.64614.511
0.065
0.077

K-13

(mmol/kg)

Molecular
Weight(g/mole)IC50 (µg/mL)1

0.442
0.018
0.018
0.018
0.068
0.068
0.068
0.012
0.012
0.012
245.800
245.800
245.800
115.351
115.351
115.351
0.217
0.217
0.217
17.43617.43617.436
2.761
2.761
2.761
214.681214.681214.681
0.878
0.878
0.878
0.202
0.202
0.202
40.35340.35340.3530.344
0.344

362.100
274.420274.420274.420406.910406.910406.910333.300
333.300
333.300
46.07046.07046.07062.08062.08062.080349.430
349.430
349.430
346.380
346.380
346.380217.300
217.300
217.300
92.09092.09092.090375.900
375.900
375.900
406.910406.910406.91090.08090.08090.080290.800
290.800

3.829
138.250788.255
180.302
3.411
1.307
2.157
114.463
78.58474.2458285.779
11875.904
10217.234
37808.041
49683.006
39715.137
3.645
2.171
1.806
2831.392
2930.893
2807.588
177.964168.655
174.18132063.852
16380.733
28796.077
3.601
3.329
3.157
0.027
0.042
0.021
1285.822
1319.269
1307.174
18.88022.439Reference LD50
2Reference LD50
(mg/kg)2
160.0
5.0
5.0
5.0
27.8
27.8
27.8
4.0
4.0
4.0
11324.011324.011324.07161.07161.07161.0
75.7
75.7
75.7
6039.56039.56039.5
600.0
600.0
600.0
19770.0
19770.0
19770.0
330.0
330.0
330.0
82.0
82.0
82.0
3635.03635.03635.0100.0
100.0

In Vitro Cytotoxicity Test Methods BRD K1

November 2006

IC50 and LD50 Values Used for Laboratory-Specific Regressions

NRU Test
Method
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK

SubstanceLab

Log IC50 (mM)1

Lindane
Lithium I carbonate
Lithium I carbonate
Lithium I carbonate
Meprobamate
Meprobamate
Meprobamate
Mercury II chloride
Mercury II chloride
Mercury II chloride
Methanol
MethanolNicotine
Nicotine
Nicotine
Paraquat
Paraquat
Paraquat
Parathion
Parathion
Parathion
Phenobarbital
Phenobarbital
Phenobarbital
PhenolPhenolPhenolPhenylthiourea
Phenylthiourea
Phenylthiourea
Physostigmine
Physostigmine
Physostigmine
Potassium cyanide
Potassium cyanide
Potassium cyanide
Potassium I chloride
Potassium I chloride
Potassium I chloride

IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS

-1.273
0.733
0.812
0.859
0.539
-0.3530.454-1.600-1.706-1.7051.543
1.815-0.246-0.129-0.172-0.729-0.449-0.684-0.945-0.993-1.012
0.466
0.179
0.210
-0.224-0.005-0.067
0.374
0.415
0.244
-0.226-0.954-0.299-0.356-0.112-0.589
1.531
1.475
1.425

Log Reference2

LD50 (mmol/kg)

IC50 (mM)1-0.464
0.902
0.902
0.902
0.803
0.803
0.803
-0.830-0.830-0.8302.434
2.434-0.367-0.367-0.367-0.443-0.443-0.443-1.679-1.679-1.679-0.016-0.016-0.016
0.908
0.908
0.908
-1.705-1.705-1.705-1.741-1.741-1.741-0.956-0.956-0.956
1.575
1.575
1.575

0.053
5.413
6.482
7.228
3.459
0.444
2.842
0.025
0.020
0.020
34.88565.259
0.568
0.742
0.673
0.187
0.356
0.207
0.114
0.102
0.097
2.922
1.512
1.622
0.598
0.989
0.857
2.367
2.600
1.754
0.594
0.111
0.502
0.441
0.773
0.258
33.99929.83726.634K-14(mmol/kg)

Molecular
Weight(g/mole)IC50 (µg/mL)1

0.344
7.985
7.985
7.985
6.353
6.353
6.353
0.148
0.148
0.148
271.835
271.835
0.430
0.430
0.430
0.360
0.360
0.360
0.021
0.021
0.021
0.965
0.965
0.965
8.097
8.097
8.097
0.020
0.020
0.020
0.018
0.018
0.018
0.111
0.111
0.111
37.58637.58637.586290.800
73.89073.89073.890218.300
218.300
218.300
271.500
271.500
271.500
32.04032.040162.200
162.200
162.200
257.200
257.200
257.200
291.300
291.300
291.300
232.230232.230232.230
94.11094.11094.110152.200
152.200
152.200
275.400
275.400
275.400
65.12065.12065.12074.55074.55074.55015.500399.969478.918534.059755.092
96.902620.393
6.815
5.339
5.352
1117.721
2090.900
92.116120.411109.14448.01091.48253.21133.09029.58228.316678.683
351.021376.704
56.23493.11180.662360.302395.670266.891
163.682
30.617138.250
28.70850.35016.7882534.622
2224.317
1985.553

Reference LD50
2Reference LD50
(mg/kg)2
100.0
590.0
590.0
590.0
1386.81386.81386.8
40.2
40.2
40.2
8709.68709.6
69.7
69.7
69.7
92.7
92.7
92.7
6.1
6.1
6.1
224.0
224.0
224.0
762.0
762.0
762.0
3.0
3.0
3.0
5.0
5.0
5.0
7.2
7.2
7.2
2802.02802.02802.0In Vitro Cytotoxicity Test Methods BRD K1

November 2006

IC50 and LD50 Values Used for Laboratory-Specific Regressions

NRU Test
Method
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK

SubstanceLab

Procainamide HCl
ECBC
Procainamide HCl
FAL
Procainamide HCl
IIVS
Propranolol
ECBC
Propranolol
FAL
Propranolol
IIVS
Propylparaben
ECBC
Propylparaben
FAL
Propylparaben
IIVS
Sodium arsenite
ECBC
Sodium arsenite
FAL
Sodium arsenite
IIVS
Sodium chloride
ECBC
Sodium chloride
FAL
Sodium chloride
IIVS
Sodium dichromate dihydrate ECBC
Sodium dichromate dihydrate FAL
Sodium dichromate dihydrate IIVS
Sodium hypochlorite
ECBC
Sodium hypochlorite
FAL
Sodium hypochlorite
IIVS
Sodium oxalate
ECBC
Sodium oxalate
FAL
Sodium oxalate
IIVS
Sodium I fluoride
ECBC
Sodium I fluoride
FAL
Sodium I fluoride
IIVS
Sodium selenate
ECBC
Sodium selenate
FAL
Sodium selenate
IIVS
Strychnine
ECBC
Strychnine
FAL
Strychnine
IIVS
Thallium II sulfate
ECBC
Thallium II sulfate
FAL
Thallium II sulfate
IIVS
Trichloroacetic acid
ECBC
Trichloroacetic acid
FAL
Trichloroacetic acid
IIVS

Log IC50 (mM)1
0.733
0.816
0.871
-0.890-0.830-1.017-1.000-0.991-1.115-2.231-2.631-2.444
1.787
1.042
1.772
-2.583-2.565-2.718
1.384
1.192
1.340
0.420
0.373
0.418
0.061
0.032
0.105
-1.405-1.117-1.279-0.601-0.844-0.784-3.440-3.525-3.602
0.323
0.507
0.379

Log Reference2

LD50 (mmol/kg)

IC50 (mM)1

0.856
0.856
0.856
0.197
0.197
0.197
1.546
1.546
1.546
-0.474-0.474-0.474
1.841
1.841
1.841
-0.771-0.771-0.771
2.142
2.142
2.142
0.674
0.674
0.674
0.480
0.480
0.480
-1.799-1.799-1.799-1.725-1.725-1.725-1.305-1.305-1.305
1.282
1.282
1.282

5.413
6.543
7.426
0.129
0.148
0.096
0.100
0.102
0.077
0.00590.00230.003661.22911.01459.1960.00260.00270.001924.203
15.54321.854
2.632
2.360
2.616
1.152
1.078
1.272
0.039
0.076
0.053
0.251
0.143
0.164
0.000360.000300.00025
2.103
3.214
2.394

K-15

(mmol/kg)

Molecular
Weight(g/mole)IC50 (µg/mL)1

7.175
7.175
7.175
1.575
1.575
1.575
35.13935.13935.139
0.336
0.336
0.336
69.30269.30269.302
0.169
0.169
0.169
138.737138.737138.737
4.724
4.724
4.724
3.020
3.020
3.020
0.016
0.016
0.016
0.019
0.019
0.019
0.050
0.050
0.050
19.13719.13719.137271.790271.790271.790295.840295.840295.840
180.200
180.200
180.200
129.900
129.900
129.900
58.44058.44058.440298.000
298.000
298.000
74.44074.44074.440134.000
134.000
134.000
41.99041.99041.990188.940
188.940
188.940
334.400
334.400
334.400
504.800
504.800
504.800
163.400
163.400
163.400

1471.183
1778.2802018.366
38.08443.75828.46518.01618.39413.825
0.763
0.304
0.467
3578.217643.675
3459.394
0.779
0.811
0.571
1801.634
1157.000
1626.797
352.641316.228350.483
48.36345.25053.4237.439
14.4409.935
83.89847.86354.996
0.183
0.151
0.126
343.558525.210391.141

Reference LD50
2Reference LD50
(mg/kg)2
1950.01950.01950.0
466.0
466.0
466.0
6332.06332.06332.0
43.6
43.6
43.6
4050.04050.04050.0
50.5
50.5
50.5
10327.6
10327.6
10327.6
633.0
633.0
633.0
126.8
126.8
126.8
3.0
3.0
3.0
6.3
6.3
6.3
25.0
25.0
25.0
3127.03127.03127.0In Vitro Cytotoxicity Test Methods BRD K1

November 2006

IC50 and LD50 Values Used for Laboratory-Specific Regressions

NRU Test
Method
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK

Substance
Triethylenemelamine
Triethylenemelamine
Triethylenemelamine
Triphenyltin hydroxide
Triphenyltin hydroxide
Triphenyltin hydroxide
Valproic acid
Valproic acid
Valproic acid
Verapamil HCl
Verapamil HCl
Verapamil HCl
XyleneLab

Log IC50 (mM)1

ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
IIVS

-2.126-2.012-1.988-4.250-4.885-4.552
0.501
0.679
0.470
-0.917-0.817-0.871
0.642

Log Reference2

LD50 (mmol/kg)

IC50 (mM)1-1.708-1.708-1.708-0.047-0.047-0.047
0.839
0.839
0.839
-0.646-0.646-0.6461.643

0.00750.00970.01030.000056
0.000013
0.000028
3.172
4.779
2.954
0.121
0.152
0.134
4.385

(mmol/kg)

Molecular
Weight(g/mole)IC50 (µg/mL)1

0.020
0.020
0.020
0.896
0.896
0.896
6.907
6.907
6.907
0.226
0.226
0.226
43.939204.230204.230204.230
367.020367.020367.020144.200
144.200
144.200
491.080491.080491.080
106.170

1.527
1.986
2.097
0.021
0.00480.010
457.439689.181425.92559.38474.87466.019465.586

Reference LD50
2Reference LD50
(mg/kg)2
4.0
4.0
4.0
329.0
329.0
329.0
996.0
996.0
996.0
111.0
111.0
111.0
4665.0Abbreviations: 3T3=Neutral red uptake with mouse fibroblast 3T3 cell line; NHK=Neutral red uptake with normal human epidermal keratinocytes; ECBC=US Army Chemical Biological Center;
FAL=FRAME Alternatives Lab; IIVS=Institute for In Vitro Sciences.
1
IC50 values are the geometric mean IC50 values for each substance in each lab.
2
Reference rat oral LD50 values from Table 4-2. Reference values were developed from rat acute oral LD50 studies located using literature searches, secondary references, and electronic database searches.

K-16

In Vitro Cytotoxicity Test Methods BRD Appendix K2

November 2006

Appendix K2

IC50 and LD50 Values Used for Combined-Laboratory Regressions

K-17

In Vitro Cytotoxicity Test Methods BRD Appendix K2

November 2006

[This Page Intentionally Left Blank]

K-18

In Vitro Cytotoxicity Test Methods BRD K2

November 2006

IC50 and LD50 Values Used for Combined-Laboratory Regressions

NRU
Test
Method
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3

Substance
1,1,1-Trichloroethane
2-Propanol
5-Aminosalicylic Acid
Acetaminophen
Acetonitrile
Acetylsalicylic Acid
Aminopterin
Amitriptyline HCl
Arsenictrioxide
Atropine Sulfate
Boric Acid
BusulfanCadmium chloride
Caffeine
Carbamazepine
Chloral Hydrate
Chloramphenicol
Citric Acid
Cupric Sulfate Pentahydrate
Cycloheximide
Dibutyl Phthalate
Dichlorvos (DDVP)
Diethyl Phthalate
DigoxinDimethylformamide
Diquat Dibromide Monohydrate
Disulfoton
Endosulfan
Ethanol
Ethyleneglycol
Fenpropathrin
Gibberellic Acid
Glutethimide
GlycerolHaloperidol
Hexachlorophene
Lactic Acid

Log IC50

Log Reference
LD50

(mM)1

(mmol/kg)2

2.186
1.764
1.037
-0.501
2.287
0.574-4.839-1.648-1.980-0.9611.476-0.501-2.549-0.105-0.3600.044-0.3950.600-0.822-3.177-0.807-1.095-0.316-0.2441.854-1.6540.163-1.8402.151
2.595-1.1751.353-0.0792.422-1.788-1.987
1.529

Molecular
Weight(g/mole)

1.957
1.929
1.350
1.155
1.942
0.922
-1.7990.046-0.8970.071
1.744-1.308-0.132
0.203
1.075
0.586
1.033
1.489
0.279
-2.1481.504-0.5761.622-1.4411.861-0.355-1.739-1.1652.391
2.062-0.664
1.241
0.441
2.332
-0.057-0.6961.606

133.41
60.11
153.10151.2041.05
180.20440.47313.90197.84694.8061.83
246.31
183.30194.20236.30165.40323.15
192.10249.70281.40278.30220.98222.20780.9073.10
362.10274.42406.91
46.07
62.08
349.43346.38217.3092.09
375.90406.91
90.08

K-19

Reference
1

IC50 (mM)

LD50

IC50 (µg/mL)1(mmol/kg)2
153.307
58.037
10.8870.315
193.701
3.754
0.000
0.022
0.010
0.109
29.924
0.315
0.003
0.785
0.437
1.107
0.403
3.981
0.151
0.001
0.156
0.080
0.483
0.570
71.463
0.022
1.456
0.014
141.588393.6150.067
22.5480.833
264.3650.016
0.010
33.79290.53484.92822.39114.29987.576
8.357
0.016
1.112
0.127
1.179
55.410
0.049
0.738
1.596
11.8793.857
10.80030.8641.902
0.007
31.9510.266
41.9040.036
72.572
0.442
0.018
0.068
245.800
115.3510.217
17.4362.761
214.6810.878
0.202
40.353

20453
3489
1667
47.7
7951
676
0.006
7.05
2.07
76.0
1850
77.7
0.518
153
103
183
130
765
37.6
0.187
43.4
17.7
107
445
5224
8.04
400
5.88
6523
24436
23.3
7810
181
24345
6.13
4.19
3044

Reference
LD50 (mg/kg)2
12078.1
5105.03428.02162.03595.01506.0
7.0
349.0
25.1
819.0
3426.0
12.1
135.2
310.0
2807.0638.03490.05929.0475.0
2.0
8892.058.7
9311.028.3
5305.0
160.0
5.0
27.8
11324.07161.075.76039.5600.0
19770.0330.0
82.0
3635.0In Vitro Cytotoxicity Test Methods BRD K2

November 2006

IC50 and LD50 Values Used for Combined-Laboratory Regressions

NRU
Test
Method
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3

Substance
Lindane
Lithium carbonate
Meprobamate
Mercury Chloride
Nicotine
Paraquat
Parathion
Phenobarbital
PhenolPhenylthiourea
Physostigmine
Potassium Cyanide
Potassium chloride
Procainamide HCl
Propranolol
Sodium Arsenite
Sodium Chloride
Sodium Dichromate Dihydrate
Sodium Hypochlorite
Sodium Oxalate
Sodium fluoride
Sodium selenate
Strychnine
Thallium Sulfate
Trichloroacetic Acid
Triethylenemelamine
Triphenyltin Hydroxide
Valproic Acid
Verapamil HCl
XyleneLog IC50

Log Reference
LD50

(mM)1

(mmol/kg)2-0.4160.881
0.351-1.8190.347-1.106-0.891
0.402-0.152-0.285-1.015-0.2741.678
0.210-1.321-2.2341.910-2.7061.145-0.5570.269-0.814-0.326-1.9680.742-2.875-4.3290.758-1.1480.832

Molecular
Weight(g/mole)-0.4640.902
0.803-0.830-0.367-0.443-1.679-0.0160.765-1.705-1.741-0.956
1.575
0.856
0.197
-0.4741.841-0.771
2.142
0.674
0.480
-1.799-1.725-1.3051.505-1.708-0.0470.839-0.6461.643

290.8073.89
218.30271.50162.20257.20291.30232.23
94.11
152.20275.4065.12
74.55
271.79295.84129.9058.44
298.0074.44
134.0041.99
188.94334.40504.80163.40204.23367.02144.20491.08106.17

K-20

Reference
1

IC50 (mM)

LD50

IC50 (µg/mL)1(mmol/kg)2
0.384
7.601
2.245
0.015
2.225
0.078
0.128
2.524
0.705
0.519
0.097
0.532
47.682
1.621
0.048
0.006
81.2070.002
13.971
0.277
1.858
0.154
0.472
0.011
5.519
0.001
0.000
5.727
0.071
6.787

0.344
7.985
6.353
0.148
0.430
0.360
0.021
0.965
5.823
0.020
0.018
0.111
37.586
7.175
1.575
0.336
69.3020.169
138.737
4.724
3.020
0.016
0.019
0.050
32.001
0.020
0.896
6.907
0.226
43.939

112
562
490
4.12
361
20.1
37.4
586
66.3
79.0
26.6
34.6
3555
441
14.1
0.759
4746
0.587
1040
37.1
78.0
29.0
158
5.43
902
0.272
0.017
826
34.9
721

Reference
LD50 (mg/kg)2
100.0
590.0
1386.8
40.2
69.7
92.7
6.1
224.0
548.0
3.0
5.0
7.2
2802.01950.0466.0
43.6
4050.050.510327.6
633.0
126.8
3.0
6.3
25.0
5229.0
4.0
329.0
996.0
111.0
4665.0In Vitro Cytotoxicity Test Methods BRD K2

November 2006

IC50 and LD50 Values Used for Combined-Laboratory Regressions

NRU
Test
Method
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK

Substance
1,1,1-Trichloroethane
2-Propanol
5-Aminosalicylic Acid
Acetaminophen
Acetonitrile
Acetylsalicylic Acid
Aminopterin
Amitriptyline HCl
Arsenictrioxide
Atropine Sulfate
Boric Acid
BusulfanCadmium chloride
Caffeine
Carbamazepine
Chloral Hydrate
Chloramphenicol
Citric Acid
Cupric Sulfate Pentahydrate
Cycloheximide
Dibutyl Phthalate
Dichlorvos (DDVP)
Diethyl Phthalate
DigoxinDimethylformamide
Diquat Dibromide Monohydrate
Disulfoton
Endosulfan
Ethanol
Ethyleneglycol
Fenpropathrin
Gibberellic Acid
Glutethimide
GlycerolHaloperidol
Hexachlorophene
Lactic Acid

Log IC50

Log Reference
LD50

(mM)1

(mmol/kg)2

1.784
1.951
-0.516
0.535
2.367
0.526
0.177
-1.545-1.461-0.9290.833
0.024-2.0090.516-0.453-0.0940.028
0.331-0.104-3.584-0.987-1.315-0.266-5.8892.026-1.922-0.007-2.2822.337
2.831-2.1580.916-0.0982.429-2.049-4.1491.161

Molecular
Weight(g/mole)

1.957
1.929
1.350
1.155
1.942
0.922
-1.7990.046-0.8970.071
1.744-1.308-0.132
0.203
1.075
0.586
1.033
1.489
0.279
-2.1481.504-0.5761.622-1.4411.861-0.355-1.739-1.1652.391
2.062-0.664
1.241
0.441
2.332
-0.057-0.6961.606

133.41
60.11
153.10151.2041.05
180.20440.47313.90197.84694.8061.83
246.31
183.30194.20236.30165.40323.15
192.10249.70281.40278.30220.98222.20780.9073.10
362.10274.42406.91
46.07
62.08
349.43346.38217.3092.09
375.90406.91
90.08

K-21

Reference
1

IC50 (mM)

LD50

IC50 (µg/mL)1(mmol/kg)2
60.88189.2420.305
3.426
232.612
3.360
1.503
0.029
0.035
0.118
6.813
1.056
0.010
3.283
0.352
0.805
1.068
2.142
0.787
0.000
0.103
0.048
0.542
0.000
106.163
0.012
0.983
0.005
217.450678.106
0.007
8.246
0.799
268.544
0.009
0.000
14.47690.53484.92822.39114.29987.576
8.357
0.016
1.112
0.127
1.179
55.410
0.049
0.738
1.596
11.8793.857
10.80030.8641.902
0.007
31.9510.266
41.9040.036
72.572
0.442
0.018
0.068
245.800
115.3510.217
17.4362.761
214.6810.878
0.202
40.353

8122
5364
46.7
518
9549
605
662
8.96
6.84
81.8
421
260
1.80
638
83.2
133
345
411
197
0.073
28.7
10.7
120
0.001
7760
4.33
270
2.13
10018
42097
2.43
2856
174
24730
3.36
0.029
1304

Reference
LD50 (mg/kg)2
12078.1
5105.03428.02162.03595.01506.0
7.0
349.0
25.1
819.0
3426.0
12.1
135.2
310.0
2807.0638.03490.05929.0475.0
2.0
8892.058.7
9311.028.3
5305.0
160.0
5.0
27.8
11324.07161.075.76039.5600.0
19770.0330.0
82.0
3635.0In Vitro Cytotoxicity Test Methods BRD K2

November 2006

IC50 and LD50 Values Used for Combined-Laboratory Regressions

NRU
Test
Method
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK

Substance
Lindane
Lithium carbonate
Meprobamate
Mercury Chloride
MethanolNicotine
Paraquat
Parathion
Phenobarbital
PhenolPhenylthiourea
Physostigmine
Potassium Cyanide
Potassium chloride
Procainamide HCl
Propranolol
Sodium Arsenite
Sodium Chloride
Sodium Dichromate Dihydrate
Sodium Hypochlorite
Sodium Oxalate
Sodium fluoride
Sodium selenate
Strychnine
Thallium Sulfate
Trichloroacetic Acid
Triethylenemelamine
Triphenyltin Hydroxide
Valproic Acid
Verapamil HCl
XyleneLog IC50

Log Reference
LD50

(mM)1

(mmol/kg)2-1.1910.801
0.213-1.6711.679-0.182-0.621-0.9830.285-0.0980.344-0.493-0.3521.477
0.807-0.912-2.4351.534-2.622
1.305
0.404
0.066
-1.267-0.743-3.5220.403-2.042-4.5620.550-0.8690.642

Molecular
Weight(g/mole)-0.4640.902
0.803-0.8302.434-0.367-0.443-1.679-0.0160.765-1.705-1.741-0.956
1.575
0.856
0.197
-0.4741.841-0.771
2.142
0.674
0.480
-1.799-1.725-1.3051.505-1.708-0.0470.839-0.6461.643

290.8073.89
218.30271.5032.04
162.20257.20291.30232.23
94.11
152.20275.4065.12
74.55
271.79295.84129.9058.44
298.0074.44
134.0041.99
188.94334.40504.80163.40204.23367.02144.20491.08106.17

Reference
1

IC50 (mM)

LD50

IC50 (µg/mL)1(mmol/kg)2
0.064
6.330
1.634
0.021
47.713
0.657
0.239
0.104
1.928
0.797
2.210
0.321
0.445
30.007
6.407
0.122
0.004
34.1770.002
20.182
2.533
1.165
0.054
0.181
0.000
2.529
0.009
0.000
3.551
0.135
4.385

0.344
7.985
6.353
0.148
271.835
0.430
0.360
0.021
0.965
5.823
0.020
0.018
0.111
37.586
7.175
1.575
0.336
69.3020.169
138.737
4.724
3.020
0.016
0.019
0.050
32.001
0.020
0.896
6.907
0.226
43.939

18.7
468
357
5.80
1529
107
61.6
30.3
448
75.0
336
88.5
29.0
2237
1741
36.2
0.477
1997
0.712
1502
339
48.9
10.2
60.4
0.152
413
1.85
0.010
512
66.5
466

Reference
LD50 (mg/kg)2
100.0
590.0
1386.840.2
8709.6
69.7
92.7
6.1
224.0
548.0
3.0
5.0
7.2
2802.01950.0466.0
43.6
4050.050.510327.6
633.0
126.8
3.0
6.3
25.0
5229.0
4.0
329.0
996.0
111.0
4665.0Abbreviations: 3T3=Neutral red uptake with mouse fibroblast 3T3 cell line; NHK=Neutral red uptake with normal human epidermal keratinocytes; ECBC=US Army Chemical
Biological Center; FAL=FRAME Alternatives Lab; IIVS=Institute for In Vitro Sciences.
1
IC50 values are the geometric mean IC50 values for each substance in each lab.
2
Reference rat oral LD50 values from Table 4-2. Reference values were developed from rat acute oral LD50 studies located using literature searches, secondary references, andelectronic database searches.

K-22

In Vitro Cytotoxicity Test Methods BRD Appendix K3

November 2006

Appendix K3

RC IC50 and LD50 Values for RC Substances with Rat Oral LD50 Data

K-23

In Vitro Cytotoxicity Test Methods BRD Appendix K3

November 2006

[This Page Intentionally Left Blank]

K-24

In Vitro Cytotoxicity Test Methods BRD Appendix K3

November 2006

RC IC50 and LD50 Values for RC Substances with Rat Oral LD50 Data
Substance

Molecular
Weight(g/mole)Formaldehyde
Methanol
Acetonitrile
Sodium I fluoride
Lithium I chloride
Acetaldehyde
Ethanol
Ammonium sulfide
Acrylonitrile
Ammonium chloride
Acrolein
Propionaldehyde
Allylalcohol
AcetonePotassium I fluoride
Sodium chloride
Acetic acid
1-Propanol
2-Propanol
Ethylene glycol
Sodium azide
Potassium cyanide
Acrylamide
n-Butanal
Isobutanal
Ethyl methyl ketone
Dimethylformamide
Isobutanol
1-Butanol
Potassium I chloride
Thioacetamide
2-Methoxyethanol
Propylene glycol
Thiourea
Dimethyl sulfoxide
Pyridine
Dichloromethane
Piperazine
N,N-Dimethylacetamide
1,4-Dioxane

30.03
32.05
41.06
41.99
42.39
44.06
46.08
51.12
53.07
53.5
56.07
58.09
58.09
58.09
58.1
58.44
60.06
60.11
60.11
62.08
65.02
65.12
71.09
72.12
72.12
72.12
73.11
74.14
74.14
74.55
75.14
76.11
76.11
76.13
78.14
79.11
84.93
86.16
87.14
88.12

IC50x
(mM)1
0.12
930
368
1.85
38.6
2.45
379
0.42
2.42
5.52
0.047
3.25
6.94
444
3.13
75.9
24.3
96.5
167
555
0.71
1.12
1.61
12.8
13.5
104
114
40.1
52.5
82
4.17
251
342
86
252
46.9
34.9
67.2
24.2
38.1

K-25

IC50x
(mg/mL)1

Rat Oral
LD50(mmol/kg)2

Rat Oral
LD50(mg/kg)2

3.60
29806.5
15110.1
77.68
1636.3107.9517464.3
21.47
128.43295.322.64
188.79403.1425792.0
181.854435.61459.55800.610038.434454.4
46.16
72.93
114.45923.1
973.627500.58334.52973.03892.4
6113.1
313.3319103.6
26029.66547.2
19691.3
3710.32964.15790.02108.83357.4

26.6
406
92.5
4.29
17.9
43.8
304
3.29
1.54
30.8
0.82
24.3
1.1
168
4.22
51.3
55.1
89.8
97.2
138
0.69
0.15
2.39
34.5
39
47.1
38.3
33.2
10.7
34.9
4.01
32.3
263
1.64
252
11.3
18.8
22.1
58.4
47.7

798.8
13012.3
3798.1
180.1
758.8
1929.814008.3
168.2
81.7
1647.846.0
1411.663.9
9759.1245.22998.03309.35397.95842.78567.0
44.9
9.8
169.9
2488.12812.73396.92800.1
2461.4793.3
2601.8301.32458.4
20016.9
124.919691.3
893.9
1596.71904.15089.04203.3

In Vitro Cytotoxicity Test Methods BRD Appendix K3

November 2006

RC IC50 and LD50 Values for RC Substances with Rat Oral LD50 Data
Substance
Ethyl acetate
1-Pentanol
1-Nitropropane
Lactic acid
1,3,5-Trioxane
GlycerolTolueneAnilinePhenolSulfuric acid
Chromium VI trioxide
2-Ethylbutanal
Cyclohexanol
Tetrahydrofurfuryl alcohol
1-Hexanol
StyreneSodium I bromide
Beryllium II sulfate
Diethylene glycol
Xylenep-Cresolo-Cresolm-Cresol
Benzylalcohol
Anisolep-Phenylenediamineo-Phenylenediamine
p-Aminophenol
m-Aminophenol
Catechol
Resorcinol
Calcium II chloride
Trifluoroacetic acid
2,5-Hexanedione
1-Heptanol
Sodium monochloroacetate
2-Butoxyethanol
Chloroform
Benzoic acid
Nicotinamide

Molecular
Weight(g/mole)
88.12
88.17
89.11
90.09
90.09
92.11
92.15
93.14
94.12
98.08
100
100.18100.18102.15102.2
104.16104.92105.07106.14106.18108.15108.15108.15108.15108.15108.16108.16109.14109.14110.12110.12110.98114.03114.16116.23116.48118.2
119.37122.13122.14

IC50x
(mM)1
128
24.9
57.9
66
213
624
17.1
6.9
3.01
36
0.0027
13.2
26.3
111
15.4
3.3
77.4
0.61
62.1
12
0.22
0.52
0.66
5.81
13.2
0.05
0.31
0.062
0.86
0.2
0.8
12.4
20.5
8.45
6.25
1.45
26
13.4
15.7
44.4

K-26

IC50x
(mg/mL)1

Rat Oral
LD50(mmol/kg)2

Rat Oral
LD50(mg/kg)2

11279.4
2195.45159.55945.919189.2
57476.6
1575.8642.67283.303530.90.27
1322.42634.711338.7
1573.9343.738120.864.09
6591.31274.2
23.79
56.24
71.38
628.351427.6
5.41
33.53
6.77
93.86
22.02
88.10
1376.22337.6964.65726.44168.903073.2
1599.6
1917.45423.0

125
34.4
5.11
41.4
8.88
137
54.3
4.72
4.4
21.8
0.8
39.7
20.6
24.5
7.04
48
33.4
0.78
139
40.5
1.91
1.12
2.24
11.4
34.2
0.74
9.89
15.2
15.2
35.3
2.73
9.01
1.75
23.7
28
0.65
12.5
7.61
20.7
28.7

11015.03033.0455.43729.7800.012619.1
5003.7439.6
414.1
2138.180.03977.12063.72502.7719.5
4999.73504.382.014753.5
4300.3
206.6
121.1
242.3
1232.93698.780.0
1069.71658.91658.93887.2
300.6
999.9
199.6
2705.63254.475.7
1477.5908.4
2528.13505.4

In Vitro Cytotoxicity Test Methods BRD Appendix K3

November 2006

RC IC50 and LD50 Values for RC Substances with Rat Oral LD50 Data
Substancep-Toluylendiamine
Nitrobenzene
p-Anisidine
2-Thiouracil
Dichloroacetic acid
Nickel II chloride
Cobalt II chloride
5-Fluorouracil
1,1,1-Trichloroethane
Sodium oxalate
1,2,6-Hexanetriol
Cupric chloride
Zinc II chloride
Salicylamide
Isoniazid
Salicylic acidp-Nitrophenol
Isononylaldehyde
8-Hydroxyquinoline
Coumarin
N-Methyl-N'-nitro-N-nitrosoguanidine
Isobenzoic furanodione
ThymolAcetaminophen
Ferrous sulfate
Methyl salicylate
Phenylthiourea
2-Nitro-p-phenylenediamine
Carbon tetrachloride
MentholBromobenzene
Dimethylaminoethyl methacrylate(polymer)
Strontium II chloride
Sodium salicylate
6-MethylcoumarinHydralazine
Nicotine
2,4-Dichlorophenol
Trichloroacetic acid

Molecular
Weight(g/mole)

IC50x
(mM)1

IC50x
(mg/mL)1

Rat Oral
LD50(mmol/kg)2

Rat Oral
LD50(mg/kg)2

122.19123.12
123.17128.16128.94129.61129.83130.09
133.4
134
134.2
134.44136.27137.15137.16138.13139.12
142.27145.17146.15

0.094
12.2
0.73
0.32
11.5
0.27
0.16
0.0026
10.3
0.44
123
0.11
0.13
1.08
7.49
3.38
0.2
1.52
0.0033
1.71

11.49
1502.1
89.91
41.01
1482.8
34.99
20.77
0.34
1374.058.96
16506.6
14.79
17.72
148.121027.3
466.9
27.8
216.3
0.48
249.9

0.83
5.2
11.4
7.8
21.9
0.81
0.62
1.77
77.2
1.16
119
1.04
2.57
13.8
4.74
6.45
2.52
22.8
8.27
2

101.4
640.2
1404.1999.6
2823.8
105.0
80.5
230.3
10298.5
155.4
15969.8
139.8
350.2
1892.7
650.1
890.9
350.6
3243.81200.6292.3

147.12

0.012

1.8

0.61

89.7

148.12150.24151.18151.91152.16152.23153.16153.81156.3
157.02

17
0.23
2.71
1.85
1.7
0.54
0.39
8.51
0.95
3.46

2518.0
34.6
409.7
281.0
258.7
82.2
59.7
1308.9
148.5
543.3

27.1
6.52
15.9
2.1
5.83
0.02
20.1
18.2
20.3
17.2

4014.1979.6
2403.8
319.0
887.1
3.0
3078.52799.33172.92700.7157.24

0.11

17.3

11.1

1745.4158.52160.11160.18160.2
162.26163
163.38

36.4
4.33
0.31
0.33
1.79
0.055
8.19

5770.1
693.3
49.7
52.9
290.4
9.0
1338.1

14.2
9.99
10.5
0.56
0.31
3.56
30.6

2251.01599.51681.9
89.7
50.3
580.3
4999.4

K-27

In Vitro Cytotoxicity Test Methods BRD Appendix K3

November 2006

RC IC50 and LD50 Values for RC Substances with Rat Oral LD50 Data
Substance
Chloral hydrate
Tetrachloroethene
t-Butyl hydroquinone(-)-Phenylephrine
m-Dinitrobenzene
Azaserine
1,2-Dibromomethane
L-Ascorbic acidn-Butyl benzoate
Phenacetin
Iproniazid
Acetylsalicylic acid
D-Glucose
Butylated hydoxyanisole
1,2,4-Trichlorobenzene
Cadmium II chloride
2,4-Dinitrophenol
Undecylenic acidTributylamine
Paraquat
Amrinone
Antipyrine
Tin II chloride
Nitrilotriacetic acid
Nitrogen mustard * HCl
Dimethyl phthalate
Caffeine
4-Hexylresorcinol
L-DopaHalothane
Arsenic III trioxide
Manganese II chloride *4 H2O
Carbaryl
Sodium cyclamate
Magnesium II chloride * 6 H2OPhenylephrine * HCl
Triethylene melamine
Ibuprofen
Milrinone
1,3-Bis(2-chloroethyl)- 1-

Molecular
Weight(g/mole)165.4
165.82
166.24167.23168.12
173.15173.85176.14178.25179.24179.25180.17180.18180.27181.44
183.3
184.12
184.31
185.4
186.25187.22
188.25
189.59191.16
192.53194.2
194.22
194.3
197.21
197.39197.84
197.92201.24201.24203.33203.69204.27206.31211.24214.07

IC50x
(mM)1
2.65
6.54
0.069
4.45
0.39
0.002
4.2
1.52
0.41
1.27
0.79
2.27
226
0.24
0.71
0.0064
0.21
0.18
15.4
0.54
0.28
11.6
1.51
3.61
0.0026
23.4
2.64
0.064
0.13
31.1
0.0042
0.13
0.26
35.4
70.4
4.16
0.00078
0.52
4.77
0.078

K-28

IC50x
(mg/mL)1

Rat Oral
LD50(mmol/kg)2

Rat Oral
LD50(mg/kg)2

438.3
1084.5
11.5
744.2
65.6
0.35
730.2
267.7
73.1
227.6
141.6
409.0
40720.7
43.3
128.8
1.2
38.7
33.2
2855.2100.6
52.4
2183.7
286.3
690.1
0.50
4544.3
512.7
12.4
25.6
6138.8
0.8
25.7
52.3
7123.914314.4
847.4
0.16
107.3
1007.6
16.7

2.9
53.4
4.81
2.09
0.49
0.98
0.62
67.6
28.8
9.21
2.04
5.55
143
12.2
4.17
0.48
0.16
13.6
2.91
0.31
0.54
9.56
3.69
7.69
0.052
35.5
0.99
2.83
9.03
28.8
0.1
7.5
1.24
75.8
39.8
1.72
0.005
4.89
0.43
0.093

479.7
8854.8
799.6
349.5
82.4
169.7
107.8
11907.1
5133.61650.8365.7
999.9
25765.7
2199.3
756.6
88.0
29.5
2506.6
539.5
57.7
101.1
1799.7699.6
1470.010.06894.1
192.3
549.9
1780.85684.819.81484.4249.515254.08092.5350.3
1.0
1008.990.8
19.9

In Vitro Cytotoxicity Test Methods BRD Appendix K3

November 2006

RC IC50 and LD50 Values for RC Substances with Rat Oral LD50 Data
Substance

Molecular
Weight(g/mole)

IC50x
(mM)1

IC50x
(mg/mL)1

Rat Oral
LD50(mmol/kg)2

Rat Oral
LD50(mg/kg)2

nitrosourea
Clofibric acid
Glutethimide
Butylated hydroxytoluene
2,4-Dichlorophenoxyacetic acid
Diethyl phthalate
Bendiocarb
Diethyldithiocarbamate sodium*
3H20
Ammonium persulfate
Cygon
Aminophenazone
Nalidixic acid
Phenobarbital
Ambazone
Mefenamic acid
Triethyltin chloride
Busulphan
Isoproterenol * HClPentobarbital sodium
Cupric sulfate * 5 H2O
2,4,5-Trichlorophenoxyacetic acid
Nabam
Trichlorfon
Natulan * HCl
Diethyl sebacate
Versalide
Secobarbital sodium
Barium II nitrate
Sodium bichromate VI
Theophylline sodium acetate
Maneb
3-Cyano-2-morpholino-5-(pyrid-4yl)-pyridine (Chemical 122)
Pentachlorophenol
Isoxepac
Dichlorophene
Mercury II chloride
Hexachlorocyclopentadiene
Disulfoton
Zineb

214.66217.29220.39221.04222.26223.25

2.61
1.56
0.056
0.77
5.52
0.18

560.3
339.0
12.3
170.2
1226.9
40.2

5.82
2.76
4.04
1.67
38.7
0.8

1249.3
599.7
890.4
369.1
8601.5178.6

225.330.00039

0.088

6.66

1500.7228.22229.27231.33232.26232.26237.32241.31241.35246.32247.75248.29249.7
255.48256.34257.44257.8
258.4
258.44260.3
261.36261.98262.23266.31

0.23
1.24
5.39
1.5
3.81
0.038
0.087
0.00046
0.046
0.022
0.71
0.33
0.44
0.035
0.27
2.74
1.63
0.15
0.21
0.81
0.00093
4.19
0.004252.5
284.3
1246.9
348.4
884.9
9.0
21.0
0.11
11.3
5.5
176.3
82.4
112.4
9.0
69.5
706.4
421.2
38.8
54.7
211.7
0.24
1098.7
1.1

3.59
0.66
4.32
5.81
0.7
3.16
3.27
0.021
0.0076
8.96
0.81
1.2
1.17
1.54
1.75
3.04
56
1.22
0.48
1.36
0.19
2.22
16.9

819.3
151.3
999.3
1349.4
162.6
749.9
789.1
5.1
1.9
2219.8
201.1
299.6
298.9
394.8
450.5
783.7
14470.4
315.3
124.9
355.4
49.8
582.2
4500.6266.31

0.96

255.7

1.3

346.2

266.32268.28269.13271.49272.75274.42275.730.036
1.33
0.00830.015
0.0031
0.11
0.059

9.6
356.8
2.2
4.1
0.85
30.2
16.3

0.19
0.74
10
0.00370.41
0.0073
18.9

50.6
198.5
2691.3
1.0
111.8
2.0
5211.3K-29

In Vitro Cytotoxicity Test Methods BRD Appendix K3

November 2006

RC IC50 and LD50 Values for RC Substances with Rat Oral LD50 Data
Substance
Triethyl citrate
Azathioprine
Amitriptyline
Imidazolidinyl urea
Dibutyl phthalate
Cyclophosphamide * H2OFlufenamic acid
Cycloheximide
Diazepam
Retinol
Dihydralazine sulfate
Sodium dodecyl sulfate
LindaneParathion
Diphenhydramine * HCl
Naftipramide
Cis-platinum
all-trans-Retinoic acid
CaptanChlorambucil
Orphenadrine * HCl
Buflomedil
Warfarin
Phenylbutazone
Aflatoxin B1
Refortan
Imipramine * HClp,p'-DDE
Chlorpromazinep,p'-DDD
Chloramphenicol
Oxyphenbutazone
Tributyltin chloride
Malathion
Frusemide
Mitomycin C
Metamizol
Dicoumarol
Caffeine sodium benzoate
Papaverine

Molecular
Weight(g/mole)276.32277.29277.44278.26278.38279.13281.25281.39284.76286.5
288.32289.43290.82291.28291.85298.47300.07300.48300.59304.24305.88307.43308.35308.41
312.29313.1
316.91318.02318.89320.04323.15324.41325.53330.38330.76334.37334.38336.31
338.33339.42

IC50x
(mM)1
14.7
0.14
0.056
0.36
0.76
3.12
0.029
0.000590.16
0.00054
0.14
0.27
0.41
0.093
0.24
0.084
0.00280.11
0.0039
0.076
0.49
1.35
0.67
0.32
0.034
0.25
0.054
0.1
0.014
0.024
0.79
0.19
0.000540.2
2.33
0.00084
0.58
0.027
5.67
0.045

K-30

IC50x
(mg/mL)1

Rat Oral
LD50(mmol/kg)2

Rat Oral
LD50(mg/kg)2

4061.9
38.8
15.5
100.2
211.6
870.9
8.2
0.17
45.6
0.15
40.4
78.1
119.2
27.1
70.0
25.1
0.84
33.1
1.2
23.1
149.9
415.0
206.6
98.7
10.6
78.3
17.1
31.8
4.5
7.7
255.3
61.6
0.18
66.1
770.7
0.28
193.9
9.1
1918.3
15.3

25.3
1.93
1.15
9.34
43.1
0.34
0.97
0.0071
2.49
6.98
2.84
4.45
0.26
0.0069
2.93
3.45
0.086
6.66
33.3
0.25
1.39
1.19
1.05
1.22
0.016
10.1
0.96
2.77
0.44
0.35
10.5
3.08
0.37
2.68
7.86
0.042
21.5
2.11
2.54
0.96

6990.9535.2
319.1
2598.911998.2
94.9
272.8
2.0
709.1
1999.8818.81288.0
75.6
2.0
855.1
1029.7
25.8
2001.210009.6
76.1
425.2
365.8
323.8
376.3
5.0
3162.3
304.2
880.9
140.3
112.0
3393.1
999.2
120.4
885.4
2599.814.0
7189.2
709.6
859.4
325.8

In Vitro Cytotoxicity Test Methods BRD Appendix K3

November 2006

RC IC50 and LD50 Values for RC Substances with Rat Oral LD50 Data
Substance
Diquat dibromide
Gibberellic acid
Dodecylbenzene sodiumsulfonate
Triisooctylaminep,p'-DDT
Benzylpenicillin sodium
Indomethacin
Quinine * HCl
Cetyltrimethylammonium chloride
Hexadecyltrimethylammonium
bromideAldrinBenzalkonium chloride
Triphenyltin hydroxide
Potassium hexacyanoferrate II
Amphetamine sulfate
Homatropine methylbromide
KelthaneDi(2-ethylhexyl)adipate
Ioxynil
Heptachlor
Dextropropoxyphene * HCl
Dieldrin
Scopolamine * HBr
Di(2-ethylhexyl)phthalate
Rotenone
Hexachlorophene
ChlordanHydroxyzine * HCl
Chloroquine sulfate
Quinidine sulfate
Oxatomide
Xanthinol nicotinate
Mitoxantrone
Amethopterin
Dimenhydrinate
EmetineTetracycline * HCl
VerapamilHCl
Chlorhexidine

Molecular
Weight(g/mole)

IC50x
(mM)1

IC50x
(mg/mL)1

Rat Oral
LD50(mmol/kg)2

Rat Oral
LD50(mg/kg)2

344.08346.41348.52353.76354.48356.4
357.81360.92362.16

0.16
2.3
0.42
0.023
0.16
5.73
0.16
0.075
0.021

55.1
796.7
146.4
8.1
56.7
2042.2
57.2
27.1
7.6

0.67
18.2
3.62
4.58
0.32
19.4
0.034
1.72
1.31

230.5
6304.71261.6
1620.2113.4
6914.2
12.2
620.8
474.4

364.530.0089

3.2

1.12

408.3

364.9
365
367.03368.37368.54370.33370.48370.64370.91373.3
375.98380.9
384.31
390.62394.45406.89409.76411.41418
422.54426.61434.51444.54454.5
470.02480.71480.94491.13505.520.067
0.00520.000049
42.3
1.97
9
0.012
3.15
0.11
0.059
0.49
0.18
1.08
0.84
0.000130.0079
0.06
0.067
0.06
0.12
0.019
15.8
0.00240.000140.076
0.00016
0.14
0.1
0.015

24.4
1.9
0.018015582.1
726.0
3333.04.41167.5
40.8
22.0
184.2
68.6
415.1
328.1
0.051
3.2
24.6
27.6
25.1
50.7
8.1
6865.3
1.07
0.064
35.7
0.077
67.3
49.1
7.6

0.11
1.1
0.12
17.4
0.15
3.24
1.55
24.6
0.3
0.11
0.22
0.12
3.3
79.4
0.33
0.15
1.12
2.31
2.6
1.08
3.31
32.5
1.32
0.3
2.81
0.14
13.4
0.22
18.2

40.1
401.5
44.0
6409.655.3
1199.9574.2
9117.7
111.3
41.1
82.7
45.7
1268.231015.2
130.2
61.0
458.9
950.4
1086.8456.31412.114121.6
586.8
136.4
1320.867.3
6444.6108.0
9200.5K-31

In Vitro Cytotoxicity Test Methods BRD Appendix K3

November 2006

RC IC50 and LD50 Values for RC Substances with Rat Oral LD50 Data
Substance
Chloroquine diphosphate
Triton X-100
Atropine sulfate
Digitoxin
Trypan blue
Actinomycin D

Molecular
Weight(g/mole)515.92647
676.9
765.05964.881255.6

IC50x
(mM)1
0.017
0.055
0.22
0.000110.095
0.0000081

IC50x(mg/mL)1

Rat Oral
LD50(mmol/kg)2

Rat Oral
LD50(mg/kg)2

8.8
35.6
148.9
0.084291.7
0.0102

1.88
2.78
0.92
0.073
6.43
0.0057969.9
1798.7622.7
55.8
6204.2
7.2

Abbreviations: RC=Registry of Cytotoxicity.
1
Geometric mean of the IC50 values collected from the literature for various in vitro basal cytotoxicity endpoints and cell
types (from the RC [Halle 1998, 2003]).
2
Rat oral LD50 values used in the RC (Halle 1998, 2003), which generally came from the 1983/1984 Registry of Toxic
Effects for Chemical Substances®.

K-32

In Vitro Cytotoxicity Test Methods BRD Appendix K4

November 2006

Appendix K4

Individual Laboratory LD50 Predictions: RC Rat-Only Millimole
Regression

K-33

In Vitro Cytotoxicity Test Methods BRD Appendix K4

November 2006

[This Page Intentionally Left Blank]

K-34

In Vitro Cytotoxicity Test Methods BRD K4

November 2006

Individual Laboratory LD50 Predictions: RC Rat-Only Millimole Regression

NRU
Test Method
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3

SubstanceLab

Log Reference
LD50(mmol/kg)1

1,1,1-Trichloroethane
1,1,1-Trichloroethane
1,1,1-Trichloroethane
2-Propanol
2-Propanol
2-Propanol
5-Aminosalicylic acid
5-Aminosalicylic acid
5-Aminosalicylic acidAcetaminophen
Acetaminophen
Acetaminophen
Acetonitrile
Acetonitrile
Acetonitrile
Acetylsalicylic acid
Acetylsalicylic acid
Acetylsalicylic acid
Aminopterin
Aminopterin
Aminopterin
Amitriptyline HCl
Amitriptyline HCl
Amitriptyline HCl
Arsenic III trioxide
Arsenic III trioxide
Arsenic III trioxide
Atropine sulfate
Atropine sulfate
Atropine sulfate
Boric acid
Boric acid
Boric acid
BusulfanBusulfanBusulfanCadmium II chloride
Cadmium II chloride
Cadmium II chloride
Caffeine
Caffeine

ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL

1.957
1.957
1.957
1.929
1.929
1.929
1.350
1.350
1.350
1.155
1.155
1.155
1.942
1.942
1.942
0.922
0.922
0.922
-1.799-1.799-1.799
0.046
0.046
0.046
-0.897-0.897-0.897
0.071
0.071
0.071
1.744
1.744
1.744
-1.308-1.308-1.308-0.132-0.132-0.1320.203
0.203

Reference LD50
(mg/kg)

1

12078
12078
12078
5105
5105
5105
3428
3428
3428
2162
2162
2162
3595
3595
3595
1506
1506
1506
7
7
7
349
349
349
25
25
25
819
819
819
3426
3426
3426
12
12
12
135
135
135
310
310

K-35

Observed LD50
Toxicity
2

Category(mg/kg)> 5000> 5000> 5000> 5000> 5000> 50002000-5000
2000-5000
2000-5000
2000-5000
2000-5000
2000-5000
2000-5000
2000-5000
2000-5000
300-2000
300-2000
300-2000
5-50
5-50
5-50
300-2000
300-2000
300-2000
5-50
5-50
5-50
300-2000
300-2000
300-2000
2000-5000
2000-5000
2000-5000
5-50
5-50
5-50
50-30050-30050-300300-2000
300-2000

Log Predicted
LD50(mmol/kg)3
51.71238.62127.60421.85526.29326.69411.24113.05011.540
2.333
2.859
2.390
38.42543.23545.155
7.307
9.635
5.915
0.029
0.039
0.027
0.731
0.820
0.821
0.589
0.418
0.731
1.306
1.989
1.524
16.68625.892
14.839
1.811
4.505
1.955
0.306
0.280
0.374
3.537
3.616

Predicted LD50
(mg/kg)

3

6899
5152
3683
1314
1580
1605
1721
1998
1767
353
432
361
1577
1775
1854
1317
1736
1066
13
17
12
229
257
258
117
83
145
907
1382
1059
1032
1601
918
446
1110
482
56
51
69
687
702

Predicted LD50
Toxicity
2

Category(mg/kg)> 5000> 50002000-5000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
5-50
5-50
5-50
50-30050-30050-30050-30050-30050-300300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
50-30050-30050-300300-2000
300-2000

Log IC50
(mM)4
2.489
2.200
1.868
1.637
1.820
1.835
0.979
1.127
1.005
-0.577-0.375-0.553
2.195
2.312
2.355
0.553
0.827
0.344
-4.926-4.612-4.980-1.724-1.611-1.609-1.937-2.278-1.724-1.151-0.734-0.998
1.370
1.804
1.254
-0.8270.075-0.751-2.585-2.675-2.387-0.165-0.143

In Vitro Cytotoxicity Test Methods BRD K4

November 2006

Individual Laboratory LD50 Predictions: RC Rat-Only Millimole Regression

NRU
Test Method
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3

SubstanceLab

Log Reference
LD50(mmol/kg)1

Caffeine
Carbamazepine
Carbamazepine
Carbamazepine
Carbon Tetrachloride
Carbon Tetrachloride
Carbon Tetrachloride
Chloral hydrate
Chloral hydrate
Chloral hydrate
Chloramphenicol
Chloramphenicol
Chloramphenicol
Citric acid
Citric acid
Citric acidColchicine
Colchicine
Colchicine
Cupric sulfate pentahydrate
Cupric sulfate pentahydrate
Cupric sulfate pentahydrate
Cycloheximide
Cycloheximide
Cycloheximide
Dibutyl phthalate
Dibutyl phthalate
Dibutyl phthalate
Dichlorvos
Dichlorvos
Dichlorvos
Diethyl phthalate
Diethyl phthalate
Diethyl phthalate
DigoxinDigoxinDigoxinDimethylformamide
Dimethylformamide
Dimethylformamide
Diquat dibromide monohydrate
Diquat dibromide monohydrate

IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL

0.203
1.075
1.075
1.075
1.391
1.391
1.391
0.586
0.586
0.586
1.033
1.033
1.033
1.489
1.489
1.489
-1.425-1.425-1.425
0.279
0.279
0.279
-2.148-2.148-2.148
1.504
1.504
1.504
-0.576-0.576-0.576
1.622
1.622
1.622
-1.441-1.441-1.441
1.861
1.861
1.861
-0.355-0.355

Reference LD50
(mg/kg)

1

310
2807
2807
2807
3783
3783
3783
638
638
638
3490
3490
3490
5929
5929
5929
15
15
15
475
475
475
2
2
2
8892
8892
8892
59
59
59
9311
9311
9311
28
28
28
5305
5305
5305
160
160

K-36

Observed LD50
Toxicity
2

Category(mg/kg)300-2000
2000-5000
2000-5000
2000-5000
2000-5000
2000-5000
2000-5000
300-2000
300-2000
300-2000
2000-5000
2000-5000
2000-5000> 5000> 5000> 5000
5-50
5-50
5-50
300-2000
300-2000
300-2000<5<5<5> 5000> 5000> 500050-30050-30050-300> 5000> 5000> 5000
5-50
5-50
5-50
> 5000> 5000> 500050-30050-300

Log Predicted
LD50(mmol/kg)3
4.149
2.631
3.381
2.754
NA
NA
NA
4.009
4.924
4.224
1.907
3.824
3.021
6.124
9.136
8.042
0.055
0.102
0.062
2.572
2.985
0.786
0.137
0.266
0.132
1.405
3.365
1.334
1.043
1.807
1.397
2.706
3.444
3.000
2.866
4.345
2.797
27.45427.77026.4640.566
1.312

Predicted LD50
(mg/kg)

3

806
622
799
651
NA
NA
NA
663
814
699
616
1236
976
1176
1755
1545
22
41
25
642
745
196
38
75
37
391
936
371
230
399
309
601
765
667
2238
3393
2184
2007
2030
1934
205
475

Predicted LD50
Toxicity
2

Category(mg/kg)300-2000
300-2000
300-2000
300-2000
NANA
NA
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
5-50
5-50
5-50
300-2000
300-2000
50-3005-5050-3005-50
300-2000
300-2000
300-2000
50-300300-2000
300-2000
300-2000
300-2000
300-2000
2000-5000
2000-5000
2000-5000
2000-5000
2000-5000
300-2000
50-300300-2000

Log IC50(mM)4-0.007-0.457-0.209-0.412NANA
NA-0.0410.162
0.011-0.776-0.088-0.321
0.378
0.774
0.648
-4.292-3.671-4.158-0.480-0.333-1.653-3.384-2.726-3.420-1.079-0.214-1.129-1.373-0.829-1.084-0.430-0.191-0.328-0.3730.039-0.397
1.862
1.874
1.826
-1.978-1.146

In Vitro Cytotoxicity Test Methods BRD K4

November 2006

Individual Laboratory LD50 Predictions: RC Rat-Only Millimole Regression

NRU
Test Method
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3

SubstanceLab

Log Reference
LD50(mmol/kg)1

Diquat dibromide monohydrate
Disulfoton
Disulfoton
Disulfoton
Endosulfan
Endosulfan
Endosulfan
Epinephrine bitartrate
Epinephrine bitartrate
Epinephrine bitartrate
Ethanol
Ethanol
Ethanol
Ethylene glycol
Ethylene glycol
Ethylene glycol
Fenpropathrin
Fenpropathrin
Fenpropathrin
Gibberellic acid
Gibberellic acid
Gibberellic acid
Glutethimide
Glutethimide
Glutethimide
Glycerol
Glycerol
GlycerolHaloperidol
Haloperidol
Haloperidol
Hexachlorophene
Hexachlorophene
Hexachlorophene
Lactic acid
Lactic acid
Lactic acid
LindaneLindaneLindane
Lithium I carbonate
Lithium I carbonate

IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL

-0.355-1.739-1.739-1.739-1.165-1.165-1.165-1.921-1.921-1.921
2.391
2.391
2.391
2.062
2.062
2.062
-0.664-0.664-0.664
1.241
1.241
1.241
0.441
0.441
0.441
2.332
2.332
2.332
-0.057-0.057-0.057-0.696-0.696-0.696
1.606
1.606
1.606
-0.464-0.464-0.4640.902
0.902

Reference LD50
(mg/kg)

1

160
5
5
5
28
28
28
4
4
4
11324
11324
11324
7161
7161
7161
76
76
76
6039
6039
6039
600
600
600
19770
19770
19770
330
330
330
82
82
82
3635
3635
3635
100
100
100
590
590

K-37

Observed LD50
Toxicity
2

Category(mg/kg)50-300
<5
<5
<5
5-50
5-50
5-50
<5
<5
<5
> 5000> 5000> 5000> 5000> 5000> 500050-30050-30050-300> 5000> 5000> 5000300-2000
300-2000
300-2000> 5000> 5000> 5000300-2000
300-2000
300-2000
50-30050-30050-3002000-5000
2000-5000
2000-5000
50-30050-30050-300300-2000
300-2000

Log Predicted
LD50(mmol/kg)3
0.652
2.900
21.284
1.939
0.592
0.907
0.512
1.837
2.013
2.016
33.21640.98936.46650.67563.89958.942
1.253
1.502
1.098
16.573NA
16.242
3.720
4.701
3.279
44.22651.10049.997
0.643
0.770
0.651
0.589
0.615
0.487
19.27920.72018.836
2.530
3.903
2.091
10.179NA

Predicted LD50
(mg/kg)

3

236
796
5841
532
241
369
209
612
671
672
1530
1888
1680
3146
3967
3659
438
525
384
5741
NA
5626
808
1022
712
4073
4706
4604
242
289
245
240
250
198
1737
1866
1697
736
1135
608
752
NA

Predicted LD50
Toxicity
2

Category(mg/kg)50-300300-2000> 5000300-2000
50-300300-2000
50-300300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
2000-5000
2000-5000
2000-5000
300-2000
300-2000
300-2000> 5000NA> 5000300-2000
300-2000
300-2000
2000-5000
2000-5000
2000-5000
50-30050-30050-30050-30050-30050-300300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000NA

Log IC50(mM)4-1.837-0.3611.611-0.760-1.933-1.511-2.076-0.813-0.723-0.721
2.051
2.259
2.143
2.469
2.698
2.618
-1.191-1.012-1.322
1.363
NA
1.343
-0.1150.117-0.240
2.334
2.477
2.455
-1.851-1.673-1.840-1.939-1.896-2.126
1.513
1.584
1.490
-0.496-0.067-0.6850.881
NAIn Vitro Cytotoxicity Test Methods BRD K4

November 2006

Individual Laboratory LD50 Predictions: RC Rat-Only Millimole Regression

NRU
Test Method
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3

SubstanceLab

Log Reference
LD50(mmol/kg)1

Lithium I carbonate
Meprobamate
Meprobamate
Meprobamate
Mercury II chloride
Mercury II chloride
Mercury II chloride
Methanol
Methanol
MethanolNicotine
Nicotine
Nicotine
Paraquat
Paraquat
Paraquat
Parathion
Parathion
Parathion
Phenobarbital
Phenobarbital
Phenobarbital
PhenolPhenolPhenolPhenylthiourea
Phenylthiourea
Phenylthiourea
Physostigmine
Physostigmine
Physostigmine
Potassium cyanide
Potassium cyanide
Potassium cyanide
Potassium I chloride
Potassium I chloride
Potassium I chloride
Procainamide HCl
Procainamide HCl
Procainamide HCl
Propranolol
Propranolol

IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL

0.902
0.803
0.803
0.803
-0.830-0.830-0.830
2.430
2.430
2.430
-0.367-0.367-0.367-0.443-0.443-0.443-1.679-1.679-1.679-0.016-0.016-0.016
0.908
0.908
0.908
-1.705-1.705-1.705-1.741-1.741-1.741-0.956-0.956-0.956
1.575
1.575
1.575
0.856
0.856
0.856
0.197
0.197

Reference LD50
(mg/kg)

1

590
1387
1387
1387
40
40
40
8710
8710
8710
70
70
70
93
93
93
6
6
6
224
224
224
762
762
762
3
3
3
5
5
5
7
7
7
2802
2802
2802
1950
1950
1950
466
466

K-38

Observed LD50
Toxicity
2

Category(mg/kg)300-2000
300-2000
300-2000
300-2000
5-50
5-50
5-50
> 5000> 5000> 500050-30050-30050-30050-30050-30050-300
5-50
5-50
5-50
50-30050-30050-300300-2000
300-2000
300-2000
<5
<5
<5
<5
<5
<5
5-50
5-50
5-50
2000-5000
2000-5000
2000-5000
300-2000
300-2000
300-2000
300-2000
300-2000

Log Predicted
LD50(mmol/kg)3
NA
5.149
7.663
5.363
0.614
0.772
0.619
NA
NA
NA
5.196
6.170
6.525
1.371
1.361
1.365
1.310
2.793
1.336
6.449
6.743
5.678
3.147
4.332
3.375
1.870
5.025
3.271
1.463
1.747
1.313
2.195
5.970
2.425
22.14923.06223.182
4.952
5.115
5.442
1.063
1.037

Predicted LD50
(mg/kg)

3

NA
1124
1673
1171
167
210
168
NA
NA
NA
843
1001
1058
353
350
351
382
814
389
1498
1566
1319
296
408
318
285
765
498
403
481
361
143
389
158
1651
1719
1728
1346
1390
1479
314
307

Predicted LD50
Toxicity
2

Category(mg/kg)NA
300-2000
300-2000
300-2000
50-30050-30050-300NA
NA
NA
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
50-300300-2000
300-2000
50-300300-2000
300-2000
300-2000
300-2000
300-2000
50-300300-2000
50-300300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000

Log IC50
(mM)4
NA
0.207
0.600
0.247
-1.897-1.670-1.889
NA
NA
NA
0.216
0.386
0.441
-1.103-1.109-1.107-1.147-0.398-1.128
0.429
0.473
0.303
-0.2800.036-0.211-0.7950.183-0.242-1.038-0.863-1.145-0.6370.353-0.538
1.650
1.690
1.695
0.168
0.200
0.261
-1.354-1.378In Vitro Cytotoxicity Test Methods BRD K4

November 2006

Individual Laboratory LD50 Predictions: RC Rat-Only Millimole Regression

NRU
Test Method
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3
3T3

SubstanceLab

Log Reference
LD50(mmol/kg)1

Propranolol
Propylparaben
Propylparaben
Propylparaben
Sodium arsenite
Sodium arsenite
Sodium arsenite
Sodium chloride
Sodium chloride
Sodium chloride
Sodium dichromate dihydrate
Sodium dichromate dihydrate
Sodium dichromate dihydrate
Sodium hypochlorite
Sodium hypochlorite
Sodium hypochlorite
Sodium oxalate
Sodium oxalate
Sodium oxalate
Sodium I fluoride
Sodium I fluoride
Sodium I fluoride
Sodium selenate
Sodium selenate
Sodium selenate
Strychnine
Strychnine
Strychnine
Thallium II sulfate
Thallium II sulfate
Thallium II sulfate
Trichloroacetic acid
Trichloroacetic acid
Trichloroacetic acid
Triethylenemelamine
Triethylenemelamine
Triethylenemelamine
Triphenyltin hydroxide
Triphenyltin hydroxide
Triphenyltin hydroxide
Valproic acid
Valproic acid

IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL

0.197
1.546
1.546
1.546
-0.474-0.474-0.474
1.841
1.841
1.841
-0.771-0.771-0.771
2.142
2.142
2.142
0.674
0.674
0.674
0.480
0.480
0.480
-1.799-1.799-1.799-1.725-1.725-1.725-1.305-1.305-1.305
1.505
1.505
1.505
-1.708-1.708-1.708-0.047-0.047-0.0470.839
0.839

Reference LD50
(mg/kg)

1

466
6332
6332
6332
44
44
44
4050
4050
4050
50
50
50
10328
10328
10328
633
633
633
127
127
127
3
3
3
6
6
6
25
25
25
5229
5229
5229
4
4
4
329
329
329
996
996

K-39

Observed LD50
Toxicity
2

Category(mg/kg)300-2000> 5000> 5000> 5000
5-50
5-50
5-50
2000-5000
2000-5000
2000-5000
50-30050-30050-300> 5000> 5000> 5000300-2000
300-2000
300-2000
50-30050-30050-300
<5
<5
<5
5-50
5-50
5-50
5-50
5-50
5-50
> 5000> 5000> 5000<5<5<5300-2000
300-2000
300-2000
300-2000
300-2000

Log Predicted
LD50(mmol/kg)3
1.203
1.615
2.390
1.481
0.362
0.554
0.415
28.90228.38829.098
0.274
0.278
0.262
11.97011.43017.184
2.434
2.168
2.552
4.927
5.977
5.601
1.273
2.401
2.025
4.435
2.695
2.271
0.424
0.730
0.602
8.195
10.085
8.371
0.137
0.474
0.183
0.062
0.051
0.046
7.487
12.661

Predicted LD50
(mg/kg)

3

356
291
431
267
47
72
54
1689
1659
1700
82
83
78
891
851
1279
326
291
342
207
251
235
240
454
383
1483
901
760
214
368
304
1339
1648
1368
28
97
37
23
19
17
1080
1826

Predicted LD50
Toxicity
2

Category(mg/kg)300-2000
50-300300-2000
50-3005-5050-30050-300300-2000
300-2000
300-2000
50-30050-30050-300300-2000
300-2000
300-2000
300-2000
50-300300-2000
50-30050-30050-30050-300300-2000
300-2000
300-2000
300-2000
300-2000
50-300300-2000
300-2000
300-2000
300-2000
300-2000
5-50
50-300
5-50
5-50
5-50
5-50
300-2000
300-2000

Log IC50(mM)4-1.232-0.940-0.553-1.026-2.419-1.998-2.284
1.913
1.896
1.920
-2.697-2.680-2.740
1.041
0.996
1.399
-0.535-0.649-0.488
0.163
0.354
0.290
-1.176-0.548-0.7170.059-0.434-0.603-2.263-1.726-1.916
0.666
0.872
0.687
-3.378-2.153-3.095-4.161-4.366-4.4590.577
1.097

In Vitro Cytotoxicity Test Methods BRD K4

November 2006

Individual Laboratory LD50 Predictions: RC Rat-Only Millimole Regression

NRU
Test Method
3T3
3T3
3T3
3T3
3T3
3T3
3T3

SubstanceLab

Log Reference
LD50(mmol/kg)1

Valproic acid
Verapamil HCl
Verapamil HCl
Verapamil HCl
XyleneXyleneXylene

IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS

0.839
-0.646-0.646-0.646
1.643
1.643
1.643

Reference LD50
(mg/kg)

1

996
111
111
111
4667
4667
4667

K-40

Observed LD50
Toxicity
2

Category(mg/kg)300-2000
50-30050-30050-3002000-5000
2000-5000
2000-5000

Log Predicted
LD50(mmol/kg)3
7.663
1.257
1.302
1.370
NA
NA
9.685

Predicted LD50
(mg/kg)

3

1105
617
640
673
NA
NA
1028

Predicted LD50
Toxicity
2

Category(mg/kg)300-2000
300-2000
300-2000
300-2000NANA
300-2000

Log IC50
(mM)4
0.600
-1.188-1.153-1.103NANA
0.832

In Vitro Cytotoxicity Test Methods BRD K4

November 2006

Individual Laboratory LD50 Predictions: RC Rat-Only Millimole Regression

NRU
Test Method
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK

SubstanceLab

Log Reference
LD50(mmol/kg)1

1,1,1-Trichloroethane
1,1,1-Trichloroethane
1,1,1-Trichloroethane
2-Propanol
2-Propanol
2-Propanol
5-Aminosalicylic acid
5-Aminosalicylic acid
5-Aminosalicylic acidAcetaminophen
Acetaminophen
Acetaminophen
Acetonitrile
Acetonitrile
Acetonitrile
Acetylsalicylic acid
Acetylsalicylic acid
Acetylsalicylic acid
Aminopterin
Aminopterin
Aminopterin
Amitriptyline HCl
Amitriptyline HCl
Amitriptyline HCl
Arsenic III trioxide
Arsenic III trioxide
Arsenic III trioxide
Atropine sulfate
Atropine sulfate
Atropine sulfate
Boric acid
Boric acid
Boric acid
BusulfanBusulfanBusulfanCadmium II chloride
Cadmium II chloride
Cadmium II chloride
Caffeine
Caffeine
Caffeine

ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS

1.957
1.957
1.957
1.929
1.929
1.929
1.350
1.350
1.350
1.155
1.155
1.155
1.942
1.942
1.942
0.922
0.922
0.922
-1.799-1.799-1.799
0.046
0.046
0.046
-0.897-0.897-0.897
0.071
0.071
0.071
1.744
1.744
1.744
-1.308-1.308-1.308-0.132-0.132-0.132
0.203
0.203
0.203

Reference LD50
(mg/kg)

1

12078
12078
12078
5105
5105
5105
3428
3428
3428
2162
2162
2162
3595
3595
3595
1506
1506
1506
7
7
7
349
349
349
25
25
25
819
819
819
3426
3426
3426
12
12
12
135
135
135
310
310
310

K-41

Observed LD50
Toxicity
2

Category(mg/kg)> 5000> 5000> 5000> 5000> 5000> 50002000-5000
2000-5000
2000-5000
2000-5000
2000-5000
2000-5000
2000-5000
2000-5000
2000-5000
300-2000
300-2000
300-2000
5-50
5-50
5-50
300-2000
300-2000
300-2000
5-50
5-50
5-50
300-2000
300-2000
300-2000
2000-5000
2000-5000
2000-5000
5-50
5-50
5-50
50-30050-30050-300300-2000
300-2000
300-2000

Log Predicted
LD50(mmol/kg)3
25.374NANA
29.70826.85033.892
2.025
2.994
2.519
7.388
6.693
7.468
45.26946.71845.140
7.243
7.532
6.598
5.652
4.583
4.817
0.936
0.753
0.957
0.989
0.572
1.535
1.662
1.610
1.630
9.755
9.249
10.120
4.189
4.039
4.633
0.583
0.509
0.556
7.731
6.715
6.724

Predicted LD50
(mg/kg)

3

3385
NA
NA
1786
1614
2037
310
458
386
1117
1012
1129
1858
1918
1853
1305
1357
1189
2490
2018
2122
294
236
300
196
113
304
1155
1119
1132
603
572
626
1032
995
1141
107
93
102
1501
1304
1306

Predicted LD50
Toxicity
2

Category(mg/kg)2000-5000
NANA
300-2000
300-2000
2000-5000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
2000-5000
2000-5000
2000-5000
50-30050-30050-30050-30050-300300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
50-30050-30050-300300-2000
300-2000
300-2000

Log IC50
(mM)4
1.784
NA
NA
1.940
1.840
2.071
-0.717-0.330-0.501
0.564
0.466
0.574
2.357
2.388
2.354
0.544
0.583
0.452
0.299
0.091
0.141
-1.480-1.696-1.458-1.426-1.968-0.991-0.912-0.943-0.932
0.839
0.786
0.875
0.003
-0.0330.102-1.948-2.083-1.995
0.609
0.469
0.471

In Vitro Cytotoxicity Test Methods BRD K4

November 2006

Individual Laboratory LD50 Predictions: RC Rat-Only Millimole Regression

NRU
Test Method
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK

SubstanceLab

Log Reference
LD50(mmol/kg)1

Carbamazepine
Carbamazepine
Carbamazepine
Carbon tetrachloride
Carbon tetrachloride
Carbon tetrachloride
Chloral hydrate
Chloral hydrate
Chloral hydrate
Chloramphenicol
Chloramphenicol
Chloramphenicol
Citric acid
Citric acid
Citric acidColchicine
Colchicine
Colchicine
Cupric sulfate pentahydrate
Cupric sulfate pentahydrate
Cupric sulfate pentahydrate
Cycloheximide
Cycloheximide
Cycloheximide
Dibutyl phthalate
Dibutyl phthalate
Dibutyl phthalate
Dichlorvos
Dichlorvos
Dichlorvos
Diethyl phthalate
Diethyl phthalate
Diethyl phthalate
DigoxinDigoxinDigoxinDimethylformamide
Dimethylformamide
Dimethylformamide
Diquat dibromide monohydrate
Diquat dibromide monohydrate
Diquat dibromide monohydrate

ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS

1.075
1.075
1.075
1.391
1.391
1.391
0.586
0.586
0.586
1.033
1.033
1.033
1.489
1.489
1.489
-1.425-1.425-1.425
0.279
0.279
0.279
-2.148-2.148-2.148
1.504
1.504
1.504
-0.576-0.576-0.576
1.622
1.622
1.622
-1.441-1.441-1.441
1.861
1.861
1.861
-0.355-0.355-0.355

Reference LD50
(mg/kg)

1

2807
2807
2807
3783
3783
3783
638
638
638
3490
3490
3490
5929
5929
5929
15
15
15
475
475
475
2
2
2
8892
8892
8892
59
59
59
9311
9311
9311
28
28
28
5305
5305
5305
160
160
160

K-42

Observed LD50
Toxicity
2

Category(mg/kg)2000-5000
2000-5000
2000-5000
2000-5000
2000-5000
2000-5000
300-2000
300-2000
300-2000
2000-5000
2000-5000
2000-5000> 5000> 5000> 5000
5-50
5-50
5-50
300-2000
300-2000
300-2000<5<5<5> 5000> 5000> 500050-30050-30050-300> 5000> 5000> 5000
5-50
5-50
5-50
> 5000> 5000> 500050-30050-30050-300

Log Predicted
LD50(mmol/kg)3
2.383
3.296
2.350
NA
NA
NA
3.848
4.049
3.521
4.042
4.484
4.387
6.478
5.147
5.965
0.029
0.035
0.036
3.697
3.743
3.846
0.096
0.132
0.110
1.513
1.763
1.372
0.992
1.163
1.171
3.745
2.244
3.876
0.023
0.003
0.020
35.15732.49129.746
0.547
0.692
0.567

Predicted LD50
(mg/kg)

3

563
779
555
NA
NA
NA
636
670
582
1306
1449
1418
1244
989
1146
12
14
14
923
935
960
27
37
31
421
491
382
219
257
259
832
499
861
18
2
15
2570
2375
2174
198
251
205

Predicted LD50
Toxicity
2

Category(mg/kg)300-2000
300-2000
300-2000
NANA
NA
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
5-50
5-50
5-50
300-2000
300-2000
300-2000
5-50
5-50
5-50
300-2000
300-2000
300-2000
50-30050-30050-300300-2000
300-2000
300-2000
5-50
<5
5-50
2000-5000
2000-5000
2000-5000
50-30050-30050-300Log IC50(mM)4-0.555-0.235-0.569NANA
NA-0.082-0.031-0.169-0.033
0.070
0.048
0.434
0.206
0.352
-4.918-4.720-4.699-0.121-0.109-0.082-3.732-3.418-3.601-1.005-0.854-1.102-1.423-1.265-1.258-0.108-0.615-0.074-5.164-7.209-5.293
2.107
2.029
1.942
-2.012-1.779-1.976

In Vitro Cytotoxicity Test Methods BRD K4

November 2006

Individual Laboratory LD50 Predictions: RC Rat-Only Millimole Regression

NRU
Test Method
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK

SubstanceLab

Log Reference
LD50(mmol/kg)1

Disulfoton
Disulfoton
Disulfoton
Endosulfan
Endosulfan
Endosulfan
Epinephrine bitartrate
Epinephrine bitartrate
Epinephrine bitartrate
Ethanol
Ethanol
Ethanol
Ethylene glycol
Ethylene glycol
Ethylene glycol
Fenpropathrin
Fenpropathrin
Fenpropathrin
Gibberellic acid
Gibberellic acid
Gibberellic acid
Glutethimide
Glutethimide
Glutethimide
Glycerol
Glycerol
GlycerolHaloperidol
Haloperidol
Haloperidol
Hexachlorophene
Hexachlorophene
Hexachlorophene
Lactic acid
Lactic acid
Lactic acid
LindaneLindaneLindane
Lithium I carbonate
Lithium I carbonate
Lithium I carbonate

ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS

-1.739-1.739-1.739-1.165-1.165-1.165-1.921-1.921-1.921
2.391
2.391
2.391
2.062
2.062
2.062
-0.664-0.664-0.664
1.241
1.241
1.241
0.441
0.441
0.441
2.332
2.332
2.332
-0.057-0.057-0.057-0.696-0.696-0.696
1.606
1.606
1.606
-0.464-0.464-0.464
0.902
0.902
0.902

Reference LD50
(mg/kg)

1

5
5
5
28
28
28
4
4
4
11324
11324
11324
7161
7161
7161
76
76
76
6039
6039
6039
600
600
600
19770
19770
19770
330
330
330
82
82
82
3635
3635
3635
100
100
100
590
590
590

K-43

Observed LD50
Toxicity
2

Category(mg/kg)

<5
<5
<5
5-50
5-50
5-50
<5
<5
<5
> 5000> 5000> 5000> 5000> 5000> 500050-30050-30050-300> 5000> 5000> 5000300-2000
300-2000
300-2000> 5000> 5000> 5000300-2000
300-2000
300-2000
50-30050-30050-3002000-5000
2000-5000
2000-5000
50-30050-30050-300300-2000
300-2000
300-2000

Log Predicted
LD50(mmol/kg)3
3.092
6.640
3.475
0.512
0.336
0.419
2.614
2.216
2.161
40.82347.81244.75769.73578.61971.258
0.564
0.449
0.414
10.50910.67010.470
3.828
3.738
3.792
54.55740.62652.042
0.543
0.525
0.513
0.061
0.074
0.055
13.423
13.57513.520
1.258
1.357
1.154
8.770
9.491
9.956

Predicted LD50
(mg/kg)

3

849
1822
954
208
137
170
871
739
720
1881
2203
2062
4329
4881
4424
197
157
145
3640
3696
3627
832
812
824
5024
3741
4793
204
197
193
25
30
22
1209
1223
1218
366
395
335
648
701
736

Predicted LD50
Toxicity
2

Category(mg/kg)300-2000
300-2000
300-2000
50-30050-30050-300300-2000
300-2000
300-2000
300-2000
2000-5000
2000-5000
2000-5000
2000-5000
2000-5000
50-30050-30050-3002000-5000
2000-5000
2000-5000
300-2000
300-2000
300-2000> 50002000-5000
2000-5000
50-30050-30050-300
5-50
5-50
5-50
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000

Log IC50(mM)4-0.2980.458-0.182-2.077-2.493-2.276-0.464-0.628-0.652
2.255
2.411
2.346
2.785
2.903
2.806
-1.982-2.207-2.287
0.912
0.927
0.909
-0.087-0.110-0.096
2.542
2.250
2.495
-2.019-2.053-2.076-4.179-3.984-4.285
1.155
1.166
1.162
-1.188-1.113-1.273
0.733
0.812
0.859

In Vitro Cytotoxicity Test Methods BRD K4

November 2006

Individual Laboratory LD50 Predictions: RC Rat-Only Millimole Regression

NRU
Test Method
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK

SubstanceLab

Log Reference
LD50(mmol/kg)1

Meprobamate
Meprobamate
Meprobamate
Mercury II chloride
Mercury II chloride
Mercury II chloride
Methanol
Methanol
MethanolNicotine
Nicotine
Nicotine
Paraquat
Paraquat
Paraquat
Parathion
Parathion
Parathion
Phenobarbital
Phenobarbital
Phenobarbital
PhenolPhenolPhenolPhenylthiourea
Phenylthiourea
Phenylthiourea
Physostigmine
Physostigmine
Physostigmine
Potassium cyanide
Potassium cyanide
Potassium cyanide
Potassium I chloride
Potassium I chloride
Potassium I chloride
Procainamide HCl
Procainamide HCl
Procainamide HCl
Propranolol
Propranolol
Propranolol

ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS

0.803
0.803
0.803
-0.830-0.830-0.830
2.434
2.434
2.434
-0.367-0.367-0.367-0.443-0.443-0.443-1.679-1.679-1.679-0.016-0.016-0.016
0.908
0.908
0.908
-1.705-1.705-1.705-1.741-1.741-1.741-0.956-0.956-0.956
1.575
1.575
1.575
0.856
0.856
0.856
0.197
0.197
0.197

Reference LD50
(mg/kg)

1

1387
1387
1387
40
40
40
8710
8710
8710
70
70
70
93
93
93
6
6
6
224
224
224
762
762
762
3
3
3
5
5
5
7
7
7
2802
2802
2802
1950
1950
1950
466
466
466

K-44

Observed LD50
Toxicity
2

Category(mg/kg)300-2000
300-2000
300-2000
5-50
5-50
5-50
> 5000> 5000> 500050-30050-30050-30050-30050-30050-300
5-50
5-50
5-50
50-30050-30050-300300-2000
300-2000
300-2000
<5
<5
<5
<5
<5
<5
5-50
5-50
5-50
2000-5000
2000-5000
2000-5000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000

Log Predicted
LD50(mmol/kg)3
7.204
2.925
6.609
0.829
0.745
0.745
NA
19.87126.159
3.259
3.666
3.511
2.000
2.654
2.092
1.608
1.531
1.502
6.691
5.009
5.167
3.333
4.159
3.905
6.100
6.355
5.347
3.325
1.593
3.088
2.916
3.732
2.304
19.64818.55317.651
8.770
9.531
10.075
1.699
1.806
1.495

Predicted LD50
(mg/kg)

3

1573
639
1443
225
202
202
NA
637
838
529
595
570
514
683
538
468
446
437
1554
1163
1200
314
391
367
928
967
814
916
439
850
190
243
150
1465
1383
1316
2383
2590
2738
503
534
442

Predicted LD50
Toxicity
2

Category(mg/kg)300-2000
300-2000
300-2000
50-30050-30050-300NA
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
50-30050-30050-300300-2000
300-2000
300-2000
2000-5000
2000-5000
2000-5000
300-2000
300-2000
300-2000

Log IC50
(mM)4
0.539
-0.3530.454-1.600-1.706-1.705
NA
1.543
1.815
-0.246-0.129-0.172-0.729-0.449-0.684-0.945-0.993-1.012
0.466
0.179
0.210
-0.224-0.005-0.067
0.374
0.415
0.244
-0.226-0.954-0.299-0.356-0.112-0.589
1.531
1.475
1.425
0.733
0.816
0.871
-0.890-0.830-1.017

In Vitro Cytotoxicity Test Methods BRD K4

November 2006

Individual Laboratory LD50 Predictions: RC Rat-Only Millimole Regression

NRU
Test Method
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK
NHK

SubstanceLab

Log Reference
LD50(mmol/kg)1

Propylparaben
Propylparaben
Propylparaben
Sodium arsenite
Sodium arsenite
Sodium arsenite
Sodium chloride
Sodium chloride
Sodium chloride
Sodium dichromate dihydrate
Sodium dichromate dihydrate
Sodium dichromate dihydrate
Sodium hypochlorite
Sodium hypochlorite
Sodium hypochlorite
Sodium oxalate
Sodium oxalate
Sodium oxalate
Sodium I fluoride
Sodium I fluoride
Sodium I fluoride
Sodium selenate
Sodium selenate
Sodium selenate
Strychnine
Strychnine
Strychnine
Thallium II sulfate
Thallium II sulfate
Thallium II sulfate
Trichloroacetic acid
Trichloroacetic acid
Trichloroacetic acid
Triethylenemelamine
Triethylenemelamine
Triethylenemelamine
Triphenyltin hydroxide
Triphenyltin hydroxide
Triphenyltin hydroxide
Valproic acid
Valproic acid
Valproic acid

ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS
ECBC
FAL
IIVS

1.546
1.546
1.546
-0.474-0.474-0.474
1.841
1.841
1.841
-0.771-0.771-0.771
2.142
2.142
2.142
0.674
0.674
0.674
0.480
0.480
0.480
-1.799-1.799-1.799-1.725-1.725-1.725-1.305-1.305-1.305
1.505
1.505
1.505
-1.708-1.708-1.708-0.047-0.047-0.047
0.839
0.839
0.839

Reference LD50
(mg/kg)

1

6332
6332
6332
44
44
44
4050
4050
4050
50
50
50
10328
10328
10328
633
633
633
127
127
127
3
3
3
6
6
6
25
25
25
5229
5229
5229
4
4
4
329
329
329
996
996
996

K-45

Observed LD50
Toxicity
2

Category(mg/kg)> 5000> 5000> 5000
5-50
5-50
5-50
2000-5000
2000-5000
2000-5000
50-30050-30050-300> 5000> 5000> 5000300-2000
300-2000
300-2000
50-30050-30050-300
<5
<5
<5
5-50
5-50
5-50
5-50
5-50
5-50
> 5000> 5000> 5000<5<5<5300-2000
300-2000
300-2000
300-2000
300-2000
300-2000

Log Predicted
LD50(mmol/kg)3
1.520
1.534
1.354
0.438
0.292
0.353
25.437
11.97925.063
0.307
0.312
0.268
16.92413.93416.183
6.390
6.091
6.372
4.446
4.318
4.644
1.010
1.351
1.147
2.277
1.780
1.892
0.129
0.118
0.110
5.790
6.976
6.129
0.487
0.547
0.560
0.057
0.030
0.042
6.936
8.303
6.722

Predicted LD50
(mg/kg)

3

274
276
244
57
38
46
1487
700
1465
92
93
80
1260
1037
1205
856
816
854
187
181
195
191
255
217
761
595
633
65
60
55
946
1140
1002
99
112
114
21
11
15
1000
1197
969

Predicted LD50
Toxicity
2

Category(mg/kg)50-30050-30050-30050-3005-50
5-50
300-2000
300-2000
300-2000
50-30050-30050-300300-2000
300-2000
300-2000
300-2000
300-2000
300-2000
50-30050-30050-30050-30050-30050-300300-2000
300-2000
300-2000
50-30050-30050-300300-2000
300-2000
300-2000
50-30050-30050-300
5-50
5-50
5-50
300-2000
300-2000
300-2000

Log IC50(mM)4-1.000-0.991-1.115-2.231-2.631-2.444
1.787
1.042
1.772
-2.583-2.565-2.718
1.384
1.192
1.340
0.420
0.373
0.418
0.061
0.032
0.105
-1.405-1.117-1.279-0.601-0.844-0.784-3.440-3.525-3.602
0.323
0.507
0.379
-2.126-2.012-1.988-4.250-4.885-4.552
0.501
0.679
0.470

In Vitro Cytotoxicity Test Methods BRD K4

November 2006

Individual Laboratory LD50 Predictions: RC Rat-Only Millimole Regression

NRU
Test Method
NHK
NHK
NHK
NHK
NHK
NHK

SubstanceLab

Log Reference
LD50(mmol/kg)1

Verapamil HCl
Verapamil HCl
Verapamil HCl
XyleneXyleneXylene

ECBC
FAL
IIVS
ECBC
FAL
IIVS

-0.646-0.646-0.646
1.643
1.643
1.643

Reference LD50
(mg/kg)

1

111
111
111
4665
4665
4665

Observed LD50
Toxicity
2

Category(mg/kg)50-30050-30050-3002000-5000
2000-5000
2000-5000

Log Predicted
LD50(mmol/kg)3
1.653
1.830
1.731
NA
NA
7.995

Predicted LD50
(mg/kg)3

Predicted LD50
Toxicity
2

Category(mg/kg)
812
899
850
NA
NA
849

300-2000
300-2000
300-2000NANA
300-2000

Log IC50(mM)4-0.917-0.817-0.871NANA
0.642

Abbreviations: 3T3=Mouse fibroblast 3T3 cell line; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; ECBC=U.S. Army Edgewood Chemical Biological Center; FAL=Fund for the Replacementof Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences.
1
Reference rat oral LD50 values from Table 4-2. Reference values were developed from rat acute oral LD50 studies located using literature searches, secondary references, and electronic database searches.
2
Globally Harmonized System (GHS) hazard classification (UN 2005):
Category
Abbreviation
Oral LD50 Limits<5
1
LD50 ≤ 5 mg/kg
5-50
25 < LD50 ≤ 50 mg/kg
50-3003
50 < LD50 ≤ 300 mg/kg
300-2000
4300 < LD50 ≤ 2000 mg/kg
2000-5000
52000 < LD50 ≤ 5000 mg/kg>5000
Unclassified
LD50 > 5000 mg/kg
3
LD50 determined using NRU IC50 value in RC rat-only millimole regression: Log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
4IC50 values are the geometric mean IC50 values for each substance in each lab.

K-46

In Vitro Cytotoxicity Test Methods BRD Appendix L

November 2006

Appendix L

Outlier Information
L1

Outlier Characterization for the 3T3 and NHK NRU Test
Methods with the RC Millimole Regression .................................... L-3

L2

Discordant Substances for GHS Acute Oral Toxicity Category
Predictions Using the 3T3 and NHK NRU Test Methods and RC
Rat-Only Regressions........................................................................ L-9

L3

Analysis of Outliers by Halle (1998, 2003) for the RC Millimole
Regression........................................................................................ L-17

L-1

In Vitro Cytotoxicity Test Methods BRD Appendix L

November 2006

[This Page Intentionally Left Blank]

L-2

November 2006

In Vitro Cytotoxicity Test Methods BRD Appendix L1

Appendix L1

Outlier Characterization for the 3T3 and NHK NRU Test Methods with
the RC Millimole Regression

L-3

November 2006

In Vitro Cytotoxicity Test Methods BRD Appendix L1[This Page Intentionally Left Blank]

L-4

November 2006

In Vitro Cytotoxicity Test Methods BRD Appendix L1

L.1

Outlier Analysis for the 3T3 and NHK NRU Test Methods and RC Millimole
Regression

The RC millimole regression and each in vitro NRU test method were used to identify outlier
substances among the reference substances tested in the validation study (i.e., those for which
the rodent LD50 was not accurately predicted by the in vitro NRU IC50) (see Section 6.2).
The outliers, identified for each test method in Table 6-3, were evaluated for common
characteristics that may assist in determining the types of chemicals that are not suited foruse in the 3T3 and NHK NRU test methods to determine starting doses for acute systemic
toxicity test methods.
A number of physico-chemical characteristics were evaluated for their frequency ofoccurrence among the 28 outlier substances for the 3T3 NRU test method and 31 outlier
substances for the NHK NRU test method versus the entire set of reference substances. The
frequency of occurrence of outliers versus the total number of reference substances for eachcategory of each characteristic examined is shown in Table L1-1.

L-5

November 2006

In Vitro Cytotoxicity Test Methods BRD Appendix L1

Table L1-1

Outliers per Category and NRU Test Method

Category
Boiling Point (BP) [in degrees C]
No information< 100
100-200
200-300
300-400
465
960
1500
decompose, sublime, or BPs were
provided at less than atmosphericpressure
Molecular Weight (g/mol)< 100
100-200
200-300
300-400
400-500
500-600
600-700
700-800
IC50 (mM)≤ 0.0001
0.0001 – 0.001
0.001 – 0.01
0.01 – 0.1
0.1 – 1
1 – 10
10 – 100
> 100
pH
< 7.1
7.1
7.2
7.3
7.4
7.5
< 7.6
7.6
7.7
7.8
7.9
8.0
8.1
8.2
8.3

3T3 NRU Test Method1
Total
Number of
Substances in
Outliers
Category

NHK NRU Test Method2
Total
Number of
Substances in
Outliers
Category

13
1
1
3
5
1
0
0

34
6
5
4
6
1
1
1

13
2
2
3
4
1
0
0

34
7
5
4
6
1
1
1

4

12

6

12

3
6
12
3
2
1
0
1

14
18
20
11
4
1
1
1

4
9
12
3
3
0
0
0

15
18
20
11
4
1
1
1

0
1
1
8
13
3
1
1

3
1
4
14
21
13
9
5

0
1
3
5
12
7
2
1

4
2
7
8
19
19
7
5

0
0
0
0
0
0
0
0
1
0
2
5
10
3
3

0
0
0
0
0
0
9
0
1
1
6
11
18
6
8

0
0
1
0
1
4
0
4
8
11
0
0
0
1
0

6
0
1
0
4
7
0
7
22
17
3
1
0
1
0

L-6

November 2006

In Vitro Cytotoxicity Test Methods BRD Appendix L1

Table L1-1

Outliers per Category and NRU Test Method

Category
8.4
8.5
> 8.5
log Kow< -4> -4 to < -3
> -3 to < -2
-2 to -1
-1 to 00 to 1
1 to 2
2 to 33 to 44 to 55 to 6
6 to 7
No information
Chemical Class
Organic Compounds
Acyclic hydrocarbon
Alcohol
Alkalies
Amide
Amine
Carbohydrate
Carboxylic acid
Cyclic hydrocarbon
Ester
Ether
Halogenated hydrocarbon
Heterocyclic compound
KetoneLipidsNitrileNitro compound
Sodium compound
Sulfur compound
Organometallic compound
Organophosphorous compound
PhenolPolycyclic compound
Urea
Inorganic Compounds
Arsenical
Boron compound
Cadmium compound
Chlorine compound

3T3 NRU Test Method1
Total
Number of
Substances in
Outliers
Category
1
5
0
1
3
4

NHK NRU Test Method2
Total
Number of
Substances in
Outliers
Category
0
0
1
1
0
1

0
0
0
1
3
4
5
1
5
2
1
0
6

1
1
0
5
6
7
13
4
8
2
2
1
20

1
0
0
1
5
3
5
1
5
2
1
0
7

1
1
0
5
7
7
13
4
8
2
2
1
20

1
3
0
1
2
1
4
0
1
1
1
7
0
0
1
0
0
5
0
3
1
1
1

1
9
1
3
3
1
14
3
1
1
3
14
1
1
2
1
1
5
1
3
5
5
1

1
4
0
0
2
0
6
1
1
1
0
10
0
0
1
0
1
5
0
3
2
0
1

1
10
1
3
3
1
14
3
1
1
3
14
1
1
2
1
1
5
1
5
5
5
1

1
0
0
2

2
1
1
5

1
0
0
2

2
1
1
5

L-7

November 2006

In Vitro Cytotoxicity Test Methods BRD Appendix L1

Table L1-1

Outliers per Category and NRU Test Method

Category
Chromium compound
Fluorine compound
Inorganic acid
Inorganic carbon compound
Lithium compound
Mercury compound
Metal
Nitrogen compound
Oxygen compound
Potassium compound
Selenium compound
Sodium compound
Sulfur compound
Substance Physical Form
Solid
Liquid3T3 NRU Test Method1
Total
Number of
Substances in
Outliers
Category
0
1
0
1
0
1
0
1
0
1
1
1
1
2
1
1
1
1
1
2
1
1
2
6
1
2
21
7

54
16

NHK NRU Test Method2
Total
Number of
Substances in
Outliers
Category
0
1
0
1
0
1
0
1
0
1
1
1
0
2
1
1
1
1
1
2
1
1
2
6
0
2
22
9

54
17

Abbreviations: NRU=Neutral red uptake; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes;
BP=Boiling point; Kow= Octanol:water partition coefficient.
1
28 discordant chemicals (i.e., outliers) are characterized for the 3T3 NRU test method by counting the number of outliers ineach category and comparing to the total number of chemicals in the category. Analysis excludes carbon tetrachloride andmethanol since no IC50 values were obtained. Total chemicals = 70.
2
31 discordant chemicals (i.e., outliers) are characterized for the NHK NRU test method by counting the number of outliersin each category and comparing to the total number of chemicals in the category. Analysis excludes carbon tetrachloride
since no IC50 values were obtained. Total chemicals = 71.

L-8

In Vitro Cytotoxicity Test Methods BRD Appendix L2

November 2006

Appendix L2

Discordant Substances for GHS Acute Oral Toxicity Category Predictions
Using the 3T3 and NHK NRU Test Methods and RC Rat-Only Regressions

L-9

In Vitro Cytotoxicity Test Methods BRD Appendix L2

November 2006

[This Page Intentionally Left Blank]

L-10

In Vitro Cytotoxicity Test Methods BRD Appendix L2

L.2

November 2006

Discordant Substances for GHS Acute Toxicity Category Predictions Using
the 3T3 and NHK NRU Test Methods and RC Rat-Only Regressions

This appendix provides a more detailed discussion of the discordant substances identified for
the GHS acute oral toxicity category predictions using the NRU test methods and the RC ratonly regressions evaluated in Section 6.4.
L.2.1

Discordant Substances for Prediction of GHS Acute Oral Toxicity Category by the
3T3 and NHK NRU Test Methods and the RC Rat-Only Millimole Regression

Table L2-1 identifies the discordant substances for which the in vitro predicted GHS toxicity
category (using the 3T3 and NHK NRU test methods with the RC rat-only millimole
regression) did not match the GHS toxicity category assigned based on the reference rat oral
LD50 data. For the 3T3 NRU test method, the toxicity category was underpredicted for 23(34%) and overpredicted for 23 (34%) of the 46 discordant substances. Of the 23 substances
for which toxicity was underpredicted,•

15 (65%) were underpredicted by one toxicity category•

2 (9%) were underpredicted by two toxicity categories•

6 (26%) were underpredicted by three toxicity categories

For the 23 substances for which toxicity was overpredicted,•

14 (61%) were overpredicted by one toxicity category•

9 (39%) were overpredicted by two toxicity categories

For the NHK NRU test method, toxicity was underpredicted for 21 (54%) and overpredicted
for 27 (46%) of the 48 discordant substances. Of the 21 substances for which toxicity was
underpredicted,•

12 (57%) were underpredicted by one toxicity category•

5 (24%) were underpredicted by two toxicity categories•

4 (19%) were underpredicted by three toxicity categories

For the 27 substances for which toxicity was overpredicted,•

18 (67%) were overpredicted by one toxicity category•

9 (33%) were overpredicted by two toxicity categories

L-11

In Vitro Cytotoxicity Test Methods BRD Appendix L2

Table L2-1

November 2006

Discordant Substances1 for the Prediction of GHS Acute Oral Toxicity Categories by the 3T3 and NHK NRU
Test Methods and the RC Rat-Only Millimole Regression2

In Vivo GHS
Toxicity Category3(mg/kg)

LD50 <5

5 < LD50 ≤50

50 < LD50 ≤300

300 < LD50 ≤2000

2000 < LD50 ≤5000

3T3 NRU Test Method
Toxicity
Toxicity
Overpredicted
Underpredicted
Cycloheximide (1)Disulfoton (3)Phenylthiourea (3)Physostigmine (3)Sodium selenate (3)Triethylenemelamine (1)Arsenic trioxide (1)Busulfan (2)Digoxin (3)Endosulfan (1)Mercury chloride (1)Parathion (2)Potassium cyanide (1)Sodium arsenite (1)Strychnine (3)Thallium sulfate (1)Dichlorvos (1)Fenpropathrin (1)Lindane (1)Nicotine (1)Paraquat (1)Phenobarbital (1)Verapamil HCl (1)Amitriptyline HCl (1)Haloperidol (1)Triphenyltin hydroxide (2)Acetaminophen (1)Acetonitrile (1)5-Aminosalicylic acid (1)Boric acid (1)Carbamazepine (1)Chloramphenicol (1)L-12

NHK NRU Test Method
Toxicity
Toxicity
Overpredicted
Underpredicted
Cycloheximide (1)Disulfoton (3)Phenylthiourea (3)Physostigmine (3)Sodium selenate (2)Triethylenemelamine (2)Aminopterin (3)Arsenic trioxide (1)Busulfan (2)Endosulfan (1)Mercury chloride (1)Parathion (2)Potassium cyanide (1)Strychnine (2)Thallium sulfate (1)Hexachlorophene (1)Amitriptyline HCl (1)Haloperidol (1)Triphenyltin hydroxide (2)Acetaminophen (1)Acetonitrile (1)5-Aminosalicylic acid (1)Boric acid (1)Carbamazepine (1)Chloramphenicol (1)Lindane (1)Nicotine (1)Paraquat (1)Phenobarbital (1)Verapamil HCl (1)Procainamide HCl (1)In Vitro Cytotoxicity Test Methods BRD Appendix L2

In Vivo GHS
Toxicity Category3(mg/kg)November 2006

3T3 NRU Test Method
Toxicity
Toxicity
Overpredicted
Underpredicted
Lactic acid (1)Potassium chloride (1)Sodium chloride (1)Xylene (1)NHK NRU Test Method
Toxicity
Toxicity
Overpredicted
Underpredicted
Lactic acid (1)Potassium chloride (1)Sodium chloride (1)Xylene (1)Citric acid (2)Citric acid (2)Dibutyl phthalate (2)Dibutyl phthalate (2)Diethyl phthalate (2)Diethyl phthalate (2)Dimethylformamide (2)Dimethylformamide (2)Ethanol (1)Ethanol (2)Gibberellic Acid (1)LD50 >5000
Ethylene glycol (1)Glycerol (1)Glycerol (1)Methanol (2)2-Propanol (2)2-Propanol (2)Sodium hypochlorite (2)Sodium hypochlorite (2)Trichloroacetic acid (2)Trichloroacetic acid (2)1,1,1-Trichloroethane (1)Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); 3T3=BALB/c 3T3 fibroblasts; NHK=Normal
human epidermal keratinocytes; NRU=Neutral red uptake; RC=Registry of Cytotoxicity
1
Substances for which the in vitro predicted GHS acute oral toxicity category was different from the category assigned to the substance based on reference rat
oral LD50 data. Numbers in parentheses indicate the number of categories different. Three substances were excluded because no rat LD50 was identified:epinephrine bitartrate, colchicine, and propylparaben. Carbon tetrachloride was excluded from the 3T3 and NHK NRU analyses because no laboratory attained
sufficient toxicity for the calculation of an IC50. Methanol was excluded from the 3T3 analysis because no laboratory attained sufficient toxicity for the
calculation of an IC50.
2
The RC rat-only millimole regression is log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
3
Reference rat oral LD50 values from Table 4-2.

L-13

In Vitro Cytotoxicity Test Methods BRD Appendix L2

L.2.2

November 2006

Discordant Substances for Prediction of Toxicity Category by the 3T3 and NHK NRU
Test Methods and the RC Rat-Only Weight Regression

Table L2-2 shows the discordant substances for which the in vitro predicted GHS toxicity
category (using the 3T3 and NHK NRU test methods with the RC rat-only weight regression) did
not match that based on the reference rat oral LD50 data. The two in vitro NRU cytotoxicity test
methods over- and under-predicted the GHS toxicity category for a similar number ofsubstances. For the 3T3 NRU test method, the GHS toxicity category of 22 of 46 (48%)
discordant substances was overpredicted, with:•

16 (73%) overpredicted by one GHS toxicity category•

6 (27%) overpredicted by two GHS toxicity categories

The toxicity of 24 substances (52%) was underpredicted by this test method, with:•

13 (54%) underpredicted by one GHS toxicity category•

7 (29%) underpredicted by two GHS toxicity categories•

4 (17%) underpredicted by three GHS toxicity categories

For the NHK NRU test method, the GHS toxicity category of 25 (53%) of the 47 discordant
substances was overpredicted. Of these,•

18 (72%) were overpredicted by one GHS toxicity category•

7 (28%) were overpredicted by two GHS toxicity categories

For this assay, the toxicity of 22 (47%) of the discordant substances was underpredicted, with•

12 (55%) underpredicted by one GHS toxicity category•

7 (32%) underpredicted by two GHS toxicity categories•

3 (14%) underpredicted by three toxicity categories

L-14

In Vitro Cytotoxicity Test Methods BRD Appendix L2

Table L2-2

November 2006

Discordant Substances1 for the Prediction of GHS Acute Oral Toxicity Categories by the 3T3 and NHK NRU
Test Methods and the RC Rat-Only Weight Regression2

In Vivo GHS
Category3(mg/kg)

LD50 <5

5 < LD50 ≤50

50 < LD50 ≤300

300 < LD50 ≤2000

2000 < LD50 ≤5000

3T3 NRU Test Method
Toxicity
Toxicity
Overpredicted
Underpredicted
Cycloheximide (2)Disulfoton (3)Phenylthiourea (3)Physostigmine (3)Sodium selenate (3)Triethylenemelamine (2)Arsenic trioxide (1)Busulfan (2)Digoxin (2)Endosulfan (1)Mercury chloride (1)Parathion (2)Potassium cyanide (2)Sodium arsenite (1)Strychnine (2)Thallium sulfate (1)Dichlorvos (1)Fenpropathrin (1)Lindane (1)Nicotine (1)Paraquat (1)Phenobarbital (1)Sodium fluoride (1)Verapamil HCl (1)Amitriptyline HCl (1)Haloperidol (1)Propranolol HCl (1)Triphenyltin hydroxide (2)Acetaminophen (1)5-Aminosalicylic acid (1)Boric acid (1)Carbamazepine (1)Chloramphenicol (1)L-15

NHK NRU Test Method
Toxicity
Toxicity
Overpredicted
Underpredicted
Cycloheximide (1)Disulfoton (3)Phenylthiourea (3)Physostigmine (3)Sodium selenate (2)Triethylenemelamine (2)Aminopterin (2)Arsenic trioxide (1)Busulfan (2)Endosulfan (1)Mercury chloride (1)Parathion (2)Potassium cyanide (2)Sodium arsenite (1)Strychnine (2)Thallium sulfate (1)Hexachlorophene (1)Amitriptyline HCl (1)Haloperidol (1)Triphenyltin hydroxide (2)Acetaminophen (1)5-Aminosalicylic acid (1)Boric acid (1)Carbamazepine (1)Chloramphenicol (1)Lindane (1)Nicotine (1)Paraquat (1)Phenobarbital (1)Sodium fluoride (1)Verapamil HCl (1)In Vitro Cytotoxicity Test Methods BRD Appendix L2

In Vivo GHS
Category3(mg/kg)November 2006

3T3 NRU Test Method
Toxicity
Toxicity
Overpredicted
Underpredicted
Xylene (1)NHK NRU Test Method
Toxicity
Toxicity
Overpredicted
Underpredicted
Lactic acid (1)Potassium chloride (1)Sodium chloride (1)Xylene (1)Citric acid (2)Citric acid (2)Dibutyl phthalate (2)Dibutyl phthalate (2)Diethyl phthalate (2)Diethyl phthalate (2)Dimethylformamide (1)Dimethylformamide (1)Ethanol (1)Ethanol (1)Ethylene glycol (1)Gibberellic acid (1)LD50 >5000
Gibberellic acid (1)Glycerol (1)Glycerol (1)Methanol (2)2-Propanol (1)2-Propanol (1)Sodium hypochlorite (2)Sodium hypochlorite (2)Trichloroacetic acid (2)Trichloroacetic acid (2)1,1,1-Trichloroethane (1)1,1,1-Trichloroethane (1)Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); 3T3=BALB/c 3T3 fibroblasts; NHK=Normal
human epidermal keratinocytes; NRU=Neutral red uptake; RC=Registry of Cytotoxicity
1
Substances for which the in vitro predicted GHS acute oral toxicity category was different from the category assigned to the substance based on reference rat
oral LD50 data. Numbers in parentheses indicate the number of categories different. Three substances were excluded because no rat LD50 was identified:epinephrine bitartrate, colchicine, and propylparaben. Carbon tetrachloride was excluded from the 3T3 and NHK NRU analyses because no laboratory attained
sufficient toxicity for the calculation of an IC50. Methanol was excluded from the 3T3 analysis because no laboratory attained sufficient toxicity for the
calculation of an IC50.
2
The RC rat-only weight regression is log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024.
3
Reference rat oral LD50 values from Table 4-2.

L-16

In Vitro Cytotoxicity Test Methods BRD Appendix L3

November 2006

Appendix L3

Analysis of Outliers by Halle (1998, 2003) for the RC Millimole Regression

L-17

In Vitro Cytotoxicity Test Methods BRD Appendix L3

November 2006

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L-18

In Vitro Cytotoxicity Test Methods BRD Appendix L3

L.3

November 2006

Analysis of Outliers for the RC Millimole Regression

The RC millimole regression was constructed from the in vitro IC50X cytotoxicity data frommultiple cell lines and the in vivo acute toxicity data from rats and mice (i.e., LD50 values) for
347 chemicals (Halle 1998, 2003). Halle (1998, 2003) investigated the 95 (27.4%) chemicals
for which the observed log LD50 values were greater than 0.699 (i.e., 0.5 log) from predicted
log LD50 values. Of the 95 outliers, 46 were positive outliers and 49 were negative outliers.
The positive outliers have IC50X values that predict a far higher in vivo toxicity (i.e., lowerLD50) than the actual animal experiment. The negative outliers are more important since the
IC50X values predict lower toxicity (i.e. higher LD50) than the observed in vivo toxicity. It
seems that Halle (1998, 2003) was not concerned about the positive outliers since theprediction erred in a health protective direction. Halle (1998, 2003) was much moreconcerned about trying to explain the reasons for the negative outliers since the error was in anonconservative direction.
Halle (1998, 2003) investigated three factors that could have explained the negative outliers.
1.

Variation in the oral LD50 values.
They reported oral LD50 values for a particular chemical might vary by afactor of 4 to 14 even when experiments were highly standardized. LD50
values were found from other sources for 23 of the 95 outliers. They foundthat the variations in the LD50 values (difference between the RTECS® value
and the “new” value found for the 23 chemicals) were larger for the negative
outliers than for the positive outliers.

2.

Species-specificity of the oral LD50 values.
Halle (1998, 2003) compared an IC50x–LD50 regression using mouse LD50
values (242 values) with a regression using rat LD50 values (285 values) andfound no significant difference between the two regressions. The RC
millimole regression with 347 chemicals has 285 rat values and 62 mouse
values and is not statistically different from either the rat or mouseregressions.

3.

The cell culture(s) used may have been unsuitable for the detection ofcytotoxic potential or it may have been unable to simulate the complex
L-19

In Vitro Cytotoxicity Test Methods BRD Appendix L3

November 2006

process of toxicity in vivo. Halle (1998, 2003) expected, a priori, that threeclasses of compounds, insecticides (Table L3-1), neurotoxins (Table L3-2),
and those requiring metabolic activation for toxicity (Table L3-3), would not
fit the RC millimole regression (i.e., cytotoxicity data would not predict invivo toxicity). Sixty-two of the 347 chemicals belong to these three classes.
Twenty-three (37.1%) of the 62 chemicals were negative outliers. Of the 23,
10 were insecticides, five were neurotoxins, and eight required metabolic
activation. No positive outliers were identified in the three classes.
Of the 49 negative outliers, 23 (46.9%) belonged to the three classes of concern. Examinationof these classes showed that the RC millimole prediction was accurate (i.e., predicted log
LD50 [mmol/kg] was within 0.699 of observed log LD50 in [mmol/kg]) for 50% of theinsecticides (Table L3-1) and chemicals that required metabolic activation (Table L3-3). For
neurotoxins (Table L3-2), the results were even better, since 21 (80.8%) fell within theprediction interval. Halle (1998, 2003) felt that the ability to predict the acute LD50 for 50%of the insecticides and xenobiotics requiring metabolic activation and for 81% of theneurotoxic xenobiotics was sufficiently accurate for practical purposes.
Of the 49 negative outliers in the RC millimole regression, 23 (46.9%) of these belonged tothe three classes of concern that may explain the false negative IC50X values. Findings were
contrary to Halle’s assumption that in vitro cytotoxicity would not predict in vivo toxicity forthese types of chemicals. The RC millimole prediction of LD50 was applicable to 50% of theinsecticides and chemicals that required metabolic activation. For neurotoxic chemicals theresults were even better, since 21 (80.8%) fell within the prediction interval. Halle felt thatthe ability to predict the acute LD50 for 50% of the insecticides and chemicals requiring
metabolic activation and for 81% of the neurotoxic chemicals was sufficiently accurate forpractical purposes.
In separate analyses, Halle (1998, 2003) considered the physicochemical properties ofchemicals (i.e., molecular weight and the octanol/water partition coefficient) as independent

L-20

In Vitro Cytotoxicity Test Methods BRD Appendix L3

November 2006

variables in a multiple regression analysis, but they did not improve the prediction of LD50 by
IC50.

L3-1

The Error of Predictiona of 20 of The Most Important Insecticides
in the RC Ordered According to Their Chemical Characteristicsb
Chemical Class

RC No

Name

LD50 Error of
Predictiona

26
40
43
61
167
185
195
197
207
223

Kelthane
ChlordanAldrinDDTDDD
Heptachlor
DDA
DDE
Dieldrin
Lindane0.340-0.046-1.074b-0.775-0.378-1.0500.133
0.251-1.223-1.043

49
51
67
75
96

Parathion
Disulfoton
Malathion
Trichlorfon
Cygon-2.339-2.3460.106-0.136-0.84873
186

Carbaryl
Zineb-0.279
1.185

134
173
235

Rotenone
Pentachlorophenol
Paraquat

0.583-0.720-1.019Chlorinated hydrocarbon

Organophosphorus compounds

Carbamate compounds

Other compounds

Abbreviations: RC=Registry of Cytotoxicity; No=RC number; DDA=p,p'-DDA [2,2-bis(4-chlorophenyl)acetic acid];
DDD=p,p'-DDD [1,1-dichloro-2,2-bis(4-chlorophenyl)ethane]; DDE= p,p'-DDE [1,1-dichloro-2,2-bis(4chlorophenyl)ethylene]; DDT=p,p'-DDT [1,1,1-trichloro-2,2-bis(2-chlorophenyl-4-chlorophenyl)ethane]aDefined as observed log LD50 (mmol/kg) - predicted log LD50 (mmol/kg).
b
Modified from Table 10 of Halle (1998, 2003) .
Bold numbers: outliers (i.e., observed log LD50 [mmol/kg] - predicted log LD50 [mmol/kg] > 0.699).

L-21

In Vitro Cytotoxicity Test Methods BRD Appendix L3

Table L3-2

November 2006

The Error of Prediction a of 26 Neurotoxic Xenobiotics in the RC Ordered
According to Their In Vivo Potencyb
Chemical Class

LD50 Error of
Predictiona

RC No

Name

69
83
84
87
101
118
247
264
317

Secobarbital sod.
Thiopental
Amobarbital
Pentobarbital sodium
Gluthetimide
Phenobarbital(+)-Thalidomide
Chloral hydrate
Barbital sodium-1.035b-0.397-0.349-0.59138
90
183

Imipramine HCl
Iproniazid
Amitriptyline-0.093-0.273
0.021

27
44
63
170

Chlorpromazine
Hydroxyzine HCl
DiazepamThioridazine HCl-0.1760.248
0.116-0.013112
262

Caffeine
Amphetamine sulfate-0.815-1.57982

Diphenylhydantoin-0.551229

Dextropropoxyphene HCl-1.150251
296

Scopolamine * HBr
Homatropine methylbromide-0.123-0.532

102
137
142
316

Acrylamide
Triethyltin chloride
Methylmercury chloride
Toluene-0.338Sedative, hypnotic, CNS depressants-0.651-0.119-0.335-0.654-0.270AntidepressantAntipsychotic, anxiolytic

Stimulants

AnticonvulsantsAnalgetic (general anesthesia)
Anticholinergic

Other Neurotoxins (not insecticide)

Abbreviations: RC=Registry of Cytotoxicity; No=RC number; CNS=Central nervous system.
aDefined as observed log LD50 (mmol/kg) - predicted log LD50 (mmol/kg).
b
Modified from Table 11 of Halle (1998, 2003).
Bold numbers: outliers (i.e., observed log LD50 [mmol/kg] - predicted log LD50 [mmol/kg] >0.699).

L-22-0.8520.105
0.571

In Vitro Cytotoxicity Test Methods BRD Appendix L3

Table L3-3

RC No
13
33
37
68
97
109
113
116
123
125
192
260
273
279
281
292

November 2006

The Error of Predictiona of the 16 Xenobiotics in the RC that Require
Metabolic Activationb
LD50 Error of Predictiona

Name-1.370b-1.077-1.783-1.128
0.292
0.109
0.386
-1.310-0.3320.229-1.176-0.4270.374-0.294-1.106-0.952

Cycloheximide
p-Chloromercuribenzoic acid
Aflatoxin B1
2.4-Dinitrophenol
Phenacetin
Frusemide
Acetaminophen
Cyclophosphamide * H2OIsoniazid
Carbon tetrachloride
1.3-Bis(2-chloroethyl)-1-nitrosourea
Coumarin
Bromobenzene
Thioacetamide
1.2-Dibromomethane
Allylalcohol

Abbreviations: RC=Registry of Cytotoxicity; No=RC number.
a
Defined as observed log LD50 (mmol/kg) - predicted log LD50 (mmol/kg).
b
Modified from Table 12 of Halle (1998, 2003).
Bold numbers: outliers (i.e., observed log LD50 [mmol/kg] - predicted log LD50 [mmol/kg] >0.699.

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November 2006

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L-24

In Vitro Cytotoxicity Test Methods BRD Appendix M

November 2006

Appendix M

Acute Oral Toxicity Test Guidelines

M1

OECD UDP Test Guideline ................................................................M-3

M2

EPA UDP Test Guideline..................................................................M-31

M3

OECD ATC Method Test Guideline................................................M-71

M4

OECD FDP Test Guideline...............................................................M-87

M5OECD Guidance on Acute Oral Toxicity Testing ....................... M-103

M-1

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M-2

In Vitro Cytotoxicity Test Methods BRD Appendix M1

November 2006

Appendix M1OECD UDP Test Guideline

M-3

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November 2006

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M-4In Vitro Cytotoxicity Test Methods BRD Appendix M2Appendix M2

EPA UDP Test Guideline

M-31

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M-32

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M-70

November 2006

In Vitro Cytotoxicity Test Methods BRD Appendix M3

Appendix M3OECD ATC Method Test Guideline

M-71

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M-72

November 2006In Vitro Cytotoxicity Test Methods BRD Appendix M4Appendix M4OECD FDP Test Guideline

M-87

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M-88

November 2006In Vitro Cytotoxicity Test Methods BRD Appendix M5

Appendix M5OECD Guidance on Acute Oral Toxicity Testing

M-103

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M-104

November 200In Vitro Cytotoxicity Test Methods BRD Appendix N

November 2006

Appendix N
UDP/ATC Simulation Modeling Results
N1

UDP Simulation Results Using Starting Doses One Default Dose
Lower than the LD50 Predicted by the 3T3 and NHK NRU IC50
and the RC Rat-Only Millimole Regression - 5000 mg/kg
Upper Limit Dose ........................................................................... N-3

N2

UDP Simulation Results Using Starting Doses One Default Dose
Lower than the LD50 Predicted by the 3T3 and NHK NRU IC50
and the RC Rat-Only Weight Regression - 5000 mg/kg Upper
Limit Dose ..................................................................................... N-13

N3

ATC Simulation Results Starting at the Next Fixed Dose Below
the LD50 Predicted by the 3T3 and NHK NRU IC50 and the RC
Rat-Only Millimole Regression - 2000 mg/kg Upper
Limit Dose ..................................................................................... N-23

N4

ATC Simulation Results Starting at the Next Fixed Dose Below
the LD50 Predicted by the 3T3 and NHK NRU IC50 and the RC
Rat-Only Weight Regression - 2000 mg/kg Upper
Limit Dose ..................................................................................... N-33

N-1

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November 2006

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N-2

In Vitro Cytotoxicity Test Methods BRD Appendix N1

November 2006

Appendix N1

UDP Simulation Results Using Starting Doses One Default Dose Lower
than the LD50 Predicted by the 3T3 and NHK NRU IC50 and the RC RatOnly Millimole Regression - 5000 mg/kg Upper Limit DoseN-3

In Vitro Cytotoxicity Test Methods BRD Appendix N1

November 2006

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N-4

In Vitro Cytotoxicity Test Methods BRD Appendix N1

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by NRU Test Method1
NRU
Test
Method

Sigma
0.12
0.25

3T3

0.50
1.25
2.00

0.12
0.25
NHK

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Animals Used
Std.
Error

Number2

0.196
7.42
0.176
7.95
0.189
8.15
0.178
8.68
0.169
8.80
0.163
9.35
0.135
9.34
0.131
9.95
0.112
9.48
0.096
10.01
Average Difference
0.203
7.43
0.176
7.92
0.197
8.18
0.174
8.66
0.176
8.86
0.157
9.36
0.145
9.41
0.125
9.96
0.121
9.53
0.092
10.01
Average Difference

Difference3

P4

0.00

0.6675%
Savings Animals
Used
6.6%

0.00

0.9311

6.0%

0.1%

0.02

0.6341

5.8%

0.5%

0.07

0.0238

6.1%

1.5%

0.07

0.0506

5.3%

1.5%

0.03
-0.01

0.7372

6.2%

-0.2%

0.00

0.3125

5.6%

-0.1%

0.01

0.2841

5.3%

0.2%

0.04

0.0129

5.5%

1.0%

0.05

0.0206

4.9%

1.1%

Animals Died

Difference3

P4

0.53

0.00020.52

0.00050.54

0.00080.61

0.00010.53

0.00030.55
0.49

0.00030.48

0.00050.50

0.00060.55

0.00020.49

0.0001Std.
Error

Number2

0.204
3.43
0.200
3.44
0.203
3.76
0.197
3.76
0.191
4.09
0.185
4.11
0.165
4.48
0.152
4.55
0.145
4.60
0.129
4.67
Average Difference
0.215
3.39
0.202
3.39
0.212
3.72
0.198
3.72
0.199
4.07
0.183
4.08
0.173
4.48
0.150
4.52
0.151
4.61
0.127
4.66
Average Difference

0.50%
Difference Animals
Died
0.1%

0.02

Abbreviations: UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a); NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope; Cyto=NRU-determined starting dose
(i.e., one dose lower than the LD50 predicted by the NRU IC50 in the RC rat-only millimole regression (log LD50 [mmol/kg] = 0.439 log IC50 [mM] + 0.621); Default=Default starting doseof 175 mg/kg; Std. Error=Standard error for number of animals; 3T3= BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity..
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit
dose =5000 mg/kg.
2
Mean number of animals for 10,000 simulations.
3
Difference between mean number of animals for the default starting dose and mean number of animals for the NRU-based starting dose.
4
P-value is from one-sided Wilcoxon signed rank test for difference in animals between the default and NRU-based starting doses. Significant values at p <0.05.

N-5

In Vitro Cytotoxicity Test Methods BRD Appendix N1

November 2006

Summary of Stopping Rules Used for the UDP Simulations by NRU Test Method1
NRU Test
MethodSigma

Starting Dose

3 Animals at
Limit Dose2

5 Reversals2

Likelihood
Ratio2

Maximum
Number of
Animals Used2

0.12

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

15.6%
15.4%
15.0%
14.7%
13.4%
13.0%
9.8%
9.1%
8.5%
7.4%
16.8%
16.6%
16.1%
15.8%
14.3%
13.8%
10.1%
9.5%
8.6%
7.6%

56.0%
56.9%
33.6%
34.1%
19.8%
20.0%
13.5%
13.6%
12.3%
12.5%
55.3%
56.0%
33.3%
33.5%
19.7%
19.9%
13.5%
13.5%
12.3%
12.5%

26.4%
25.3%
47.4%
46.0%
59.0%
57.3%
64.0%
60.9%
65.2%
62.6%
26.0%
25.0%
46.5%
45.5%
58.0%
56.6%
63.1%
60.4%
64.6%
62.3%

2.0%
2.4%
4.0%
5.3%
7.8%
9.7%
12.7%
16.4%
14.0%
17.5%
1.8%
2.4%
4.1%
5.2%
8.1%
9.7%
13.3%
16.5%
14.5%
17.6%

0.25

3T3

0.5
1.25
2
0.12
0.25

NHK

0.5
1.25
2

Abbreviations: UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a); NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope;
Cyto=NRU-determined starting dose (i.e., one dose lower than the LD50 predicted by the NRU IC50 in the RC rat-only millimole regression (logLD50 [mmol/kg] = 0.439 log IC50 [mM] + 0.621); Default=Default starting dose of 175 mg/kg; 3T3= BALB/c 3T3 mouse fibroblasts;
NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in
the NHK NRU test method. Upper limit dose =5000 mg/kg.
2
Percentage of the 10,000 test simulations that satisfied the specified condition for completion of testing (see OECD [2001a]; EPA [2002a]).

N-6

In Vitro Cytotoxicity Test Methods BRD Appendix N1

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

3T3

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

1
0.12
0.25
NHK

0.50
1.25
2.00

0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

2
0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

0.431
8.74
0.277
9.70
0.660
9.56
0.179
10.58
0.697
10.19
0.201
11.32
0.664
10.68
0.156
11.75
0.548
10.65
0.146
11.47
Average Difference
0.516
8.95
0.268
9.66
0.699
9.77
0.217
10.53
0.707
10.47
0.241
11.21
0.692
10.92
0.169
11.70
0.627
10.81
0.159
11.47
Average Difference
0.467
8.54
0.278
8.46
0.426
9.21
0.210
9.08
0.453
9.74
0.230
9.68
0.413
10.25
0.236
10.17
0.328
10.34
0.177
10.20
Average Difference
0.488
8.77
0.260
8.43
0.428
9.44
0.166
9.08
0.448
9.99
0.164
9.65
0.424
10.46
0.183
10.14
0.348
10.49
0.148
10.18
Average Difference

Animals Died

Difference3

P4

0.96

0.62501.02

0.62501.14

0.62501.07

0.62500.82

0.62501.00
0.71

0.37500.77

0.37500.75

0.37500.78

0.37500.66

0.37500.73-0.08

0.8926-0.13

0.8926-0.07

1.0000-0.08

0.9460-0.14

0.5879

-0.10
-0.33

0.3757-0.36

0.4143-0.34

0.3757-0.32

0.3396-0.32

0.4143

-0.33

N-7

Std.
Error

Number2

0.459
5.58
0.170
6.39
0.581
6.06
0.155
6.90
0.609
6.46
0.197
7.37
0.598
6.70
0.169
7.57
0.506
6.54
0.152
7.24
Average Difference
0.531
5.79
0.169
6.37
0.626
6.26
0.177
6.87
0.638
6.69
0.224
7.31
0.636
6.91
0.179
7.56
0.578
6.70
0.157
7.24
Average Difference
0.426
5.16
0.239
5.11
0.404
5.54
0.202
5.47
0.417
5.83
0.211
5.77
0.394
6.06
0.218
5.97
0.335
6.01
0.178
5.91
Average Difference
0.476
5.26
0.232
5.11
0.444
5.64
0.187
5.46
0.453
5.94
0.185
5.75
0.440
6.16
0.196
5.95
0.381
6.09
0.166
5.89
Average Difference

Difference3

P4

0.81

0.62509.9%%
Difference Animals
Died
12.7%

0.84

0.6250

9.7%

12.2%

0.91

0.6250

10.0%

12.3%

0.87

0.6250

9.1%

11.5%

0.71

0.6250

7.1%

9.8%

0.58

0.3750

7.3%

9.1%

0.61

0.3750

7.3%

8.9%

0.63

0.3750

6.7%

8.6%

0.65

0.3750

6.7%

8.6%

0.53

0.3750

5.7%

7.4%

-0.05

0.9460

-1.0%

-1.0%

-0.07

0.9460

-1.4%

-1.3%

-0.06

1.0000

-0.7%

-1.0%

-0.09

0.8926

-0.8%

-1.5%

-0.10

0.7354

-1.4%

-1.8%

-0.15

0.5879

-3.9%

-3.0%

-0.17

0.6848

-4.0%

-3.1%

-0.18

0.5417

-3.5%

-3.2%

-0.21

0.4973

-3.2%

-3.5%

-0.20

0.5417

-3.1%

-3.4%

% Savings- Animals
Used

0.83

0.60

-0.07

-0.18

In Vitro Cytotoxicity Test Methods BRD Appendix N1

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

3T3

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

3
0.12
0.25
NHK

0.50
1.25
2.00

0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

4
0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

0.189
6.90
0.188
6.61
0.220
7.53
0.152
7.20
0.213
8.18
0.101
7.76
0.141
8.98
0.059
8.62
0.084
9.33
0.040
9.10
Average Difference
0.190
6.85
0.190
6.57
0.229
7.48
0.159
7.17
0.206
8.12
0.109
7.78
0.120
8.93
0.061
8.65
0.079
9.31
0.036
9.11
Average Difference
0.191
7.15
0.235
7.46
0.186
7.66
0.201
7.94
0.210
8.14
0.212
8.53
0.180
8.82
0.145
9.16
0.133
9.16
0.084
9.38
Average Difference
0.196
7.00
0.247
7.49
0.213
7.53
0.207
7.97
0.234
8.03
0.221
8.55
0.218
8.76
0.147
9.17
0.163
9.12
0.086
9.40
Average Difference

Animals Died

Difference3

P4

-0.29

0.0425-0.33

0.0522-0.42

0.0522-0.35

0.0522-0.23

0.0522

-0.33
-0.28

0.1514-0.31

0.0425-0.34

0.0923-0.28

0.0522-0.20

0.0923-0.28
0.31

0.04430.28

0.05070.38

0.10460.33

0.02500.22

0.05770.31
0.49

0.00730.45

0.01310.52

0.03350.41

0.01820.27

0.04430.43

N-8Difference3

P4

0.149
3.60
0.125
3.37
0.169
3.96
0.103
3.71
0.163
4.31
0.080
4.04
0.123
4.69
0.057
4.47
0.094
4.85
0.050
4.70
Average Difference-0.23

0.0522-4.3%%
Difference Animals
Died
-6.8%

-0.24

0.0640

-4.6%

-6.6%

-0.27

0.0640

-5.5%

-6.7%

-0.22

0.0771

-4.1%

-4.9%

-0.15

0.2036

-2.5%

-3.3%

0.133
3.52
0.127
3.35
0.152
3.86
0.106
3.69
0.143
4.20
0.082
4.04
0.108
4.60
0.060
4.48
0.088
4.77
0.048
4.70
Average Difference-0.16

0.2334

-4.2%

-4.9%

-0.17

0.1099

-4.4%

-4.6%

-0.16

0.2036

-4.4%

-4.1%

-0.12

0.4697

-3.2%

-2.6%

-0.07

0.7334

-2.2%

-1.5%

0.063
3.39
0.066
3.40
0.032
3.61
0.048
3.60
0.040
3.80
0.049
3.86
0.049
4.10
0.022
4.13
0.042
4.26
0.019
4.25
Average Difference

0.01

0.93994.1%

0.2%-0.0030.2522

3.5%

-0.1%

0.05

0.1591

4.5%

1.4%

0.03

0.0934

3.6%

0.8%

-0.01

0.8603

2.3%

-0.2%

0.06

0.1439

6.5%

1.7%

0.05

0.0577

5.6%

1.4%

0.09

0.0654

6.1%

2.4%

0.04

0.1297

4.5%

1.1%

-0.02

0.8999

2.9%

-0.4%

Std.
Error

Number2

0.064
3.36
0.071
3.42
0.036
3.58
0.048
3.63
0.041
3.78
0.052
3.88
0.051
4.10
0.023
4.14
0.042
4.28
0.018
4.26
Average Difference% Savings- Animals
Used

-0.22

-0.14

0.02

0.05

In Vitro Cytotoxicity Test Methods BRD Appendix N1

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

3T3

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

5
0.12
0.25
NHK

0.50
1.25
2.00

0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

6
0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

0.308
7.96
0.232
9.17
0.196
9.01
0.157
10.34
0.148
9.46
0.102
10.73
0.131
9.29
0.065
10.66
0.107
9.20
0.061
10.36
Average Difference
0.285
8.06
0.233
9.17
0.241
9.12
0.152
10.31
0.200
9.54
0.082
10.75
0.167
9.40
0.052
10.66
0.131
9.28
0.037
10.35
Average Difference
0.685
6.18
0.587
7.76
0.647
7.10
0.541
8.67
0.486
8.29
0.342
9.87
0.301
9.01
0.058
10.89
0.246
8.94
0.030
10.75
Average Difference
0.630
6.19
0.560
7.66
0.585
7.16
0.499
8.63
0.440
8.41
0.317
9.87
0.276
9.14
0.056
10.87
0.234
9.06
0.022
10.74
Average Difference

Animals Died

Difference3

P4

1.21

0.00201.33

0.00391.28

0.00391.38

0.00201.16

0.00391.27
1.11

0.00201.19

0.00201.21

0.00201.27

0.00391.06

0.00201.17
1.58

0.00051.57

0.00051.58

0.00051.88

0.00051.81

0.00051.68
1.47

0.00021.47

0.00021.47

0.00021.73

0.00021.69

0.0002

1.56

N-9

Std.
Error

Number2

0.042
3.25
0.034
3.30
0.049
3.46
0.062
3.57
0.051
3.56
0.059
3.65
0.038
3.67
0.030
3.87
0.032
3.78
0.013
3.96
Average Difference
0.030
3.25
0.038
3.31
0.048
3.47
0.061
3.56
0.046
3.55
0.064
3.65
0.030
3.68
0.037
3.86
0.029
3.79
0.022
3.96
Average Difference

Difference3

P4

0.06

0.0137

13.2%%
Difference Animals
Died
1.7%

0.11

0.0195

12.8%

3.1%

0.09

0.0195

11.9%

2.5%

0.20

0.0020

12.9%

5.2%

0.18

0.0039

11.2%

4.6%

0.06

0.0273

12.1%

1.7%

0.10

0.0273

11.5%

2.8%

0.10

0.0098

11.3%

2.7%

0.18

0.0039

11.9%

4.7%

0.17

0.0020

10.3%

4.2%

% Savings- Animals
Used

0.13

0.12

0.314
0.88
0.304
0.85
0.316
1.33
0.309
1.30
0.255
2.04
0.255
2.03
0.126
3.00
0.121
3.19
0.088
3.33
0.066
3.60
Average Difference-0.02

0.0923

20.3%

-2.8%

-0.03

0.0342

18.1%

-2.1%

-0.01

0.1294

16.0%

-0.4%

0.19

0.0005

17.3%

6.0%

0.28

0.0005

16.8%

7.7%

0.298
0.82
0.289
0.80
0.295
1.28
0.287
1.26
0.236
2.03
0.236
2.02
0.112
3.02
0.114
3.18
0.078
3.36
0.062
3.61
Average Difference-0.02

0.0281

19.2%

-3.1%

-0.02

0.1099

17.0%

-1.7%

-0.01

0.0942

14.8%

-0.7%

0.16

0.0002

16.0%

5.0%

0.25

0.0002

15.7%

7.0%

0.08

0.07

In Vitro Cytotoxicity Test Methods BRD Appendix N1

November 2006

Abbreviations: UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a); Toxcat=Category from Globally Harmonized System of Classification and Labeling of Chemicals5 (GHS; UN
2005); NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope; Cyto=NRU-determined starting dose (i.e., one dose lower than the LD50 predicted by the NRU IC50 in the RC
rat-only millimole regression (log LD50 [mmol/kg] = 0.439 log IC50 [mM] + 0.621); Default=Default starting dose of 175 mg/kg; Std. Error=Standard error for number of animals; 3T3=
BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit
dose =5000 mg/kg.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit
dose =5000 mg/kg.
2
Mean number of animals for 10,000 simulations.
3
Difference between mean animals used for the default starting dose and mean animals used for the NRU-based starting dose.
4
P-value is from one-side Wilcoxon signed rank test for difference in animals between the default and NRU-based starting doses. Significant values at p <0.05.
5
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

N-10

In Vitro Cytotoxicity Test Methods BRD Appendix N1

November 2006

Concordance of NRU-Based Starting Dose with Default Starting Dose for GHS Acute Oral Toxicity Category Outcome
Based on Simulated UDP LD501
GHS Category Based on
LD50 Outcome with
Default Starting Dose

1

2

1
2
3
4
5
6
Total

4
0
0
0
0
0
4

0
13
1
0
0
0
14

GHS Category2 Based on LD50 Outcome with NHK NRU-Based Starting DoseHigherCategory
3
4
5
6
Total
NRU
Match
Category
0
0
0
0
4
100%
0%
0
0
0
0
13
100%
0%
11
0
0
0
12
92%
0%
1
15
1
0
17
88%
6%
0
0
22
0
22
100%
0%
0
0
0
0
0
NA
0%
12
15
23
0
68
96%
1%

Lower
NRU
Category
0%
0%
8%
6%
0%
NA
3%

GHS Category2 Based on LD50 Outcome with 3T3 NRU-Based Starting DoseGHS Category Based on
LD50 Outcome with
Default Starting Dose

1

2

3

4

5

6

Total

Category
Match

1
2
3
4
5
6
Total

4
0
0
0
0
0
4

0
13
1
0
0
0
14

0
0
11
0
0
0
11

0
0
0
16
0
0
16

0
0
0
1
21
0
22

0
0
0
0
0
0
0

4
13
12
17
21
0
67

100%
100%
92%
94%
100%
NA
97%

HigherNRU
Category
0%
0%
0%
6%
0%
0%
1%

Lower
NRU
Category
0%
0%
8%
0%
0%
NA
1%

Abbreviations: NRU=Neutral red uptake; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a);
NHK=Normal human epidermal keratinocytes; 3T3= BALB/c 3T3 mouse fibroblasts; NA=Not applicable; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit
dose =5000 mg/kg. The NRU-based starting dose was one dose lower than the LD50 predicted by the NRU IC50 in the RC rat-only millimole regression (log LD50 [mmol/kg] = 0.439 log
IC50 [mM] + 0.621). The default starting dose = 175 mg/kg. Shaded cells are those containing the correct predictions.
2
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

N-11

In Vitro Cytotoxicity Test Methods BRD Appendix N1

November 2006

Discordant Substances for GHS Category Outcomes of UDP Simulations1
NRU
Test
Method3T3

NHK

Substance
Acetaminophen
Sodium Dichromate Dihydrate
Acetaminophen
Caffeine
Sodium Dichromate Dihydrate

NRU-Based Starting Dose2

Default Starting Dose3

LD50

Toxcat4

LD50

Toxcat4

2046.7843.70
2173.95
279.63
45.09

5
2
5
3
2

1765.4451.87
1755.26
357.17
51.77

4
3
4
4
3

LD50 Difference-281.348.17-418.69
77.55
6.69

Abbreviations: Toxcat=Globally Harmonized System of Classification and Labelling of Chemicals (GHS; UN 2005); UDP= Up-and-Down Procedure (OECD 2001a, EPA 2002a);
NRU=Neutral red uptake; 3T3=BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes.
1
Substances for which the simulated UDP outcome (in terms of GHS category) at the NRU-based starting dose did not match the simulated UDP outcome at thedefault starting dose. Simulations were performed with 10,000 runs at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and68 substances in the NHK NRU test method. Upper limit dose =5000 mg/kg.
2
NRU-based starting dose was one dose lower than the LD50 predicted by the NRU IC50 in the RC rat-only millimole regression (log LD50 [mmol/kg] = 0.439 log IC50[mM] + 0.621).
3
The default starting dose = 175 mg/kg. Shaded cells are those containing the correct predictions.
4
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

N-12

In Vitro Cytotoxicity Test Methods BRD Appendix N2

November 2006

Appendix N2

UDP Simulation Results Using Starting Doses One Default Dose Lower
than the LD50 Predicted by the 3T3 and NHK NRU IC50 and the RC RatOnly Weight Regression - 5000 mg/kg Upper Limit Dose

N-13

In Vitro Cytotoxicity Test Methods BRD Appendix N2

November 2006

[This Page Intentionally Left Blank]

N-14

In Vitro Cytotoxicity Test Methods BRD Appendix N2

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by NRU Test Method1
NRU
Test
Method

Sigma
0.12
0.25

3T3

0.50
1.25
2.00

0.12
0.25
NHK

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Animals Used
Std.
Error

Number2

0.193
7.32
0.178
7.94
0.186
8.04
0.180
8.67
0.164
8.70
0.164
9.36
0.132
9.26
0.130
9.96
0.110
9.41
0.095
10.01
Average Difference
0.195
7.38
0.176
7.92
0.189
8.12
0.175
8.66
0.169
8.80
0.159
9.36
0.136
9.36
0.125
9.96
0.114
9.48
0.092
10.02
Average Difference

Difference3

P4

0.04

0.9360%
Savings Animals
Used
7.8%

0.04

0.5758

7.2%

1.2%

0.06

0.3430

7.0%

1.5%

0.11

0.0119

7.0%

2.3%

0.10

0.0371

6.0%

2.1%

0.07
0.04

0.8066

6.8%

1.1%

0.05

0.3274

6.3%

1.2%

0.05

0.3154

6.0%

1.3%

0.09

0.0044

6.1%

2.0%

0.09

0.00897.8%

1.9%

Animals Died
Std.
Error

Difference3

P4

0.62

0.00003

0.63

0.00010.66

0.00010.70

0.00003

0.60

0.000050.64
0.54

0.00020.54

0.00020.56

0.00030.61

0.00010.53

0.0001

0.56

Number2

0.200
3.39
0.200
3.43
0.198
3.72
0.197
3.76
0.186
4.05
0.185
4.11
0.161
4.44
0.152
4.55
0.141
4.58
0.129
4.67
Average Difference
0.208
3.35
0.203
3.39
0.204
3.67
0.199
3.72
0.191
4.02
0.184
4.08
0.164
4.43
0.151
4.52
0.144
4.56
0.127
4.66
Average Difference%
Difference Animals
Died
1.2%

0.06

Abbreviations: UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a); NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope; Cyto=NRU-determined starting dose (i.e., one doselower than the LD50 predicted by the NRU IC50 in the RC rat-only weight regression (log LD50 [mg/kg] = 0.372 log IC50 [mg/mL] + 2.024); Default=Default starting dose of 175 mg/kg; Std
Error=Standard error for number of animals; 3T3= BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit dose =5000
mg/kg.
2
Mean number of animals for 10,000 simulations.
3
Difference between mean number of animals for the default starting dose and mean number of animals for the NRU-based starting dose.
4
P-value is from one-sided Wilcoxon signed rank test for difference in animals between the default starting dose and the NRU-based starting dose. Significant values at p <0.05.

N-15

In Vitro Cytotoxicity Test Methods BRD Appendix N2

November 2006

Summary of Stopping Rules Used for the UDP Simulations by NRU Test Method1
NRU Test
MethodSigma

Starting Dose

3 Animals at
Limit Dose2

5 Reversals2

Likelihood
Ratio2

Maximum
Number of
Animals Used2

0.12

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

15.6%
15.4%
15.0%
14.7%
13.4%
13.0%
9.9%
9.1%
8.6%
7.4%
16.8%
16.6%
16.2%
15.8%
14.3%
13.8%
10.2%
9.5%
8.7%
7.6%

55.8%
56.8%
33.4%
34.1%
19.7%
20.1%
13.4%
13.6%
12.3%
12.5%
55.4%
55.9%
33.3%
33.5%
19.7%
19.9%
13.5%
13.5%
12.3%
12.5%

26.8%
25.3%
47.9%
46.0%
59.6%
57.3%
64.6%
60.8%
65.6%
62.5%
26.0%
25.0%
46.7%
45.4%
58.3%
56.6%
63.5%
60.4%
64.9%
62.3%

1.8%
2.4%
3.7%
5.3%
7.2%
9.7%
12.1%
16.4%
13.5%
17.6%
1.8%
2.4%
3.8%
5.2%
7.7%
9.7%
12.8%
16.5%
14.1%
17.6%

0.25

3T3

0.5
1.25
2
0.12
0.25

NHK

0.5
1.25
2

Abbreviations: UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a); NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope;
Cyto=NRU-determined starting dose (i.e., one dose lower than the LD50 predicted by the NRU IC50 in the RC rat-only weight regression (log LD50
[mg/kg] = 0.372 log IC50 [µg/mL] + 2.024); Default=Default starting dose of 175 mg/kg; 3T3= BALB/c 3T3 mouse fibroblasts; NHK=Normal
human epidermal keratinocytes; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in
the NHK NRU test method. Upper limit dose =5000 mg/kg.
2
Percentage of the 10,000 test simulations that satisfied the specified condition for completion of testing (see OECD [2001a]; EPA [2002a]).

N-16

In Vitro Cytotoxicity Test Methods BRD Appendix N2

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

3T3

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

1
0.12
0.25
NHK

0.50
1.25
2.00

0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

2
0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

0.366
8.92
0.278
9.70
0.587
9.75
0.181
10.55
0.623
10.38
0.197
11.29
0.594
10.86
0.147
11.72
0.503
10.80
0.142
11.45
Average Difference
0.515
8.97
0.268
9.66
0.703
9.79
0.218
10.53
0.711
10.49
0.242
11.21
0.694
10.94
0.168
11.70
0.632
10.83
0.159
11.47
Average Difference
0.442
8.41
0.276
8.47
0.393
9.07
0.201
9.11
0.419
9.58
0.219
9.71
0.381
10.11
0.225
10.19
0.297
10.22
0.170
10.21
Average Difference
0.439
8.59
0.267
8.45
0.384
9.24
0.178
9.12
0.413
9.78
0.177
9.70
0.385
10.28
0.187
10.17
0.306
10.35
0.149
10.19
Average Difference

Animals Died

Difference3

P4

0.78

0.62500.81

0.62500.90

0.62500.86

0.62500.66

0.62500.80
0.69

0.37500.74

0.37500.72

0.62500.76

0.62500.63

0.62500.71
0.06

1.00000.05

1.00000.13

0.94600.08

0.9460-0.01

0.73540.06-0.13

0.3757-0.12

0.5879-0.07

0.5417-0.11

0.4973-0.16

0.4973

-0.12

N-17

Std.
Error

Number2

0.404
5.74
0.171
6.39
0.521
6.22
0.158
6.88
0.549
6.63
0.196
7.35
0.540
6.86
0.166
7.55
0.466
6.66
0.151
7.24
Average Difference
0.531
5.81
0.169
6.37
0.629
6.28
0.178
6.87
0.641
6.71
0.224
7.31
0.638
6.93
0.179
7.56
0.581
6.72
0.157
7.24
Average Difference
0.398
5.04
0.240
5.12
0.370
5.41
0.202
5.48
0.381
5.70
0.210
5.78
0.359
5.93
0.214
5.98
0.302
5.91
0.174
5.91
Average Difference
0.427
5.10
0.235
5.11
0.396
5.46
0.193
5.47
0.409
5.76
0.192
5.78
0.391
5.99
0.198
5.96
0.334
5.95
0.166
5.89
Average Difference

Difference3

P4

0.65

0.62508.0%%
Difference Animals
Died
10.2%

0.66

0.6250

7.7%

9.6%

0.72

0.6250

8.0%

9.8%

0.70

0.6250

7.3%

9.2%

0.57

0.6250

5.7%

7.9%

0.56

0.3750

7.1%

8.8%

0.59

0.3750

7.0%

8.6%

0.60

0.6250

6.4%

8.2%

0.62

0.6250

6.5%

8.3%

0.52

0.6250

5.5%

7.1%

0.08

1.0000

0.8%

1.5%

0.06

1.0000

0.5%

1.1%

0.09

1.0000

1.3%

1.5%

0.05

0.9460

0.8%

0.8%

0.00

0.7869

-0.1%

0.0%

0.01

0.6848

-1.6%

0.2%

0.01

1.0000

-1.4%

0.2%

0.02

0.8394

-0.8%

0.3%

-0.03

0.8394

-1.1%

-0.4%

-0.05

0.7869

-1.6%

-0.9%

% Savings- Animals
Used

0.66

0.58

0.05

-0.01

In Vitro Cytotoxicity Test Methods BRD Appendix N2

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

3T3

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

3
0.12
0.25
NHK

0.50
1.25
2.00

0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

4
0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

0.181
6.76
0.190
6.58
0.182
7.33
0.147
7.13
0.180
7.99
0.100
7.74
0.119
8.86
0.056
8.64
0.069
9.25
0.039
9.11
Average Difference
0.205
6.75
0.194
6.58
0.225
7.33
0.160
7.11
0.209
7.99
0.110
7.75
0.123
8.85
0.058
8.67
0.083
9.26
0.035
9.13
Average Difference
0.176
7.17
0.236
7.46
0.173
7.68
0.202
7.93
0.193
8.16
0.208
8.52
0.159
8.83
0.142
9.15
0.115
9.17
0.084
9.38
Average Difference
0.160
7.17
0.234
7.48
0.194
7.71
0.189
7.98
0.223
8.20
0.206
8.54
0.196
8.86
0.141
9.14
0.143
9.18
0.083
9.38
Average Difference

Animals Died

Difference3

P4

-0.18

0.0923-0.20

0.1514-0.25

0.1514-0.22

0.1294-0.15

0.1294

-0.20
-0.18

0.2036-0.22

0.1099-0.24

0.1294-0.18

0.1294-0.14

0.1294-0.19
0.28

0.03350.25

0.05070.35

0.05770.32

0.02500.21

0.03350.28
0.31

0.05770.27

0.05070.34

0.08330.28

0.05770.19

0.06540.28

N-18

Difference3

P4

0.136
3.50
0.127
3.36
0.146
3.83
0.102
3.68
0.146
4.18
0.080
4.02
0.112
4.61
0.057
4.48
0.084
4.78
0.049
4.70
Average Difference-0.14

0.1294-2.7%%
Difference Animals
Died
-4.2%

-0.15

0.2061

-2.8%

-4.0%

-0.16

0.1763

-3.3%

-4.0%

-0.13

0.3804

-2.5%

-2.8%

-0.09

0.5186

-1.6%

-1.8%

0.137
3.41
0.129
3.35
0.145
3.74
0.108
3.66
0.141
4.09
0.082
4.01
0.106
4.52
0.060
4.49
0.088
4.70
0.048
4.70
Average Difference-0.06

0.2334

-2.7%

-1.8%

-0.08

0.1763

-3.2%

-2.1%

-0.07

0.1763

-3.1%

-1.9%

-0.03

0.8501

-2.1%

-0.7%

0.00

0.9097

-1.5%

-0.1%

0.063
3.39
0.067
3.39
0.032
3.61
0.049
3.60
0.039
3.80
0.047
3.85
0.048
4.10
0.020
4.13
0.043
4.26
0.020
4.25
Average Difference

0.00

0.8999

3.8%

0.0%

-0.01

0.1928

3.1%

-0.3%

0.05

0.1167

4.1%

1.2%

0.03

0.1046

3.5%

0.8%

-0.01

0.7820

2.3%

-0.2%

0.03

0.2744

4.1%

0.8%

0.02

0.1591

3.4%

0.4%

0.05

0.1167

3.9%

1.3%

0.02

0.2744

3.1%

0.4%

-0.02

0.7820

2.1%

-0.6%

Std.
Error

Number2

0.060
3.38
0.066
3.41
0.028
3.62
0.042
3.63
0.040
3.82
0.046
3.87
0.050
4.11
0.022
4.13
0.043
4.28
0.019
4.25
Average Difference% Savings- Animals
Used

-0.13

-0.05

0.01

0.02

In Vitro Cytotoxicity Test Methods BRD Appendix N2

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by GHS Toxicity Category and NRU Test Method1
Animals Used
Toxcat

Cell
Type

Sigma

Method

Std.
Error

0.12

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

0.365
7.61
0.235
9.20
0.285
8.67
0.159
10.39
0.242
9.14
0.106
10.78
0.204
9.08
0.071
10.69
0.161
9.05
0.064
10.38
Average Difference

0.25
3T3

0.50
1.25
2.00

5
0.12
0.25
NHK

0.50
1.25
2.00

0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

6
0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Number2

0.326
7.90
0.234
9.18
0.307
8.93
0.146
10.34
0.251
9.40
0.084
10.77
0.194
9.30
0.055
10.67
0.155
9.20
0.038
10.36
Average Difference
0.686
6.14
0.587
7.76
0.653
7.05
0.542
8.67
0.484
8.23
0.343
9.87
0.305
8.93
0.058
10.89
0.251
8.87
0.028
10.75
Average Difference
0.625
6.12
0.560
7.66
0.581
7.10
0.500
8.63
0.435
8.34
0.318
9.88
0.277
9.07
0.057
10.88
0.235
8.99
0.022
10.74
Average Difference

Animals Died

Difference3

P4

1.59

0.00201.72

0.00201.64

0.00391.61

0.00201.33

0.00391.58
1.28

0.00201.41

0.00201.38

0.00201.37

0.00201.15

0.00201.32
1.63

0.00051.62

0.00051.65

0.00051.96

0.00051.88

0.00051.75
1.53

0.00051.53

0.00021.54

0.00021.81

0.00021.75

0.0002

1.63

N-19

Std.
Error

Number2

0.046
3.20
0.037
3.31
0.056
3.41
0.065
3.59
0.055
3.52
0.063
3.67
0.044
3.65
0.031
3.88
0.037
3.77
0.015
3.97
Average Difference
0.035
3.23
0.038
3.31
0.052
3.43
0.066
3.58
0.047
3.54
0.067
3.66
0.033
3.67
0.038
3.86
0.031
3.79
0.023
3.96
Average Difference

Difference3

P4

0.12

0.005917.3%%
Difference Animals
Died
3.6%

0.18

0.0098

16.6%

5.1%

0.16

0.0137

15.2%

4.2%

0.23

0.0020

15.0%

6.0%

0.21

0.0039

12.8%

5.2%

0.08

0.0273

14.0%

2.5%

0.15

0.0098

13.6%

4.2%

0.13

0.0098

12.8%

3.5%

0.19

0.0020

12.8%

5.0%

0.18

0.0020

11.1%

4.4%

% Savings- Animals
Used

0.18

0.15

0.316
0.88
0.304
0.85
0.317
1.33
0.309
1.30
0.254
2.04
0.256
2.03
0.126
2.99
0.122
3.20
0.089
3.32
0.067
3.61
Average Difference-0.03

0.1294

21.0%

-3.1%

-0.03

0.0210

18.7%

-2.2%

-0.01

0.3394

16.7%

-0.4%

0.20

0.0005

18.0%

6.3%

0.29

0.0005

17.5%

8.0%

0.298
0.82
0.289
0.80
0.296
1.28
0.287
1.26
0.236
2.03
0.236
2.02
0.112
3.01
0.114
3.18
0.078
3.34
0.062
3.61
Average Difference-0.02

0.0398

20.0%

-3.1%

-0.02

0.1099

17.7%

-1.8%

-0.01

0.1677

15.6%

-0.6%

0.17

0.0002

16.7%

5.4%

0.27

0.0005

16.3%

7.4%

0.09

0.08

In Vitro Cytotoxicity Test Methods BRD Appendix N2

November 2006

Abbreviations: UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a); Toxcat=Category from Globally Harmonized System of Classification and Labeling of Chemicals5 (GHS; UN
2005); NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope; Cyto=NRU-determined starting dose (i.e., one dose lower than the LD50 predicted by the NRU IC50 in the RC
rat-only weight regression (log LD50 [mmol/kg] = 0.372 log IC50 [mM] + 2.024); Default=Default starting dose of 175 mg/kg; Std. Error=Standard error for number of animals; 3T3=
BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit
dose =5000 mg/kg.
2
Mean number of animals for 10,000 simulations.
3
Difference between mean animals used for the default starting dose and mean animals used for the NRU-based starting dose.
4
P-value is from one-side Wilcoxon signed rank test for difference in animals between the default and NRU-based starting doses. Significant values at p <0.05.
5
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

N-20

In Vitro Cytotoxicity Test Methods BRD Appendix N2

November 2006

Concordance of NRU-Based Starting Dose with Default Starting Dose for GHS Acute Oral Toxicity Category Outcome
Based on Simulated UDP LD501
GHS Category Based on
LD50 Outcome with
Default Starting Dose

d

2

1
2
3
4
5
6
Total

4
0
0
0
0
0
4

0
13
1
0
0
0
14

GHS Category Based on
LD50 Outcome with
Default Starting Dose

1

2

1
2
3
4
5
6
Total

4
0
0
0
0
0
4

0
13
1
0
0
0
14

GHS Category Based on LD50 Outcome with NHK NRU-Based Starting DoseHigherCategory
3
4
5
6
Total
NRU
Match
Category
0
0
0
0
4
100%
0%
0
0
0
0
13
100%
0%
11
0
0
0
12
92%
0%
1
16
0
0
17
94%
0%
0
0
22
0
22
100%
0%
0
0
0
0
0
NA
0%
12
16
22
0
68
97%
0%

Lower
NRU
Category
0%
0%
8%
6%
0%
NA
3%

GHS Category Based on LD50 Outcome with 3T3 NRU-Based Starting DoseHigherCategory
3
4
5
6
Total
NRU
Match
Category
0
0
0
0
4
100%
0%
0
0
0
0
13
100%
0%
11
0
0
0
12
92%
0%
1
16
0
0
17
94%
0%
0
0
21
0
21
100%
0%
0
0
0
0
0
NA
0%
12
16
21
0
67
97%
0%

Lower
NRU
Category
0%
0%
8%
6%
0%
NA
3%

Abbreviations: NRU=Neutral red uptake; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a);
NHK=Normal human epidermal keratinocytes; 3T3= BALB/c 3T3 mouse fibroblasts; NA=Not applicable; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit
dose =5000 mg/kg. The NRU-based starting dose was one dose lower than the LD50 predicted by the NRU IC50 in the RC rat-only weight regression (log LD50 [mg/kg] = 0.372 log IC50[µg/mL] + 2.024). The default starting dose = 175 mg/kg. Shaded cells are those containing the correct predictions.
2
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

N-21

In Vitro Cytotoxicity Test Methods BRD Appendix N2

November 2006

Discordant Substances for GHS Category Outcomes of UDP Simulations1
NRU
Test
Method3T3
NHK

Substance
Caffeine
Sodium dichromate dihydrate
Caffeine
Sodium dichromate dihydrate

NRU-Based Starting Dose2

Default Starting Dose3

LD50

Toxcat4

LD50

Toxcat4

271.5443.70
269.85
48.52

3
47.97
3
2

338.162
339.43
50.64

4
50.66
4
3

LD50 Difference
66.62
3
69.59
2.12

Abbreviations: Toxcat=Globally Harmonized System of Classification and Labelling of Chemicals (GHS; UN 2005); UDP= Up-and-Down Procedure (OECD 2001a, EPA 2002a);
NRU=Neutral red uptake; 3T3=BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes.
1
Substances for which the simulated UDP outcome (in terms of GHS category) at the NRU-based starting dose did not match the simulated UDP outcome at thedefault starting dose. Simulations were performed with 10,000 runs at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and68 substances in the NHK NRU test method. Upper limit dose =5000 mg/kg.
2
NRU-based starting dose was one dose lower than the LD50 predicted by the NRU IC50 in the RC rat-only weight regression (log LD50 [mg/kg] = 0.372 log IC50[µg/mL] + 2.024).
3
The default starting dose = 175 mg/kg. Shaded cells are those containing the correct predictions.
4
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

N-22

In Vitro Cytotoxicity Test Methods BRD Appendix N3

November 2006

Appendix N3

ATC Simulation Results Starting at the Next Fixed Dose Below the LD50
Predicted by the 3T3 and NHK NRU IC50 and the RC Rat-Only Millimole
Regression - 2000 mg/kg Upper Limit Dose

N-23

In Vitro Cytotoxicity Test Methods BRD Appendix N3

November 2006

[This Page Intentionally Left Blank]

N-24

In Vitro Cytotoxicity Test Methods BRD Appendix N3

November 2006

Summary of Animals Used and Animals Dead for ATC Simulations by NRU Test Method1
NRU
Test
Method

Sigma
0.12
0.25

NHK

0.50
1.25
2.00

0.12
0.25
3T3

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Animals Used
Std.
Error

Number2

Difference3

P4

0.48

0.1061%
Savings Animals
Used
6.6%

0.50

0.5613

7.1%

14.7%

0.53

0.0002

7.3%

14.2%

0.55

0.0002

7.1%

12.4%

0.53

0.0012

7.3%

11.2%

0.43

0.0283

4.8%

13.4%

0.45

0.0139

5.4%

13.0%

0.46

0.0237

5.7%

12.2%

0.48

0.00003

5.8%

10.8%

0.47

0.00002

6.1%

9.9%

Animals Died
Std.
Error

Difference3

P4

0.290
9.96
0.169
10.67
0.269
9.98
0.149
10.75
0.239
10.11
0.114
10.91
0.183
10.31
0.068
11.10
0.163
10.43
0.050
11.25
Average Difference

0.70

0.01130.77

0.01270.80

0.00050.79

0.00350.82

0.0003

0.273
10.13
0.170
10.64
0.257
10.15
0.151
10.73
0.238
10.27
0.115
10.89
0.193
10.46
0.067
11.10
0.166
10.56
0.049
11.25
Average Difference

0.51

0.02260.58

0.00750.62

0.00380.64

0.01540.69

0.00020.78

0.61

Number2

0.286
2.67
0.334
3.15
0.283
2.88
0.324
3.38
0.261
3.19
0.297
3.72
0.201
3.86
0.228
4.40
0.168
4.20
0.189
4.73
Average Difference
0.291
2.77
0.335
3.20
0.281
2.99
0.325
3.43
0.257
3.31
0.299
3.77
0.201
3.96
0.228
4.43
0.168
4.28
0.190
4.76
Average Difference%
Difference Animals
Died
15.3%

0.52

0.46

Abbreviations: ATC=Acute Toxic Class method (OECD 2001d); NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope; Cyto=NRU-determined starting dose (i.e., one doselower than the LD50 predicted by the NRU IC50 in the RC rat-only millimole regression (log LD50 [mmol/kg] = 0.439 log IC50 [mM] + 0.621); Default=Default starting dose of 300 mg/kg;
Std. Error=Standard error for number of animals; 3T3= BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
1
For 2000 ATC simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit dose
=2000 mg/kg.
2
Mean number of animals for 2000 simulations.
3
Difference between mean number of animals for the default starting dose and mean number of animals for the NRU-based starting dose.
4
P-value is from one-sided Wilcoxon signed rank test for difference in animals between the default and NRU-based starting doses. Significant values at p <0.05.

N-25

In Vitro Cytotoxicity Test Methods BRD Appendix N3

November 2006

Summary of Animals Used and Animals Dead for ATC Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

NHK

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

1
0.12
0.25
3T3

0.50
1.25
2.00

0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

2
0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

1.228
6.09
0.083
9.09
1.284
6.37
0.178
9.35
1.311
6.78
0.158
9.74
1.247
7.48
0.111
10.39
1.285
7.86
0.066
10.85
Average Difference
1.088
6.38
0.081
9.08
1.068
6.68
0.174
9.36
1.087
7.09
0.170
9.77
1.106
7.75
0.093
10.42
1.113
8.16
0.060
10.84
Average Difference
0.448
10.42
0.165
11.76
0.395
10.35
0.180
11.64
0.352
10.38
0.212
11.56
0.400
10.26
0.156
11.54
0.478
10.21
0.089
11.62
Average Difference
0.433
10.60
0.163
11.75
0.471
10.46
0.189
11.64
0.522
10.39
0.214
11.56
0.550
10.30
0.148
11.51
0.555
10.31
0.083
11.58
Average Difference

Animals Died

Difference3

P4

2.99

0.25002.99

0.25002.96

0.25002.91

0.25002.99

0.25002.97
2.70

0.12502.68

0.12502.68

0.12502.67

0.12502.68

0.12502.68
1.33

0.03221.29

0.01711.18

0.03981.28

0.04791.41

0.03981.30
1.15

0.04791.19

0.03981.17

0.04791.21

0.06811.27

0.0574

1.20

N-26

Std.
Error

Number2

1.209
5.87
0.080
8.86
1.183
5.68
0.070
8.67
1.192
5.52
0.060
8.50
1.052
5.20
0.066
7.92
0.973
5.05
0.052
7.51
Average Difference
1.163
6.15
0.081
8.86
1.089
6.01
0.073
8.67
1.073
5.85
0.049
8.50
0.975
5.49
0.081
7.93
0.887
5.32
0.058
7.54
Average Difference
0.702
5.21
0.256
6.55
0.764
5.40
0.313
6.71
0.739
5.66
0.312
6.88
0.590
5.85
0.191
6.91
0.526
5.77
0.142
6.77
Average Difference
0.645
5.39
0.255
6.56
0.662
5.52
0.314
6.72
0.647
5.71
0.313
6.89
0.538
5.90
0.194
6.89
0.474
5.82
0.146
6.77
Average Difference

Difference3

P4

2.99

0.125033.0%%
Difference Animals
Died
33.8%

2.99

0.1250

31.9%

34.5%

2.98

0.2500

30.4%

35.0%

2.72

0.1250

28.0%

34.4%

2.46

0.2500

27.6%

32.7%

2.71

0.1250

29.7%

30.5%

2.66

0.1250

28.7%

30.7%

2.65

0.1250

27.4%

31.2%

2.43

0.1250

25.6%

30.7%

2.22

0.1250

24.7%

29.4%

1.34

0.0266

11.4%

20.5%

1.31

0.0327

11.1%

19.5%

1.22

0.0479

10.2%

17.7%

1.06

0.0681

11.1%

15.3%

1.01

0.0479

12.1%

14.9%

1.16

0.0479

9.8%

17.7%

1.21

0.0398

10.2%

17.9%

1.18

0.0398

10.2%

17.1%

0.99

0.0681

10.5%

14.3%

0.95

0.0398

10.9%

14.0%

% Savings- Animals
Used

2.83

2.54

1.19

1.10

In Vitro Cytotoxicity Test Methods BRD Appendix N3

November 2006

Summary of Animals Used and Animals Dead for ATC Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

NHK

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

3
0.12
0.25
3T3

0.50
1.25
2.00

0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

4
0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

Animals Died

Difference3

P4

0.489
9.63
0.264
9.44
0.407
9.86
0.275
9.89
0.288
10.39
0.207
10.83
0.254
10.80
0.083
11.73
0.290
10.63
0.038
11.82
Average Difference-0.20

0.37500.03

0.30130.44

0.15140.93

0.01221.19

0.0015

0.110
9.27
0.258
9.42
0.153
9.65
0.271
9.90
0.172
10.39
0.202
10.81
0.237
11.05
0.084
11.73
0.261
11.03
0.037
11.80
Average Difference

0.15

0.75200.25

0.14750.42

0.05220.69

0.04250.77

0.0640

0.625
10.11
0.098
9.26
0.560
10.14
0.095
9.43
0.494
10.37
0.062
9.77
0.290
10.88
0.043
10.57
0.095
11.13
0.048
11.10
Average Difference
0.619
10.56
0.102
9.26
0.543
10.52
0.098
9.42
0.483
10.67
0.065
9.75
0.283
10.99
0.040
10.57
0.099
11.11
0.047
11.11
Average Difference

0.48

0.45
-0.85

0.1133-0.71

0.1089-0.60

0.7960-0.31

0.0730-0.03

0.6051

-0.50
-1.30

0.0210-1.09

0.0806-0.92

0.0262-0.42

0.00380.00

0.8040

-0.75

N-27

Std.
Error

Number2

0.073
3.12
0.217
3.41
0.138
3.37
0.237
3.80
0.160
3.59
0.171
4.26
0.201
4.19
0.119
5.03
0.217
4.51
0.091
5.41
Average Difference
0.102
3.18
0.213
3.40
0.171
3.49
0.237
3.80
0.170
3.81
0.169
4.26
0.202
4.45
0.119
5.04
0.198
4.82
0.095
5.41
Average Difference
0.069
3.05
0.067
3.04
0.093
3.14
0.092
3.12
0.062
3.18
0.057
3.18
0.051
3.66
0.067
3.70
0.061
4.08
0.080
4.16
Average Difference
0.070
3.05
0.068
3.04
0.093
3.13
0.095
3.13
0.060
3.20
0.056
3.18
0.057
3.64
0.069
3.71
0.062
4.02
0.077
4.17
Average Difference

Difference3

P4

0.29

0.2749-2.1%%
Difference Animals
Died
8.4%

0.44

0.0098

0.3%

11.5%

0.67

0.0015

4.0%

15.8%

0.85

0.0049

7.9%

16.8%

0.90

0.0015

10.0%

16.6%

0.23

0.9097

1.6%

6.6%

0.31

0.0830

2.5%

8.1%

0.45

0.0425

3.9%

10.5%

0.59

0.0361

5.8%

11.8%

0.59

0.0522

6.5%

10.9%

-0.01

0.1627

-9.2%

-0.2%

-0.02

0.0013

-7.5%

-0.7%

-0.001

0.9229

-6.1%

0.1%

0.04

0.5520

-2.9%

1.2%

0.08

0.5871

-0.3%

1.9%

-0.01

0.5520-14.0%

-0.2%

0.00

0.4690-11.6%

0.0%

-0.02

0.0787

-9.5%

-0.7%

0.07

0.0787

-4.0%

1.9%

0.15

0.08320.0%

3.6%% Savings- Animals
Used

0.63

0.43

0.02

0.04

In Vitro Cytotoxicity Test Methods BRD Appendix N3

November 2006

Summary of Animals Used and Animals Dead for ATC Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

NHK

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

5
0.12
0.25
3T3

0.50
1.25
2.00

0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

6
0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

0.148
11.89
0.101
11.87
0.138
11.65
0.119
11.63
0.119
11.25
0.083
11.22
0.062
10.81
0.038
10.77
0.041
10.87
0.011
10.83
Average Difference
0.096
11.77
0.103
11.88
0.113
11.53
0.117
11.62
0.080
11.14
0.083
11.22
0.039
10.75
0.037
10.77
0.018
10.84
0.010
10.85
Average Difference
0.804
9.34
0.000
12.00
0.801
9.35
0.002
11.99
0.732
9.43
0.034
11.86
0.462
9.70
0.058
11.23
0.288
10.06
0.031
10.98
Average Difference
0.842
9.81
0.000
12.00
0.839
9.80
0.002
11.99
0.779
9.82
0.035
11.85
0.509
9.98
0.060
11.22
0.332
10.19
0.029
10.99
Average Difference

Animals Died

Difference3

P4

-0.02

0.6328-0.02

0.6250-0.03

0.3750-0.04

0.7695-0.04

0.2422-0.03
0.11

0.07810.09

0.43160.08

0.06450.02

0.69530.01

0.69530.06
2.66

0.01952.65

0.03222.43

0.00241.53

0.00240.92

0.01052.04
2.19

0.01952.19

0.01372.03

0.02101.24

0.05220.79

0.0425

1.69

N-28

Difference3

P4

0.185
0.39
0.189
0.39
0.157
1.10
0.157
1.08
0.096
1.82
0.097
1.82
0.040
2.89
0.041
2.89
0.033
3.36
0.026
3.35
Average Difference

0.001

0.6953-0.2%%
Difference Animals
Died
0.2%

-0.02

0.0625

-0.2%

-1.5%

0.00

1.0000

-0.3%

0.0%

-0.01

0.6426

-0.4%

-0.2%

-0.01

0.6250

-0.3%

-0.2%

0.188
0.39
0.188
0.39
0.155
1.10
0.158
1.09
0.098
1.82
0.093
1.82
0.043
2.87
0.041
2.88
0.032
3.36
0.027
3.35
Average Difference

0.005

0.2324

0.9%

1.2%

-0.01

0.3848

0.8%

-0.8%

-0.0020.8457

0.7%

-0.1%

0.01

1.00000.2%

0.2%-0.0050.6250

0.1%

-0.1%

Std.
Error

Number2% Savings- Animals
Used

-0.01

-0.0010.00020.00040.00020.0004
0.033
0.11
0.033
0.10
0.099
0.73
0.099
0.73
0.106
2.14
0.086
2.27
0.080
2.85
0.053
2.93
Average Difference

0.00004

1.0000

22.2%

9.1%

-0.002

0.4824

22.1%

-1.6%

0.01

0.0398

20.5%

1.2%

0.12

0.0479

13.6%

5.5%

0.08

0.14658.4%

2.7%

0.00020.004
0.0002
0.003
0.034
0.11
0.035
0.12
0.107
0.74
0.106
0.74
0.110
2.17
0.096
2.27
0.091
2.86
0.057
2.95
Average Difference-0.0010.750018.3%-25.0%

0.003

0.2334

18.3%

2.3%

-0.0020.6773

17.1%

-0.3%

0.11

0.0923

11.0%

4.6%

0.09

0.0425

7.2%

3.0%

0.04

0.04

In Vitro Cytotoxicity Test Methods BRD Appendix N3

November 2006

Abbreviations: ATC=Acute Toxic Class method (OECD 2001d); Toxcat=Category from Globally Harmonized System of Classification and Labeling of Chemicals5 (GHS; UN 2005);
NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope; Cyto=NRU-determined starting dose (i.e., one dose lower than the LD50 predicted by the NRU IC50 in the RC rat-only
millimole regression (log LD50 [mmol/kg] = 0.439 log IC50 [mM] + 0.621); Default=Default starting dose of 300 mg/kg; Std. Error=Standard error for number of animals; 3T3= BALB/c
3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity..
1
For 2000 ATC simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit dose
=2000 mg/kg.
2
Mean number of animals for 2000 simulations.
3
Difference between mean animals used for the default starting dose and mean animals used for the NRU-based starting dose.
4
P-value is from one-side Wilcoxon signed rank test for difference in animals between the default and NRU-based starting doses. Significant values at p <0.05.
5
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

N-29

In Vitro Cytotoxicity Test Methods BRD Appendix N3

November 2006

Concordance of NRU-Based Starting Dose with Default Starting Dose for GHS Acute Oral Toxicity Category Outcome for
ATC Simulations1
GHS Category Outcome with NHK NRU-Based Starting DoseGHS Category Outcome
with Default Starting Dose

1

2

3

4

5

6

Total

Category
Match

1
2
3
4
5
6
Total

8
0
0
0
0
0
8

0
11
1
0
0
0
12

0
0
13
0
0
0
13

0
0
0
13
0
0
13

0
0
0
0
21
0
21

0
0
0
0
0
1
1

8
11
14
13
21
1
68

100%
100%
93%
100%
100%
100%
99%

HigherNRU
Category
0%
0%
0%
0%
0%
0%
0%

Lower
NRU
Category
0%
0%
7%
0%
0%
0%
1%

HigherNRU
Category
0%
0%
0%
0%
0%
0%
0%

Lower
NRU
Category
0%
0%
0%
8%
0%
0%
1%

GHS Category Outcome with 3T3 NRU-Based Starting DoseGHS Category Outcome
with Default Starting Dose

1

2

3

4

5

6

Total

Category
Match

1
2
3
4
5
6
Total

8
0
0
0
0
0
8

0
11
0
0
0
0
11

0
0
15
1
0
0
16

0
0
0
11
0
0
11

0
0
0
0
20
0
20

0
0
0
0
0
1
1

8
11
15
12
20
1
67

100%
100%
100%
92%
100%
100%
99%

Abbreviations: NRU=Neutral red uptake; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); ATC=Acute Toxic Class method (OECD 2001d); NHK=Normal
human epidermal keratinocytes; 3T3= BALB/c 3T3 mouse fibroblasts; RC=Registry of Cytotoxicity.
1
For 2000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit dose
=2000 mg/kg. The NRU-based starting dose was one dose lower than the LD50 predicted by the NRU IC50 in the RC rat-only millimole regression (log LD50 [mmol/kg] = 0.439 log IC50[mM] + 0.621). The default starting dose = 300 mg/kg. Shaded cells are those containing the correct predictions.
2
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

N-30

In Vitro Cytotoxicity Test Methods BRD Appendix N3

November 2006

Discordant Substances1 for GHS Category2 Outcomes of ATC Simulations
NRU Test
Method3T3
NHK

Substance
Cupric sulfate pentahydrate
Hexachlorophene

NRU-Based Starting Dose3
Toxicity Category
32

Default Starting Dose4
Toxicity Category43Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); ATC= Acute
Toxic Class method (OECD 2001d): NRU=Neutral red uptake; 3T3=BALB/c 3T3 mouse fibroblasts; NHK=Normal humanepidermal keratinocytes.
1
Substances for which the simulated ATC outcome for the NRU-based starting dose did not match the simulated
ATC outcome for the default starting dose. Simulations were performed with 2000 runs at each starting doseand dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU
test method. Upper limit dose =2000 mg/kg.
2
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg
3
NRU-based starting dose was one dose lower than the LD50 predicted by the NRU IC50 in the RC rat-only
millimole regression (log LD50 [mmol/kg] = 0.439 log IC50 [mM] + 0.621).
4
The default starting dose = 300 mg/kg.

N-31

In Vitro Cytotoxicity Test Methods BRD Appendix N3

November 2006

[This Page Intentionally Left Blank]

N-32

In Vitro Cytotoxicity Test Methods BRD Appendix N4

November 2006

Appendix N4

ATC Simulation Results Starting at the Next Fixed Dose Below the LD50
Predicted by the 3T3 and NHK NRU IC50 and the RC Rat-Only Weight
Regression - 2000 mg/kg Upper Limit DoseN-33

In Vitro Cytotoxicity Test Methods BRD Appendix N4

November 2006

[This Page Intentionally Left Blank]

N-34

In Vitro Cytotoxicity Test Methods BRD Appendix N4

November 2006

Summary of Animals Used and Animals Dead for ATC Simulations by NRU Test Method1
NRU
Test
Method

Sigma
0.12
0.25

NHK

0.50
1.25
2.00

0.12
0.25
3T3

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Animals Used
Std.
Error

Number2

Difference3

P4

0.48

0.0999%
Savings Animals
Used
8.6%

0.50

0.3451

9.1%

14.7%

0.52

0.0030

8.8%

13.8%

0.54

0.0000

7.8%

12.3%

0.53

0.0001

7.6%

11.3%

0.47

0.0001

10.2%

14.6%

0.48

0.0024

10.4%

14.1%

0.50

0.0007

9.6%

13.2%

0.51

0.0000

7.9%

11.4%

0.49

0.0000

7.2%

10.3%

Animals Died
Std.
Error

Difference3

P4

0.297
9.75
0.169
10.67
0.274
9.77
0.149
10.75
0.242
9.95
0.114
10.91
0.180
10.24
0.068
11.10
0.152
10.39
0.050
11.25
Average Difference

0.91

0.00250.98

0.00150.96

0.00000.86

0.00050.86

0.0000

0.293
9.55
0.170
10.64
0.273
9.61
0.151
10.73
0.244
9.85
0.115
10.89
0.187
10.22
0.067
11.10
0.153
10.43
0.049
11.25
Average Difference

1.09

0.00061.11

0.00021.04

0.00010.88

0.00000.81

0.00000.91

0.99

Number2

0.280
2.67
0.334
3.15
0.276
2.88
0.324
3.38
0.254
3.21
0.297
3.72
0.193
3.86
0.228
4.40
0.160
4.19
0.189
4.73
Average Difference
0.283
2.73
0.335
3.20
0.275
2.95
0.325
3.43
0.251
3.27
0.299
3.77
0.192
3.93
0.228
4.43
0.160
4.27
0.190
4.76
Average Difference%
Difference Animals
Died
15.2%

0.51

0.49

Abbreviations: ATC=Acute Toxic Class method (OECD 2001d); NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope; Cyto=NRU-determined starting dose (i.e., one doselower than the LD50 predicted by the NRU IC50 in the RC rat-only weight regression (log LD50 [mg/kg] = 0.372 log IC50 [ g/mL] + 2.024); Default=Default starting dose of 300 mg/kg;
Std. Error=Standard error for number of animals; NHK=Normal human epidermal keratinocytes; 3T3= BALB/c 3T3 mouse fibroblasts; RC=Registry of Cytotoxicity.
1
For 2000 ATC simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit dose
=2000 mg/kg.
2
Mean number of animals for 2000 simulations.
3
Difference between mean number of animals for the default starting dose and mean number of animals for the NRU-based starting dose.
4
P-value is from one-sided Wilcoxon signed rank test for difference in animals between the default and NRU-based starting doses. Significant values at p <0.05.

N-35

In Vitro Cytotoxicity Test Methods BRD Appendix N4

November 2006

Summary of Animals Used and Animals Dead for ATC Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

NHK

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

1
0.12
0.25
3T3

0.50
1.25
2.00

0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

2
0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

1.195
6.18
0.083
9.09
1.250
6.45
0.178
9.35
1.277
6.87
0.158
9.74
1.215
7.52
0.111
10.39
1.225
7.94
0.066
10.85
Average Difference
0.987
6.85
0.081
9.08
0.980
7.12
0.174
9.36
1.029
7.56
0.170
9.77
1.011
8.16
0.093
10.42
1.017
8.61
0.060
10.84
Average Difference
0.333
10.40
0.165
11.76
0.266
10.31
0.180
11.64
0.192
10.31
0.212
11.56
0.261
10.21
0.156
11.54
0.344
10.21
0.089
11.62
Average Difference
0.329
10.27
0.163
11.75
0.350
10.13
0.189
11.64
0.384
10.06
0.214
11.56
0.425
9.98
0.148
11.51
0.445
10.03
0.083
11.58
Average Difference

Animals Died

Difference3

P4

2.91

0.12502.91

0.25002.87

0.25002.87

0.25002.90

0.25002.89
2.24

0.12502.24

0.12502.21

0.12502.26

0.12502.22

0.12502.23
1.36

0.00491.33

0.00341.25

0.00611.33

0.01051.41

0.00341.33
1.48

0.00611.51

0.00241.51

0.00611.52

0.00611.55

0.0046

1.51

N-36

Std.
Error

Number2

1.180
5.96
0.080
8.86
1.156
5.77
0.070
8.67
1.157
5.61
0.060
8.50
1.033
5.26
0.066
7.92
0.940
5.10
0.052
7.51
Average Difference
1.057
6.62
0.081
8.86
1.008
6.44
0.073
8.67
0.998
6.28
0.049
8.50
0.911
5.89
0.081
7.93
0.841
5.68
0.058
7.54
Average Difference
0.618
5.20
0.256
6.55
0.690
5.38
0.313
6.71
0.668
5.66
0.312
6.88
0.504
5.82
0.191
6.91
0.438
5.74
0.142
6.77
Average Difference
0.597
5.06
0.255
6.56
0.645
5.19
0.314
6.72
0.630
5.41
0.313
6.89
0.486
5.64
0.194
6.89
0.426
5.60
0.146
6.77
Average Difference

Difference3

P4

2.91

0.125032.0%%
Difference Animals
Died
32.8%

2.90

0.2500

31.1%

33.5%

2.89

0.1250

29.4%

34.0%

2.66

0.1250

27.6%

33.6%

2.41

0.1250

26.8%

32.1%

2.24

0.1250

24.6%

25.3%

2.23

0.1250

23.9%

25.7%

2.22

0.1250

22.6%

26.1%

2.04

0.1250

21.7%

25.7%

1.86

0.1250

20.5%

24.7%

1.36

0.0144

11.5%

20.7%

1.33

0.0046

11.5%

19.8%

1.22

0.0134

10.8%

17.8%

1.09

0.0046

11.5%

15.8%

1.03

0.0012

12.1%

15.3%

1.49

0.0024

12.6%

22.8%

1.53

0.0024

13.0%

22.8%

1.48

0.0022

13.0%

21.5%

1.25

0.0061

13.2%

18.2%

1.17

0.0024

13.4%

17.3%

% Savings- Animals
Used

2.75

2.12

1.21

1.39

In Vitro Cytotoxicity Test Methods BRD Appendix N4

November 2006

Summary of Animals Used and Animals Dead for ATC Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

NHK

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

3
0.12
0.25
3T3

0.50
1.25
2.00

0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

4
0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

Animals Died

Difference3

P4

0.489
9.63
0.264
9.44
0.407
9.86
0.275
9.89
0.279
10.41
0.207
10.83
0.248
10.91
0.083
11.73
0.302
10.74
0.038
11.82
Average Difference-0.20

0.46880.04

0.30130.42

0.10990.82

0.03421.09

0.0034

0.099
9.20
0.258
9.42
0.155
9.60
0.271
9.90
0.176
10.35
0.202
10.81
0.228
11.11
0.084
11.73
0.253
11.09
0.037
11.80
Average Difference

0.22

0.26470.31

0.04490.47

0.02250.62

0.02100.71

0.0449

0.645
10.23
0.098
9.26
0.565
10.23
0.095
9.43
0.500
10.46
0.062
9.77
0.296
10.91
0.043
10.57
0.093
11.12
0.048
11.10
Average Difference
0.664
10.65
0.102
9.26
0.586
10.56
0.098
9.42
0.496
10.67
0.065
9.75
0.279
10.95
0.040
10.57
0.105
11.12
0.047
11.11
Average Difference

0.43

0.47
-0.97

0.1445-0.80

0.1089-0.69

0.6416-0.34

0.0256-0.02

0.4851

-0.57
-1.39

0.0762-1.14

0.0437-0.93

0.0229-0.38

0.0928-0.01

0.4212

-0.77

N-37

Std.
Error

Number2

0.073
3.12
0.217
3.41
0.139
3.35
0.237
3.80
0.155
3.62
0.171
4.26
0.193
4.27
0.119
5.03
0.218
4.60
0.091
5.41
Average Difference
0.104
3.17
0.213
3.40
0.165
3.50
0.237
3.80
0.160
3.83
0.169
4.26
0.180
4.51
0.119
5.04
0.186
4.88
0.095
5.41
Average Difference

Difference3

P4

0.28

0.0713-2.1%%
Difference Animals
Died
8.3%

0.45

0.0210

0.4%

11.8%

0.64

0.0093

3.8%

15.1%

0.76

0.0210

7.0%

15.1%

0.81

0.0342

9.2%

15.1%

0.23

0.1294

2.4%

6.7%

0.30

0.1060

3.1%

7.9%

0.43

0.0522

4.3%

10.1%

0.52

0.0210

5.3%

10.4%

0.53

0.0640

6.0%

9.8%% Savings- Animals
Used

0.59

0.40

0.068
3.045
0.067
3.040
0.093
3.13
0.092
3.12
0.058
3.20
0.057
3.18
0.057
3.64
0.067
3.70
0.070
4.04
0.080
4.16
Average Difference-0.0050.2444-10.4%

-0.1%

-0.01

0.0229

-8.5%

-0.4%

-0.02

0.0744

-7.1%

-0.6%

0.06

0.3259

-3.3%

1.6%

0.13

0.6791

-0.2%

3.0%

0.068
3.04
0.068
3.04
0.094
3.13
0.095
3.13
0.057
3.18
0.056
3.18
0.053
3.62
0.069
3.71
0.061
4.00
0.077
4.17
Average Difference

0.00

0.8160-15.0%

0.0%

0.00

0.5871-12.1%

0.0%

-0.01

0.4691

-9.5%

-0.2%

0.08

0.1208

-3.6%

2.2%

0.17

0.1089

-0.1%

4.2%

0.03

0.05

In Vitro Cytotoxicity Test Methods BRD Appendix N4

November 2006

Summary of Animals Used and Animals Dead for ATC Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

NHK

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

5
0.12
0.25
3T3

0.50
1.25
2.00

0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

6
0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

0.604
11.03
0.101
11.87
0.528
10.88
0.119
11.63
0.365
10.69
0.083
11.22
0.175
10.52
0.038
10.77
0.094
10.69
0.011
10.83
Average Difference
0.876
9.44
0.103
11.88
0.751
9.54
0.117
11.62
0.514
9.80
0.083
11.22
0.260
10.08
0.037
10.77
0.127
10.49
0.010
10.85
Average Difference
0.853
8.75
0.000
12.00
0.847
8.75
0.002
11.99
0.776
8.91
0.034
11.86
0.481
9.44
0.058
11.23
0.318
9.89
0.031
10.98
Average Difference
0.912
8.67
0.000
12.00
0.912
8.68
0.002
11.99
0.833
8.83
0.035
11.85
0.542
9.41
0.060
11.22
0.346
9.86
0.029
10.99
Average Difference

Animals Died

Difference3

P4

0.84

0.88670.75

0.32230.53

0.15230.24

0.19340.14

0.19340.50
2.43

0.07422.08

0.19341.43

0.02730.69

0.02730.36

0.02731.40
3.25

0.00683.25

0.01052.94

0.00811.78

0.00851.09

0.0266

2.46
3.33

0.00983.31

0.00683.02

0.00681.81

0.01221.12

0.0161

2.52

N-38

Difference3

P4

0.188
0.393
0.189
0.390
0.156
1.10
0.157
1.08
0.098
1.819
0.097
1.817
0.041
2.87
0.041
2.89
0.034
3.37
0.026
3.35
Average Difference-0.0030.1055

7.1%%
Difference Animals
Died
-0.9%

-0.02

0.0098

6.4%

-1.8%

-0.0020.7891

4.7%

-0.1%

0.01

0.3223

2.2%

0.5%

-0.01

0.7695

1.3%

-0.3%

0.184
0.38
0.188
0.39
0.150
1.09
0.158
1.09
0.095
1.80
0.093
1.82
0.052
2.87
0.041
2.88
0.046
3.41
0.027
3.35
Average Difference

0.01

0.1856

20.5%

2.1%

-0.01

0.5566

17.9%

-0.5%

0.02

0.0488

12.7%

1.1%

0.01

0.6953

6.4%

0.4%

-0.06

0.1055

3.3%

-1.6%

Std.
Error

Number2% Savings- Animals
Used

0.00

0.00

0.000220.00050.000210.0004
0.033
0.11
0.033
0.10
0.099
0.72
0.099
0.73
0.106
2.12
0.086
2.27
0.090
2.82
0.053
2.93
Average Difference-0.0001

0.531327.1%-27.3%-0.0004

0.1099

27.1%

-3.8%

0.02

0.0327

24.8%

2.3%

0.15

0.0085

15.9%

6.4%

0.11

0.02889.9%

3.8%

0.000200.00050.00018
0.003
0.034
0.11
0.035
0.12
0.106
0.74
0.106
0.74
0.117
2.12
0.096
2.27
0.095
2.83
0.057
2.95
Average Difference-0.00017

0.250027.7%-50.0%

0.01

0.0210

27.6%

4.4%

0.00

0.8057

25.5%

-0.3%

0.15

0.0269

16.1%

6.6%

0.12

0.0269

10.2%

4.0%

0.05

0.05

In Vitro Cytotoxicity Test Methods BRD Appendix N4

November 2006

Abbreviations: ATC=Acute Toxic Class method (OECD 2001d); Toxcat=Category from Globally Harmonized System of Classification and Labeling of Chemicals5 (GHS; UN 2005);
NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope; Cyto=NRU-determined starting dose (i.e., one dose lower than the LD50 predicted by the NRU IC50 in the RC rat-only
weight regression (log LD50 [mg/kg] = 0.372 log IC50 [µg/mL] + 2.024); Default=Default starting dose of 300 mg/kg; Std. Error=Standard error for number of animals; NHK=Normal
human epidermal keratinocytes; 3T3= BALB/c 3T3 mouse fibroblasts; RC=Registry of Cytotoxicity.
1
For 2000 ATC simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit dose
=2000 mg/kg.
2
Mean number of animals for 2000 simulations.
3
Difference between mean animals used for the default starting dose and mean animals used for the NRU-based starting dose.
4
P-value is from one-side Wilcoxon signed rank test for difference in animals between the default and NRU-based starting doses. Significant values at p <0.05.
5
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

N-39

In Vitro Cytotoxicity Test Methods BRD Appendix N4

November 2006

Concordance of NRU-Based Starting Dose with Default Starting Dose for GHS Acute Oral Toxicity Category Outcome for
ATC Simulations1
GHS Category Outcome with NHK NRU-Based Starting DoseGHS Category Outcome
with Default Starting Dose

1

2

3

4

5

6

Total

Category
Match

1
2
3
4
5
6
Total

8
0
0
0
0
0
8

0
11
1
0
0
0
12

0
0
13
1
0
0
14

0
0
0
12
0
0
12

0
0
0
0
21
0
21

0
0
0
0
0
1
1

8
11
14
13
21
1
68

100%
100%
93%
92%
100%
100%
97%

HigherNRU
Category
0%
0%
0%
0%
0%
0%
0%

Lower
NRU
Category
0%
0%
7%
8%
0%
0%
3%

HigherNRU
Category
0%
0%
0%
0%
0%
0%
0%

Lower
NRU
Category
0%
0%
0%
8%
0%
0%
1%

GHS Category Outcome with 3T3 NRU-Based Starting DoseGHS Category Outcome
with Default Starting Dose

1

2

3

4

5

6

Total

Category
Match

1
2
3
4
5
6
Total

8
0
0
0
0
0
8

0
11
0
0
0
0
11

0
0
15
1
0
0
16

0
0
0
11
0
0
11

0
0
0
0
20
0
20

0
0
0
0
0
1
1

8
11
15
12
20
1
67

100%
100%
100%
92%
100%
100%
99%

Abbreviations: NRU=Neutral red uptake; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); ATC=Acute Toxic Class method (OECD 2001d); NHK=Normal
human epidermal keratinocytes; 3T3= BALB/c 3T3 mouse fibroblasts; RC=Registry of Cytotoxicity.
1
For 2000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit dose
=2000 mg/kg. The NRU-based starting dose was one dose lower than the LD50 predicted by the NRU IC50 in the RC rat-only weight regression (log LD50 [mg/kg] = 0.372 log IC50 [µg/mL]+ 2.024).. The default starting dose = 300 mg/kg. Shaded cells are those containing the correct predictions.
2
GHS Toxicity Category
Oral LD50 Limit
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

N-40

In Vitro Cytotoxicity Test Methods BRD Appendix N4

November 2006

Discordant Substances1 for GHS Category2 Outcomes of ATC Simulations
NRU Test
Method3T3
NHKNHK

Substance
Cupric sulfate pentahydrate
Hexachlorophene
Propranolol

NRU-Based Starting Dose3
Toxicity Category323

Default Starting Dose4
Toxicity Category434Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); ATC= Acute
Toxic Class method (OECD 2001d); 3T3=BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes;
NRU=Neutral red uptake.
1
Substances for which the simulated ATC outcome for the NRU-based starting dose did not match the simulated
ATC outcome for the default starting dose. Simulations were performed with 2000 runs at each starting doseand dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU
test method. Upper limit dose =2000 mg/kg.
2
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg
3
NRU-based starting dose was one dose lower than the LD50 predicted by the NRU IC50 in the RC rat-only
weight regression (log LD50 [mg/kg] = 0.372 log IC50 [µg/mL] + 2.024).
4
The default starting dose = 300 mg/kg.

N-41

In Vitro Cytotoxicity Test Methods BRD Appendix N4

November 2006

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N-42

In Vitro Cytotoxicity Test Methods BRD Appendix O

November 2006

Appendix O

Federal Register Notices

O1

70FR14473 Request for Nominations for an Independent Peer Review
Panel To Evaluate In Vitro Testing Methods for Estimating Acute Oral
Systemic Toxicity and Request for In Vivo and In Vitro Data...........................O-3

O2

69FR61504 Availability of Updated Standardized In Vitro Cytotoxicity
Test Method Protocols for Estimating Acute Oral Systemic Toxicity;
Request for Existing In Vivo and In Vitro Acute Toxicity Data ........................O-5

O3

69FR1148 Notice of the Availability of Agency Responses to ICCVAM Test
Recommendations for the Revised Up-and-Down Procedure for Determining
Acute Oral Toxicity and In Vitro Methods for Assessing Acute Systemic
Toxicity...........................................................................................................O-7

O4

66FR49686 Report of the International Workshop on In Vitro Methods forAssessing Acute Systemic Toxicity; Guidance Document on Using In Vitro
Data to Estimate In Vivo Starting Doses for Acute Toxicity: Notice of
Availability and Request for Public Comment .................................................O-9

O5

65FR57203 Notice of an International Workshop on In Vitro Methods forAssessing Acute Systemic Toxicity, co-sponsored by NIEHS, NTP and
the U.S. Environmental Protection Agency (EPA): Workshop Agenda andRegistration Information................................................................................O-11

O6

65FR37400 Notice of an International Workshop on In Vitro Methods forAssessing Acute Systemic Toxicity, co-sponsored by NIEHS, NTP and
the U.S. Environmental Protection Agency (EPA): Request for Data and
Suggested Expert Scientists ...........................................................................O-15

O-1

In Vitro Cytotoxicity Test Methods BRD Appendix O

November 2006

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O-2In Vitro Cytotoxicity Test Methods BRD Appendix P

November 2006

Appendix P

In Vitro Cytotoxicity Test Methods and the High Production Volume(HPV) Challenge Program
P1

Supplemental Acute Toxicity Protocol ..........................................P-3

P2

Office of Pollution Prevention and Toxics (OPPT)
Letters to Manufacturers/Importers .............................................P-9

P-1

In Vitro Cytotoxicity Test Methods BRD Appendix P

November 2006

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P-2

In Vitro Cytotoxicity Test Methods BRD Appendix P1

November 2006

Appendix P1

Supplemental Acute Toxicity Protocol

P-3

In Vitro Cytotoxicity Test Methods BRD Appendix P1

November 2006

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P-4

In Vitro Cytotoxicity Test Methods BRD Appendix P1

November 2006

U.S. EPA/OPPTS/OPPT/High Production Volume (HPV) Challenge Program[NOTE: This statement was extracted from the EPA web site. The original can be visited at:
http://www.epa.gov/chemrtk/toxprtcl.htm]
Supplemental Acute Toxicity Protocol
The EPA, along with the National Toxicology Program and the National Institute ofEnvironmental Health Sciences (NIEHS), sponsored an International Workshop on In Vitro
Methods held on October 17-20, 2000, to review the validation status of available in vitro
methods for predicting acute oral toxicity, among other goals.
The October 2000 Workshop concluded that in vitro cytotoxicity data could be useful inestimating starting doses for in vivo acute toxicity testing, and in this way could also reduce
the number of animals used in subsequent in vivo tests. The two candidate cytotoxicity tests
recommended for use with the regression model for estimating starting doses from in vitro
cytotoxicity data are neutral red uptake assays using BALB/c 3T3 mouse fibroblasts andnormal human keratinocytes. Other cell lines/cells could also be used with the regression
model to estimate starting doses, but first the correlation between the in vitro test and the invivo test must be established quantitatively. Guidance on these in vitro tests, protocols for useof recommended tests, and a reporting template for results of in vitro tests are all contained in
the ICCVAM Guidance Document (2001), which is one of the products of the OctoberWorkshop. Further background on the October workshop can be found in the ICCVAM
Workshop Report (2001).
While the formal request to EPA from NIH that would ask the Agency to accept or reject
these protocols has not yet been received (nor have these methods been incorporated inOECD or the EPA acute toxicity test guidelines), the findings of this workshop included a
recommendation to all Agencies participating in ICCVAM to consider the use of these invitro cytotoxicity tests as supplements to the current acute oral in vivo acute toxicity
protocols. These in vitro cytotoxicity protocols were recognized earlier in Steven Johnson's
letter of October 30, 2001. The in vitro tests are supplements to, not replacements for, theP-5

In Vitro Cytotoxicity Test Methods BRD Appendix P1

November 2006

OECD acute toxicity test guideline 425 (known as the Up-and-Down Procedure) which iscurrently recommended for use in the HPV Challenge Program. The new in vitro tests areintended to better estimate the starting doses for new in vivo acute oral toxicity studies
conducted under the HPV Challenge.
We encourage those participating in the HPV Challenge Program to consider using therecommended in vitro tests noted here as a supplemental component in conducting any new
in vivo acute oral toxicity studies under the HPV Challenge Program, to note the intention to
use these protocols in HPV Challenge test plans submitted to EPA, and to summarize theresults using the recommended reporting template. This information on the in vitro templateshould accompany results from the in vivo acute oral tests, and be provided to EPA as part of
the HPV Challenge Program. The October workshop documents and the recommended
reporting template for the in vitro tests can be found below. The ICCVAM website - In Vitro
methods page - should be consulted for any future updates to the in vitro guidance
methodologies prior to proceeding with testing.
In order to gain more familiarity with these methods, technical experts from industry andother organizations were invited to a workshop sponsored by EPA, NIEHS, and others onthese in vitro methods. The workshop was held February 19-21, 2001 (see the ICCVAM
website at http://iccvam.niehs.nih.gov/meetings/schedule.htm for more details).
ICCVAM (Interagency Coordinating Committee on the Validation of Alternative
Methods)
Report of the International Workshop on In Vitro Methods for Assessing Acute
Systemic Toxicity. 2001. NIH Publication No. 01-4499. National Institute of Environmental
Health Sciences (NIEHS)ICCVAM (Interagency Coordinating Committee on the Validation of Alternative
Methods)

P-6

In Vitro Cytotoxicity Test Methods BRD Appendix P1

November 2006

Guidance Document on Using In Vitro Data to Estimate In Vivo Starting Doses for
Acute Toxicity. 2001. NIH Publication No. 01-4500. National Institute of Environmental
Health Sciences (NIEHS)
Standard Test Reporting Template
Any updates to this methodology can be found under In Vitro Methods on the Interagency
Coordinating Committee on the Validation of Alternative Methods (ICCVAM) web
site.
-----------------------------------------------------------------------Last updated on September 16, 2002
Visit the ICCVAM Home Page

P-7

In Vitro Cytotoxicity Test Methods BRD Appendix P1

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P-8

In Vitro Cytotoxicity Test Methods BRD Appendix P2

November 2006

Appendix P2

Office of Pollution Prevention and Toxics (OPPT)
Letters to Manufacturers/Importers

P-9

In Vitro Cytotoxicity Test Methods BRD Appendix P2

November 2006

[This Page Intentionally Left Blank]

P-10

In Vitro Cytotoxicity Test Methods BRD Appendix P2

November 2006

Office of Pollution Prevention
And Toxics

Letters to Manufacturers/Importers[High Production Volume Voluntary Challenge Program]
October 14, 1999
Company nameStreet #City, State, Zip
Dear Company Contact:
On behalf of the Environmental Protection Agency (EPA), I would like to thank you for yourcommitment to participate in the voluntary High Production Volume Challenge (HPV)
program. We look forward to working with you over the coming years as we achieve ourgoals for this important program.
As you may be aware, a number of animal protection organizations and the public haveraised concerns that the HPV Challenge program may lead to the excessive use of animals in
tests and to inadequate attention to existing information and alternative testing methods that
do not require animals as test subjects. As a general matter, animal experiments should not be
performed if another validated method -- not involving the use of animals -- is reasonably
and practically available for use in the HPV Challenge program. To respond to theseconcerns, and after consultation with the organizations involved in developing the framework
for this initiative, I am asking you and your fellow HPV Challenge participants to observe thefollowing principles as we proceed with the program:
1. In analyzing the adequacy of existing data, participants shall conduct a thoughtful,qualitative analysis rather than use a rote checklist approach. Participants may conclude that
there is sufficient data, given the totality of what is known about a chemical, including
human experience, that certain endpoints need not be tested.
2. Participants shall maximize the use of existing and scientifically adequate data to minimize
further testing. To reinforce this approach, EPA will consider information contained in the
databases identified in the enclosure, or in databases maintained by the organizations
identified in the enclosure, to have been known to the Agency within the meaning of Section
8(e) of the Toxic Substances Control Act (TSCA), 42 U.S.C. 2607(e). This policy is limited
to information reported by participants under the HPV Challenge
program and generated for or contained in these databases as of the date of this letter. In
addition, any other potential liability under TSCA Section 8(e) for existing data on HPVChallenge program chemicals will be limited according to the terms of the “Registration

P-11

In Vitro Cytotoxicity Test Methods BRD Appendix P2

November 2006

Agreement for TSCA Section 8(e) Compliance Audit Program (56 Fed. Reg. 4128, Feb. 1,
1991).” This policy does not affect prior 8(e) enforcement actions.
3. Participants shall maximize the use of scientifically appropriate categories of related
chemicals and structure activity relationships.
4. Consistent with the Screening Information Data Set (SIDS) program of the Organization
for Economic Cooperation and Development (OECD), participants shall not conduct any
terrestrial toxicity testing.
5. Participants are encouraged to use in vitro genetic toxicity testing to generate any needed
genetic toxicity screening data, unless known chemical properties preclude its use.
6. Consistent with the OECD/SIDS program, participants generally should not develop any
new dermal toxicity data.
7. Participants shall not develop sub-chronic or reproductive toxicity data for the HPV
chemicals that are solely closed system intermediates, as defined by the OECD/SIDS
guidelines.
8. In analyzing the adequacy of screening data for chemicals that are substances Generally
Recognized as Safe (GRAS) for a particular use by the Food and Drug Administration(FDA), participants should consider all relevant and available information supporting the
FDA's conclusions. Participants reviewing the adequacy of existing data for these chemicals
should specifically consider whether the information available makes it unnecessary toproceed with further testing involving animals. As with all chemicals, before generating new
information, participants should further consider whether any additional information obtained
would be useful or relevant.
9. Because validated non-animal tests for some SIDS endpoints may be available soon,
participants shall make the following revisions to the sequence of testing:(a) Testing of closed system intermediates, which present less risk of exposure, shall bedeferred until 2003;(b) Individual chemicals (i.e., those HPV chemicals not proposed for testing in a category)that require further testing on animals shall be deferred until November 2001.
These revisions should not be construed to suggest that delay or deferral is appropriate withrespect to testing of scientifically appropriate categories of related chemicals.
10. Companies shall allow 120 days between the posting of test plans and the implementationof any testing plans.
To promote the availability and use of alternatives to tests involving animals, the National
Institute of Environmental Health Sciences (NIEHS) and the National Toxicology Program

P-12

In Vitro Cytotoxicity Test Methods BRD Appendix P2

November 2006

(NTP) will commit at least $1.5 million in FY 2000, and $3 Million in FY 2001, and any
further funds appropriated by Congress, to the development and validation of non-animalalternative test methods and protocols. EPA will provide an additional $250,000 this year andwill seek to provide a similar amount next year to these efforts. The Multicenter Evaluationof In Vitro Cytotoxicity (MEIC), on the agenda for the October 14 meeting of NTP's
Advisory Committee on Alternative Toxicological Methods, will be given priority attention.
EPA will promptly incorporate, as appropriate, the work of NIEHS and NTP into the HPVChallenge program.
EPA recognizes that the HPV Challenge is a voluntary program that includes substantial
public review and involvement. The successful implementation of the changes described inthis letter will depend upon the good faith effort and cooperation of all parties. We appreciate
the spirit of cooperation and commitment that has characterized this initiative to date. Thechanges to the HPV Challenge program outlined above present the opportunity to advanceour shared goals of expanding the basic health data available to the public, while
incorporating certain animal welfare concerns and scientific principles. It is the intention of
the Agency that the HPV Challenge program, including the test rule(s), should proceed in amanner that is consistent with these principles and concerns.
Again, I thank you for your commitment to participate in the HPV Challenge program. If you
need further clarification or assistance with this program, please contact Barbara Leczynski
at 202-260-3749 or visit the website at www.epa.gov/chemrtk.
Sincerely,/s/Susan H. Wayland
Deputy Assistant Administrator
Enclosure

P-13

In Vitro Cytotoxicity Test Methods BRD Appendix P2

November 2006

ENCLOSURE A
The IUCLID database administered by the European Union’s Existing Chemicals Bureau
Aquatic Information Retrieval (AQUIRE)
Catalog of Teratogenic Agents (CTA)
Chemical Carcinogenesis Research Information System (CCRIS)
Chemical Information System (CIS)The ChemID database of the National Library of Medicine (NLM)
Datalog
Developmental and Reproductive Technology (DART)
Envirofate Environmental Mutagen Information Center (EMIC)
Environmental Teratology Information Center (ETIC/ETICBACK)
GENE-TOX
Hazardous Substances Data Bank (HSDB)
Integrated Risk Management System (IRIS)
Merck Index National Institute for Occupational Safety and Health (NIOSH)
National Library of Medicine TOXLINE and TOXNETNational Toxicology Program (NTP) Testing Information and Study Results
NTP Technical Reports
NTP Chemical Health and Safety Data
Phytotox Registry of Toxic Effects of Chemical Substances
Structure and Nomenclature Search System (SANSS)
Toxics Substances Control Act Test Submissions (TSCATS)
WHO/IPCS Documents (CICADS and Environmental Health Criteria Documents) BIODEG
BIOLOGCANCERLITCHEMFATECHRISFIFRA Database/MRID
IRAC Documents
MEDLINE
National Cancer Institute Journal
POISINDEX
Shepard’s Catalog
STN (Chemical Abstracts Service)
Document Source: http://www.epa.gov/opptintr/chemrtk/ceoltr.htm

P-14

In Vitro Cytotoxicity Test Methods BRD Appendix Q

November 2006

Appendix Q

Additional UDP Simulation Modeling Results
Q1

UDP Simulation Results for the RC Rat-Only Millimole Regression
Starting at the LD50 Predicted by the 3T3 and NHK NRU
IC50 - 5000 mg/kg Upper Limit Dose ............................................ Q-3

Q2

UDP Simulation Results for the RC Rat-Only Weight Regression
Starting at the LD50 Predicted by the 3T3 and NHK NRU
IC50 - 5000 mg/kg Upper Limit Dose .......................................... Q-13

Q-1

In Vitro Cytotoxicity Test Methods BRD Appendix Q

November 2006

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Q-2

In Vitro Cytotoxicity Test Methods BRD Appendix Q1

November 2006

Appendix Q1

UDP Simulation Results for the RC Rat-Only Millimole Regression
Starting at the LD50 Predicted by the 3T3 and NHK NRU IC50 5000 mg/kg Upper Limit DoseQ-3

In Vitro Cytotoxicity Test Methods BRD Appendix Q1

November 2006

[This Page Intentionally Left Blank]

Q-4

In Vitro Cytotoxicity Test Methods BRD Appendix Q1

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by NRU Test Method1
NRU
Test
Method

Sigma
0.12
0.25

3T3

0.50
1.25
2.00

0.12
0.25
NHK

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Animals Used
Std.
Error

Number2

Difference3

P4

-0.16

0.1577%
Savings Animals
Used
7.9%

-0.19

0.1615

7.2%

-4.9%

-0.19

0.2406

6.8%

-4.6%

-0.12

0.8288

6.7%

-2.7%

-0.08

0.8530

6.0%

-1.8%

-0.18

0.1299

6.3%

-5.3%

-0.19

0.1848

6.3%

-5.0%

-0.20

0.1974

5.9%

-4.9%

-0.13

0.7671

6.2%

-2.8%

-0.09

0.7533

5.6%

-1.9%

Animals Died
Std.
Error

Difference3

P4

0.20997.35
0.17507.97
0.20538.06
0.17468.69
0.19048.72
0.16149.35
0.16499.27
0.13109.94
0.14219.41
0.0956
10.02
Average Difference

0.63

0.00170.63

0.00360.63

0.00440.67

0.00220.60

0.00110.22257.44
0.1741
7.94
0.21248.12
0.16978.67
0.19198.79
0.15439.35
0.16339.34
0.12419.96
0.14059.47
0.0921
10.03
Average Difference

0.50

0.00600.54

0.00500.56

0.00450.62

0.00100.56

0.00050.63

0.56

Number2

0.22703.62
0.19993.45
0.2257
3.97
0.1955
3.78
0.21664.31
0.1821
4.12
0.19174.67
0.14914.55
0.16784.76
0.1265
4.68
Average Difference
0.23573.58
0.20233.40
0.2317
3.91
0.19673.73
0.21914.28
0.18124.08
0.19314.66
0.14784.53
0.1696
4.75
0.1249
4.66
Average Difference%
Difference Animals
Died
-4.7%

-0.15

-0.16

Abbreviations: UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a); NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope; Cyto=NRU-determined starting dose
(i.e., LD50 predicted by the NRU IC50 in the RC rat-only millimole regression (log LD50 [mmol/kg] = 0.439 log IC50 [mM] + 0.621); Default=Default starting dose of 175 mg/kg; Std.
Error=Standard error for number of animals; 3T3= BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit
dose =5000 mg/kg.
2
Mean number of animals for 10,000 simulations.
3
Difference between mean number of animals used for the default starting dose and mean number of animals used for the NRU-based starting dose.
4
P-value is from one-sided Wilcoxon signed rank test for difference in animals between the default and NRU-based starting doses. Significant values at p <0.05.

Q-5

In Vitro Cytotoxicity Test Methods BRD Appendix Q1

November 2006

Summary of Stopping Rules Used for the UDP Simulations by NRU Test Method1
NRU Test
MethodSigma

Starting Dose

3 Animals at
Limit Dose2

5 Reversals2

Likelihood
Ratio2

Maximum
Number of
Animals Used2

0.12

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

15.8%
15.4%
15.2%
14.6%
13.8%
13.0%
10.5%
9.1%
9.4%
7.4%
17.0%
16.6%
16.3%
15.8%
14.4%
13.8%
10.5%
9.5%
9.2%
7.6%

58.7%
57.3%
33.9%
34.3%
19.7%
20.0%
13.2%
13.6%
12.1%
12.5%
54.8%
56.3%
32.7%
33.7%
19.3%
19.9%
13.3%
13.5%
12.0%
12.5%

24.3%
24.9%
48.3%
45.9%
60.4%
57.5%
64.7%
60.9%
65.4%
62.5%
26.7%
24.8%
48.0%
45.5%
59.6%
56.9%
64.2%
60.5%
65.2%
62.1%

1.1%
2.4%
2.7%
5.2%
6.1%
9.6%
11.6%
16.3%
13.2%
17.6%
1.5%
2.3%
3.0%
5.1%
6.6%
9.5%
11.9%
16.4%
13.6%
17.7%

0.25

3T3

0.5
1.25
2
0.12
0.25

NHK

0.5
1.25
2

Abbreviations: UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a); NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope;
Cyto=NRU-determined starting dose (i.e., the LD50 predicted by the NRU IC50 in the RC rat-only millimole regression (log LD50 [mmol/kg] =0.439 log IC50 [mM] + 0.621); Default=Default starting dose of 175 mg/kg; 3T3= BALB/c 3T3 mouse fibroblasts; NHK=Normal humanepidermal keratinocytes; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in
the NHK NRU test method. Upper limit dose =5000 mg/kg.
2
Percentage of the 10,000 test simulations that satisfied the specified condition for completion of testing (see OECD [2001a]; EPA [2002a]).

Q-6

In Vitro Cytotoxicity Test Methods BRD Appendix Q1

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

3T3

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

1
0.12
0.25
NHK

0.50
1.25
2.00

0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

2
0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

0.650
9.65
0.273
9.65
0.642
10.24
0.192
10.47
0.646
10.87
0.201
11.22
0.624
11.27
0.141
11.67
0.563
11.16
0.123
11.41
Average Difference
0.815
9.91
0.292
9.61
0.693
10.44
0.267
10.39
0.629
11.05
0.257
11.14
0.583
11.43
0.176
11.65
0.561
11.26
0.155
11.40
Average Difference
0.433
9.04
0.284
8.44
0.460
9.66
0.208
8.98
0.491
10.24
0.227
9.53
0.449
10.71
0.236
10.06
0.364
10.70
0.178
10.12
Average Difference
0.494
9.17
0.263
8.41
0.473
9.79
0.160
8.96
0.498
10.33
0.153
9.52
0.471
10.77
0.179
10.07
0.392
10.77
0.147
10.14
Average Difference

Animals Died

Difference3

P4

0.01

0.87500.23

0.62500.35

0.62500.40

0.62500.25

0.62500.25-0.30

0.8750-0.05

1.00000.09

0.87500.22

0.87500.15

0.87500.02-0.60

0.1272-0.68

0.1099-0.71

0.1272-0.65

0.0942-0.58

0.0942

-0.64
-0.76

0.0942-0.83

0.0803-0.81

0.0942-0.71

0.0803-0.63

0.0574

-0.75

Q-7

Std.
Error

Number2

0.586
6.31
0.170
6.37
0.579
6.69
0.162
6.84
0.596
7.14
0.198
7.31
0.587
7.30
0.161
7.52
0.532
7.05
0.140
7.21
Average Difference
0.682
6.46
0.178
6.35
0.603
6.84
0.195
6.81
0.578
7.28
0.232
7.28
0.565
7.44
0.188
7.53
0.532
7.15
0.153
7.20
Average Difference
0.417
5.64
0.241
5.11
0.428
5.99
0.201
5.43
0.447
6.31
0.209
5.71
0.425
6.52
0.216
5.92
0.361
6.41
0.177
5.87
Average Difference
0.486
5.66
0.231
5.09
0.478
6.02
0.183
5.41
0.495
6.33
0.179
5.70
0.480
6.53
0.192
5.91
0.417
6.42
0.164
5.87
Average Difference

Difference3

P4

0.06

0.87500.1%%
Difference Animals
Died
1.0%

0.15

0.8750

2.2%

2.2%

0.18

0.6250

3.1%

2.4%

0.22

0.6250

3.4%

2.9%

0.17

0.6250

2.2%

2.3%

-0.11

1.0000

-3.1%

-1.7%

-0.03

1.0000

-0.5%

-0.4%

0.00

1.0000

0.8%

0.0%

0.09

0.8750

1.9%

1.1%

0.05

0.8750

1.3%

0.7%

-0.53

0.0942-7.1%-10.4%-0.56

0.1099-7.6%-10.4%-0.61

0.0942-7.4%-10.6%-0.61

0.0942-6.5%-10.2%-0.53

0.1099

-5.7%

-9.1%

-0.57

0.1677-9.1%-11.1%-0.60

0.0942-9.2%-11.1%-0.63

0.0942-8.5%-11.1%-0.62

0.0681-7.0%-10.4%-0.55

0.0803

-6.2%

-9.4%

% Savings- Animals
Used

0.15

0.00

-0.67

-0.59

In Vitro Cytotoxicity Test Methods BRD Appendix Q1

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

3T3

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

3
0.12
0.25
NHK

0.50
1.25
2.00

0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

4
0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

0.255
7.32
0.212
6.71
0.269
8.07
0.138
7.28
0.274
8.71
0.094
7.76
0.193
9.35
0.059
8.56
0.120
9.54
0.038
9.07
Average Difference
0.258
7.11
0.222
6.67
0.297
7.78
0.173
7.23
0.271
8.45
0.107
7.77
0.168
9.13
0.061
8.61
0.104
9.42
0.037
9.09
Average Difference
0.156
6.76
0.259
7.54
0.181
7.33
0.231
8.04
0.197
7.85
0.237
8.64
0.162
8.61
0.154
9.24
0.121
9.01
0.089
9.44
Average Difference
0.202
6.95
0.257
7.54
0.208
7.44
0.219
8.07
0.221
7.93
0.233
8.66
0.188
8.68
0.150
9.24
0.136
9.04
0.090
9.45
Average Difference

Animals Died

Difference3

P4

-0.61

0.0522-0.78

0.0093-0.94

0.0093-0.79

0.0049-0.48

0.0068

-0.72
-0.44

0.0923-0.56

0.0269-0.68

0.0269-0.52

0.0093-0.33

0.0425-0.51
0.78

0.00920.71

0.00890.79

0.00920.63

0.00920.43

0.00520.67
0.59

0.08330.63

0.03860.72

0.02900.57

0.02900.41

0.0155

0.58

Q-8

Std.
Error

Number2

0.213
4.02
0.133
3.42
0.221
4.42
0.094
3.76
0.220
4.75
0.077
4.05
0.170
5.04
0.056
4.45
0.128
5.10
0.047
4.69
Average Difference
0.196
3.80
0.139
3.40
0.222
4.17
0.112
3.73
0.210
4.51
0.083
4.04
0.154
4.83
0.059
4.47
0.118
4.95
0.048
4.69
Average Difference
0.053
3.31
0.078
3.45
0.050
3.58
0.060
3.67
0.053
3.81
0.059
3.93
0.051
4.17
0.022
4.19
0.045
4.35
0.018
4.29
Average Difference
0.092
3.43
0.077
3.45
0.087
3.66
0.057
3.69
0.087
3.88
0.059
3.94
0.073
4.23
0.022
4.19
0.056
4.39
0.017
4.29
Average Difference

Difference3

P4

-0.60

0.0161-9.1%%
Difference Animals
Died-17.5%-0.66

0.0068-10.8%-17.4%-0.70

0.0068-12.2%-17.3%-0.58

0.0068-9.2%-13.1%-0.42

0.0122

-5.3%

-8.9%

-0.40

0.0269-6.6%-11.8%-0.44

0.0640-7.7%-11.8%-0.47

0.1294-8.8%-11.7%-0.36

0.0923

-6.0%

-8.1%

-0.26

0.0923

-3.7%

-5.6%

0.13

0.1754

10.3%

3.9%

0.09

0.0386

8.8%

2.5%

0.13

0.0443

9.2%

3.2%

0.02

0.1754

6.8%

0.5%

-0.06

0.0577

4.6%

-1.4%

0.02

0.4637

7.8%

0.5%

0.03

0.0739

7.8%

0.8%

0.06

0.1167

8.4%

1.5%

-0.04

0.3755

6.1%

-0.9%

-0.10

0.0443

4.3%

-2.4%

% Savings- Animals
Used

-0.59

-0.39

0.06

-0.01

In Vitro Cytotoxicity Test Methods BRD Appendix Q1

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

3T3

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

5
0.12
0.25
NHK

0.50
1.25
2.00

0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

6
0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

0.299
7.16
0.216
9.19
0.233
8.10
0.141
10.39
0.178
8.54
0.090
10.79
0.141
8.60
0.062
10.75
0.118
8.68
0.055
10.45
Average Difference
0.358
7.38
0.218
9.19
0.314
8.26
0.111
10.38
0.240
8.75
0.062
10.77
0.156
8.81
0.049
10.72
0.123
8.86
0.038
10.42
Average Difference
0.561
5.71
0.576
7.74
0.536
6.56
0.531
8.64
0.399
7.65
0.337
9.84
0.245
8.41
0.062
10.89
0.196
8.45
0.022
10.78
Average Difference
0.561
5.87
0.548
7.63
0.534
6.80
0.486
8.59
0.392
7.95
0.309
9.83
0.226
8.67
0.059
10.87
0.180
8.67
0.021
10.78
Average Difference

Animals Died

Difference3

P4

2.03

0.00202.29

0.00202.25

0.00202.15

0.00201.77

0.00202.10
1.81

0.00202.12

0.00202.02

0.00201.91

0.00201.56

0.00201.89
2.03

0.00052.08

0.00052.19

0.00052.48

0.00052.34

0.00052.22
1.76

0.00021.79

0.00021.88

0.00022.20

0.00022.11

0.0002

1.95

Q-9

Std.
Error

Number2

0.035
3.18
0.045
3.32
0.031
3.43
0.075
3.59
0.050
3.53
0.071
3.68
0.045
3.62
0.034
3.91
0.040
3.74
0.017
4.00
Average Difference
0.058
3.22
0.056
3.32
0.049
3.44
0.081
3.60
0.041
3.57
0.079
3.66
0.035
3.67
0.041
3.89
0.036
3.79
0.024
3.99
Average Difference

Difference3

P4

0.14

0.064522.1%%
Difference Animals
Died
4.2%

0.16

0.0645

22.1%

4.6%

0.14

0.0488

20.9%

3.8%

0.28

0.0020

20.0%

7.3%

0.26

0.0020

16.9%

6.5%

0.10

0.3750

19.7%

2.9%

0.16

0.1934

20.5%

4.4%

0.10

0.3750

18.7%

2.6%

0.22

0.0020

17.9%

5.7%

0.20

0.0020

15.0%

5.1%

% Savings- Animals
Used

0.20

0.15

0.325
0.90
0.300
0.85
0.326
1.37
0.305
1.29
0.249
2.07
0.254
2.02
0.120
2.97
0.124
3.20
0.083
3.27
0.070
3.62
Average Difference-0.06

0.1294

26.2%

-6.6%

-0.08

0.0049

24.1%

-6.2%

-0.05

0.0640

22.2%

-2.4%

0.23

0.0034

22.7%

7.1%

0.35

0.0005

21.7%

9.6%

0.309
0.85
0.285
0.79
0.317
1.36
0.283
1.25
0.245
2.12
0.233
2.00
0.116
3.04
0.115
3.18
0.080
3.35
0.064
3.62
Average Difference-0.06

0.0500

23.0%

-8.0%

-0.11

0.0034

20.8%

-9.1%

-0.12

0.0024

19.1%

-5.9%

0.14

0.0134

20.3%

4.3%

0.27

0.0002

19.6%

7.5%

0.08

0.02

In Vitro Cytotoxicity Test Methods BRD Appendix Q1

November 2006

Abbreviations: UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a); Toxcat=Category from Globally Harmonized System of Classification and Labeling of Chemicals5 (GHS; UN
2005); NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope; Cyto=NRU-determined starting dose (i.e., the LD50 predicted by the NRU IC50 in the RC rat-only millimole
regression (log LD50 [mmol/kg] = 0.439 log IC50 [mM] + 0.621); Default=Default starting dose of 175 mg/kg; Std. Error=Standard error for number of animals; 3T3= BALB/c 3T3 mouse
fibroblasts; NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit
dose =5000 mg/kg.
2
Mean number of animals for 10,000 simulations.
3
Difference between mean animals used for the default starting dose and mean animals used for the NRU-based starting dose.
4
P-value is from one-side Wilcoxon signed rank test for difference in animals between the default and NRU-based starting doses. Significant values at p <0.05.
5
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

Q-10

In Vitro Cytotoxicity Test Methods BRD Appendix Q1

November 2006

Concordance of NRU-Based Starting Dose with Default Starting Dose for GHS Acute Oral Toxicity Category Outcome
Based on Simulated UDP LD501
GHS Category Based on
LD50 Outcome with
Default Starting Dose

1

2

1
2
3
4
5
6
Total

4
0
0
0
0
0
4

0
13
1
0
0
0
14

GHS Category Based on LD50 Outcome with NHK NRU-Based Starting DoseHigherCategory
3
4
5
6
Total
NRU
Match
Category
0
0
0
0
4
100%
0%
0
0
0
0
13
100%
0%
11
0
0
0
12
92%
0%
1
15
1
0
17
88%
6%
0
0
22
0
22
100%
0%
0
0
0
0
0
NA
0%
12
15
23
0
68
96%
1%

Lower
NRU
Category
0%
0%
8%
6%
0%
NA
3%

GHS Category Based on LD50 Outcome with 3T3 NRU-Based Starting DoseGHS Category Based on
LD50 Outcome with
Default Starting Dose

1

2

3

4

5

6

Total

Category
Match

1
2
3
4
5
6
Total

4
0
0
0
0
0
4

0
13
1
0
0
0
14

0
0
11
0
0
0
11

0
0
0
16
0
0
16

0
0
0
1
21
0
22

0
0
0
0
0
0
0

4
13
12
17
21
0
67

100%
100%
92%
94%
100%
NA
97%

HigherNRU
Category
0%
0%
0%
6%
0%
0%
1%

Lower
NRU
Category
0%
0%
8%
0%
0%
NA
1%

Abbreviations: NRU=Neutral red uptake; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a);3T3= BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes; NA=Not applicable; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit
dose =5000 mg/kg. The NRU-based starting dose was the LD50 predicted by the NRU IC50 in the RC rat-only millimole regression (log LD50 [mmol/kg] = 0.439 log IC50 [mM] + 0.621).
The default starting dose = 175 mg/kg. Shaded cells are those containing the correct predictions.
2
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

Q-11

In Vitro Cytotoxicity Test Methods BRD Appendix Q1

November 2006

Discordant Substances for GHS Category Outcomes of UDP Simulations1
NRU
Test
Method3T3

NHK

Substance
Acetaminophen
Sodium dichromate dihydrate
Acetaminophen
Caffeine
Sodium dichromate dihydrate

NRU-Based Starting Dose2

Default Starting Dose3

LD50

Toxcat4

LD50

Toxcat4

2046.7843.70
2173.95
279.63
45.09

5
2
5
3
2

1765.4451.87
1755.26
357.17
51.77

4
3
4
4
3

LD50 Difference-281.348.17-418.69
77.55
6.69

Abbreviations: Toxcat=Globally Harmonized System of Classification and Labelling of Chemicals (GHS; UN 2005); UDP= Up-and-Down Procedure (OECD 2001a, EPA 2002a);
NRU=Neutral red uptake; 3T3=BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes.
1
Substances for which the simulated UDP outcome (in terms of GHS category) at the NRU-based starting dose did not match the simulated UDP outcome at thedefault starting dose. Simulations were performed with 10,000 runs at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and68 substances in the NHK NRU test method. Upper limit dose =5000 mg/kg.
2
NRU-based starting dose was the LD50 predicted by the NRU IC50 in the RC rat-only millimole regression (log LD50 [mmol/kg] = 0.439 log IC50 [mM] + 0.621).
3
The default starting dose = 175 mg/kg. Shaded cells are those containing the correct predictions.
4
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

Q-12

In Vitro Cytotoxicity Test Methods BRD Appendix Q2

November 2006

Appendix Q2

UDP Simulation Results for the RC Rat-Only Weight Regression Startingat the LD50 Predicted by the 3T3 and NHK NRU IC50 5000 mg/kg Upper Limit Dose

Q-13

In Vitro Cytotoxicity Test Methods BRD Appendix Q2[This Page Intentionally Left Blank]

Q-14

November 2006

In Vitro Acute Toxicity Test Methods BRD Appendix Q2

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by NRU Test Method1
NRU
Test
Method

Sigma
0.12
0.25

3T3

0.50
1.25
2.00

0.12
0.25
NHK

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Animals Used
Std.
Error

Number2

Difference3

P4

-0.15

0.2888%
Savings Animals
Used
8.2%

-0.16

0.1284

7.6%

-4.3%

-0.16

0.2071

7.2%

-3.9%

-0.10

0.9458

7.1%

-2.2%

-0.07

0.8240

6.2%

-1.4%

-0.15

0.1185

7.4%

-4.3%

-0.16

0.2017

7.0%

-4.3%

-0.17

0.1974

6.6%

-4.2%

-0.10

0.8704

6.8%

-2.3%

-0.07

0.9230

6.0%

-1.5%

Animals Died
Std.
Error

Difference3

P4

0.210
7.33
0.177
7.98
0.202
8.03
0.174
8.70
0.184
8.67
0.160
9.35
0.159
9.24
0.130
9.95
0.137
9.39
0.095
10.02
Average Difference

0.66

0.00130.66

0.00150.68

0.00230.71

0.00090.63

0.0005

0.216
7.37
0.175
7.96
0.209
8.07
0.169
8.68
0.189
8.73
0.153
9.35
0.161
9.28
0.124
9.96
0.139
9.43
0.092
10.03
Average Difference

0.59

0.00210.61

0.00170.62

0.00190.68

0.00040.60

0.00040.66

0.62

Number2

0.224
3.60
0.201
3.46
0.221
3.94
0.196
3.78
0.211
4.28
0.182
4.12
0.187
4.65
0.149
4.55
0.163
4.75
0.127
4.68
Average Difference
0.230
3.55
0.203
3.41
0.227
3.90
0.197
3.74
0.215
4.26
0.181
4.08
0.190
4.63
0.148
4.53
0.167
4.74
0.125
4.66
Average Difference%
Difference Animals
Died
-4.2%

-0.13

-0.13

Abbreviations: UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a); NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope; Cyto=NRU-determined starting dose
(i.e., the LD50 predicted by the NRU IC50 in the RC rat-only weight regression (log LD50 [mg/kg] = 0.372 log IC50 [mg/mL] + 2.024); Default=Default starting dose of 175 mg/kg; Std.
Error=Standard error for number of animals; 3T3= BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit
dose =5000 mg/kg.
2
Mean number of animals for 10,000 simulations.
3
Difference between mean number of animals for the default starting dose and mean number of animals for the NRU-based starting dose.
4
P-value is from one-sided Wilcoxon signed rank test for difference in animals between the default and NRU-based starting doses. Significant values at p <0.05.

Q-15

In Vitro Acute Toxicity Test Methods BRD Appendix Q2

November 2006

Summary of Stopping Rules Used for the UDP Simulations by NRU Test Method1
NRU Test
MethodSigma

Starting Dose

3 Animals at
Limit Dose2

5 Reversals2

Likelihood
Ratio2

Maximum
Number of
Animals Used2

0.12

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

15.8%
15.4%
15.1%
14.6%
13.7%
12.9%
10.4%
9.1%
9.3%
7.4%
17.0%
16.6%
16.2%
15.8%
14.5%
13.8%
10.5%
9.6%
9.2%
7.6%

60.2%
57.4%
34.2%
34.3%
19.6%
20.1%
13.3%
13.6%
12.1%
12.5%
56.2%
56.4%
33.1%
33.8%
19.3%
19.9%
13.2%
13.6%
12.0%
12.5%

22.9%
24.8%
48.1%
45.9%
60.8%
57.5%
65.1%
61.0%
65.7%
62.5%
25.5%
24.6%
47.8%
45.4%
60.0%
56.8%
64.7%
60.4%
65.5%
62.1%

1.1%
2.4%
2.6%
5.2%
5.8%
9.5%
11.2%
16.3%
12.9%
17.6%
1.2%
2.3%
2.8%
5.1%
6.2%
9.5%
11.6%
16.4%
13.2%
17.7%

0.25

3T3

0.5
1.25
2
0.12
0.25

NHK

0.5
1.25
2

Abbreviations: UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a); NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope;
Cyto=NRU-determined starting dose (i.e., the LD50 predicted by the NRU IC50 in the RC rat-only weight regression (log LD50 [mg/kg] = 0.372 log
IC50 [µg/mL] + 2.024); Default=Default starting dose of 175 mg/kg; 3T3= BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal
keratinocytes; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in
the NHK NRU test method. Upper limit dose =5000 mg/kg.
2
Percentage of the 10,000 test simulations that satisfied the specified condition for completion of testing (see OECD [2001a]; EPA [2002a]).

Q-16

In Vitro Acute Toxicity Test Methods BRD Appendix Q2

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

3T3

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

1
0.12
0.25
NHK

0.50
1.25
2.00

0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

2
0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

0.581
9.85
0.263
9.64
0.560
10.45
0.188
10.42
0.582
11.06
0.202
11.18
0.559
11.45
0.141
11.65
0.513
11.31
0.116
11.40
Average Difference
0.773
10.35
0.284
9.56
0.614
10.66
0.259
10.36
0.550
11.24
0.247
11.11
0.510
11.60
0.174
11.62
0.493
11.42
0.149
11.40
Average Difference
0.423
8.84
0.307
8.49
0.422
9.54
0.214
9.02
0.449
10.13
0.218
9.55
0.416
10.60
0.227
10.07
0.335
10.61
0.174
10.13
Average Difference
0.423
8.74
0.287
8.51
0.434
9.64
0.175
9.02
0.465
10.25
0.158
9.54
0.445
10.70
0.182
10.08
0.364
10.70
0.147
10.13
Average Difference

Animals Died

Difference3

P4

-0.21

0.6250-0.03

1.00000.12

0.87500.20

0.62500.09

0.62500.03-0.80

0.6250-0.30

0.8750-0.13

0.87500.03

0.8750-0.02

0.8750

-0.24
-0.35

0.3054-0.52

0.0942-0.58

0.1272-0.54

0.1099-0.47

0.1272

-0.49
-0.23

0.4548-0.62

0.0803-0.71

0.1099-0.61

0.1099-0.57

0.0681

-0.55

Q-17

Std.
Error

Number2

0.532
6.49
0.167
6.36
0.515
6.87
0.163
6.82
0.541
7.30
0.198
7.29
0.535
7.47
0.161
7.51
0.488
7.19
0.136
7.21
Average Difference

Difference3

P4

-0.12

0.6250-2.2%%
Difference Animals
Died
-1.9%

-0.05

1.0000

-0.3%

-0.7%

-0.01

1.0000

1.1%

-0.1%

0.05

1.0000

1.7%

0.6%

0.02

1.00000.8%

0.3%% Savings- Animals
Used

-0.02

0.632
6.77
0.176
6.33
0.538
7.02
0.190
6.80
0.512
7.45
0.226
7.27
0.506
7.59
0.189
7.51
0.479
7.30
0.150
7.20
Average Difference-0.44

0.6250

-8.3%

-7.0%

-0.22

0.8750

-2.9%

-3.2%

-0.18

0.8750

-1.2%

-2.5%

-0.08

0.8750

0.2%

-1.0%

-0.09

0.8750

-0.2%

-1.3%

0.396
5.48
0.250
5.13
0.390
5.88
0.204
5.44
0.406
6.21
0.205
5.72
0.390
6.42
0.213
5.92
0.330
6.31
0.175
5.88
Average Difference-0.36

0.1677

-4.1%

-6.9%

-0.44

0.0942

-5.7%

-8.1%

-0.49

0.1272

-6.1%

-8.6%

-0.50

0.1099

-5.3%

-8.4%

-0.44

0.1272

-4.7%

-7.4%

-0.27

0.3054

-2.7%

-5.3%

-0.47

0.1677

-6.9%

-8.6%

-0.54

0.1465

-7.4%

-9.4%

-0.53

0.1099

-6.1%

-9.0%

-0.48

0.0803

-5.6%

-8.2%

0.434
5.40
0.239
5.13
0.442
5.90
0.188
5.43
0.460
6.25
0.183
5.71
0.447
6.46
0.194
5.92
0.385
6.35
0.164
5.87
Average Difference

-0.20

-0.44

-0.46

In Vitro Acute Toxicity Test Methods BRD Appendix Q2

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

3T3

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

3
0.12
0.25
NHK

0.50
1.25
2.00

0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

4
0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

0.255
7.32
0.212
6.71
0.269
8.07
0.138
7.28
0.274
8.71
0.094
7.76
0.193
9.35
0.059
8.56
0.120
9.54
0.038
9.07
Average Difference
0.256
7.24
0.217
6.70
0.260
7.77
0.165
7.29
0.228
8.38
0.102
7.79
0.136
9.07
0.056
8.62
0.086
9.40
0.035
9.09
Average Difference
0.179
6.73
0.259
7.53
0.173
7.34
0.224
8.03
0.180
7.86
0.227
8.63
0.144
8.64
0.147
9.23
0.104
9.03
0.084
9.44
Average Difference
0.202
6.92
0.256
7.53
0.189
7.43
0.216
8.06
0.201
7.92
0.226
8.65
0.168
8.65
0.147
9.24
0.123
9.02
0.087
9.45
Average Difference

Animals Died

Difference3

P4

-0.61

0.0522-0.78

0.0093-0.94

0.0093-0.79

0.0049-0.48

0.0068

-0.63
-0.54

0.1514-0.49

0.0425-0.58

0.0342-0.46

0.0342-0.31

0.0122-0.47
0.80

0.00920.69

0.00920.77

0.00920.59

0.00920.41

0.00520.65
0.61

0.09340.63

0.04430.73

0.02500.59

0.01550.43

0.0155

0.60

Q-18

Std.
Error

Number2

0.213
4.02
0.133
3.42
0.221
4.42
0.094
3.76
0.220
4.75
0.077
4.05
0.170
5.04
0.056
4.45
0.128
5.10
0.047
4.69
Average Difference
0.193
3.88
0.136
3.42
0.193
4.16
0.107
3.76
0.178
4.47
0.080
4.06
0.130
4.80
0.058
4.48
0.102
4.94
0.048
4.69
Average Difference
0.053
3.30
0.079
3.44
0.050
3.58
0.057
3.66
0.052
3.80
0.055
3.93
0.050
4.16
0.020
4.18
0.043
4.34
0.018
4.28
Average Difference
0.098
3.41
0.077
3.44
0.076
3.64
0.056
3.68
0.076
3.86
0.056
3.94
0.067
4.20
0.021
4.19
0.056
4.37
0.017
4.29
Average Difference

Difference3

P4

-0.60

0.0161-9.1%%
Difference Animals
Died-17.5%-0.66

0.0068-10.8%-17.4%-0.70

0.0068-12.2%-17.3%-0.58

0.0068-9.2%-13.1%-0.42

0.0122

-5.3%

-8.9%

-0.46

0.0923-8.0%-13.6%-0.40

0.0771-6.7%-10.6%-0.41

0.0923-7.5%-10.1%-0.33

0.0771

-5.3%

-7.3%

-0.25

0.1099

-3.4%

-5.3%

0.15

0.0739

10.7%

4.3%

0.09

0.0386

8.6%

2.4%

0.12

0.0507

8.9%

3.1%

0.02

0.2744

6.4%

0.4%

-0.06

0.1167

4.3%

-1.4%

0.03

0.3484

8.2%

1.0%

0.04

0.0833

7.8%

1.0%

0.08

0.1046

8.4%

2.0%

-0.01

0.3755

6.4%

-0.3%

-0.08

0.0934

4.6%

-1.8%

% Savings- Animals
Used

-0.53

-0.37

0.06

0.01

In Vitro Acute Toxicity Test Methods BRD Appendix Q2

November 2006

Summary of Animals Used and Animals Dead for UDP Simulations by GHS Toxicity Category and NRU Test Method1
ToxcatNRU
Test
MethodAnimals Used
Sigma
0.12
0.25

3T3

0.50
1.25
2.00

Starting
Dose
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

5
0.12
0.25
NHK

0.50
1.25
2.00

0.12
0.25
3T3

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

6
0.12
0.25
NHK

0.50
1.25
2.00

Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default
Cyto
Default

Std.
Error

Number2

0.287
7.12
0.220
9.19
0.228
8.01
0.145
10.40
0.186
8.45
0.091
10.81
0.133
8.55
0.061
10.76
0.105
8.64
0.051
10.46
Average Difference
0.335
7.31
0.219
9.21
0.301
8.17
0.114
10.38
0.224
8.62
0.065
10.79
0.148
8.73
0.051
10.74
0.114
8.78
0.039
10.44
Average Difference
0.596
5.75
0.575
7.74
0.574
6.61
0.529
8.63
0.411
7.69
0.335
9.83
0.241
8.42
0.062
10.88
0.194
8.47
0.021
10.78
Average Difference
0.588
5.79
0.548
7.63
0.561
6.72
0.486
8.59
0.413
7.85
0.309
9.83
0.240
8.56
0.059
10.87
0.194
8.57
0.021
10.78
Average Difference

Animals Died

Difference3

P4

2.07

0.00202.39

0.00202.36

0.00202.21

0.00201.81

0.00202.17
1.90

0.00202.21

0.00202.16

0.00202.01

0.00201.66

0.00201.99
1.99

0.00052.02

0.00052.15

0.00052.46

0.00052.31

0.00052.19
1.84

0.00021.87

0.00021.97

0.00022.31

0.00022.21

0.0002

2.04

Q-19

Std.
Error

Number2

0.039
3.19
0.042
3.32
0.038
3.43
0.074
3.60
0.047
3.52
0.071
3.68
0.035
3.62
0.034
3.91
0.027
3.75
0.019
4.01
Average Difference
0.048
3.22
0.057
3.33
0.047
3.44
0.081
3.60
0.039
3.56
0.077
3.67
0.038
3.67
0.041
3.89
0.036
3.79
0.023
4.00
Average Difference

Difference3

P4

0.13

0.084022.5%%
Difference Animals
Died
4.0%

0.17

0.0488

23.0%

4.8%

0.16

0.0488

21.8%

4.4%

0.29

0.0020

20.6%

7.3%

0.26

0.0020

17.4%

6.5%

0.11

0.3223

20.6%

3.3%

0.16

0.2324

21.3%

4.4%

0.11

0.2754

20.1%

3.1%

0.22

0.0039

18.7%

5.6%

0.21

0.0020

15.9%

5.3%

% Savings- Animals
Used

0.20

0.16

0.327
0.91
0.300
0.84
0.335
1.40
0.305
1.29
0.258
2.10
0.253
2.02
0.125
2.98
0.123
3.19
0.088
3.29
0.069
3.62
Average Difference-0.06

0.0923

25.7%

-7.5%

-0.10

0.0015

23.4%

-8.1%

-0.08

0.0068

21.8%

-3.7%

0.21

0.0010

22.6%

6.6%

0.33

0.0005

21.4%

9.0%

0.310
0.85
0.285
0.79
0.318
1.36
0.283
1.25
0.247
2.11
0.232
2.00
0.121
3.02
0.115
3.18
0.085
3.33
0.063
3.62
Average Difference-0.06

0.0327

24.1%

-7.7%

-0.11

0.0012

21.8%

-8.9%

-0.11

0.0046

20.1%

-5.4%

0.16

0.0061

21.2%

5.0%

0.30

0.0005

20.5%

8.2%

0.06

0.03

In Vitro Acute Toxicity Test Methods BRD Appendix Q2

November 2006

Abbreviations: UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a); Toxcat=Category from Globally Harmonized System of Classification and Labeling of Chemicals5 (GHS; UN
2005); NRU=Neutral red uptake; Sigma=Reciprocal of dose-mortality slope; Cyto=NRU-determined starting dose (i.e., the LD50 predicted by the NRU IC50 in the RC rat-only weight
regression (log LD50 [mmol/kg] = 0.372 log IC50 [mM] + 2.024); Default=Default starting dose of 175 mg/kg; Std. Error=Standard error for number of animals; 3T3= BALB/c 3T3 mouse
fibroblasts; NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit
dose =5000 mg/kg.
2
Mean number of animals for 10,000 simulations.
3
Difference between mean animals used for the default starting dose and mean animals used for the NRU-based starting dose.
4
P-value is from one-side Wilcoxon signed rank test for difference in animals between the default and NRU-based starting doses. Significant values at p <0.05.
5
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

Q-20

In Vitro Acute Toxicity Test Methods BRD Appendix Q2

November 2006

Concordance of NRU-Based Starting Dose with Default Starting Dose for GHS Acute Oral Toxicity Category Outcome
Based on Simulated UDP LD501
GHS Category Based on
LD50 Outcome with
Default Starting Dose

1

2

1
2
3
4
5
6
Total

4
0
0
0
0
0
4

0
13
1
0
0
0
14

GHS Category Based on
LD50 Outcome with
Default Starting Dose

1

2

1
2
3
4
5
6
Total

4
0
0
0
0
0
4

0
13
1
0
0
0
14

GHS Category Based on LD50 Outcome with NHK NRU-Based Starting DoseHigherCategory
3
4
5
6
Total
NRU
Match
Category
100%
0%
0
0
0
0
4
100%
0%
0
0
0
0
13
92%
0%
11
0
0
0
12
88%
6%
15
1
0
17
1
100%
0%
22
0
22
0
0
NA
0%
0
0
0
0
0
96%
1%
12
15
23
0
68

Lower
NRU
Category
0%
0%
8%
6%
0%
NA
3%

GHS Category Based on LD50 Outcome with 3T3 NRU-Based Starting DoseHigherCategory
3
4
5
6
Total
NRU
Match
Category
100%
0%
0
0
0
0
4
100%
0%
0
0
0
0
13
92%
0%
11
0
0
0
12
82%
12%
14
2
0
17
1
100%
0%
21
0
21
0
0
NA
0%
0
0
0
0
0
94%
3%
12
14
23
0
67

Lower
NRU
Category
0%
0%
8%
6%
0%
NA
3%

Abbreviations: NRU=Neutral red uptake; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); UDP=Up-and-Down Procedure (OECD 2001a, EPA 2002a);3T3= BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes; NA=Not applicable; RC=Registry of Cytotoxicity.
1
For 10,000 UDP simulations at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test method. Upper limit
dose =5000 mg/kg. The NRU-based starting dose was the LD50 predicted by the NRU IC50 in the RC rat-only weight regression (log LD50 [mg/kg] = 0.372 log IC50 [µg/mL] + 2.024). The
default starting dose = 175 mg/kg. Shaded cells are those containing the correct predictions.
2
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

Q-21

In Vitro Acute Toxicity Test Methods BRD Appendix Q2

November 2006

Discordant Substances for GHS Category Outcomes of UDP Simulations1
NRU
Test
Method3T3

NHK

Substance
Acetaminophen
Caffeine
Procainamide HCl
Sodium dichromate dihydrate
Acetaminophen
Caffeine
Sodium dichromate dihydrate

NRU-Based Starting Dose2

Default Starting Dose3

LD50

Toxcat4

LD50

Toxcat4

2146.93297.82
2000.2444.48
2171.18292.06
45.85

5
3
5
2
5
3
2

1768.39342.76
1529.98
52.17
1755.21353.96
51.91

4
4
4
3
4
4
3

LD50 Difference-378.5444.95-470.26
7.69-415.96
61.91
6.06

Abbreviations: Toxcat=Globally Harmonized System of Classification and Labelling of Chemicals (GHS; UN 2005); UDP= Up-and-Down Procedure (OECD 2001a, EPA 2002a);
NRU=Neutral red uptake; 3T3=BALB/c 3T3 mouse fibroblasts; NHK=Normal human epidermal keratinocytes.
1
Substances for which the simulated UDP outcome (in terms of GHS category) at the NRU-based starting dose did not match the simulated UDP outcome at thedefault starting dose. Simulations were performed with 10,000 runs at each starting dose and dose-mortality slope for 67 substances in the 3T3 NRU test method and68 substances in the NHK NRU test method. Upper limit dose =5000 mg/kg.
2
NRU-based starting dose was the LD50 predicted by the NRU IC50 in the RC rat-only weight regression (log LD50 [mg/kg] = 0.372 log IC50 [µg/mL] + 2.024).
3
The default starting dose = 175 mg/kg. Shaded cells are those containing the correct predictions.
4
GHS Toxicity Category
Oral LD50 Limits
1
LD50 ≤5 mg/kg
2
5 < LD50 ≤50 mg/kg
350 < LD50 ≤300 mg/kg
4300 < LD50 ≤2000 mg/kg
52000 < LD50 ≤5000 mg/kg
6
LD50 >5000 mg/kg

Q-22