Altern. Animal Test. EXperiment. 5, 1 - 38 (1998)

Validation Study on Five Cytotoxicity Assays by JSAAE
I.
Overview of the Study and Analyses of Variations of ED50 Values
Tadao Ohno*1, Masumi Asakura2, Takumi Awogi3, Yoshihiro Futamura4, Akihiro
Harihara5, Masato Hatao6, Chiharu Hattori7, Akira Hayasaka8, Makoto Hayashi9,
Takashi Hayashi10, Zenzou Hirata4, Hiroshi Hori11, Hiroyoshi Hoshi12, Koichi
Imai13, Ikuo Imazeki8, Takuya Ishibashi11, Hiroshi Itagaki6, Tatsuo Iwata14, Miyako
Kakuma15, Shinya Kaneda16, Ikuo Kato5, Mayako Kato1, Tooru Kawahatsu17, Akio
Kawakami18, Akemi Kazama19, Akihiko Kido20, Shigemi Kimura21, Michiyo
Kitazawa22, Shigefumi Kogiso23, Hajime Kojima24, Mayumi Kotani25, Masahiro
Kuramochi21, Daijyo Maki26, Masako Matsuda27, Chihomi Mitsuoka26, Satoru
Miyazaki28, Fumio Mizuno20, Minako Mori29, Katsunori Morimoto30, Matsuko
Moriyasu26, Keiya Nakajima29, Madoka Nakajima22, Masaaki Nakamura13, Masaki
Nakamura4, Nahoko Nakano26, Shinobu Nakamura26, Akira I. Negami31, Masato
Nishino19, Tamotsu Nishitomi20, Kenji Ohkoshi32, Yuuko Okamoto32, Takashi
Omori33, Hiroshi Ono34, Makoto Ono15, Yuko Osanai15, Kaoru Saijo1, Yuka Sano35,
Kyoko Saotome36, Kazunori Sasaki5, Tetsuji Sasaki1, Hidetaka Sato37, Shinichiro
Sato38, Hiroyasu Shimada7, Shoichiro Shimogo6, Kazuyuki Shimono16, Hiroshi
Shionoya14, Hideaki Sugawara28, Yasuko Sugiki39, Shigeo Sugimoto40, Shigeru
Sugimoto 37, Junko Suzuki 37, Kinya Takagaki 4, Ken Takahashi 41, Minoru
Takizawa42, Chieko Tamaki23, Noriho Tanaka34, Jirou Taniya43, Noriko Teramoto1,
Hisashi Torishima44, Toshiyuki Tsuchiya45, Motoo Uejima16, Hayao Ueno46, Yuji
Ugai22, Shogo Wada47, Shinobu Wakuri34, Xinhai Wang1, Ikuo Watanabe29, Masami
Watanabe48, Satoshi Yajima,49 Yoshiki Yamagata19, Yuuzou Yamaguchi49, Osamu
Yamakita40, Ryohei Yamamoto44, Masanori Yoshida46, Isao Yoshimura,33 Kouichi
Yuhki43, Shiro Yukiyama40
1

RIKEN Cell Bank, The Institute of Physical and Chemical Research (RIKEN), 31-1 Koyadai, Tsukuba Science City, Ibaraki 305, 2Japan Bioassay Laboratory,* Correspondence : The Working Group of JSAAE for the
1st Validation Study on Cytotoxicity Assays, c/o T.
Ohno, RIKEN Cell Bank, 3-1-1 Koyadai, Tsukuba Science City, Ibaraki 305, Japan
Key words: alternatives, cytotoxicity assay, colony formation assay, crystal-violet staining assay, LDH assay,
neutral red uptake assay, MTT assay, inter-laboratory
validation, Draize eye irritation test
Abbreviations: CF, colony formation; CV, crystal-violet

staining; ED50, 50% effective dose; FRLA, factor forremaining LDH activity; JSAAE, Japanese Society of
Alternatives to Animal Experiments; LDH, lactate dehydrogenase release; MAS, maximum average score;
MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide; NR, neutral red uptake; PFD, powerfor distinction..
Received: July 4, 1997. Accepted: September 10, 1997.

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Validation Article I. Overview

2445 Hirasawa, Hadano-shi, Kanagawa 257, 3Drug Safety Research Center, Tokushima
Research Institute, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno, Kawauchicho, Tokushima-shi, 771-01, 4Toxicology Laboratory, Mochida Pharmaceutical Co.,
Ltd., 342 Gensuke, Fujieda-shi, Shizuoka 426, 5Shionogi Research Laboratories,
Shionogi & Co., Ltd., 3-1-1 Futaba-cho, Toyonaka-shi, Osaka 561, 6Shiseido Safety &Analytical Research Center,1050 Nippa-cho, Kohoku-Ku, Yokohama-shi 223, 7Developmental Research Laboratory, Daiich Pharmaceutical Co., Ltd., 1-16-13 Kita-Kasai,
Edogawa-ku, Tokyo 134, 8Chugai Pharmaceutical Co., Ltd., 3-41-8 Takada, Toshimaku, Tokyo 171, 9Division of Genetics and Mutagenesis, National Institute of Health
Sciences (NIHS), 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158, 10Ina Research Inc., 8047
Nishiminowa, Ina-shi, Nagano 399-45, 11Tsuruga Institute of Biotechnology, Toyobo
Co., Ltd., 10-24 Toyo-cho, Tsuruga-shi, Fukui 914, 12Research Institute for the Functional Peptides, 11-26 Minamisanban-cho, Yamagata-shi 990, 13Department of
Biomaterials, Osaka Dental University, 1-5-31 Otemae, Chuo-ku, Osaka-shi 540, 14Department of Drug Safety Research, R & D Division, Eisai Co., Ltd., Takehaya,
Kawashima-cho, Hashima-gun, Gifu 501-61, 15Beauty Care Products Laboratory,
Nagase, Co., Ltd., 5-1 Kobuna-cho, Nihonbashi, Chuo-ku, Tokyo 103, 16Naruto Research Institute, Otsuka Pharmaceutical Factory, Inc., 115 kuguhara, Tateiwa, Muyacho, Naruto-shi, Tokushima 772, 17Pharmaceutical Research Center, Meiji Seika Kaisha,Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama-shi 222, 18Safety Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., 1-1-8 Azusawa, Itabashi-ku, Tokyo 174,19
Biochemical Development & Production, Oriental Yeast Co., Ltd., 4-4-1 Minamisuita,
Suita-shi, Osaka 564, 20Kashima Laboratory, Mitsubishi-kagaku Institute ofEnviromental and Toxicological Sciences, 14 Sunayama, Hasaki-machi, Kashima-gun,Ibaraki 314-02, 21BML Inc., 1361-1 Matoba, Kawagoe-shi, Saitama 350, 22Biosafety
Research Center,Foods, Drugs and Pesticides, 582-2 Arahama, Shioshinden, Fukudecho, Iwata-gun, Shizuoka 437-12, 23Environmental Health Science Laboratory, Sumitomo
Chemical Co., Ltd., 3-1-98 Kasugadanaka, Konohana-ku, Osaka-shi 554, 24Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd., 2-7 Torimi-cho, Nishi-ku,
Nagoya-shi 451, 25R&D Planning HQ, Sunstar Inc., 3-1 Asahi-machi, Takatsuki-shi,
Osaka 569, 26Panapharm Laboratories Co., Ltd., 1285 Kurisaki-cho, Uto-shi, Kumamoto
869-04, 27Research and Development Center, Duskin Co., Ltd., 4-19-8 Minamisuita,
Suita-shi, Osaka 564, 28Life Science Research Information Laboratory, The Institute ofPhysical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01,
29
The Center of Japan Biological Chemistry, 52 Fukue, Kaizu-cho, Kaizu-gun, Gifu
503-06, 30Takara Belmont Co., Ltd., 5-1 Takamatsu, Kouzai-cho, Koga-gun, Shiga 52032, 31Deparment of Obstetrics and Gynaecology, Fukui Medical School, 23-3
Shimoaizuki, Matsuoka-cho, Yoshida-gun, Fukui 910-11, 32Division of Fundamental
Research, KOSÉ Co., 1-18-4 Azusawa, Itabashi-ku, Tokyo 174, 33Department of Management Science, Faculty of Engineering, Science University of Tokyo, 1-3 Kagurazaka,–2–

Shinjuku-ku, Tokyo 162, 34Hatano Research Institute, Food and Drug Safety Center,729-5 Ochiai, Hadano-shi, Kanagawa 257, 35Biotechnology Research Laboratories, TAKARA SHUZO Co., Ltd., 3-4-1 Seta, Ohtsu-shi, Shiga 520-21, 36Yokohama
City Institute of Health, 1-2-17 Takigashira, Isogo-ku, Yokohama-shi 235, 37Japan Food Research Laboratories, 6-11-10 Nagayama, Tama-shi, Tokyo 206, 38Division of Radiation Medicine, National Institute of Radiological Sciences, 4-9-1
Anagawa, Inage-ku, Chiba-shi 263, 39Menicon Co., Ltd., 5-1-10 Takamoridai,
Kasugai-shi, Aichi 487, 40Drug Safety Laboratory, Taiho Pharmaceutical Co.,
Ltd., 224-2 Ebisuno, Hiraishi, Kawauchi-Cho, Tokushima-shi 771-01, 41The Institute of Environmental Toxicology, 4321 Uchimoriya-cho, Mitsukaido-shi,
Ibaraki 303, 42Hakko Electric Machine Works Co. Ltd., 3055 Togura, Toguramachi, Hanishina-gun, Nagano 389-08, 43Koken Bioscience Institute, Koken Co.
Ltd., 2-11-12 Nakane, Meguro-ku, Tokyo 152, 44Kurabo Industries Ltd., 14-41
Shimokida-cho, Neyagawa-shi, Osaka 572, 45Safety Evaluation Center, Central
Research Laboratory, Showa Denko K. K., 1-1-1 Ohnodai, Midori-ku, Chiba-shi
267, 46Takeda Analytical Research Laboratories, Ltd., 2-17-85 Juso-honmachi,
Yodogawa-ku, Osaka 532, 47Wako Pure Chemical Industries, Ltd., 6-1 Takadacho, Amagasaki-shi, Hyogo 661, 48Division of Radiation Biology, Department of
Health Science, Faculty of Pharmaceutical Sciences, Nagasaki University, 1-14
Bunkyo-machi, Nagasaki-shi 852, 49Central Research Laboratory, Takasago International Co., 1-4-11 Nishiyawata, Hiratsuka-shi, Kanagawa 254, Japan
Abstract
In October 1992, the Japanese Society of Alternatives to Animal Experiments(JSAAE) organized an inter-laboratory validation study on cytotoxicity assays
for development of an alternative(s) to the in vivo Draize eye irritation test. The
main purpose is to evaluate practicability of five proposed cytotoxicity assays
with two cell lines each through a large scale inter-laboratory assessment. The
five assays were colony formation (CF) assay, crystal-violet staining (CV) assay,
lactate dehydrogenase release (LDH) assay, neutral red uptake (NR) assay, and
MTT assay. They were selected because of their popularity in Japan. We chose
six detergents as model chemicals for this first step validation. One of the six
chemicals, which was revealed to be Tween 20 after breaking the code, was additionally included in the doubly-mask-coded chemical set for simultaneous evaluation of intra-laboratory variation. Technology transfer for all the assays wasmade cumulatively 118 times to less-experienced laboratories. Concomitantly,
we performed the Draize test to confirm toxicity of the coded chemicals in vivo.
A total of 3,810 final data files including preliminary test results were submitted from 42 laboratories. Of 1,535 raw data files with final definitive assay results, 292 files were rejected because of not only apparent misunderstanding of
the protocols provided by the Working Group but also for violating pre-set common rules. Acceptability of data files was also examined by a computer-assisted–3–

Validation Article I. Overview

logistic analysis program (LAP-JSAAE) with a six step-wise check code to detect abnormality in the data file. After generating ED50 values through the program, 5 data file sets of 7 tested chemicals were judged unreliable since the large
differences in ED50 values were found for the same but differently coded test
chemical, Tween 20. This clearly indicates considerable intra-laboratory variation. After excluding these data files, analyses of inter-laboratory variation weremade on 969 data files with the box-whiskand-er plot analysis.
The important results of our study are as follows. (1) CF, CV, MTT, and NR
assays are recommendable from the view point of performance of these assays.
Performance rate of each assay was calculated on the number of finally accepted
assay data files divided by the expected number of data files. The highest rate
was for the CF assay with BALB/3T3 A31-1-1 cells followed by the CV assay
with two cell lines. Lower performance rates were observed in the sub-divided
LDH assays. The performance rate was considered to reflect simplicity of themethod and labor needed for the assay; (2) From the view point of the intralaboratory variation of the same but differently coded chemical Tween 20, medians of the log(ED50) values of each assay were satisfactorily close; (3) Aftereliminating the sub-divided LDH assays which gave a small number of acceptable data files per assay and therefore resulted in unstable hinge-spread oflog(ED50) values, the CV assay with CHL cells and the MTT assay with SQ-5
cells were found to have given the smallest mean hinge-spread of log(ED50)
followed closely by the CF assay with HeLa S3 (SC) cells and the CV assay with
HeLa S3 (SC) cells. These assays were therefore considered to give small interlaboratory variation; (4) the CF assay with HeLa S3 (SC) cells resulted in thelargest "power for distinction (PFD)" of toxicities between the least and the most
toxic chemicals defined as the ratio of difference in medians of log(ED50) valuesof two chemicals to the mean hinge-spread, followed by the CV assay with HeLa
S3 (SC) cells. However, the CF assay is not necessarily advantageous as far as
distinguishing moderately irritating chemicals from non-irritating chemicals; (5)Medians of log(ED50) values enabled us to classify tested chemicals into at least
three categories, namely, non-, moderately-, and highly-cytotoxic chemicals. Nonand highly-cytotoxic chemicals corresponded to non- and severe-irritants in thein vivo Draize test.
(6) Considering performance rate, inter-laboratory variation of data reflectedon the mean hinge-spread, power for distinction of chemical cytotoxicity, timeneeded for an assay (i.e. the CF assay requires longer incubation time than others), and the data on the common cell line HeLa S3 (SC), we concluded that theCV assay is the most practical and recommendable as a part of alternatives to thein vivo Draize test.
Many problems that were revealed during the present validation study including human factors were discussed. The assay results will be further described in
the ensuing articles in this issue, i. e., the calculation of ED50 values by LAPJSAAE, problems on the CF, CV, LDH, MTT, and NR assays. A fact data basewas constructed on the data files of this validation study which will be available
on request.

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Introduction
Validation is necessary to confirm the practicability of any newly developed alternatives toin vivo experimentation. Many in vitro cytotoxicity assays have been proposed as alternatives
to the Draize eye irritation test (Ekwall and
Ekwall, 1988; Ekwall et al., 1989; Balls et al.,
1990; Ekwall et al., 1990; Bruner et al., 1991;
Spielmann et al. 1991) and to the acute systemic
toxicity test (Clemedson et al., 1996a, 1996b).
However, well-organized, comparative, interlaboratory validation studies on reported multiple cytotoxicity assays have not been carriedout in Japan except for one which commenced
in 1990 (Ohno, Y. et al., 1994, 1995). Independently to this validation study, The JapaneseSociety of Alternatives to Animal Experiments(JSAAE) organized an early phase inter-laboratory validation study on five cytotoxicity assays
in October 1992, since we assume that a properbattery of cytotoxicity assays should be the core
alternative to the Draize test, and, as discussed
in CAAT/ERGATT Workshop (Balls et al.,1990), inter-laboratory assessment should consist of two major steps, an early phase and a definitive phase.
The purpose of the present study is to evaluate practicability of the five proposed cytotoxicity assays through a large scale inter-laboratory
assessment in Japan without strict restriction oncompetence for participation, such as manufacturers of cosmetics, to avoid potential bias in thecollected data. Performance of the assay is therefore one of the key markers of the present validation.
The five assays validated were the crystalviolet staining (CV) assay (Saotome et al., 1989),
the neutral red uptake (NR) assay (Borenfreundand Puerner, 1985), the MTT assay (Mosmann,1983), the colony formation (CF) assay (Sasaki
et al., 1991), and the lactate dehydrogenase release (LDH) assay (Wang et al., 1993). These
were selected for their popularity and were expected to be standardized in Japan by the Working Group (The Office is located at RIKEN,
Ibaraki) for this validation study organized under the Validation Committee of JSAAE (chairedby H. Ono, Hatano Research Institute, Food andDrug Safety Center, Kanagawa). We thought that
their protocols should be standardized in the near
future since any one of them is highly likely to
be selected as a component of the future official
alternatives to the Draize test in Japan.
We selected the strategy where (1) any laboratory interested in the present validation study
would be accepted as a participant, since it was
impossible to expect as participants well-qualified laboratories which were able to carry out
the expected assays with highly skillful techniques following good laboratory practice(GLP), (2) technology transfer on each assay
would be given free of charge through 2-5 day
hands-on workshop courses to less-experiencedlaboratories, (3) the collected data would be thoroughly reviewed and recalculated by expert toxicologists to determine whether or not there are
any abnormalities or problems, (4) data files
which passed this rigorous examination ("datacleaning") are then analyzed for performance
rates of the assays from several view points andfor the intra- and inter-laboratory variations of
the 50% effective doses (ED50) values. (5) We
also tried to evaluate the cytotoxicity assays with
the newly defined "power for distinction" (PFD)
among log(ED50) values of test chemicals.
(6) We chose six test chemicals with known
irritancy based on the reported Draize test and
set up a chemical bank for quality control andstable supply of the test chemicals. Each chemical was once given a mask-code number in thechemical bank. At this step, one of the codedchemicals was duplicated and the duplicate wasgiven a new mask-code number. Thus, seven test
chemicals were sent to The Working Group for
the first validation study on cytotoxicity. They
were again given new mask-code numbers(therefore, doubly masked), and sent to the participating laboratories. By this process, we expected to obtain intra-laboratory variation of dataon a chemical together with inter-laboratory
variation of data on 6 chemicals.
(7) We also selected 4 cell lines that are widelyused and easily available in Japan. They werequality-controlled by an established cell bank.
Additionally, rabbit cornea cells were selected–5–Validation Article I. Overview

since the cells were primary-cultured in serumfree medium and supplied with the medium as akit. The serum-free cultured cells have been generally considered as being more sensitive thanthose cultured in serum-containing medium(Torishima et al., 1990, 1995). (8) A subline of
HeLa was tested in every assay for mutual comparison of the five assay methods. (9) Simultaneously with these in vitro assays, the in vivo
Draize test was carried out on the same coded
seven chemicals for confirmation of the toxicity.
Common rules on experimentation for thefive assays that were previously discussed andaccepted in the orientation meeting before thestart of this validation study are as follows;[1] Chemicals should be stored in the dark at 4
°C and should be diluted and recorded on a%(w/v) basis;[2] A 10-ml volume-certified flask should be
used for initial preparation of a chemical solution except in the case where the initial concentration was less than 1%(w/v);[3] For an observation point*, the data should
be collected from 3 independent 96-multiwellplates. This is to make it clear that in an assay** one plate corresponds to a single runof an expected test possible to draw a doseresponse curve and the test was repeated 3
times for an assay. In a single plate, any number of replications for an observation point isallowed, but the plate must contain negative
control (untreated) wells and, when indicated,
positive control (definitively treated) wells.
[4] Assays should be repeated until data for atleast 3 observed points (a point as an averageof replicate observations for a concentrationof a chemical) ranging between 20% and 80%of the maximum effect were obtained.
[5] After the assays, a floppy disk of raw data
(observed values of optical density, not calculated percentages) written in a preset format should be submitted to the Working
Group together with a print-out of a data filewhich corresponds to one assay.
The Working Group that included specialists
on statistical analyses of collected data consistedof T. Ohno, M. Hayashi, H. Itagaki, M. Kato, S.

Miyazaki, T. Omori, H. Ono, K. Saijo, H.
Sugawara, N. Tanaka, N. Teramoto, S. Wakuri
and I. Yoshimura. A total of 50 laboratories has
co-worked in this study and three staffs were required for the data cleaning and the other two
for computer-software programming and statistical analyses of the results. After the preliminary data cleaning, a follow-up meeting was heldin Tokyo on November 30, 1994, to discuss problems on this validation study. These problems
are occasionally described in the following paragraphs.
This paper presents the overview of the firstvalidation study, describes performance of theassays, and compares their variations on eachtested chemical. General problems and points tobe improved for further validation studies are
discussed. In the following article (hereafter, abbreviated as Validation Article***), we will describe the algorithm of the logistic analysis program LAP-JSAAE (see Validation Article II),and human errors found in the analysis (see Validation Article III). Moreover, five articles will
follow to describe representative hand-plotted
dose-response curves, precise descriptions on
ED50 values, comments and discussions on eachcytotoxicity assay (see Validation Article IV VIII). Protocols for each assay will be given in
the short articles in this issue. A fact data baseconstructed on the present results will be available on request.
Materials and Methods
Chemicals tested
The following 6 chemicals used in this study
are given below. Since one of the six chemicals
was added after mask-coding to determine intra-laboratory variation of the final data, a totalof 7 chemicals was tested. These chemicals wereselected to reflect a range from non-irritating toseverely irritating as reported in in vivo Draize* : An observation point was based on the mean of raw
data at a concentration of the tested chemical.
** : An assay consists of 3 tests carried out in triplicate
plates.
*** : A series of articles on validation study on five cytotoxicity assays by JSAAE consisting of I through
VIII.

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Table 1. Cell lines used in the first validation study
Number ofparticipating laboratories
Assay

Colony formation(CF)
Crystal violet
staining (CV)#LDH release
LDH-1
LDH-2A
LDH-2B
LDH-2CMTT assay(MTT)
Neutral red uptake(NR)

Cell lines

Characteristics

coworked*

datasubmitted**

HeLa S3 (SC)BALB/3T3 A31-1-1
HeLa S3 (SC)CHL

Human cervix carcinoma
Mouse embryonic fibroblasts
Human cervix carcinoma
Chinese hamster lung cells

24
24
16
16

19
22
14
14

HeLa S3 (SC)SQ-5
HeLa S3 (SC)SQ-5
HeLa S3 (SC)SQ-5
HeLa S3 (SC)SQ-5
HeLa S3 (SC)SQ-5
HeLa S3 (SC)NRCE

Human cervix carcinoma
Human lung squamous carcinoma
Human cervix carcinoma
Human lung squamous carcinoma
Human cervix carcinoma
Human lung squamous carcinoma
Human cervix carcinoma
Human lung squamous carcinoma
Human cervix carcinoma
Human lung squamous carcinoma
Human cervix carcinoma
Rabbit corneal cells, serum-free

18
18
18
18
18
18
18
18
20
20
24
24

11
11
10
10
9
9
10
9
18
17
22
20

* Number of participating laboratories involved in each assay.
** Number of laboratories which submitted final data files to The Working Group.
# Characters in the parentheses of the column, Assay, indicate abbreviations used in the text.

tests (Draize, 1944). They are:Tween 20 (#1); the maximum average score(MAS) at 10% is 5.7 to 6.0 (Catroux et al.,
1993; Bagley et al., 1992a). In ocular irritancy
classification, this chemical has been reported
as 'non' (Watanabe et al., 1989), 'minimal'(Gordon, 1992), 'less than mild' (Gordon andBergman, 1988), 'mild' (Silverman andPennisi, 1987), 'mild' at 100% (Rachui et al.,1994).
Tween 80 (#2); MAS at 10% is 3.8 to 4.0(Catroux et al., 1993; Bagley et al., 1992b),
modified MAS is 4.00 (Bagley et al., 1992a).
In ocular irritancy classification, this chemical has been reported as 'non'(Watanabe et al.,1989), 'no' (Marinovich et al., 1990), 'minimal' (Gordon, 1992), 'less than mild' (Gordon and Bergman, 1988), 'mild' at 100%
(Rachui et al., 1994).
Sucrose fatty acid ester (#3); in ocular irritancy
classification, this chemical has been reported
as 'mild' (Watanabe et al., 1989).

Propylene glycol (#4); modified MAS is 1.33
(Bagley et al., 1992a). In ocular irritancy classification, this chemical has been reported as'non' (Watanabe et al., 1989), 'less than mild'
at 15% (Gordon and Bergman, 1988), 'minimal' (Silverman and Pennisi, 1987), 'slightly
irritant' (Guillot et al., 1982).
Cetylpyridinium chloride monohydrate (#5); in
ocular irritancy classification, this chemicalhas been reported as 'severe' (Gordon, 1992),'moderate' at 0.5% (Gordon and Bergman,1988). Otherwise as cetylpyridinium bromide,
modified MAS at 10% is 89.67 (Bagley et al.,1992a), MAS at 10% is 57.20 (Catroux et al.,1993), thus as 'severe' (Marinovich et al.,1990).
Sodium lauryl sulfate (#6); MAS at 10% is 37.0
(Bagley et al., 1992a) and 37.30 and 22.30(Catroux et al., 1993), modified MAS at 15%
is 59.17 (Bagley et al., 1992b), maximum at
10% is 40 (Griffith et al., 1980). This chemical has been reported as 'moderate' (Watanabe–7–Validation Article I. Overview

et al., 1989), 'moderate' (Marinovich et al.,
1990; Gordon, 1992), 'Moderate' at 10%(w/v) (Silverman and Pennisi, 1987), 'Moderate'
at 10% (Rachui et al., 1994).
Tween 20 (#7, equal to #1, an intrinsic masked
reference chemical).
The # numbers in parentheses are doublymask-codes given before the transfer of thechemicals to each laboratory. The Chemical
Bank was previously set up in The ResearchLaboratory of Wako Pure Chemical Industries
Ltd. (Hyogo) which supplied all the chemicals
except sucrose fatty acid ester purchased from
Tokyo Chemical Industry Co., Ltd. (Tokyo). A
single lot of each chemical was used and original stocks of the lot were stored at 4°C in theChemical Bank. These chemicals were sent to
the 45 participating laboratories (two laboratories dropped out before receiving the chemicals)
and also to Hatano Reseach Institute, Food andDrug Safety Center (Kanagawa) for the in vivo
Draize test for confirmation of the Draize scores.
Purity and chemical properties assured in theChemical Bank revealed that these were stablethrough the whole period of the present validation study.
Cell lines
Two cells lines were used in each assay as
shown in Table 1. HeLa S3 (SC) cells (a sublineof HeLa) were used as a common cell line in all
assays.
Each cell line except serum-free cultured
NRCE was supplied from RIKEN Cell Bank aslive cultures derived from a single lot. Eagle's
minimum essential medium (MEM) supplemented with 10% bovine calf serum (CS) wasused for HeLa S3 (SC) and CHL cells. MEM
containing 10% fetal bovine serum (FBS) wasused for SQ-5 and BALB/3T3 A31-1-1 cells.
Since serum-lots are known to greatly influence
the cloning efficiency of BALB/3T3 A31-1-1
cells, a specified lot of FBS (lot. U15600,
NescoBio, Co., Tokyo) was purchased by theparticipating laboratories. Lots of CS and FBS,
however, were freely chosen by each laboratory
for assays with HeLa S3 (SC) cells and othercell lines. NRCE cells, the serum-free primary-cultured Japanese white rabbit cornea epithelial
cells, were provided in a commercially available
frozen kit (Corne-Pack) together with the serumfree culture medium RCGM from Kurabo Industries Ltd. (Osaka).
Cytotoxicity assays
Precise protocols on each assay will be described in subsequent articles in this issue written by the Working Group. The following paragraphs are briefly digested descriptions of theassays. Manufacturers of plastic plates and otherlab-wares were not specified but were freely selected by the participants.
Colony formation (CF) assay has been done
to determine directly the proliferation ability of
individual cells (Sasaki and Tanaka, 1991). Twokinds of cells, BALB/3T3 A31-1-1 and HeLa S3(SC) were used. Cells were thoroughly dissociated and seeded into 60-mm dishes. After 18-24
hr culture, 20 µl of apropriately diluted test
chemical solution was added. Incubation was
continued without medium change at 37°C under 5% CO2 in air for 7 days for BALB/3T3 A311-1 cells and 13 days for HeLa S3 (SC) cells.
Then the cells were fixed and stained with Giemsa solution. Colonies with 50 or more cells
were counted.
For crystal-violet staining (CV) assay(Saotome et al., 1989; Itagaki et al., 1991), 100µl samples of suspensions of HeLa S3 (SC)(10,000 cells/well) or CHL (4,000 cells/well)
cells in logarithmic growth phase were seededin each well of 96-well plates containing appropriately diluted chemicals in 100 µl of culture
medium. The cells were cultured at 37˚C under
5% CO2 in air for 3 days, and then fixed with25% glutaraldehyde for 10 minutes, and stainedwith 0.4% crystal violet for 30 minutes. The
plates were then dried and OD590 values were
determined.
Lactate dehydrogenase release (LDH) assay
has been described in detail in previous papers(Sasaki et al., 1992; Wang et al., 1993). Briefly,
SQ-5 cells or HeLa S3 (SC) cells were seeded
into 96-well culture plates at 2,000 cells/well and
4,000 cells/well, respectively, suspended in 100µl of culture medium which contain 5% FBS to–8–

Submission of the worksheet

Step 1

No

Is it strictly keeping the protocol ?
Yes

Step 2

Were both data files on
chemical #1 and #7 submitted ?

Discard all the data files ofchemical #1 - #7 on thespecific assay.

No

Yes
Step 3

Analysis on LAP-JSAAE program

Step 4Is the check code "OK" on data filesof chemical #1 and #7 ? A checkcode will appear in the order of A,
B, C, D, E, F, then "OK".

No

Yes
Step 5Is the ratio of log ED50 of chemical #1 tothat of chemical #7, or its reciprocal, lessthan 5 ?

No

Reject all the data files ofchemical #1 - #7 on thespecific assay.

Yes
Step 6Is the check code "OK" on data filesof chemical #2 - #6 ? A check code
will appear in the order of A, B, C,
D, E, F, then "OK".

No

Reject the specific data file

Yes
Step 7

Box-whisker plot analysis

Fig. 1. Data file processing in the present study
Definitions of check codes are as follows:
Code-A : Calculation of ED50 values failed because of wide variation of data.
Code-B : In the LDH assay, LDH activity is directly inhibited or stimulated by the test chemical with thecorrection factor of below 0.1 or above 2.0, respectively. With these factors, correction of the observed LDH activity to the original LDH activity in a sample is essentially meaningless.
Code-C : No observed point between 20-80% of the maximum effect was found in the data file.
Code-D : Data included response of 200% or more (negative controls set at 100%).
Code-E : Of the 95% confidence interval of ED50, the upper limit was over 100 times that of the lower
limit.
Code-F : RMS is 10 or more. RMS indicates the degree of deviation of observed dose-response relationship from the logistic model. See details in the following Validation Article II in this issue.

–9–Validation Article I. Overview

reduce the background LDH activity in the medium, and pre-cultured at 37˚C under 5% CO2
in air for 24 hr. Then the cells were treated withappropriately diluted solutions of test chemicals
in PBS(-) for 20 min at room temperature (assay
series LDH-1), for 2 hrs (assay series LDH-2A)or for 48 hrs (assay series LDH-2B and LDH2C) in culture medium at 37˚C. LDH activity
was determined with a commercially available
enzymatic test kit (Kyokuto Pharmaceutical Inc.,Tokyo). Cell lysis and growth-inhibition by thetreatments were determined from OD560 values of the culture supernatants (LDH-1, LDH2A, LDH-2C) or cell layer (LDH-2B). The possible direct inhibition of LDH activity by thechemicals was first tested, and if inhibition was
detected, the factor for remaining LDH activity(FRLA) at each concentration of the test chemicals was used to normalize the cytotoxicity data.
F o r t h e M T T a s s a y [ M T T, 3 - ( 4 , 5 dimethylthiazol-2-yl)2,5-diphenyl tetrazolium
bromide], HeLa S3 (SC) cells (3,000 cells/well)and SQ-5 cells (3,000 cells/well) were pre-cultured in 100 µl/well of culture medium in 96well plates for overnight. Then the same volumeof 2-fold concentrated test solution was added
and the culture was continued for 48 hr. MTT
metabolism was determined according toMosmann (1983) .
For the neutral red uptake assay (NR)
(Borenfreund and Puerner, 1985; Hockley andBaxter, 1986), NRCE cells (2,500 cells/well)
were pre-cultured in 100 µl/well of serum-free
RCGM-CaCl2 medium at 37˚C under 5% CO2
in air for 72 hr. HeLa S3 (SC) cells (4,000 cells/well) were pre-cultured in 100 µl/well of MEM10%CS medium at 37˚C under 5% CO2 in airfor 24 hr. Both cell lines were incubated for 48
hr with test chemicals. After uptake of NR andfixation, OD540 values derived from neutral red
were measured and the cell survival was calculated.
Technology transfer
After the initial orientation held in Tokyo on
October 30, 1992, technology transfer of the fivecytotoxicity assays was carried out according to
the protocols (standard operating procedures) at

RIKEN on the 5 assays; at Hatano Research Institute, Food and Drug Safety Center, on the CF
and NR assays; at Nagasaki University on the 5assays; at Yokohama City University on the CV
assay, and at Kurabo Industries Ltd. on the NR
assay with NRCE cells in serum-free culture.
Participants from each laboratory in this validation study received training in one of these technology-transfer courses (2-5 days) except those
who had already received training in the 5 assays at RIKEN in July 1992. After the training
courses, each laboratory received cell lines forthe assays in the present study.
Data handling at The Working Group
Fig. 1 illustrates the flow of collected data
files handled by The Working Group. One data
file written in the style of a pre-set worksheet
corresponded to one assay on a chemical carried out by a laboratory. Data files stored on
floppy disks were also submitted to The Working Group. Each data file was examined to determine whether the assay followed the common
rules and/or if it contained any problem pertaining to the protocol used. Any data file found tocontain a serious problem against even only oneof the common rules or the protocol was rejected
and no further analyses were done on it. If thedata file of chemical #1 or #7 was not includedin the set of data files for the 7 chemicals on aparticular assay submitted from a laboratory or
if the data file was rejected for any reason, theset of data files for the 7 chemicals on that particular assay from that laboratory was discarded.
Acceptable files were analyzed by the logistic analysis program, LAP-JSAAE (see details
in the Validation Articles II in this issue : thesource code of the program is available on request). If any check code from Code-A - CodeF (see below) was attached by the program tothe data file of chemical #1 or #7, the set of data
files for the 7 chemicals on a particular assay
submitted from the laboratory was rejected. If
the ratio of ED50 values of chemicals #1 to #7(or #7 to #1: the smaller ED50 was taken as thedenominator) resulted in a figure of 5 or more,again, the set of data files of the 7 chemicals on– 10 –

the assay submitted from the laboratory was rejected. If any check code was attached by the
program to a specific data file of chemicals #2 #6, the specific data file of the chemical in theassay submitted from the laboratory was rejected.
Data files that passed through the above scrutiny were further submitted to box-and-whisker
plot analysis.
Definitions of check codes are as follows:
Code-A : Calculation of ED50 values failed because ofwide variation of data.
Code-B : In the LDH assay, LDH activity is directly inhibited or stimulated by the test chemical with thecorrection factor of below 0.1 or above 2.0, respectively. With these factors, correction of the observed
LDH activity to the original LDH activity in a sample
is essentially meaningless.
Code-C : No observed point between 20-80% of the maximum effect was found in the data file.
Code-D : Data included responses of 200% or more where
the negative controls were set at 100%.
Code-E : Of the 95% confidence interval of ED50, theupper limit was over 100 times that of the lower limit.
Code-F : RMS is 10 or more. RMS indicates the degreeof deviation of observed dose-response relationshipfrom the logistic model. See details in the following
Validation Article-II in this issue.

Analysis by box-and-whisker plot
To evaluate the inter-laboratory variations, weused the method of box-and-whisker plot (Tukey,1977), since the distribution of ED50 values
among participating laboratories was rather skew
in the upper tail and frequently contained outliers. As a result, the median and the hinge-spreadof log(ED50) were calculated for each chemicaland each assay with a cell line to represent thecentral tendency and the amount of variation of
ED50s among laboratories, where the hingespread was defined as the difference between theupper and lower hinges, i.e., the inter-quartile
range (see Validation Article-II).
With this context, the capability of each assay to distinguish irritancy of chemicals wasevaluated by the range of medians among chemicals standardized by the mean of hinge-spreads,
which was referred to as the "power for
distinction"(PFD). Thus, PFD was defined as
PFD = [max(median of log(ED50)) min(median of log(ED50))] / [mean of hingespreads]

where the mean in the denominator was calculated among the chemicals.
In vivo Draize test
The Draize eye irritation test (Draize, 1944)
was carried out to evaluate the eye irritancy ofchemicals at Hatano Research Institute, Food andDrug Safety Center. Using 0.1 ml of a 10% aqueous solution, the test chemical was applied into
the conjunctival sac of one eye of each of 3 rabbits and no irrigation was performed following
the application. Effects on the eyes were gradedperiodically according to the classic scoring system (Draize, 1944). The average scores for cornea, iris and conjunctiva and the average of thetotal scores were recorded at the time of maximal responses. If the eye irritancy was found to
be too high or too low to quantify, the test was
repeated with ten-times lower or higher concentration of the test chemical, respectively.
RESULTS
Organization and processing of the present
validation study
After calling on laboratories to participate in
the present validation study by September 1992,
47 laboratories had registered. Every laboratory
was allowed to participate in any assays they selected without any prerequisites, but they wererequired to state their selected assays before thestart of the validation study. Two laboratories did
not attend the orientation meeting held in October 1992, in which common rules for assays werediscussed and accepted.
Among the remaining 45 laboratories, threelaboratories began to co-work in this study but
two of them were not able to submit any data
files by the deadline that was initially set atMarch 19, 1993, but deferred until August 31,
1993. The other one had carried out LDH assay
they participated in, but was beset by a mycoplasma contamination in their test cell lines during maintenance culture; other laboratories did
not have this problem. Thus 3 laboratories were
prompted to withdraw from the validation study.
The number of qualified laboratories was there-– 11 –

Validation Article I. Overview

Table 2. Number of participating laboratories that received the technology transfer
Technology transfer(1)
Received
and practiced

(2)
(3)
(4)
Received
Not received
Did not submit
but not practiced, but practiced*;
any data
used technician used technician(but received)

Assay

Coworkedlaboratories

CF
CV
LDH-1
LDH-2A
LDH-2B
LDH-2C
MTT
NR

24
16
18
18
18
18
20
24

15
6
9
8
7
8
10
16

1
1
1
1
1
1
2
1

6
7
1
1
1
1
6
5

2
2
7
8
9
8
2
2

156

79

9

28

40 (30)Total**

(1)
(2)
(5)
(6)
(7)
(6)
(1)
(2)

* Laboratories which have been well experienced in each assay.
** Cumulative number of laboratories. Each laboratory was allowed to participate in multiple assays. The
actual number of co-worked laboratories which carried out the assays was 45.

fore trimmed down to 42. Their data files wereanalyzed based on the following description.
The Chemical Bank and the Cell Bank wereset for this validation study in Wako Pure Chemical Industries, Ltd. (Hyogo) and in RIKEN Cell
Bank (Ibaraki), respectively. These banks provided quality-controlled chemicals and cell lines
except the serum-free cultured cell line, NRCE,
which was directly provided in a commerciallyavailable kit together with the serum-free culture medium from Kurabo Industries Ltd.
(Osaka).
Technology transfer on the assays was carried out for the participating laboratories through2-5 day courses as described in Materials andMethods. We assumed that this transfer contributed to the adjustment of technical expertise of,at least, the attendants to the course. As shownin Table 2, the cumulative number of 79 laboratories in a total of 156 co-worked laboratories
carried out the assays by those who received thetechnology transfer. However, in 9 laboratories
the assays were carried out not by researchers
who received the technology transfer but by technicians who did not directly receive the technol-

ogy transfer (the qualities of these technicians
are unknown). Twenty-eight laboratories that are
well experienced in all assays did not attend thetraining. There were 40 laboratories which did
not submit any data, 30 of which had received
the technology transfer.
Through a questionnaire, we asked a total of
90 participants about the length of their experience in cell culture . Among the 61 who had over
one year’s experience, 34 received the technology transfer, while among 5 with less than one
year’s experience, 4 received the technology
transfer.
Cell lines
Two cell lines per assay (Table 1) were sent
to participating laboratories as live cultures (except NRCE cells that have been frozen) between
November 24 and December 1, 1992. HeLa S3(SC), a subline of HeLa, was set as a common
cell line in the 5 assays since it has been adopted
in the standardized protocol for quality controlof Eagle’s minimum essential medium labeledwith the Japanese Industrial Standard (JIS) K-– 12 –

3602. Other cell lines were selected because of
their wide-spread use in corresponding assays.
Since a single lot of cells was sent to eachlaboratory, no essential difference of characteristics was observed in the cells during the present
validation study except in Lab-15 where mycoplasma contamination was found as mentioned
above.
Common rules for assays and data collection
The five cytotoxicity assays were carried out
from November 1992 to August 1993. From theexpected 2,184 data files of final definitive assays where one data file corresponded to one
assay (i.e., triplicate tests carried out in three
separate plates) on a chemical, we collected
1,535 data files, although a total of 3,810 data
files were submitted including 2,275 data filesof preliminary assays (Table 3).
Participating laboratories were expected tosubmit data files written in the preset format oncomputer software. However, many of the submitted data formats were different from the preset ones. Due to the wide variety of data formats, The Working Group should have examined precisely and thoroughly all the input positions in every data file. The data files were finally shaped up to the format for a personal computer software program, EXCEL version 4.0(Microsoft Co. Ltd.) with considerable datacleaning work.
Although no violation of the common rules[1] and [2] was observed, violation of the common rule [3] was found. There were 98 data files
for the CV, LDH, MTT, and NR assays from Lab24 which were derived from one-plate tests (not3 independent-plate tests), and 68 data files on
the LDH and MTT assays from Lab-23 obtained
from tests in which negative- and/or positivecontrol measurements had been done in separate (not in the same) plates. Assays in whichcell lines were cultured in the medium with different type of serum and/or basal medium comprised 77 data files. These data files were notaccepted for the calculation of ED50 values.
A laboratory (Lab-2) carried out the CV assay with two cell lines on the 7 test chemicals

using two plates each. Lab-17 performed the CF
assay with BALB/3T3 A31-1-1 cells using 3
dishes for an observation point although 4 dishes
were required. For these two rare cases, however, The Working Group included their data files
as possibly acceptable candidate data files sincemeasurements for an observation point in duplicate plates or triplicate dishes allowed us to detect their intra-assay variations which were revealed to be generally far smaller than the interlaboratory variation.
Other apparent misunderstandings of the protocols were observed. Such misunderstandings
were evident in 49 data files that consisted of
the following : 1) the correction factor for calculation of original LDH activity resulting fromdirect inhibition on LDH activity of each chemical tested only with HeLa S3 (SC) cells was applied to the tests with SQ-5 cells; 35 data files;
2) the chemical concentrations tested in each
LDH-2B and LDH-2C assay should have been
the same since these assays were to be carriedout in tandem but the concentrations were different in 12 data files. Two files were short of
LDH-2C assay. Taken altogether, 292 files were
judged unacceptable before calculation of thedose of 50% effectiveness (ED50) (Table 3, column c).
On the common rule [4], only 797 data files
recorded 3 or more observation points between
20% and 80% of the maximum effect, that comprising 64% of the 1,243 possibly acceptable
candidate files (Table 4). The lowest was 46%
of possibly acceptable candidate files observedin the LDH-2A assay with HeLa S3 (SC) cells,
while the highest was 82% in the LDH-2B assay with SQ-5 cells. Although hand-plotted doseresponse curves of data drawn for each laboratory (See subsequent Validation Articles IV - VIII
for each assay in this issue) did not conform tothe common rule [4], we found that many of themwhich did not contain 3 observed points between
20-80% of the maximum effect, but 2 (238 files)
or 1 (122 files), were practically informative tocalculate ED50 values.
Therefore The Working Group reconsidered* Source code of LAP-JSAAE is available on request.

– 13 –

Validation Article I. Overview

Table 3. Data files of final definitive tests submitted to The Working Group

Number of data files
Assay

Cells

a*

b

c

Performance rate of

d

e

runs$
b/a

CF

CV
LDH-1#
LDH-2A
LDH-2B
LDH-2C
MTTNR

HeLa S3 (SC)BALB/3T3
A31-1-1
HeLa S3 (SC)CHL
HeLa S3 (SC)SQ-5
HeLa S3 (SC)SQ-5
HeLa S3 (SC)SQ-5
HeLa S3 (SC)SQ-5
HeLa S3 (SC)SQ-5
HeLa S3 (SC)NRCE

Total number of files

candidate finallyfiles$$ accepted
d/a
e/a

168
168

130
149

14
0

116
149

100
126

77%
89

69%
89

60%
75

112
112
126
126
126
126
126
126
126
126
140
140
168
168

98
97
70
70
68
67
62
62
68
61
123
117
154
139

14
14
13
20
14
28
21
28
14
21
28
28
28
7

84
83
57
50
54
39
41
34
54
40
95
89
126
132

75
78
39
25
26
28
18
20
26
25
89
83
108
103

88
87
56
56
54
53
49
49
54
48
88
84
92
83

75
74
45
40
43
31
33
27
43
32
68
64
75
79

67
70
31
20
21
22
14
16
21
20
64
59
64
61

2184

1535

292

1243

969

* a, expected; b, submitted; c, unacceptable; d, acceptable before ED50 calculation; e, finally accepted by
the logistic analysis program, LAP-JSAAE, and the intra-laboratory variation analysis (see Table 5 and7).
$ runs = b/a x 100$$ candidate files = d/a x 100, accepted by The Working Group before ED50 calculation.
# Different series in the LDH release assay, see Materials and Methods.
The figures in the table were updated and amended after our presentation in the meeting INVITOX’94(Zurich, Switzerland, 1994) to which data were submitted and published in the proceedings (Ohno, T., et al.,1995). Definition of performance rates were changed in order to reflect reality from that described in theproceedings. Other than these final test data files, 157 laboratories submitted 2,275 data files of preliminary
tests.

that, by using a computer-assisted program forstatistical analysis to calculate ED50, these files
should be accepted if dose-response curves weresuccessfully drawn with regular sigmoidal shape.
The curves were calculated by the logistic analysis program, named LAP-JSAAE*, which was
newly developed and based on the general statistical data analysis software, SAS (SAS Institute Japan Co., Tokyo) (see Validation Article IIin this issue for the algorithm). However, 86 data
files containing no observation point between 2080% of the maximum effect were discarded since
47 of them were found unable to give any reasonable sigmoidal dose-response curves by the

LAP-JSAAE program. This reconsideration on
the common rule [4] increased the number of
acceptable files to 1,157 (93% of the candidate
1,243 files).
Human errors
Fig. 2 illustrates numbers and percentages ofdata files in which errors, originating from data
handling before the data submission (defined as“human errors”), were found in the data cleaning process. Although precise description will
be made later (see the Validation Article III inthis issue), cumulatively 1,742 data files were

– 14 –

Table 4. Number of files including different number of observed points*
between 20 - 80 % effectiveness

Between 20 - 80 % effectiveness

Assay
CF
CV
LDH-1
LDH-2A
LDH-2B
LDH-2C
MTT
NR

Cells
HeLa S3 (SC)BALB/3T3 A31-1-1
HeLa S3 (SC)CHL
HeLa S3 (SC)SQ-5
HeLa S3 (SC)SQ-5
HeLa S3 (SC)SQ-5
HeLa S3 (SC)SQ-5
HeLa S3 (SC)SQ-5
HeLa S3 (SC)
NRCE

Total

Candidatefiles

3 points
or more%**2 points

1 point

0 points

116
149
84
83
57
50
54
39
41
34
54
40
95
89
126
132

68
95
56
59
37
34
25
20
22
28
35
25
75
59
92
67

59%
64%
67%
71%
65%
68%
46%
51%
54%
82%
65%
63%
79%
66%
73%
51%

28
32
17
14
5
5
11
5
7
3
7
14
12
20
26
32

11
12
7
7
8
3
9
9
6
3
6
1
5
5
5
25

9
10
4
3
7
8
9
5
6
0
6
0
3
5
3
8

1243

797

64%

238

122

86

* An observed point corresponds to mean of raw data derived from replicatewells for an observation ofeffectiveness of a chemical.
** % of candidate files

confirmed one by one by The Working Group as
possessing errors. The highest error (44%) was
the lack of essential data into the data files.
Rejected data files
As described in Materials and Methods, after
checking the abnormality of data files of chemical #1 and #7 (both are Tween 20) and intra-laboratory variation of ED50 values on these twocoded chemicals, the check codes worked todetect “rejectable” data files. Table 5 shows theresults. Priorities of these check codes are in theorder of A, B (for LDH assay only), C, D, E, andF.
If any error was found in one of the data filesof chemical #1 and #7 before the ED50 calculation, detection of the intra-laboratory variation
became impossible for that specific assay. The
data file set of chemicals #1 - #7 became accordingly meaningless. As a whole, 133 data
files were thus rejected before the calculation of

ED50 values. The most frequent rejection happened in the LDH-2A and -2B assays with HeLa
S3 (SC) cells where 21 data files each were rejected in 54 and 41 possibly acceptable candidate files, respectively. With analyses on the intra-laboratory variation of ED50 values determined as the ratio of ED50 values of chemical#1 and #7 (see below for the explanation of Table6), 5 more data file sets (29 files) were rejected
because the ratio exceeded 5.
In the data files for chemicals #2 - #6, a totalof 26 data files resulted in failure to calculate an
ED50 value (Code-A). Drawing of a logistic
curve was impossible in these data files due tolarge variation of the raw data. An example ofthis type of variation is shown in Fig. 3.
In the LDH assay, the observed LDH activity
is occasionally reduced or enhanced by the test
chemicals. The observed raw data for LDH activity were therefore corrected using these inhibitory- or stimulatory-factors, i. e., the factor
for remaining LDH activity (FRLA), as de-– 15 –

Validation Article I. Overview

g, 42(2%)
e, 96(6%) f, 83(5%)
d, 129(7%)
a, 754(44%)
c, 302(17%)

b, 336(19%)
Fig. 2. Data files in which errors were found in the data cleaning process.
a, Lack of essential data. b, Out-of-format inputs. c, Violation of the protocol or common rules. d, Simple
mis-recording reported from the laboratories. e, Abnormal data amended by the laboratory before any notationfrom The Working Group. f, Different data were written in the print-outs, the data stored in the submitted
floppy-disks, and the worksheet. g, others. Total files are 1742.

scribed in Materials and Methods and more precisely in the Validation Article VI on the LDH
assay in this issue. However, if the correction
factors FRLA fell below 0.1 or above 2.0 thatwere apparently out of linear range of the possible correction of the observed LDH activity tothe original LDH activity in a sample, the correction should be considered essentially impossible. The LAP-JSAAE program marked thesedata files with the Code-B. Four files in the subdivided LDH assays were therefore rejected(Table 5). An example of this type is shown in
Fig. 4 as the raw data plot.
Although calculation met the fundamental
requirements of the logistic analysis, as describedabove, the data files containing no observed point
between 20-80% of the maximum effect were
rejected (indicated with Code-C in Table 5).
There were, at this step, 47 files*. Also, thosefiles with raw data that included a response of
200% or more, where that of negative controls
was set at 100%, were rejected (Code-D in Table5). Five data files were rejected for this reason.
In 7 data files, the upper limit of the 95%
confidence interval of ED50 values was over 100
times the lower limit (Code-E in Table 5). An
example of a raw data plot is shown in Fig. 5. At
most, 3 files in the NR assay with the serumfree cultured NRCE cells were marked with theCode-E.

We set the upper limit of the RMS value at
10, which, roughly speaking, corresponds to therobustness of the drawn curve (see the subsequent Validation Article II on statistical analyses for its definition) at 10. If the value is 10 or
more, the estimated logistic curve should be considered unstable because of a shortage of rigid
data for estimation of the logistic curve. Thus,
23 files were rejected for this reason (Code-F inTable 5). As a result, 969 files appeared to be
finally acceptable for further analyses of thepresent inter-laboratory validation study (Tables
3 and 5). Therefore, as a whole, 37% of the submitted data files of the final definitive assays
were discarded before the box-and-whisker plot
analysis.
Intra-laboratory variation
The chemical #1 and #7 are the same chemical, Tween 20, derived from the same lot. Since
samples of this chemical were sent to each laboratory after doubly-mask-coding, the differencein ED50 values of chemical #1 and #7 was considered to reflect intra-laboratory variation ofeach assay. To examine this variation, we calculated the ratio of ED50 values of chemical #1* : This number does not contain the files that have been
rejected under the check code A and B. It is, therefore,
different from the number of data files shown in Table
4.

– 16 –

Table 5. Number of files rejected or finally accepted by the logistic analysis program, LAP-JSAAE.
Candidate
files

Assay

Rejected files

Finally
accepted
files

Ratio
Chemical of ED50
Chemical #2 - #6 with**#1 or #7 of #1/#7
with Code (#7/#1) is Code-A Code-B Code-C Code-D Code-E Code-F $A* ~ F 5 or more

Cells

HeLa S3 (SC)BALB/3T3
A31-1-1CV
HeLa S3 (SC)CHL
LDH-1 HeLa S3 (SC)SQ-5
LDH-2A HeLa S3 (SC)SQ-5
LDH-2B HeLa S3 (SC)SQ-5
LDH-2C HeLa S3 (SC)SQ-5
MTT
HeLa S3 (SC)SQ-5
NR
HeLa S3 (SC)
NRCE

116
149

0
7

6&
4&

2
3

7
7

0
0

1
0

0
2

100
126

84
83
57
50
54
39
41
34
54
40
95
89
126
132

7
0
7
14
21
7
21
14
14
7
0
0
7
7

0
0
0
5&
0
0
0
0
0
0
0
0
7
7

1
2
2
1
2
1
0
0
6
2
2
2
0
0

1
1
6
4
1
3
0
0
3
0
2
4
3
5

0
1
1
0
0
0
0
0
0
0
0
0
0
3

0
0
0
0
0
0
0
0
2
1
0
0
0
3

0
1
1
1
2
0
2
0
2
5
2
0
1
4

75
78
39
25
26
28
18
20
26
25
89
83
108
103

Total files

1243

133

29

26

47

5

7

23

969

CF

1
0
2
0
0
0
1
0

4

* Code-A. Calculation of an ED50 value failed because of wide variation of data.
** Calculation met the requirement by the logistic analysis program LAP-JSAAE but with the code B, D, E, orF.
Code-B indicates that, in the LDH-release assay, LDH activity is directly inhibited or stimulated by the test
chemical with the correction factor of below 0.1 or above 2.0, respectively. With these factors, correction of
the observed LDH activity to the original LDH activity in a sample is essentially meaningless.
Code-C indicates that no observed point was found between 20-80% of the maximum effect in the data file.
Note that the numbers in this column do not contain the files that have been rejected under the check codeA and B, therefore they are different from the number of data files shown in Table 4.
Code-D indicates that data include response of 200% or more where that of negative control was 100%.
Code-E indicates that, of the 95% confidence interval of ED50, the upper limit is over 100 times the lower limit.
$ Code-F indicates that RMS is 10 or more. RMS indicates the degree of deviation of observed dose-response
relationship form the logistic model. See details in the following Validation Article II in this issue.
& The data file set on this assay did not include files on one or two of chemicals #2 - #6.

and #7, wherein the smaller ED50 value wastaken as the denominator (Table 6).
In 164 data file sets of the chemical #1 and#7 paired in each assay, 75 data file sets (46%)
had a ratio in the range of 1.0 to less than 1.1.
Only 16 data file sets showed a ratio of 2.0 or
more and 159 data file sets (97%) had a ratio
less than 5.0. In one extreme case, Lab-24 submitted data files for the CF assay with HeLa S3(SC) cells that resulted in a ratio of over 100.
We defined a ratio of 5.0 or more as an indication of extraordinary large intra-laboratory
variation. Such a data file set in the assay fromthe indicated laboratory should, therefore, beeliminated, i.e., Lab-24 in the CF assay with
HeLa S3 (SC) cells, Lab-4 in the CF assay with
BALB/3T3 A31-1-1 cells, Lab-7 in the LDH-1
assay with SQ-5 cells, Lab-31 in the NR assay
with HeLa S3 (SC) cells, and Lab-1 in the NR
assay with NRCE cells (Table 6). The above
consideration resulted in 159 remaining data filesets which were thought to be acceptable.
As seen in Table 6, four data file sets resulted
in a ratio of over 2.0 out of 21 CF assays with
BALB/3T3 A31-1-1 cells, and of 18 NR assays
with serum-free NRCE cells 3 data sets had– 17 –

Validation Article I. Overview

Fig. 3. An example of data files that resulted in
calculation-failure (Code A) by the logistic analysis
program, LAP-JSAAE
Because of extremely large variation of data,
drawing of a dose-response curve was impossible
in the file of MTT assay for chemical #4 with HeLa
S3 (SC) cells.

Fig. 4. An example of data files in LDH-2A assay
which used the correction factor, FRLA, below 0.1(Code B)The data file of LDH-2A assay with HeLa S3(SC) cells used the correction factor 0.09 for thehighest concentration of the chemical #5.

Fig. 5. An example of data files in NR assay with
NRCE cells resulted in that the upper limit of 95%
confidence interval of ED50 was over 100 times
the lower limit (Code E)

ED50 ratios exceeding 2.0. The CV and MTT
assays, each with 2 cell lines, that were conducted in 11 - 14 trials resulted in ratios of lessthan 1.7. In the NR assay with HeLa S3 (SC)cells, the ratios observed were within 1.0-1.4
except for Lab-31.
Performance rate of the assays
From the data files that survived the abovementioned scrutiny, it was possible to calculate
the performance rate of each assay as shown in

Table 3. Laboratories performed the CF, CV, NR,
and MTT assays at rates of 77 - 92% on a running basis (column b/a in Table 3). On the other
hand, subdivided LDH assays revealed lower
performance rates of 48 - 56%. On the possibly
acceptable candidate file basis (column d/a inTable 3), the CF, CV, NR, and MTT assays maintained relatively higher performance rates of 64- 89%. Especially in the CF assay with BALB/
3T3 A31-1-1 cells, none of the submitted data
files was seen to be unacceptable before calculation of ED50 values.

– 18 –

Table 6. Number of data file sets which resulted in the ratio of ED50 values of chemicals #1
and #7 deviating from 1
Assays

CF

CV

LDH-1

LDH-2A

LDH-2B

LDH-2C

MTT

NR
Total

Cells#HeLa BALB HeLa CHL HeLa SQ-5 HeLa SQ-5 HeLa SQ-5 HeLa SQ-5 HeLa SQ-5 HeLa NRCE filesets
Ratio*
1.0 - <1.1
1.1 - <1.2
1.2 - <1.3
1.3 - <1.4
1.4 - <1.5
1.5 - <1.6
1.6 - <1.7
1.7 - <1.8
1.8 - <1.9
1.9 - <2.0
2.0 - <5.0
5.0 - <10
10 - <100
>100

8
3
3

7
1
3
2
1
1
1
1

1

4
5
1
1

7
2

1
2
1

1
1

4

3
1

2

1

1

1

2

2
2

8
2
2

2
1
1
1

1

1

3

9
3
2
1

1
2

1
1
1
1

1

5 12
2 2
2 1
3 1
1

1
1

1

2

1

1

1

2
1

13 17

18

1
1

Total file 17
sets

21

11

12

8

6

5

5

3

3

6

5

14

75
26
15
10
7
5
4
3
1
2
11
3
1
1
164

The file sets with the ratio of 2.0 or more
AssaysCells#

CF

CV

LDH-1

LDH-2A

LDH-2B

LDH-2C

MTTNR

HeLa BALB HeLa CHL HeLa SQ-5 HeLa SQ-5 HeLa SQ-5 HeLa SQ-5 HeLa SQ-5 HeLa NRCE

Labo No. 24
4Ratio** 173 13.0
Labo No.
Ratio

12
4.45

Labo No.
Ratio

30
2.78

Labo No.
Ratio

45
2.01

9
7
3.29 7.11

7
2.7

45 29
45
2.83 4.28 3.65
42
2.00

31
1
9.15 7.76
18
3.06
30
2.25

# Cell names were partly abbreviated as follows. HeLa, HeLa S3 (SC); BALB, BALB/3T3 A31-1-1.
* Ratio of ED50 values of chemicals #1 and #7. The smaller ED50 value derived from #1 or #7 in each assay
was taken as the denominator.
** Extraordinary figures refer to the ratio of ED50s of chemical #1 and #7 (or #7 and #1) equivalent to 5.0 or
more as indicated with bold figures.

However, since a relatively large number of
unacceptable files (274 files) was detected by
the logistic analysis program LAP-JSAAE asshown in Table 5, each assay lowered the performance rate on finally accepted file basis by
59 - 75% for the CF, CV, NR, and MTT assays
and by 14 - 31% for the subdivided LDH assays. In that, LDH-2B with HeLa S3 (SC) cells
performed on the finally accepted basis as low
as 14% of 126 data files initially expected from18 laboratories. The total number of files finallyaccepted was 969, that is 44% of 2,184 initially
expected files, 63% of 1,535 submitted files asfinal definitive assays, and 78% of 1,243 possibly acceptable candidate files.
In Fig. 6, we visualized distribution of acceptability of data files from each laboratory. Boxes
with a thick background pattern represent files
not submitted, unaccepted before the ED50 calculation, or finally rejected. Boxes with a thin

– 19 –

Validation Article I. Overview– 20 –

dot pattern (for example, Lab-44 in CV assay
shown in Fig. 6b) are the files finally accepted
but their descriptions of original “final” concentrations of the tested chemicals were miswritten
at the submission of raw data to The Working
Group.
Boxes containing a wide thick-patterned areain the subdivided LDH assay sections indicatethat many laboratories did not perform the assay. In contrast, sections with many white boxes
such as in the CV assay with two cell lines andthe CF assay with BALB/3T3 A31-1-1 cells indicate that the assays were performed relatively
efficiently. In the CF assay with HeLa S3 (SC)cells carried out by Lab-36, all the log(ED50)
values calculated from 6 accepted data files resulted in outliers of the box-and-whisker plot
analysis. Also, 5 outliers from Lab-28 were observed in the NR assay with the serum-free cultured NRCE cells.
In Fig. 6, failure of assays on chemical #4
was apparent especially in the CF assay. With
HeLa S3 (SC) cells, 16 data files from 24 expected data files were not submitted or were finally rejected. Also with BALB/3T3 A31-1-1
Fig. 6. Acceptability of data files from each laboratory
Open boxes are the files finally accepted forcomparison of inter-laboratory variation oflog(ED50) values without any difficulty in obtaining ED50 values. Asterisks in open boxes
indicate that the log(ED50) value became an
outlier in the box-whisker plot analysis (see Fig.
8).
No data file was submitted.
A data file(s) was submitted but not useful
because of severe violation of the protocol.
A check code (Code A, B, C, D, E, or F) was
found in the data file of chemical #1 or #7, thenthe corresponding data file set of chemicals#1~#7 of the assay was rejected.
The ratio of ED50s of chemical #1 and chemical #7, or vise versa, was over 5.
A check code (Code A, B, C, D, E, or F) was
found in data files of chemical #2, #3, #4, #5, or#6.
Data files accepted after amending simple
recording errors on reported concentrations ofchemicals after the discussion meeting held on
November 30, 1994.

cells, 16 data files were not submitted or were
finally rejected and 2 data files resulted in outliers of log(ED50) values in the box-and-whisker
plot analysis. This tendency was also observed
for the LDH-1 assay (Fig. 6c) but not in the CV,
MTT, and NR assays.
Typical dose-response curves
Although the performance rate of the LDH2B assay was low (Table 3), Fig. 7 shows a representative example of dose-response curves of
the chemicals in LDH-2B with the HeLa S3 (SC)cell line as determined by Lab-45. The curves
are essentially parallel between the cytotoxicity
levels of 20% and 80%, therefore relative cytotoxicity of the 7 test chemicals can be represented
by ED50.
Box-and-whisker plots of log(ED50) values
and inter-laboratory variation
While arithmetic means and standard deviation are usually used to summarize a set of measurements, they are inappropriate to represent ourdata, since the ED50 values were irregularly distributed and the distribution among laboratories
was highly asymmetric, thus outliers were likely
to exist. In this situation, statistical insight suggests the use of percentiles such as median andquartiles, invoking the use of box-and-whisker
plot as has been described by Tukey (1977). In
the box-and-whisker plot (Fig. 8a - g), the vertical line drawn in each box implies the median
and, accordingly, represents an average tendency
among laboratories, while the length of each box
implies the inter-quartile range, i.e., the hingespread, and, accordingly, represents the interlaboratory variation.
All log(ED50) values, their mean and standard deviations, and their coefficient of variations will be described in subsequent Validation
Articles IV - VIII written on each assay. In Fig.
8a on chemical #1, relatively small box sizes
were observed in the CV and in LDH-2A assays with 2 cell lines. We did not plot the box
for the LDH-2B assay since finally accepted data
files were less than 5 per chemical tested. For– 21 –

Validation Article I. Overview

#5

#3

#6

#7

#1 #2

#4

100

Cytotoxicity (%)

80
60
40
20
0
-20
.00001

.0001

.001

.01

.1

1

10

Concentration (%w/v)
Fig. 7. Typical dose-response curves of the chemicals in LDH-2B with the common
HeLa S3 (SC) cell line from Lab-45

the LDH-2C assay with HeLa S3 (SC) cells,
however, a large box and an extraordinary longupper whisker were drawn from 6 data files. The
same assay on chemical #7 (the same chemical
as #1, Tween 20), shown in Fig. 8b, revealed
that the median was almost attached to the leftedge of the box (lower hinge) and the box lostthe upper whisker but was accompanied by an
upper outlier.
The CF assay with BALB/3T3 A31-1-1 cells
resulted in the lowest median values oflog(ED50) on chemical #1, #7, #2, and #5 (Fig.
8a, b, c, and f, respectively), while for chemical#4 the lowest median value of log(ED50) was
obtained in the CF assay with HeLa S3 (SC) cells(Fig. 8e). However, for chemical #3 and #6, thelowest median value of log(ED50) was seen in
the NR assays with the serum-free cultured
NRCE cells (Fig. 8d and g). Two upper outliers
were observed in the NR assay with NRCE cells
for chemical #6 (Fig. 8g).
Evaluation of the cytotoxicity assays
To compare inter-laboratory variation of the
CF assays with two cell lines, the means of thehinge-spreads were calculated on the sevenchemicals (Table 7). The mean hinge-spread on
the CF assay with HeLa S3 (SC) cells and with
BALB/3T3 A31-1-1 cells was 0.24 and 0.48,
respectively. The latter was twice the former,
suggesting that the assay with BALB/3T3 A311-1 cells has a tendency for greater variation of
ED50 values than the assay with HeLa S3 (SC)cells.
This concept was extended to other assays
with different cell lines. However, it is wellknown that, when sample size of the box-andwhisker plot analysis is too small, box size will
become unstable and inappropriate for statistical analysis. Calculation was therefore not madeon mean hinge-spreads of the sub-divided LDH
assays, since their sample sizes, i.e., numbers offinally accepted data files, were apparently too
small on each chemical, except the LDH-1 assay with HeLa S3 (SC) cells for chemical #1 and#7 (Table 7). Thus, the smallest in calculated
mean hinge-spreads was 0.22 for the CV assay
with CHL cells and for the MTT assay with SQ5 cells, followed by 0.24 for the CF assay with
HeLa S3 (SC) cells and for the CV assay with
HeLa S3 (SC) cells. The largest, 0.48, was seen

– 22 –

Fig. 8. Box-whisker plots of cytotoxicity data on each chemical. Code number of the chemical tested was given under eachfigure.
– 23 –

Validation Article I. Overview

Fig. 8(Continued). Box-whisker plots of cytotoxicity data oneach chemical. Code number of the chemical tested was given
under each figure.
– 24 –

Fig. 8(Continued). Box-and-whisker plots of cytotoxicity dataon each chemical. Code number of the chemical tested was given
under each figure.

for the CF assay with BALB/3T3 A31-1-1 cells.
Considering the practicability of a cytotoxicity assay, it is desirable that the most cytotoxic
chemicals should give the lower ED50 value andthe least cytotoxic ones should give the higher
ED50 value. Differences in these ED50 valuesof the assay will serve as an indicator to distinguish individual chemicals from the view pointof cytotoxicity.
Comparing the difference of medianlog(ED50) values of the least toxic chemical #4
and the most severely toxic chemical #5 among
the assays, the CF assay with BALB/3T3 A311-1 cells and the CV assay with CHL cells resulted in the largest and the smallest difference,
respectively (Table 7). Then the difference wasdivided with the corresponding mean hingespread to normalize the effect of variation oflog(ED50) values. This was defined as “powerfor distinction” (PFD) (Table 7, last column) of
cytotoxicity of chemicals. The CF assay with
HeLa S3 (SC) cells gave the largest PFD of 21.0.
CV assay with HeLa S3 (SC) cells revealed also
a high PFD with a value of 20.4. The lowest PFD,
13.4, belonged to the CF assay with BALB/3T3
A31-1-1 cells.

When the performance rates, mean-hinge
spreads, and PFDs were displayed in 2-dimensional figures (Fig. 9), CV assays with the twocell lines shared the right upper corner in Fig. 9a
and b, although in Fig. 9c the CF assay with HeLa
S3 (SC) cells and the MTT assay with SQ-5 cells
were also grouped together with CV assays withthe two cell lines.
In vivo Draize test
We have conducted an in vivo Draize test to
confirm the toxicity of the 7 doubly-mask-codedchemicals in parallel with the cytotoxicity assays. At the first test in which the chemicals wereapplied to rabbit eyes as 10%(w/v) aqueous solution, chemicals #1, #2, #4, and #7 showed noirritating effect on cornea, iris, and conjunctiva.
The maximum average score (MAS) was therefore 0. When chemicals were applied at 100%(original undiluted chemical fluid) at the second
and the third tests, the MAS were 4.0, 5.3, 4.7,
and 5.3 for chemical #1, #7, #2, and #4, respectively (Fig. 10a, b, c, and e, respectively). No
abnormality was found in cornea or iris at any
observed time after the treatment, but in con-– 25 –

Validation Article I. Overview– 26 –

Performance rate (%)
on finally accepted data file basis
50

55

60

65

70

75

80

Mean hinge-spread

0.1
CV CHLCV HeLaMTT SQ-5

0.2

CV
CF
NR
MTT

CF HeLaNR NRCE

0.3

NR HeLa
MTT HeLa

0.4
CF BALB

0.5

Power for distinction

25

20

CV
HeLa

CF
HeLa
MTT
SQ-5
NR
NRCE

15

10
50

55

CV
CHL

NR
HeLa
MTT
HeLa

60

CF
BALB

65

70

75

80

Performance rate (%)
on finally accepted data file basis
Power for distinction
10

15

20

Mean hinge-spread

0.1
CV
CHL

0.2

0.3
MTT
HeLa

NR
HeLa

NR
NRCE

MTT
SQ-5

CV
CF
HeLa HeLa

0.4

0.5

CF
BALB

Fig. 9. Two dimensional displays of performance rate, mean hinge-spread, and thepower for distinction (PFD)a, performance rate vs. mean hinge-spread. b, performance rate vs. PFD. c,mean hinge-spread vs. PFD. Note that mean hinge-spread was plotted up-side down.
– 27 –

Validation Article I. Overview
b. Chemical #7

a. Chemical #1
10

10
1st step: 10% solution
2nd step: 100% chemical
3rd step: 100% chemical

6

6

4

4

2

2

0
0

24

48

72

96

120

144

1st step: 10% solution
2nd step: 100% chemical
3rd step: 100% chemcal

8

MAS

MAS

8

0

168

0

24

Time after application (hrs)

48

96

120

144

168

d. Chemical #3

c. Chemical #2
1050
1st step: 10% solution
2nd step: 10% chemical
3rd step: 100% chemical

8

6

30

4

20

2

10

0
0

24

48

72

96

120

144

1st step: 10% solution
2nd step: 100% chemical
3rd step: 20% solution

40

MAS

MAS

72

Time after application (hrs)

0

168

0

24

Time after application (hrs)

48

72

96

120

144

168

Time after application (hrs)f. Chemical #5

e. Chemical #4
10100
2nd step: 100% chemical

8

80

6

60

4

40

2

20

2nd step: 1% solution
3rd step: 0.1% solution

0
0

24

48

72

96

120

144

0

168

0

24

48

72

96

120

144

Time after application (hrs)g. Chemical #6
100
2nd step: 1% solution

80

60

40

20

0
0

24

48

72

96

120

144

168

Fig. 10. Scores of the chemicals in the in vivo Draize test
All the data for an observed point were mean values
derived from 3 rabbits. a, b, c and e: Four chemicals wereapplied at 100% (original chemical fluid) at the second
and/or the third tests, the maximum average score (MAS)were 4.0, 5.3, 4.7, and 5.3 for chemical #1, #2, #4, and#7, respectively. No abnormality was found in cornea andiris at any observed time after the treatment, but in conjunctivae, redness, chemosis, or discharge were found in
rabbits after 1, 4, and 24 hr of the treatment. d: Chemical#3 was applied at 10% aqueous solution, 100% original
fluid, and then 20% aqueous solution. f: Chemical #5
was applied at 10%, 1%, and 0.1% aqueous solutions.
MAS of the 10% solution at 24 hr was 82.3±1.2. At 48,
72, and 96 hr, no deviation of MAS was observed. g:
Chemical #6 was applied at 10%, 1%, and 0.5% aqueous
solutions. The error bar indicates Standard Deviation of
the observed MAS.
– 28 –

NR
NRC
E

S3 (
SC)

SQ5
MTT

NR
HeL
a

S3 (
SC)
HeL
a
MTT

CV
CHL

S3 (
SC)

-1
CV
HeL
a

T3 A
31-1
ALB
/3

CF
B

S3 (
SC)
CF
HeL
a

In vivo
Draize test Chemical

MAS

0
-1

log(ED50)

0-2(10% soln.)

1
#1
#2
#3
#4
#5

-3

#6

-4

#7
82.3

-5
-6
-7
Fig. 11. Correspondence of the median log(ED50) values measured by the in vitro cytotoxicity assays to the
MAS measured in the in vivo Draize test
Medians of log(ED50) were plotted on each in vitro assays. The MAS value, 0, of non-irritants, i.e.,chemical #1, #2, and #7, was adjusted to the mean of median log(ED50) values of these chemicals, -1.54.
Then the MAS of chemical #5 at 10% solution, 82.3, was adjusted to the position where the mean of medianlog(ED50) values of chemical #5 was -4.54.

junctiva, redness, chemosis, or discharge were
found in rabbits 1, 4, and 24 hr after the treatment.
For chemical #3 at the first test, 10% aqueous solution induced no change in cornea or iris,but caused redness and chemosis in all the rabbits 1-48 hr after the treatment. The MAS was
14.7. With 100% chemical application at the second test, the MAS increased to 25.3. When 20%
aqueous solution was applied to rabbit eyes at
the third test, one of the 3 rabbits exhibited opacity of cornea for 24 - 96 hr after treatment andinjection of iris for 24 - 120 hr after treatment.
The MAS was 19.7 (Fig. 10d).
Chemical #5 irritated all the 3 rabbits treatedwith 10% aqueous solution after 24 hr. Rabbits
developed opacity of cornea and injection of iris.

Redness of conjunctiva, chemosis, and discharge
were observed through the whole test period. The
MAS was 82.3. With 1% and 0.1% aqueous solutions, these values decreased to 25.0 and 4.0,
respectively (Fig. 10f).
Chemical #6 at 10% aqueous solution induced
opacity of cornea in 2 of 3 rabbits after 24 hr
and in one rabbit after 48 hr. All the rabbits developed redness of conjunctiva and chemosis
through the whole test period. The MAS was
39.3. At 1% and 0.5% aqueous solution, the values decreased to 14.7 and 4.0, respectively (Fig.
10g).
From these results, chemical #1 (Tween 20),
#2 (Tween 80), #4 (propylene glycol), and #7(Tween 20) were judged “non-irritant”; chemical #3 (sucrose fatty acid ester) , “irritant”;– 29 –

Validation Article I. Overview

chemical #5 (cetylpyridinium chloride monohydrate), “severe irritant”; chemical #6 (sodium
lauryl sulfate), “moderate irritant”. These resultsroughly corresponded to the log(ED50) values
measured by the in vitro cytotoxicity assays.
As shown in Fig. 11, medians of log(ED50)
can be possibly classified into 4 groups, namely,(1) medians of chemical #4 which were distributed near log(ED50) values of 0.0 in every assay; (2) those of chemical #2 and #1(and therefore #7) of log(ED50) between -2.0 and -1.0
except that of the CF assay with BALB/3T3 A311-1 cells which resulted in the median slightly
lower than -2.0; (3) those of chemical #3 and #6
consistently near -2.0, although chemical #6 (sodium lauryl sulphate) exhibited an extremely low
median log(ED50) in the NR assay with serumfree cultured NRCE cells; (4) those of chemical#5, the severe irritant in the Draize test, near 4.0 or lower (Fig. 11).
When the MAS value of non-irritants, i.e.,
MAS = 0, was arbitrarily adjusted to the meanof median log(ED50) values of group 2 chemicals (-1.54) and the MAS of chemical #5 at 10%
solution (82.3) to the mean median values ofgroup 4 (-4.54), the mean median values of group3 (-2.17) corresponded roughly to the MAS values of ‘mild’ irritant chemicals #3 and #6. However, the median log(ED50) values of chemical#3 and #6 from the NR assay with serum-free
cultured NRCE cells did not match the positionof MAS values of these two chemicals.
Fact data base construction
A database was developed that allows thecollaborators to cross-refer the data produced inthis project. Since most of the members use
stand-alone DOS machines or Macintosh in theirlaboratories, Excel version 4.0 was selected forthe database management system that absorbs
the difference of the platform. A user is able tosearch the database by using a combination offour data items such as the identification number of a laboratory, a range of ED50 values, atest chemical, and a type of proposed cytotoxicity assay. In the event of a query, the file namewhich contains the raw data submitted by a par-ticipating laboratory is also displayed together
with the four data items and reliability information. Thus the detailed experimental results could
be referred to at once and with ease with pointand click of the PC mouse. This fact data base isavailable at the homepage below.
http://wdcm.nig.ac.jp/validation.html
Discussion
General consideration
Since the present validation study wasplanned to determine the practicability of the
proposed cytotoxicity assay methods, no restriction was set on the competence of participating
laboratories. However the submitted data files
were rigorously examined and the performanceof the assays was seriously considered. Results
shown in Table 3 suggest that the CF, CV, NR,
and MTT assays, but not the LDH assay, are recommendable from the view point of performance
rates based on the finally accepted data files.
These four assays have essentially the same characteristic, i. e., determining the extent of cell
survival and/or growth. On the other hand, theLDH assay measures simultaneously, as shownby the subdivided LDH-2 assays, not only thesurvival and/or growth of cells but also cell deaththrough LDH activity released from the cells(LDH-2C assay). Besides the determination of
FRLA, this characteristic of the LDH assay madethe procedures apparently more complicated thanthe other 4 assays and, therefore, was assumed
to have resulted in low performance rates (Table3). This suggests that simplicity of the assay
method is an important component to give optimum performance rate.
The CV assay with CHL cells and the MTT
assay with SQ-5 cells resulted in the smallest
mean hinge-spread (Table 7). These assays may
be proposed as the most useful from the view
point of data variation. However, from the view
point of the newly defined PFD of cytotoxicities for chemicals (see the next section for its
meaning), the CF assay with HeLa S3 (SC) cells
may also be the most useful assay if one compares the least and the most toxic chemicals– 30 –

(Table 7). This assay, however, was not powerful enough to distinguish the toxicities ofslightly- to moderately-toxic chemicals, #1, #7,#2, #3, and #6, as shown in Fig. 11. Next to the
CF assay, the CV assay with HeLa S3 (SC) cells
gave the second smallest mean hinge-spread andthe second highest PFD (Table 7) with the ability to distinguish the toxicities of slightly- tomoderately-toxic chemicals (Fig. 11).
The CF assay required 8 or 14 days for a run,i. e., one day for preculture and 7 or 13 days fortreatment with chemicals and colony formation,although actual labor for the assay requires only
3 days, i.e., the preculture, the treatment, andcolony counting. The size distribution of colonies is additionally informative. Contrary to thislong period assay, the CV assay with HeLa S3(SC) cells, the second best suggested from Table
7 and Fig. 11, requires 3-day assay period and2-day actual labor. Although cells were treatedfor only 2 days in the MTT and NR assays, theseassays require a 24-hr preculture (except the NR
assay with NRCE cells that require 72-hr
preculture) for a total of 3 days with actual labor, which is equal to the entire period required
for the CV assay. Therefore we think that theCV assay is simpler than the CF, MTT, and NR
assays.
These points and the localization displayedin Fig. 9 prompted us to consider that the CV
assay will give the most robust inter-laboratory
results and therefore is the most practical cytotoxicity assay at least for surfactants as tested in
the present validation study.
Power for distinction
The following is the background explaining
the reason for the adoption by The Working
Group of the newly defined “power for distinction” (PFD) in the evaluation of cytotoxicity inan assay. In detecting cytotoxicity, in vitro cytotoxicity assays have generally higher sensitivity, on the basis of chemical concentration in test
solutions, than the in vivo Draize eye irritation
test. A typical example is shown by chemical #5
in Fig. 7 and Fig. 10 (less than 0.0002% and0.1%, respectively). Highly sensitive detectionof cytotoxicity is feasible with any one of the 5cytotoxicity assays for this purpose when compared to the in vivo Draize test. Therefore, theabsolute sensitivity of cytotoxicity among the 5assays is not necessarily important.
Considering two chemicals, the larger the difference in the median toxicity values of the two
is important in the distinction of toxicity of thesetwo chemicals. However, if variation of data foreach of the two chemicals is also large, the distinction of their toxicities will be difficult. On
the contrary, if variation of data is small enough,the assay will be useful for distinction of toxicity of the two chemicals.
Participating laboratories
Many laboratories claimed that the initiallyset duration for the cytotoxicity assays was too
short for completion of sufficiently repeated assays, even though the deadline for data file submission was postponed for five months. Considering the first step validation study and manpower cost in each assay and since the participants were called on a volunteer basis, we could
not insist on them to carry out all the assays under the principle of good laboratory practice(GLP) as recommended by the regulatory organizations. Other reasons for the failure to follow
GLP included a variety of laboratory equipment
and the insufficiency of experience in GLP in
most of the participating laboratories. These may
have affected the efficiency of data file acceptance and the variation of ED50 values. If we
had selected only the participants who are highly
skillful and sufficiently experienced, the above
mentioned conclusion could have been slightly
different.
We have partly covered this weak point by
replicating one of the 6 test chemicals in the 7
doubly-mask-coded samples and by requiring theparticipants to carry out an assay on a chemicalwith 3 separate plates including negative- and/or positive-control wells in each plate. The
former contributed to detection of the intra-laboratory variation of data (Table 6), and the latter
contributed to detect technical stability in eachlaboratory. Researchers or technicians who at-– 31 –

Validation Article I. Overview

tended the technology transfer course were madeaware that, when they found an extraordinary
difference in the control data in one of the 3plates, they should repeat the assay until the data
fall in a range of relatively small difference than
before which can be considered acceptable. Sincemany laboratories (corresponding to 64% of
possibly acceptable candidate data files) repeatedassays until they observed a dose-response curve
which included 3 or more observed points falling between 20-80% of the maximum cytotoxic
effect (Table 4), although still many other laboratories (corresponding to 36% of possibly acceptable candidate data files) could not complete
repeating the assays satisfactorily, we thoughtthat most of the participating laboratories trained
themselves until they obtained stable results
among the 3 plates in an assay. So far, in thedata files examined by The Working Group, thedifferences of observed negative- or positivecontrol values among the 3 plates in an assay
were sufficiently small compared to the differences of those control values among the laboratories (data not shown. See Validation Articles
IV - VIII on each assay). After the discussion
meeting held on November 30, 1994, The Working Group accepted simple recording errors onreported concentrations of chemicals. At the final box-and-whisker plot analyses, the jump-offlog(ED50) values among data files in each assay were excluded and further analyses wereconcentrated on the log(ED50) data that fell in
the hinge-spread. By these strategies, as manyerrors as possible were thought to be excluded.
Chemicals, cell lines and protocol of theassaysIn the present validation study, the following
six test chemicals with diverse Draize scores
reported in the literature were selected (see Materials and Methods): 3 non-irritants, 1 irritant,
1 moderate irritant, and 1 severe irritant. This
unbalanced selection was reflected in the scoresof the Draize test done on the same lot of thechemicals. For ease of further statistical analyses, there should have been a more balanced selection of chemicals including more severe irri-tants.
Although we did not indicate in the protocolthat the test chemicals should be prepared justbefore use, the 6 test chemicals are all stable atroom temperature. In case of unknown chemicals in the assays, timing will be an importantfactor in the preparation of test chemical solutions.
What is more important is that the cells in
preculture should be carefully kept in log phase.
Culture of HeLa S3 (SC) cells was stably performed in every laboratory since this cell line
was familiar and can be easily handled by almost all participants. We were not, however, able
to confirm this point from submitted data files
except the LDH-2B and -2C assays. From thepreliminary experiments, population doublingtimes for HeLa S3 (SC) cells and SQ-5 cells were
determined to be 20.3 hr and 20.8 hr, respectively. Therefore, in the 48 hr treatment period
in the LDH-2B and -2C assays, the number ofcells in the negative (untreated) control wells
should increase by about 5-fold.
Unexpectedly, growth of the cell lines in control wells was very different among laboratories,
resulting in large variability in negative control
data in the inter-laboratory assays (data notshown, see Validation Articles IV - VIII on eachassay). These variations may have influenced theresults of assays.
The Working Group considered that observation of initial cell density and a reference
chemical should have been set at a specified concentration showing near 50% effectiveness ineach assay to determine whether the assay was
valuable from the view points of cell growth
control and the effectiveness of a standard toxicant. Furthermore, in the LDH assay, we shouldhave been more careful in determining LDH
activity in FBS-containing culture medium.
Some lots of FBS increased the background levelof LDH in preliminary assays and therefore reduced cytotoxic sensitivity of the assay.
To our surprise, many laboratories could not
perform LDH assays (Table 3, Fig. 6). The LDH1 assay which includes 20 min incubation atroom temperature is the shortest one in the assays validated. This assay can be repeated 2 or 3

– 32 –

times in a day. However, presumably because
this assay requires the determination of FRLA
(the factor of direct effect on LDH activity ofthe test chemical at each concentration used),participants may have considered it tedious, thus
resulting in non submission of so many data files.
The LDH-2A and LDH-2B assays (and therefore the LDH-2C assay coupled with LDH-2A
and 2B) should be carried out on the same lot of
tests, since calculation of ED50 for the LDH-2B
assay in which cells were treated for 48 hrs withchemicals utilizes the total LDH activity of thecells in negative control wells of the LDH-2A
assay as the initial cellular LDH activity. This
point was not clearly made in the protocol. In
the present study, only 6 data file sets were foundto have come from assays which have been carried out in the same lot of the test. Many sets of
LDH-2B assays used the initial cellular LDH
activity derived from a different test for the LDH2A assay (for details, see Validation Article VI
on LDH assay in this issue). The Working Group
accepted this utilization since the initial LDH
activity per number of cells from different lotsof tests gave very small variation of data.
The CV assay includes steps of cellular attachment to the culture surface, cell proliferation, and protein synthesis in the medium containing chemicals over a 3-day incubation(Saotome et al., 1989). The attachment step is
not included in other assays. However, this characteristic of the CV assay seemed to not strongly
influence the log(ED50) values observed in thepresent study since the medians of log(ED50)
values were not necessarily lower than thoseobserved in the MTT and NR assays with HeLa
S3 (SC) cells (Table 7).
The Working Group was awared later that, ifcell-bound crystal violet dye was extracted with
70% methanol before the OD590 measurement,
as the formazane formed in the MTT assay was
also extracted, then the observed values became
free from occasional uneven localization of cells
in a well. This point will improve variation of
the observed data.
NRCE cells, the primary cultured rabbit cornea cells, sometimes ceased to increase in number over the culture period indicated in the in-struction sheet attached in the commercial kit,probably because these cells enter the so-called“crisis”. Therefore in some laboratories, shortage of cells was experienced for extensive repeating of the NR assays with NRCE cells in
one kit. The cost was found to be a potential problem in the NR assay using this kit.
Concerning the length of treatment of cellswith chemicals, the CF assay is the longest (7 or
13 days) and the LDH-1 assay is the shortest (20min). Effect of duration of the chemical treatments was markedly reflected in the mean values of log(ED50) for chemicals #1, #7, #2, and#5 (Fig. 8a, b, c, and f, respectively). However,for chemical #3 and #6, this effect was not observed (Fig. 8d and g), presumably because ofdifferent mechanism of toxic action.
Cytotoxicity assay with serum-free cultured
cells has been described as generally more sensitive than those with cells cultured in serumcontaining medium (Torishima et al., 1990;
Torishima et al., 1995). In the present assays,
this tendency was observed only on 4 out of 6
chemicals tested (not on chemical #4 and #5)
when the mean log(ED50) of the NR assay with
NRCE cells were compared to those of the NR
assay with HeLa S3 (SC) cells (Table 7, Fig. 8e
and f). Especially with chemical #6, approximately 100-fold higher sensitivity was observedin the NR assay with NRCE cells than in assays
with cells cultured in regular serum-containing
medium (Fig. 8g). This reason should be at least
partly ascribed to the strong affinity of chemical#6, sodium lauryl sulfate, to serum proteins.
Technology transfer
In the initial stage of this study, The Working
Group asked the participants their extent of cell
culture experience. However, so far as we observed during the technology transfer, qualitatively speaking, extensive experience in cell culture could not necessarily serve to guarantee that
one will obtain smaller variations in assay results. We found a problem soon after the beginning of collection of data that in some laboratories the trainees did not carry out the assays themselves but the technicians under their supervi-– 33 –

Validation Article I. Overview

sion. In such cases, it was doubtful whether these
persons were as skillful as the representative
trainees. There was a considerable number oflaboratories whose staffs were not skillful
enough to carry out the popular and easy cytotoxicity assays (Table 3 and Fig. 6). The Working Group assumes this was partly because theparticipants were called on a volunteer basis. It
is desirable by some arrangements to adopt a
minimum threshold level of skill on cytotoxicity assays to avoid the human errors (Fig. 2) asmuch as possible in future validation studies.
Common rules
On the common rule [4], all the participating
laboratories initially thought that calculation of
ED50 values and data analyses would be easy
from final data files if they contained 3 observed
points between 20% and 80% of the maximum
effectiveness on each chemical. Direct readingof ED50 values should have been possible fromhand-plotted dose-response curves. However,
only 64% of data files could provide such dataand so many laboratories could not (Table 4);
perhaps time was over-spent and work remained
incomplete in repeating assays many times todetermine the suitable narrow dose range of eachchemical to fit the desired effectiveness. Then,considering the practicability of assays, we haveloosened strict application of the common rule[4] and rescued a number of data files by passing through the LAP-JSAAE program as described in Results. However, the data files whichcontain no observed point between 20% and 80%
of maximum effectiveness were discarded sinceprecision of the calculated ED50 value will be
impaired.
Data collection
Because of the difference in application
softwares and difference of versions of the software used, The Working Group had to invest amajor effort to clean up the raw data. On collection of data, this point must be improved, if possible, through on-line systems such as theInternet in future validation studies. On the final

definitive test of assays, The Working Groupjudged a file as the final definitive one recordedwith the latest date. This point should have beenclearly indicated in each protocol of assays.
Simple mistakes on data recording were realized by the corresponding laboratories after the
discussion meeting held November 30, 1994. In
the present validation study, we accepted correction of this type of simple miswriting of concentrations after the discussion meeting. Such a
stance rescued 42 files. However, human errors
should have been excluded in the validation
study as much as possible. For this purpose, several check points had to be set during assays,
before the data submission, and during raw data
cleaning.
Abnormal data
Wang and Ohno (1995) described that some
types of chemicals such as ethylene glycol increases the LDH activity of cells to more thanthat of control cells in low-dose ranges. Slightly
above this low dose range, the chemical showed
inhibition of cell growth with a sigmoidal doseresponse. We could not judge whether the increase in the LDH activity that indicated cell
growth stimulation has actually occurred. Although we could not find typical responses ofthis type in the present study, such a phenomenon should be considered on the statistical curve
fittings in the dose-response of new chemicals
in in vitro assays.
Performance rates
From the viewpoint of practice, simplicity ofthe protocol seemed very influential on the performance rate of an assay and ultimately on thevariation of log(ED50) values (Table 3, Table
7). The apparently lower performance rates observed in every sub-divided LDH assay compared to other 4 assays (Table 3, Fig. 6) indicated that the LDH assay seemed immature practically to be carried out in a laboratory with average technical expertise.
If performed skillfully (for example, Fig. 7),
the LDH-2A, B, and C assays done in one series– 34 –

of practice were more informative on toxicological characteristics of chemicals than the other 5
assays (Wang et al., 1993; Wang and Ohno,
1995). The rapid cell lysis was detectable by
LDH-2A, and after 48-hr incubation, both attached cells and lysed cells were quantifiable inthe same culture by LDH-2B and LDH-2C, respectively. However, LDH assays required assessment of direct effect of chemicals on the
LDH activity. In these assays, determination of
the factor FRLA, for correction of observed LDH
activity to original LDH activity at each concentration of the test chemical, forced laboratories
to carry out complicated assay procedures. On
the other hand, the other 4 assays included nostep to correct initially observed data. In thiscontext, on the basis of runs, the NR assay with
HeLa S3 (SC) cells seemed the easiest amongthe 16 assays since it was run at the highest performance rate, 92% (Table 3), while, in the LDH
assay, especially LDH-2C with SQ-5 cells,
showed the lowest performance rate, 48% (Table3). To compensate for this weak point, use of
automated laboratory processors (robots) isstrongly recommended (Wang et al., 1993). Oncethe assay conditions were set precisely, the robot can be expected to reproduce data in the assay with smaller variations than in the assay performed manually (data not shown).
In vivo Draize test
Reported toxicities of the test chemicals wereconfirmed by the in vivo Draize eye irritation
test (Fig. 10). Unfortunately however, Tween 20(the chemical to detect intra-laboratory variationthat has been chosen under a doubly-masked
system) showed no toxicity in vivo. It was therefore impossible for us to know the intra-laboratory variation in the in vivo Draize test.
The assay methods examined in the present
validation study should be correlated with the invivo Draize test. However, since the actual number of chemicals is only 6 in which half are nonirritants, and there being only one severe irritant, estimation of a statistically reliable correlation was difficult. A fairly correlative tendency
was observed between the median of log(ED50)

values and the MAS values at 10% chemicalsolution in the Draize test (Table 7 and Fig. 11).
Chemical #6, sodium lauryl sulfate, has been
widely used as a reference chemical in toxicological studies in vivo and in vitro (Kojima etal., 1995). The MAS of chemical #6, 39.3, inthe present in vivo Draize test was apparently
distinguishable from the MAS of chemical #3,
14.7. At present, however, we were not able toexplain why log(ED50) values of chemical #3
and #6, except in the NR assay with serum-free
cultured NRCE cells, were not clearly distinguishable in vitro as seen in Fig. 11. This implies that the in vitro assay is not necessarily able
to reflect exactly the in vivo Draize test.
Balls et al. (1995) described that, after thelarge scale international validation study on alternatives to the Draize test, none of the ninealternative tests including the NR assay on cell
viability met any of the four performance targets (wherein the results of the alternative tests
and the modified MAS obtained in the Draize
test were examined statistically for a significant
relationship between them). Their in vivo Draize
test data were obtained from the data bank of
the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) which stores
data from many different Draize tests carried out
on different occasions and in different places.
Therefore, large variability of the data among
the in vivo Draize tests carried out in different
laboratories must be taken into account, but still
Balls et al. (1995) described their disappointing
results “with the possible exception of predicting the irritancy of surfactants”. The corresponding results for the possible exception were derived from the NR assay on cell viability tested
with surfactants. Our present results are consistent with their conclusion on surfactants.
Statistical analyses
In Fig. 6, open boxes are the files finally accepted for comparison of inter-laboratory variation of log(ED50) values obtained without any
difficulty. If required and if possible, we read
ED50 values from hand-plotted dose-response
curves (see the subsequent issue with Validation– 35 –

Validation Article I. Overview

Articles IV - VIII on each assay). However, in
this study, we calculated ED50 values by thelogistic analysis program LAP-JSAAE. When
we tried direct reading of ED50 values fromhundreds of hand-plotted dose responses, although most of the ED50 values were very close
to those derived from the LAP-JSAAE program,some of the hand-plotted curves contained observed points showing only lower or higher than
50% effectiveness and therefore did not crossover the 50% effectiveness level. Two or more
ED50 values were readable from a hand-plottedcurve since the curve crossed over the 50% effectiveness level twice or more. The LAPJSAAE program enabled us to avoid these difficulties. In the coming validation study, a statistical analysis program such as the LAP-JSAAE
should be given to each participating laboratory
beforehand in order to greatly reduce the number of rejected data files.
Fact database construction
On the final definitive data files, we havedeveloped a fact data base on a personal computer. We acquired a powerful tool for data dissemination on the network, World Wide Web(WWW). It will be useful for researchers worldwide after we have converted the database to theone on WWW. In WWW, on-demand-analysis
will be also feasible on each assay by use of thecommon browser software.
Conclusion
From the results shown in the present study,i.e., performance rate, inter-laboratory variationof data represented by mean hinge-spread, andthe power for distinction of chemical cytotoxicity, and time needed for the assays, we consider
that the CV assay is the most practical and recommendable assay as a part of alternatives tothe in vivo Draize eye irritation test.
Acknowledgments
This work was supported in part by a fund from
JSAAE and in part by Special Coordination

Funds for Promoting Science and Technology
from The Science and Technology Agency ofJapan.
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