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

3.1 Rationale for the Reference Substances Selected for Testing 3-3

> 3.1.1 Reference Substance Selection Criteria 3-3
>
> 3.1.2 Candidate Reference Substances 3-5
>
> 3.1.3 Selection of Reference Substances for Testing 3-15

3.2 Rationale for the Number of Reference Substances Selected 3-16

3.3 Characteristics of the Selected Reference Substances 3-17

> 3.3.1 Source Databases Represented by the Selected Reference
> Substances 3-17
>
> 3.3.2 Chemical Classes Represented by the Selected Reference
> Substances 3-21
>
> 3.3.3 Product/Use Classes Represented by the Selected Reference
> Substances 3-22
>
> 3.3.4 Toxicological Characteristics of the Selected Reference
> Substances 3-22
>
> 3.3.5 Selection of Reference Substances for Testing in Validation
> Study Phases Ib and II 3-27
>
> 3.3.6 Unsuitable and Challenging Reference Substances 3-29

3.4 Reference Substance Procurement, Coding, and Distribution 3-30

3.5 Reference Substances Recommended by the *Guidance Document* (ICCVAM
2001b) 3-32

> **3.6 Summary 3-32**

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

This section discusses the rationale for the selection of the 72
reference substances tested to validate the 3T3 and NHK NRU test methods
for determining starting doses for rodent acute oral systemic toxicity
testing. Information regarding chemical class and physical/chemical
characteristics is provided, as is the available information on
toxicological characteristics, such as target, organ, extent of
metabolism, and mechanism of action, for the 72 reference substances.
Such information may be useful for characterizing the performance of the
3T3 and NHK NRU test methods for various chemical types. Chemical
supplier and purity information are provided as well as the methods for
purchasing, coding, and distributing the substances to the testing
laboratories.

**3.1 Rationale for the Reference Substances Selected for Testing**

This section describes the procedure used to select the 72 reference
substances tested in the NICEATM/ECVAM validation study.

3.1.1 [Reference Substance Selection Criteria]{.underline}

The SMT selected reference substances for testing in 2001 and 2002 using
a process based primarily on general recommendations made by Workshop
2000 participants (ICCVAM 2001b). The following criteria were used:

-   the toxicities of the reference substances should be evenly
    distributed across the expected range of LD~50~ values (i.e., the
    GHS classification for acute oral toxicity \[UN 2005\])

-   the reference substances should cover a wide range of structural and
    use classes, according to the needs of various user communities

-   substances with human toxicity data and/or human exposure potential
    (i.e., substances of interest to society) should be included

**Table 3-1** shows the GHS classification scheme which classifies
chemicals into five toxicity categories or an unclassified group based
on acute oral LD~50~ values (UN 2005).

**Table 3-1 UN GHS^1^ Classificatio**n Scheme for Acute Oral Toxicity

  -------------------------------- --------------------------------------
  **Category**                     **LD~50~ (mg/kg)**

  1                                LD~50~ ≤ 5

  2                                5 \< LD~50~ ≤ 50

  3                                50 \< LD~50~ ≤ 300

  4                                300 \< LD~50~ ≤ 2000

  5                                2000 \< LD~50~ ≤ 5000

  Unclassified                     LD~50~ \> 5000
  -------------------------------- --------------------------------------

> ^1^Globally Harmonized System of Classification and Labelling of
> Chemicals (UN 2005)

For the purposes of toxicity classification, the rodent oral LD~50~
values for individual reference substances were obtained from readily
available toxicological databases. Rat LD~50~ values were preferred, but
mouse LD~50~ values were used (three reference substances) when rat data
were unavailable. However, mouse data were not used in the regression
analyses (See **Section 6**). The Registry of Cytotoxicity (RC) is a
database of acute oral LD~50~ values for rats and mice obtained from
RTECS^®^ and IC~50~ values from *in vitro* cytotoxicity assays using
multiple cell lines and cytotoxicity endpoints for chemicals with known
molecular weights (Halle 1998). The toxicological databases, in order of
preference, were:

-   the RC, which contains LD~50~ values that came largely from the
    1983/84 RTECS^®^ (Halle et al. 1998)

-   the current RTECS^®^ (MDL Information Systems 2001, 2002)

-   the current Hazardous Substances Data Bank (HSDB; U.S. National
    Library of Medicine \[NLM\] 2001, 2002).

To assure that a wide range of structural and use classes were selected,
reference substances of interest to the various U.S. regulatory
agencies, as determined from chemical lists (personal communication)
from the agencies, were included. Chemicals with human toxicity data
and/or human exposure potential (i.e., chemicals of interest to society)
were chosen by mining publicly available databases (e.g., NTP test
database) for potential candidates.

3.1.2 [Candidate Reference Substances]{.underline}

*Sources of Candidate Chemicals*

The process of identifying the 72 reference substances started with the
compilation of a database that ultimately contained 116 candidate
chemicals. The intent of the SMT was to compile a database with more
than 12 chemicals in each toxicity category that also met the other
criteria, and then to prioritize the chemicals in each category to
select the 72 reference substances to be tested. As recommended by the
Workshop 2000 participants (ICCVAM 2001a), the following publicly
available databases and other indicated sources were used to identify
candidate chemicals:

-   the MEIC program, which collected human toxicity data and *in vitro*
    toxicity data from 61 test methods for the first 50 chemicals
    (Ekwall et al. 1998)

-   the RC (Halle 1998), which contains a compilation of *in vitro*
    cytotoxicity and *in vivo* rodent LD~50~ data for 347 chemicals

-   the Toxic Exposure Surveillance System (TESS) (Litovitz et al.
    2000), which compiles reports of toxic human exposures from poison
    control centers throughout the United States

-   pesticides recommended for consideration by the EPA Office of
    Pesticide Programs (OPP)

-   the *Guidance Document* (ICCVAM 2001b), which reported the NRU
    results for 11 RC chemicals using protocols similar to those used in
    the NICEATM/ECVAM validation study

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-   the U.S. NTP test database, which contains information on the
    toxicity of chemicals relevant to human exposure (NTP 2002)

-   the EPA High Production Volume (HPV) Challenge Program, which is a
    voluntary testing program to provide the public with a complete set
    of baseline health and environmental effects data for each chemical
    that is manufactured within or imported into the United States at
    amounts \> 1 million pounds/year (EPA 2000)

*Selection of Candidate Chemicals*

The complete list of candidate chemicals is provided in **Table 3-2.**
The left side of **Table 3-2** presents selected chemicals and the right
side presents the alternate chemicals. The candidate chemicals are
grouped by GHS acute oral toxicity classification. For each candidate
chemical, the table provides the corresponding rat or mouse oral LD~50~
value, the database(s) or other source(s) used to identify the chemical
as a potential candidate, and the type of product and/or use for the
chemical. Product/use categories were identified from HSDB (NLM 2001,
2002) or RTECS (MDL Information Systems 2001, 2002).

The final list of candidate chemicals compiled by the SMT included:

-   65 MEIC chemicals. These include the first 50 chemicals evaluated by
    MEIC as well as another 15 chemicals that were identified for future
    evaluation (C. Clemedson, personal communication 2001). Twenty of
    these chemicals were identified for the EDIT program, a follow-on
    project to the MEIC study to develop supplementary toxicity and
    kinetic tests (to determine distribution of chemicals in the body
    and biotransformation of chemicals to more toxic metabolites) to
    improve the prediction of human toxicity by the battery of tests
    identified as the best predictors in the MEIC program (Clemedson et
    al. 2002). The EDIT chemicals were selected by excluding MEIC
    chemicals that were volatile, those that precipitated at the IC~50~
    dose level, and those with sparse or insufficient data on human
    toxicity or mechanism of acute toxicity

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-   16 pesticides with extensive human exposure nominated by the EPA
    OPP. These included fenpropathrin, endosulfan, bromoxynil (phenol),
    fipronil, carbaryl, rotenone, metaldehyde, molinate,
    1,3-dichloropropene, dichlorvos, chlorpyrifos, sodium arsenite,
    triphenyltin hydroxide, cycloheximide, acrolein, and boric acid.
    Pentachlorophenol was also nominated, but was already on the
    candidate list since it was a MEIC chemical

##### 

<table>
<colgroup>
<col style="width: 13%" />
<col style="width: 7%" />
<col style="width: 9%" />
<col style="width: 14%" />
<col style="width: 13%" />
<col style="width: 0%" />
<col style="width: 7%" />
<col style="width: 10%" />
<col style="width: 8%" />
<col style="width: 14%" />
</colgroup>
<thead>
<tr class="header">
<th colspan="10"><strong>Table 3-2 Candidate Chemicals for the 3T3 and
NHK NRU Test Methods Validation Study</strong></th>
</tr>
<tr class="odd">
<th colspan="4"><strong>Selected Chemicals</strong></th>
<th colspan="6"><strong>Alternate Chemicals</strong></th>
</tr>
<tr class="header">
<th><strong>GHS Category<sup>1</sup>/Chemical</strong></th>
<th><strong>Rodent Oral LD<sub>50</sub><sup>2</sup>
(mg/kg)</strong></th>
<th><strong>Source<sup>3</sup></strong></th>
<th><strong>Product/Use<sup>4</sup></strong></th>
<th><p><strong>GHS</strong></p>
<p><strong>Category<sup>1</sup>/Chemical</strong></p></th>
<th colspan="2"><strong>Rodent Oral LD<sub>50</sub><sup>2</sup>
(mg/kg)</strong></th>
<th><strong>Source<sup>3</sup></strong></th>
<th><strong>Notes<sup>5</sup></strong></th>
<th><strong>Product/Use<sup>4</sup></strong></th>
</tr>
</thead>
<tbody>
<tr class="odd">
<td colspan="10"><em><strong>LD<sub>50</sub> ≤ 5
mg/kg</strong></em></td>
</tr>
<tr class="even">
<td>Mercury II chloride</td>
<td>1</td>
<td>MEIC, EDIT, RC (outlier), TESS, NTP</td>
<td>Preservative/ manufacturing/ insecticide</td>
<td>Aflatoxin B1</td>
<td colspan="2">5.0</td>
<td>RC (outlier)</td>
<td>Prohibitively expensive</td>
<td>Food contaminant</td>
</tr>
<tr class="odd">
<td>Triethylenemelamine</td>
<td>1</td>
<td>RC (outlier), NTP</td>
<td><p>Manufacturing/</p>
<p>insect sterilant</p></td>
<td> </td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="even">
<td>Sodium selenate</td>
<td>2<sup>a</sup></td>
<td>TESS, NTP</td>
<td>Feed additive</td>
<td></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td>Busulfan</td>
<td>2</td>
<td>RC (outlier), NTP</td>
<td>Pharmaceutical (antineoplastic)</td>
<td> </td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="even">
<td>Cycloheximide</td>
<td>2</td>
<td>RC (outlier), NTP</td>
<td>Antibiotic/fungicide</td>
<td></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td>Disulfoton</td>
<td>2</td>
<td>RC (outlier), EPA, NTP</td>
<td>Pesticide (insecticide)</td>
<td> </td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="even">
<td>Parathion</td>
<td>2</td>
<td>RC (outlier), EPA, NTP</td>
<td>Pesticide (insecticide)</td>
<td></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td>Strychnine</td>
<td>2<sup>a</sup></td>
<td>MEIC, TESS, EPA</td>
<td>Pesticide (rodenticide)</td>
<td> </td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="even">
<td>Aminopterin</td>
<td>3<sup>b</sup></td>
<td>RC</td>
<td>Pharmaceutical (antineoplastic); Rodenticide</td>
<td></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td>Phenylthiourea</td>
<td>3</td>
<td>RC (outlier), NTP</td>
<td>Pesticide (rodenticide)</td>
<td> </td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="even">
<td>Epinephrine bitartrate</td>
<td>4<sup>b</sup></td>
<td>RC (outlier), NTP (HCl salt)</td>
<td>Pharmaceutical (adrenergic)</td>
<td></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td>Physostigmine</td>
<td>5<sup>a</sup></td>
<td>EHS</td>
<td>Pharmaceutical (anticholinesterase)</td>
<td> </td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="even">
<td colspan="10"><em><strong>5 &lt; LD<sub>50</sub> ≤ 50
mg/kg</strong></em></td>
</tr>
<tr class="odd">
<td>Colchicine</td>
<td>6<sup>b</sup></td>
<td>MEIC, RC, TESS, NTP</td>
<td>Pharmaceutical (gout suppressant)</td>
<td>2,4-Dinitrophenol</td>
<td colspan="2">30</td>
<td>RC (outlier), NTP, HPV</td>
<td></td>
<td>Pesticide (fungicide/ insecticide) manufacturing</td>
</tr>
<tr class="even">
<td>Potassium cyanide</td>
<td>10</td>
<td>MEIC, EDIT, RC (outlier), TESS</td>
<td>Electroplating</td>
<td>t-Butylamine</td>
<td colspan="2">44<sup>a</sup></td>
<td>EPA, NTP, HPV</td>
<td> </td>
<td>Manufacturing</td>
</tr>
<tr class="odd">
<td>Dichlorvos</td>
<td>17<sup>a</sup></td>
<td>TESS, EPA, NTP, HPV</td>
<td>Pesticide (insecticide)</td>
<td>Acrolein</td>
<td colspan="2">46</td>
<td>RC, TESS, EPA, NTP, HPV</td>
<td>Volatile (BP=52°C)</td>
<td>Pesticide (herbicide/ rodenticide/ algicide), manufacturing</td>
</tr>
<tr class="even">
<td>Digoxin</td>
<td>18<sup>b</sup></td>
<td>MEIC, EDIT, RC (outlier), TESS</td>
<td>Pharmaceutical (antiarrhythmic)</td>
<td> </td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="odd">
<td>Fenpropathrin</td>
<td>18<sup>a</sup></td>
<td>EPA</td>
<td>Pesticide (insecticide)</td>
<td></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td>Endosulfan</td>
<td>18<sup>a</sup></td>
<td>TESS, EPA, NTP</td>
<td>Pesticide (insecticide)</td>
<td> </td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="odd">
<td>Arsenic III trioxide</td>
<td>20</td>
<td>MEIC, EDIT, RC, TESS, EPA, NTP</td>
<td>Pesticide (insecticide)</td>
<td></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td>Thallium I sulfate</td>
<td>29<sup>b</sup></td>
<td>MEIC, EDIT, RC (outlier), TESS</td>
<td>Pesticide (rodenticide/ insecticide)</td>
<td> </td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="odd">
<td>Sodium arsenite</td>
<td>41<sup>a</sup></td>
<td>TESS, NTP</td>
<td>Pesticide (herbicide, insecticide, fungicide)</td>
<td></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td>Triphenyltin hydroxide</td>
<td>44</td>
<td>RC, EPA, NTP, HPV</td>
<td>Pesticide (fungicide/ insecticide)</td>
<td> </td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="odd">
<td>Sodium dichromate dihydrate</td>
<td>50</td>
<td>RC, EPA, GD, NTP</td>
<td>Oxidizing agent</td>
<td></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td>Nicotine</td>
<td>50</td>
<td>MEIC, EDIT, RC (outlier), TESS, EPA, NTP</td>
<td>Pharmaceutical (stimulant)</td>
<td> </td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="odd">
<td colspan="10"><em><strong>50 &lt; LD<sub>50</sub> ≤ 300
mg/kg</strong></em></td>
</tr>
<tr class="even">
<td>Paraquat</td>
<td>58</td>
<td>MEIC, EDIT, RC (outlier), TESS, EPA</td>
<td>Pesticide (herbicide)</td>
<td>Pentachlorophenol</td>
<td colspan="2">51</td>
<td>MEIC, RC (outlier), NTP</td>
<td></td>
<td>Disinfectant</td>
</tr>
<tr class="odd">
<td>Hexachlorophene</td>
<td>61</td>
<td>MEIC, RC, TESS, NTP</td>
<td>Disinfectant</td>
<td>Amphetamine sulfate</td>
<td colspan="2">55</td>
<td>MEIC, EDIT, RC (outlier), TESS, NTP</td>
<td>DEA</td>
<td>Pharmaceutical (stimulant)</td>
</tr>
<tr class="even">
<td>Lindane</td>
<td>76</td>
<td>MEIC, EDIT, RC (outlier), EPA, NTP</td>
<td>Pesticide (insecticide)</td>
<td>Rotenone</td>
<td colspan="2">60</td>
<td>RC, TESS, EPA, NTP</td>
<td></td>
<td>Pesticide (insecticide/ piscicide)</td>
</tr>
<tr class="odd">
<td>Cadmium II chloride</td>
<td>88</td>
<td>RC, TESS, GD, NTP</td>
<td>Consumer/ industrial products</td>
<td>Furfural</td>
<td colspan="2">65<sup>a</sup></td>
<td>NTP, HPV</td>
<td> </td>
<td>Solvent, food additive</td>
</tr>
<tr class="even">
<td>Verapamil HCl</td>
<td>108</td>
<td>MEIC, EDIT, RC (outlier), TESS. NTP</td>
<td>Pharmaceutical (antiarrhythmic)</td>
<td>p-Phenylenediamine</td>
<td colspan="2">80</td>
<td>RC, GD, NTP, HPV</td>
<td></td>
<td>Dyeing</td>
</tr>
<tr class="odd">
<td>Haloperidol</td>
<td>128<sup>a</sup></td>
<td>MEIC, TESS</td>
<td>Pharmaceutical (antipsychotic)</td>
<td>Chlorpyrifos</td>
<td colspan="2">82<sup>a</sup></td>
<td>TESS, EPA, NTP</td>
<td> </td>
<td>Pesticide (insecticide)</td>
</tr>
<tr class="even">
<td>Sodium oxalate</td>
<td>155</td>
<td>MEIC, EDIT, RC, TESS, NTP</td>
<td>Paints, cleaners</td>
<td>Dextropropoxyphene HCl</td>
<td colspan="2">83</td>
<td>MEIC, RC (outlier), TESS</td>
<td></td>
<td>Pharmaceutical (analgesic)</td>
</tr>
<tr class="odd">
<td>Phenobarbital</td>
<td>163</td>
<td>MEIC, RC (outlier), TESS, NTP</td>
<td>Pharmaceutical (anticonvulsant)</td>
<td>Methadone</td>
<td colspan="2">86<sup>a</sup></td>
<td>MEIC,TESS, NTP</td>
<td>DEA</td>
<td>Pharmaceutical (analgesic)</td>
</tr>
<tr class="even">
<td>Sodium I fluoride</td>
<td>180</td>
<td>MEIC, RC, TESS, EPA, NTP</td>
<td>Electroplating, fluoridation</td>
<td>Fipronil</td>
<td colspan="2">92<sup>a</sup></td>
<td>EPA</td>
<td></td>
<td>Pesticide (insecticide)</td>
</tr>
<tr class="odd">
<td>Caffeine</td>
<td>192</td>
<td>MEIC, RC (outlier), TESS, NTP, HPV</td>
<td>Pharmaceutical (stimulant), food additive</td>
<td>Pentobarbital</td>
<td colspan="2">125</td>
<td>MEIC, RC TESS</td>
<td>DEA</td>
<td>Pharmaceutical (sedative)</td>
</tr>
<tr class="even">
<td>Diquat dibromide</td>
<td>231</td>
<td>MEIC, RC, TESS</td>
<td>Pesticide (herbicide)</td>
<td>Bromoxynil (phenol)</td>
<td colspan="2">190<sup>a</sup></td>
<td>EPA</td>
<td></td>
<td>Pesticide (herbicide)</td>
</tr>
<tr class="odd">
<td>Cupric sulfate * 5 H2O</td>
<td>300</td>
<td>MEIC, RC, TESS, EPA, NTP</td>
<td>Pesticide (insecticide/ fungicide)</td>
<td>Diphenylhydantoin</td>
<td colspan="2">199</td>
<td>MEIC, RC, TESS, NTP</td>
<td> </td>
<td>Pharmaceutical (anticonvulsant)</td>
</tr>
<tr class="even">
<td></td>
<td></td>
<td></td>
<td></td>
<td>Metaldehyde</td>
<td colspan="2">227<sup>a</sup></td>
<td>TESS, EPA</td>
<td></td>
<td>Pesticide (molluscicide)</td>
</tr>
<tr class="odd">
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td>Carbaryl</td>
<td colspan="2">230</td>
<td>RC, EPA. NTP</td>
<td> </td>
<td>Pesticide (insecticide)</td>
</tr>
<tr class="even">
<td colspan="10"><em><strong>300 &lt; LD<sub>50</sub> ≤ 2000
mg/kg</strong></em></td>
</tr>
<tr class="odd">
<td>Amitriptyline HCl</td>
<td>319</td>
<td>MEIC, EDIT, RC, TESS</td>
<td>Pharmaceutical (antidepressant)</td>
<td>Ferrous sulfate</td>
<td colspan="2">319</td>
<td>MEIC, RC, TESS</td>
<td></td>
<td>Food additive</td>
</tr>
<tr class="even">
<td>Phenol</td>
<td>414</td>
<td>MEIC, RC, TESS, EPA, NTP, HPV</td>
<td>Disinfectant</td>
<td>Warfarin</td>
<td colspan="2">324</td>
<td>MEIC, RC, TESS, EPA</td>
<td> </td>
<td>Pharmaceutical (anticoagulant), pesticide</td>
</tr>
<tr class="odd">
<td>Propranolol HCl</td>
<td>470<sup>b</sup></td>
<td>MEIC, RC, TESS, GD</td>
<td>Pharmaceutical (antiarrhythmic)</td>
<td>Disopyramide</td>
<td colspan="2">333<sup>a</sup></td>
<td>MEIC, TESS</td>
<td></td>
<td>Pharmaceutical (antiarrythmic)</td>
</tr>
<tr class="even">
<td>Chloral hydrate</td>
<td>479</td>
<td>MEIC, RC, TESS, NTP</td>
<td>Pharmaceutical (sedative)</td>
<td>Barium II nitrate</td>
<td colspan="2">355</td>
<td>MEIC, RC, TESS, NTP</td>
<td> </td>
<td>Pyrotechnic</td>
</tr>
<tr class="odd">
<td>Glutethimide</td>
<td>600</td>
<td>MEIC, RC, TESS</td>
<td>Pharmaceutical (sedative)</td>
<td>Thioridazine HCl</td>
<td colspan="2">358</td>
<td>MEIC, RC, TESS</td>
<td></td>
<td>Pharmaceutical (antipsychotic)</td>
</tr>
<tr class="even">
<td>Atropine sulfate</td>
<td>623</td>
<td>MEIC, EDIT, RC, TESS</td>
<td>Pharmaceutical (antimuscarinic)</td>
<td>Methylphenidate</td>
<td colspan="2">367<sup>a</sup></td>
<td>NTP</td>
<td>DEA</td>
<td>Pharmaceutical (stimulant)</td>
</tr>
<tr class="odd">
<td>Valproic acid</td>
<td>1695 <sup>b</sup></td>
<td>RC, MEIC, TESS, NTP</td>
<td>Pharmaceutical (anticonvulsant)</td>
<td>Molinate</td>
<td colspan="2">369<sup>a</sup></td>
<td>EPA, NTP</td>
<td> </td>
<td>Pesticide (herbicide)</td>
</tr>
<tr class="even">
<td>Meprobamate</td>
<td>794<sup>a</sup></td>
<td>MEIC, TESS</td>
<td>Pharmaceutical (antidepressant)</td>
<td>2,4-Dichlorophenoxy-acetic acid</td>
<td colspan="2">369</td>
<td>MEIC, RC, TESS, EPA, NTP, HPV</td>
<td></td>
<td>Pesticide (herbicide)</td>
</tr>
<tr class="odd">
<td>Acetylsalicylic acid</td>
<td>1000</td>
<td>MEIC, EDIT, RC, TESS, NTP</td>
<td>Pharmaceutical (analgesic)</td>
<td>Orphenadrine HCl</td>
<td colspan="2">425</td>
<td>MEIC, RC, NTP</td>
<td> </td>
<td>Pharmaceutical (analgesic)</td>
</tr>
<tr class="even">
<td>Lithium I sulfate</td>
<td>1187<sup>b</sup></td>
<td>MEIC, RC, TESS, NTP (Cl salt)</td>
<td>Pharmaceutical (mood stabilizer)</td>
<td>Trichlorfon</td>
<td colspan="2">451</td>
<td>RC, EPA, GD, NTP</td>
<td></td>
<td>Pesticide (insecticide)</td>
</tr>
<tr class="odd">
<td>Procainamide</td>
<td>1950<sup>a</sup></td>
<td>MEIC, TESS</td>
<td>Pharmaceutical (antiarrythmic)</td>
<td>Quinidine sulfate</td>
<td colspan="2">456</td>
<td>MEIC, RC, NTP (base)</td>
<td> </td>
<td>Pharmaceutical (antiarrhythmic)</td>
</tr>
<tr class="even">
<td>Carbamazepine</td>
<td>1957<sup>a</sup></td>
<td>MEIC, TESS</td>
<td>Pharmaceutical (antiepileptic)</td>
<td>1,3-Dichloropropene</td>
<td colspan="2">470<sup>a</sup></td>
<td>TESS, EPA, NTP</td>
<td></td>
<td>Pesticide (nematocide)</td>
</tr>
<tr class="odd">
<td></td>
<td></td>
<td></td>
<td></td>
<td>Theophylline</td>
<td colspan="2">600 <sup>b</sup></td>
<td>MEIC, RC, TESS, NTP</td>
<td></td>
<td>Pharmaceutical (antiasthmatic)</td>
</tr>
<tr class="even">
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td>Isoniazid</td>
<td colspan="2">650</td>
<td>MEIC, RC, TESS, NTP</td>
<td> </td>
<td>Pharmaceutical (antibiotic)</td>
</tr>
<tr class="odd">
<td></td>
<td></td>
<td></td>
<td></td>
<td>Diazepam</td>
<td colspan="2">709</td>
<td>MEIC, EDIT, RC, TESS, NTP</td>
<td>DEA</td>
<td>Pharmaceutical (anxiolytic)</td>
</tr>
<tr class="even">
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td>Maprotiline</td>
<td colspan="2">760<sup>a</sup></td>
<td>MEIC, TESS</td>
<td> </td>
<td>Pharmaceutical (antidepressant)</td>
</tr>
<tr class="odd">
<td></td>
<td></td>
<td></td>
<td></td>
<td>Methyleugenol</td>
<td colspan="2">810<sup>a</sup></td>
<td>NTP</td>
<td></td>
<td>Food additive</td>
</tr>
<tr class="even">
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td>Diphenhydramine HCl</td>
<td colspan="2">855</td>
<td>MEIC, RC, TESS, NTP</td>
<td> </td>
<td>Pharmaceutical (antihistamine)</td>
</tr>
<tr class="odd">
<td></td>
<td></td>
<td></td>
<td></td>
<td>Malathion</td>
<td colspan="2">885</td>
<td>MEIC, EDIT, RC, TESS, EPA, NTP</td>
<td></td>
<td>Pesticide (insecticide)</td>
</tr>
<tr class="even">
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td>Salicylic acid</td>
<td colspan="2">891</td>
<td>RC, TESS, GD, NTP, HPV</td>
<td> </td>
<td>Pharmaceutical (analgesic)</td>
</tr>
<tr class="odd">
<td></td>
<td></td>
<td></td>
<td></td>
<td>Chloroform</td>
<td colspan="2">908</td>
<td>MEIC, RC, NTP, HPV</td>
<td>Volatile (BP=61°C)</td>
<td>Solvent</td>
</tr>
<tr class="even">
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td>Chloroquine diphosphate</td>
<td colspan="2">970</td>
<td>MEIC, RC</td>
<td> </td>
<td>Pharmaceutical (antimalarial))</td>
</tr>
<tr class="odd">
<td></td>
<td></td>
<td></td>
<td></td>
<td>Ibuprofen</td>
<td colspan="2">1009</td>
<td>RC, TESS, GD</td>
<td></td>
<td>Pharmaceutical (analgesic)</td>
</tr>
<tr class="even">
<td> </td>
<td> </td>
<td> </td>
<td> </td>
<td>Nalidixic acid</td>
<td colspan="2">1349</td>
<td>RC, GD, NTP</td>
<td> </td>
<td>Pharmaceutical (antibiotic)</td>
</tr>
<tr class="odd">
<td></td>
<td></td>
<td></td>
<td></td>
<td>Dichloromethane</td>
<td colspan="2">1597</td>
<td>MEIC, RC, TESS, NTP, HPV</td>
<td>Volatile (BP=40°C)</td>
<td>Solvent</td>
</tr>
<tr class="even">
<td><strong> </strong></td>
<td><strong> </strong></td>
<td><strong> </strong></td>
<td><strong> </strong></td>
<td>Antipyrene</td>
<td colspan="2">1800</td>
<td>RC, GD</td>
<td> </td>
<td>Pharmaceutical (analgesic)</td>
</tr>
<tr class="odd">
<td colspan="10"><em><strong>2000 &lt; LD<sub>50</sub> ≤ 5000
mg/kg</strong></em></td>
</tr>
<tr class="even">
<td>Acetaminophen</td>
<td>2404</td>
<td>MEIC, EDIT, RC, TESS, NTP</td>
<td>Pharmaceutical (analgesic)</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td>Potassium I chloride</td>
<td>2602</td>
<td>MEIC, RC, TESS, NTP</td>
<td>Pharmaceutical (electrolyte), manufacturing</td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="even">
<td>Boric aid</td>
<td>2660<sup>a</sup></td>
<td>TESS, EPA, NTP</td>
<td>Pesticide (insecticide)</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td>Carbon tetrachloride</td>
<td>2799</td>
<td>MEIC, RC, TESS, NTP, HPV</td>
<td>Solvent</td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="even">
<td>Dimethylformamide</td>
<td>2800</td>
<td>RC, GD, NTP, HPV</td>
<td>Solvent</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td>Sodium chloride</td>
<td>2998</td>
<td>MEIC, EDIT, RC, TESS, EPA, NTP</td>
<td>Pharmaceutical (electrolyte), food additive</td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="even">
<td>Citric Acid</td>
<td>3000<sup>a</sup></td>
<td>EPA, NTP, HPV</td>
<td>Food additive</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td>Chloramphenicol</td>
<td>3393</td>
<td>MEIC, RC, NTP</td>
<td>Pharmaceutical (antibiotic)</td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="even">
<td>Lactic acid</td>
<td>3730</td>
<td>RC, NTP, HPV</td>
<td>Food additive</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td>Acetonitrile</td>
<td>3798</td>
<td>RC, NTP, HPV</td>
<td>Solvent</td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="even">
<td>Xylene</td>
<td>4300</td>
<td>MEIC, RC, TESS, NTP, HPV</td>
<td>Solvent</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td>Trichloroacetic acid</td>
<td>4999</td>
<td>RC, NTP</td>
<td>Fixative</td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="even">
<td colspan="10"><em><strong>LD<sub>50</sub> &gt; 5000
mg/kg</strong></em></td>
</tr>
<tr class="odd">
<td>2-Propanol</td>
<td>5843</td>
<td>MEIC, RC, TESS, EPA, NTP, HPV</td>
<td>Disinfectant</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td>Gibberellic acid</td>
<td>6305</td>
<td>RC, EPA, NTP</td>
<td>Plant growth regulator</td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="odd">
<td>Propylparaben</td>
<td>6326<sup>b</sup></td>
<td>RC (outlier), NTP</td>
<td>Food additive</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td>5-Aminosalicylic acid</td>
<td>7749<sup>b</sup></td>
<td>RC (outlier), NTP</td>
<td>Pharmaceutical (antibiotic)</td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="odd">
<td>Ethylene glycol</td>
<td>8567</td>
<td>MEIC, EDIT, RC, TESS, NTP, HPV</td>
<td>Antifreeze</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td>Diethyl phthalate</td>
<td>8602</td>
<td>RC (outlier), NTP, HPV</td>
<td>Plasticizer</td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="odd">
<td>Sodium hypochlorite</td>
<td>8910<sup>d</sup></td>
<td>TESS, NTP</td>
<td>Disinfectant</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td>1,1,1-Trichloroethane</td>
<td>10298</td>
<td>MEIC, RC, NTP, HPV</td>
<td>Solvent</td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="odd">
<td>Dibutyl phthalate</td>
<td>11998</td>
<td>RC (outlier), NTP, HPV</td>
<td>Plasticizer</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td>Glycerol</td>
<td>12691</td>
<td>RC, GD, NTP, HPV</td>
<td>Solvent</td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr class="odd">
<td>Methanol</td>
<td>13012</td>
<td>MEIC, EDIT, RC, TESS, NTP, HPV</td>
<td>Solvent</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td>Ethanol</td>
<td>14008</td>
<td>MEIC, RC (outlier), TESS, EPA, NTP, HPV</td>
<td>Solvent</td>
<td colspan="2"> </td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
</tbody>
</table>

^1^GHS-Globally Harmonized System of Classification and Labelling of
Chemicals for acute oral toxicity (UN 2005).

^2^LD~50~ data are from the Registry of Cytotoxicity (Halle 1998) and
are for rats, the preferred species for oral acute toxicity studies,
unless otherwise noted. Data with decimal places are rounded to the
nearest one.

^3^Sources used to identify candidate chemicals: EDIT =
Evaluation-guided Development of New *In vitro* Test Batteries; EPA =
pesticides registered with the Environmental Protection Agency; EHS =
EPA's Extremely Hazardous Substance list; HPV = High Production Volume
chemicals (i.e., those that are imported into or produced in the United
States in amounts [\>]{.underline} 1,000,000 lbs/year; GD = *Guidance
Document* (ICCVAM 2001b); MEIC = Multicentre Evaluation of In Vitro
Cytotoxicity; NTP = National Toxicology Program; RC = Registry of
Cytotoxicity with chemicals classified as regression outliers shown in
parentheses; TESS = Toxic Exposure Surveillance System (Litovitz et al.
2000).

^4^Product/use categories from Hazardous Substances Data Bank (NLM 2002)
or Registry of Toxic Effects of Chemical Substances (\[RTECS^®^\], MDL
Information Systems 2002). Pharmaceutical uses from Gilman et al. (1985)
or Thomson PDR^®^ (2004).

^5^Only chemicals expected to be too volatile for the cytotoxicity assay
system have \"volatile\" notations. BP = Boiling point. DEA (U.S. Drug
Enforcement Agency) refers to Schedule II controlled substances.
Chemicals with no \"DEA\" notation are expected to be under less strict
control.

^a^RTECS^®^ (MDL Information Systems 2002).

^b^Mouse.

-   five chemicals associated with the highest incidence of toxic
    exposures reported by U.S. poison control centers participating in
    the TESS (Litovitz et al. 2000): hypochlorite, acetaminophen,
    ethanol, diphenhydramine, and isopropanol. The five chemicals with
    the greatest incidence of toxic exposures among children were the
    same, except that oxalate replaced ethanol. Most of these chemicals
    were already identified as candidate chemicals due to their
    inclusion in the MEIC study. Since hypochlorite (sodium salt) and
    diphenhydramine, were not already included, they were added to the
    list of candidates

-   11 chemicals recommended in the *Guidance Document* (ICCVAM 2001b)
    for qualifying *in vitro* cytotoxicity assays for the prediction of
    starting doses using the RC regression. These chemicals were
    recommended because the IC~50~ and LD~50~ data for these chemicals
    fit the RC regression line extremely well. These chemicals were
    sodium dichromate dihydrate, cadmium chloride, p-phenylenediamine,
    DL-propranolol HCl, trichlorfon, ibuprofen, nalidixic acid,
    salicylic acid, antipyrene, dimethylformamide, and glycerol

-   16 chemicals from the NTP

    -   furfural, methyleugenol, and methylphenidate, scheduled for
        testing by the NTP National Center for Toxicogenomics (NCT) (G.
        Boorman, personal communication 2001), were added.
        Acetaminophen, another hepatotoxin to be tested by the NCT, was
        already a candidate chemical because it was included in the MEIC
        study. Chromium (VI), recommended by the NTP for consideration
        due to the potential for human exposure via drinking water
        (NTP 2002) was represented in the list of candidate chemicals by
        sodium dichromate dihydrate, which was also recommended in the
        *Guidance Document* (ICCVAM 2001b)

    -   dibutyl phthalate, 5-aminosalicylic acid, propylparaben,
        gibberellic acid, and diethyl phthalate were added to increase
        the number of chemicals with LD~50~ values \> 5000 mg/kg

    -   trichloroacetic acid was added to increase the number of
        chemicals in the 2000 \< LD~50~ ≤ 5000 mg/kg category

    -   sodium selenate was added to increase the number of chemicals in
        the LD~50~ ≤ 5 mg/kg category to 12

    -   six chemicals that were also on the HPV list were added. Lactic
        acid, citric acid, and acetonitrile were added to increase the
        number of chemicals in the 2000 \< LD~50~ ≤ 5000 mg/kg category.
        Tert-butylamine, 2,4-dinitrophenol, and acrolein were added to
        increase the number of chemicals in the 5 \< LD~50~ ≤ 50 mg/kg
        category

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-   eight additional RC chemicals in the LD~50~ ≤ 5 mg/kg category.
    These were: triethylenemelamine, busulfan, disulfoton, parathion,
    aminopterin, phenylthiourea, epinephrine bitartrate, and aflatoxin
    B1

The goal to identify more than 12 candidate chemicals for each toxicity
category was unrealized for three toxicity categories. The most toxic
category (LD~50~ ≤ 5 mg/kg), and least toxic categories (2000 \< LD~50~
≤ 5000 mg/kg, LD~50~ \> 5000 mg/kg), contained only 12 candidate
chemicals each. The intermediate toxicity categories (50 \< LD~50~ ≤ 300
mg/kg, \> 300 \< LD~50~ ≤ 2000 mg/kg), however, contained two to three
times the minimum number of candidate chemicals.

3.1.3 [Selection of Reference Substances for Testing]{.underline}

Using the candidate chemical database, 72 reference substances (12
unclassified chemicals and 12 chemicals from each the five GHS acute
oral toxicity hazard categories) were selected for use in the
NICEATM/ECVAM validation study. The criteria for prioritizing the
candidate chemicals were:

-   the availability of rodent acute oral toxicity data (e.g., RC,
    RTECS^®^)

-   the availability of human acute oral toxicity data and/or relevance
    for human exposure (e.g., MEIC, EDIT, TESS, NTP)

-   the lack of excessive volatility as estimated by SMT chemical
    consultants. Since the cells are exposed for 48 hours while
    incubated at 37°C in 96-well plates, volatilization from wells with
    high reference substance concentrations would reduce the extent of
    cytotoxicity and potentially contaminate other wells in close
    proximity

-   the lack of U.S. Drug Enforcement Agency (DEA) controls. Excluding
    chemicals that are listed in DEA Schedules I and II from
    consideration obviates the requirement for U.S. laboratories to
    obtain a DEA license and adhere to strict chemical storage and
    control procedures

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-   practical considerations such as cost and disposal issues

If more than twelve candidate chemicals in a GHS category met the above
criteria, then selection was based on two further considerations. One
consideration was the distribution of chemical toxicities within each
toxicity category (i.e., the goal was to select chemicals that
represented the entire range of toxicity within each category). Another
consideration, which applied only to candidate chemicals selected from
the RC database, was the fit of the chemical to the RC regression.
Chemicals with the best fit to the RC regression were preferentially
selected to prevent the entire set of reference substances from having
proportionally more "outlier" substances (i.e., greater than one-half
log from the RC regression) than the entire RC database.

The final list of selected reference substances is provided by GHS acute
oral toxicity category on the left side of **Table 3-2.**

**3.2 Rationale for the Number of Reference Substances Selected**

Seventy-two reference substances were used to evaluate the ability of
the 3T3 and NHK NRU test methods to estimate the acute oral LD~50~ and
thus the starting dose for *in vivo* acute oral toxicity tests. The SMT
determined the number of reference substances for testing by first using
the GHS classification scheme for acute oral toxicity (UN 2005) to
assure that the candidate chemicals covered the complete range of
toxicity, (see **Table 3-1**) then deciding how many chemicals would be
tested per category. To adequately cover the range of toxicity within
each of the six toxicity groups, the SMT decided to test 12 chemicals
per group. Seventy-two reference substances (12 substances/group with
six groups) were deemed adequate by the SMT, the ICCVAM Acute Toxicity
Working Group (ATWG), ICCVAM, and ECVAM.

The total number of reference substances was comparable to the number
used in other contemporary *in vitro* test method multilaboratory
validation studies. The European Cosmetic, Toiletry, and Perfumery
Association evaluation of multiple alternatives to the Draize eye
irritation test used 55 reference substances (Brantom et al. 1997).
ECVAM's evaluations of *in vitro* dermal corrosivity test methods
(Fentem et al. 1998) and *in vitro* dermal irritation assays (Botham
2004) used 60 reference substances.

**3.3 Characteristics of the Selected Reference Substances**

The physical/chemical and toxicological information in **Appendix F**
may be useful for characterizing the performance of the *in vitro* NRU
assays for various chemical types. **Appendix F-1** lists the selected
reference substances in alphabetical order with information on the
CASRN, purity, supplier, pH, and concentrations tested in the *in vitro*
NRU cytotoxicity assays. **Appendix F-2** also provides the reference
substances in alphabetical order, but with the available information on
molecular weight, chemical class, water solubility, acid/base
dissociation constant (pK), boiling point, lipid solubility (log K~ow~),
major toxic effects, ability to pass the blood:brain barrier, metabolic
activation/inactivation, and mechanism of lethality. The remainder of
**Section 3.3** summarizes selected characteristics of the reference
substances.

3.3.1 [Source Databases Represented by the Selected Reference
Substances]{.underline}

The primary sources of chemicals, which reflect the level of societal
interest, were well represented in the final list of reference
substances. **Table 3-3** shows the distribution of reference substances
by GHS category from the MEIC, EDIT, TESS, NTP, and HPV lists. Forty-two
(58%) of the 72 selected chemicals were MEIC chemicals (17 of the 42
MEIC chemicals \[40%\] were EDIT chemicals), 46 (64%) chemicals were
involved in human poisonings report by TESS, 51 (71%) chemicals have
been evaluated by the NTP, and 18 (25%) chemicals are listed in the
EPA's HPV Challenge Program. Some chemicals were found in more than one
source.

The other major source of chemicals was the RC. As shown in **Table
3-4**, 58 (81%) of the selected chemicals were included in the RC. Since
one of the regression formulas evaluated in the NICEATM/ECVAM validation
study was the RC regression, the fit of the RC chemicals to the
regression was relevant (Halle 1998). Halle (1998) defined outliers as
those chemicals with log IC~50~-log LD~50~ points that were \> 0.699
(i.e., log 5) from the RC regression. For each toxicity category,
**Table 3-4** shows the number of RC outliers selected for testing and
the corresponding number of outliers in the RC. Although the percentage
of outliers for the selected chemicals in several GHS categories is
similar to the RC, the total percentage of RC outliers in the set of
reference substances (i.e., 38% \[22/58\]) is greater than the total
percentage of outliers in the RC (i.e., 27% \[95/347\]). For the
reference substances, the RC prediction model underpredicted toxicity
(i.e., actual LD~50~ is lower than predicted) for 17 outliers and
overpredicted toxicity (i.e., actual LD~50~ is higher than predicted)
for five outliers. **Figure 3-1** shows the 58 RC chemicals selected for
testing with the remaining 289 RC data points and the RC regression. In
the figure, the outliers are those points outside the RC prediction
interval. The 17 outlier chemicals for which toxicity is underpredicted
are below the lower prediction interval and the five outliers for which
toxicity is overpredicted are above the upper prediction interval.

**Table 3-3 Distribution of Candidate Chemicals and Reference Substances
by Source^1^ and Toxicity Category**

+---------+----------+--------+---------+---------+---------+---------+
| *       | **R      | **MEIC | **EDIT  | **TESS  | **NTP   | **HPV   |
| *GHS^2^ | eference | Refere | Ref     | Refer   | Refer   | Refer   |
| C       | Sub      | nce/** | erence/ | ence/** | ence/** | ence/** |
| ategory | stances/ |        | EDIT    |         |         |         |
| (LD~50~ | C        | **MEIC | Candi   | **TESS  | **NTP   | **HPV   |
| in      | andidate | Candid | dates** | Candi   | Candi   | Candi   |
| m       | Che      | ates** |         | dates** | dates** | dates** |
| g/kg)** | micals** |        |         |         |         |         |
+---------+----------+--------+---------+---------+---------+---------+
| LD~50~  | 12/13    | 2/2    | 1/1     | 3/3     | 5/5     | 0/0     |
| ≤ 5     |          |        |         |         |         |         |
+---------+----------+--------+---------+---------+---------+---------+
| 5 \<    | 12/15    | 6/6    | 5/5     | 9/10    | 8/11    | 2/5     |
| LD~50~  |          |        |         |         |         |         |
| ≤ 50    |          |        |         |         |         |         |
+---------+----------+--------+---------+---------+---------+---------+
| 50 \<   | 12/26    | 11/17  | 4/5     | 11/19   | 9/18    | 1/3     |
| LD~50~  |          |        |         |         |         |         |
| ≤ 300   |          |        |         |         |         |         |
+---------+----------+--------+---------+---------+---------+---------+
| 300 \<  | 12/38    | 12/29  | 3/5     | 12/27   | 5/23    | 1/5     |
| LD~50~  |          |        |         |         |         |         |
| ≤ 2000  |          |        |         |         |         |         |
+---------+----------+--------+---------+---------+---------+---------+
| 2000 \< | 12/12    | 6/6    | 2/2     | 6/6     | 12/12   | 6/6     |
| LD~50~  |          |        |         |         |         |         |
| ≤ 5000  |          |        |         |         |         |         |
+---------+----------+--------+---------+---------+---------+---------+
| LD~50~  | 12/12    | 5/5    | 2/2     | 5/5     | 12/12   | 8/8     |
| \> 5000 |          |        |         |         |         |         |
+---------+----------+--------+---------+---------+---------+---------+
| Total   | 72/116   | 42/65  | 17/20   | 46/70   | 51/81   | 18/27   |
+---------+----------+--------+---------+---------+---------+---------+

^1^Substances may be represented in more than one source (see **Table
3-2**).

^2^GHS = Globally Harmonized System of Classification and Labelling of
Chemicals (UN 2005).

MEIC = Multicentre Evaluation of In Vitro Cytotoxicity**;** EDIT=
Evaluation-Guided Development of *In vitro* Tests; TESS =Toxic Exposure
Surveillance System; NTP = U.S. National Toxicology Program; HPV = EPA
High Production Volume program.

**Table 3-4 Selected Chemicals: Distribution of Registry of Cytotoxicity
(RC) Chemicals and RC Outliers^1^** by Toxicity Category

+-------------+----------+-------+-----------------+-----------------+
| **GHS^2^    | **RC     | *     |                 |                 |
| Category**  | O        | *Cand |                 |                 |
|             | utliers/ | idate |                 |                 |
| **(LD~50~   | Total    | and   |                 |                 |
| in mg/kg)** | Che      | Sel   |                 |                 |
|             | micals** | ected |                 |                 |
|             |          | C     |                 |                 |
|             |          | hemic |                 |                 |
|             |          | als** |                 |                 |
+-------------+----------+-------+-----------------+-----------------+
|             |          | *     | **RC Reference  | **RC Reference  |
|             |          | *Cand | /**             | Outliers/**     |
|             |          | idate |                 |                 |
|             |          | C     | **RC            | **RC Reference  |
|             |          | hemic | Candidates**    | Chemicals**     |
|             |          | als** |                 |                 |
+-------------+----------+-------+-----------------+-----------------+
| LD~50~ ≤ 5  | 10/11    | 13    | 9/10            | 8/9 (89%)       |
|             | (91%)    |       |                 |                 |
+-------------+----------+-------+-----------------+-----------------+
| 5 \< LD~50~ | 15/26    | 15    | 8/10            | 4/8 (50%)       |
| ≤ 50        | (58%)    |       |                 |                 |
+-------------+----------+-------+-----------------+-----------------+
| 50 \<       | 24/70    | 26    | 11/18           | 5/11 (45%)      |
| LD~50~ ≤    | (34%)    |       |                 |                 |
| 300         |          |       |                 |                 |
+-------------+----------+-------+-----------------+-----------------+
| 300 \<      | 14/139   | 38    | 9/29            | 0/9 (0%)        |
| LD~50~ ≤    | (10%)    |       |                 |                 |
| 2000        |          |       |                 |                 |
+-------------+----------+-------+-----------------+-----------------+
| 2000 \<     | 12/57    | 12    | 10/10           | 0/10 (0%)       |
| LD~50~ ≤    | (21%)    |       |                 |                 |
| 5000        |          |       |                 |                 |
+-------------+----------+-------+-----------------+-----------------+
| LD~50~ \>   | 20/44    | 12    | 11/11           | 5/11 (45%)      |
| 5000        | (45%)    |       |                 |                 |
+-------------+----------+-------+-----------------+-----------------+
| Total       | 95/347   | 116   | 58/88           | 22/58 (38%)     |
|             | (27%)    |       |                 |                 |
+-------------+----------+-------+-----------------+-----------------+

^1^Chemicals falling outside the log 5 (i.e., \> ± 0.699) prediction
interval for the RC regression (Halle 1998).

^2^GHS: Globally Harmonized System of Classification and Labelling of
Chemicals (UN 2005).

**Figure 3-1 The Fifty-Eight (58) Selected Registry of Cytotoxicity (RC)
Chemicals on the RC Regression**

![](media/image1.pct){width="6.243055555555555in"
height="4.981944444444444in"}

The 58 RC chemicals tested in the NICEATM/ECVAM validation study are
shown by \*. The RC regression, log (LD~50~) = 0.435 x log (IC~50x~) +
0.625, is shown by the bold line. The lighter lines show the ± log 5
(i.e., ± 0.699) prediction interval (Halle 1998). The remaining 289 RC
data points are shown by the open boxes.

3.3.2 [Chemical Classes Represented by the Selected Reference
Substances]{.underline}

Medical subject headings (MeSH) from the NLM were used to determine
chemical class for the selected chemicals. Of the 72 reference
substances, 55 (76%) were organic compounds and 17 (24%) were inorganic
compounds. The most commonly represented classes of organic compounds
were heterocyclic compounds (14/55, 26%), carboxylic acids (12/55, 22%),
and alcohols (10/55, 18%). **Table 3-5** shows the distribution of the
selected chemicals among the GHS toxicity categories. The 14
heterocyclic compounds were rather evenly distributed among the first
four GHS toxicity categories for LD~50~ ≤ 2000 mg/kg with the majority
of the heterocyclics (11/14) in the categories for LD~50~ \< 300 mg/kg.
The majority of the carboxylic acids (10/12) and alcohols (8/10) had
LD~50~ \> 300 mg/kg. The majority of the inorganic compounds (12/17) had
LD~50~ \< 300 mg/kg.

3.3.3 [Product/Use Classes Represented by the Selected Reference
Substances]{.underline}

Product and use information for the selected chemicals was obtained from
HSDB (NLM 2002) or RTECS^®^ (MDL Information Systems 2002). Since more
than one use was reported for some chemicals, the number of assigned
uses (77) is greater than the number of selected chemicals. **Table
3-6** shows the distribution of products and uses of the selected
chemicals among the GHS toxicity categories. Pharmaceutical (27/77; 35%)
and pesticide (17/77; 22%) uses were observed most frequently. The
toxicity category for 300 \< LD~50~ ≤ 2000 mg/kg had the highest number
of chemicals with pharmaceutical uses. Every toxicity category except
for LD~50~ \> 5000 mg/kg had at least four chemicals with pharmaceutical
uses. The majority of chemicals (16/17; 94%) with pesticide uses had
LD~50~ \< 300 mg/kg. The next most frequent uses for the selected
chemicals were solvents (8/77; 10%) and food additives (5/77; 6%). The
toxicity categories for LD~50~ \> 2000 mg/kg contained most of the
chemicals with solvent (8/8; 100%) and food additive (4/5; 80%) uses.

3.3.4 [Toxicological Characteristics of the Selected Reference
Substances]{.underline}

*Corrosivity*

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

  -------------------------------------------------------------------------------------------
  **Table 3-5                                                                     
  Distribution of                                                                 
  Chemical Class for                                                              
  the 72 Reference                                                                
  Substances by                                                                   
  Toxicity Category**                                                             
  ------------------- ------------- ------- ------- --------- ---------- -------- -----------
                      **GHS Acute                                                 **Total**
                      Oral Toxicity                                               
                      Category^1^                                                 
                      (mg/kg)**                                                   

  **Chemical          **≤ 5**       **\>    **\>    **\>      **\>       **\>     
  Class^2^**                        5 - ≤   50 - ≤  300 - ≤   2000 - ≤   5000**   
                                    50**    300**   2000**    5000**              

  **Organic**         ** **         ** **   ** **   ** **     ** **      ** **    ** **

  Heterocyclic        4             3       4       3         0          0        14
  compound                                                                        

  Carboxylic acid     1             0       1       3         3          4        12

  Alcohol             2             0       0       2         1          5        10

  Amide               0             0       0       1         2          0        3

  Halogenated         0             0       1       0         1          1        3
  hydrocarbon                                                                     

  Cyclic hydrocarbon  0             0       1       0         1          0        2

  Hydrocarbon         0             1       0       0         0          1        2

  Organophosphorous   2             1       0       0         0          0        3
  compound                                                                        

  Polycyclic compound 0             1       0       1         0          0        2

  Amine               0             0       1       0         0          0        1

  Nitrile             0             0       0       0         1          0        1

  Organometallic      0             1       0       0         0          0        1
  compound                                                                        

  Phenol              0             0       0       1         0          0        1

  Total               9             7       8       11        9          11       55

  **Inorganic**                                                                    

  Arsenical           0             2       0       0         0          0        2

  Sulfur compound     1             0       1       0         0          0        2

  Boron compound      0             0       0       0         1          0        1

  Cadmium compound    0             0       1       0         0          0        1

  Ketone              0             0       1       0         0          0        1

  Lithium compound    0             0       0       1         0          0        1

  Mercury compound    1             0       0       0         0          0        1

  Metal               0             1       0       0         0          0        1

  Potassium, chlorine 0             0       0       0         1          0        1
  compound                                                                        

  Potassium, nitrogen 0             1       0       0         0          0        1
  compound                                                                        

  Sodium, chlorine    0             0       0       0         1          0        1
  compound                                                                        

  Sodium, chromium    0             1       0       0         0          0        1
  compound                                                                        

  Sodium, fluorine    0             0       1       0         0          0        1
  compound                                                                        

  Sodium, oxygen,     0             0       0       0         0          1        1
  chlorine compound                                                               

  Sodium, selenium    1             0       0       0         0          0        1
  compound                                                                        

  Total               3             5       4       1         3          1        17
  -------------------------------------------------------------------------------------------

^1^GHS: Globally Harmonized System of Classification and Labelling of
Chemicals based on oral LD~50~ (UN 2005).

≤ 5: LD~50~ ≤ 5 mg/kg

\> 5 - ≤ 50: 5 \< LD~50~ ≤ 50 mg/kg

\> 50 - ≤ 300: 50 \<LD~50~ ≤ 300 mg/kg

\> 300 - ≤ 2000: 300 \< LD~50~ ≤ 2000 mg/kg

\> 2000 - ≤ 5000: 2000 \< LD~50~ ≤ 5000 mg/kg

\> 5000: LD~50~ \> 5000 mg/kg

^2^Based on the Medical Subject Heading \[MeSH\] index (NLM 2005).

**Table 3-6 Distribution of Product/Use^1^** Class for the 72 Reference
Substances by Toxicity Category

  ---------------------- ------------- ------- ------- --------- ---------- -------- -----------
                         **GHS Acute                                                 **Total**
                         Oral Toxicity                                               
                         Category^2^                                                 
                         (mg/kg)**                                                   

  **Product/Use          **≤ 5**       **\>    **\>    **\>      **\>       **\>     
  Class^1^**                           5 - ≤   50 - ≤  300 - ≤   2000 - ≤   5000**   
                                       50**    300**   2000**    5000**              

  Antibiotic/fungicide   1             0       0       0         0          0        1

  Antifreeze             0             0       0       0         0          1        1

  Consumer/industrial    0             0       1       0         0          0        1
  products                                                                           

  Disinfectant           0             0       1       1         0          2        4

  Electroplating         0             2       0       0         0          0        2

  Fluoridation           0             0       1       0         0          0        1

  Feed additive          1             0       0       0         0          0        1

  Fixative               0             0       0       0         1          0        1

  Food additive          0             0       1       0         3          1        5

  Manufacturing          1             0       0       0         1          0        2

  Oxidizing agent        0             1       0       0         0          0        1

  Paints, cleaners       0             0       1       0         0          0        1

  Pesticide              5             7       4       0         1          0        17

  Pharmaceutical         4             3       4       11        4          1        27

  Plant growth regulator 0             0       0       0         0          1        1

  Plasticizer            0             0       0       0         0          2        2

  Preservative           1             0       0       0         0          0        1

  Solvent                0             0       0       0         4          4        8
  ---------------------- ------------- ------- ------- --------- ---------- -------- -----------

^1^Product/use categories from Hazardous Substances Data Bank (NLM 2002)
or Registry of Toxic Effects of Chemical Substances (\[RTECS^®^\], MDL
Information Systems 2002). Some chemicals are counted more than once due
to multiple uses.

≤ 5: LD~50~ ≤ 5 mg/kg

\> 5 - ≤ 50: 5 \< LD~50~ ≤ 50 mg/kg

\> 50 - ≤ 300: 50 \< LD~50~ ≤ 300 mg/kg

\> 300 - ≤ 2000: 300 \< LD~50~ ≤ 2000 mg/kg

\> 2000 - ≤ 5000: 2000 \< LD~50~ ≤ 5000 mg/kg

\> 5000: LD~50~ \> 5000 mg/kg

^2^GHS: Globally Harmonized System of Classification and Labelling of
Chemicals based on oral LD~50~ (UN 2005).

*Toxicity Targets*

As shown in **Appendix F**, the most common toxicological effects were
neurological (40 reference substances); 26 reference substances cause
central nervous system (CNS) depression, seven reference substances
produce CNS stimulation, four reference substances produce other CNS
affects such as encephalopathy, and three reference substances attack
the peripheral nervous system. Other common toxicity targets include the
liver (17 reference substances), kidney (15 reference substances), and
cardiovascular system (10 reference substances). No target organ
information was available for gibberellic acid. Among the 72 reference
substances, 27 had multiple toxicity targets.

*Metabolism*

**Table 3-7** shows the 22 reference substances that are known or
expected to produce active/toxic metabolites *in vivo*. In contrast,
dichlorvos, fenpropathrin, meprobamate, phenylthiourea, and sodium
dichromate are known to be rapidly inactivated by metabolism *in vivo*
to less toxic compounds. Because the NHK and 3T3 cells have little (see
Babich 1991) or no metabolic capability, respectively, metabolites of
these compound would be unavailable *in vitro*. See **Appendix F-2** for
more information on the metabolism of the selected chemicals.

Table 3-7 Reference Substances Metabolized to Active Metabolites

  ----------------- --------------- -------------- -------------- ---------------------
  **Known to Have                                                 **Active Metabolites
  Active                                                          Expected**
  Metabolites**                                                   

  Acetaminophen     Carbamazepine   Digoxin        Methanol       Carbon tetrachloride

  Acetonitrile      Chloral hydrate Disulfoton     Parathion      Triethylenemelamine

  Acetylsalicylic   Cycloheximide   Ethanol        Procainamide   Valproic acid
  acid                                             HCl            

  Amitriptyline HCl Dibutyl         Ethylene       Verapamil HCl  
                    phthalate       glycol                        

  Busulfan          Diethyl         Glutethimide                  
                    phthalate                                     
  ----------------- --------------- -------------- -------------- ---------------------

3.3.5 [Selection of Reference Substances for Testing in Validation Study
Phases Ib and II]{.underline}

Based on the *Guidance Document* (ICCVAM 2001b) recommendation that
10-20 chemicals be tested to qualify candidate *in vitro* cytotoxicity
tests for determining starting doses for acute oral systemic toxicity
assays, 12 reference substances were chosen from the 72 reference
substances for testing in Phases Ib and II of the validation study (see
**Table 3-8**). The criteria for choosing these reference substances, in
order of importance, were:

-   two reference substances must be included from each of the five GHS
    toxicity categories and the unclassified category

-   the log LD~50~ (mmol/kg) must be within 0.699 of the RC regression
    (i.e., within the RC prediction interval). The *Guidance Document*
    (ICCVAM 2001b) recommends that reference substances for evaluating a
    cytotoxicity test to use with the RC regression fit the regression
    as closely as possible

-   MEIC chemicals must be included. Cytotoxicity data from these phases
    (and Phase III of this study) and the available human toxicity
    information for the MEIC chemicals could be used to build a
    prediction model for estimating human lethal blood concentrations.
    Phase Ib reference substances arsenic trioxide and ethylene glycol
    are EDIT chemicals

If more than two chemicals in a GHS category met the above criteria,
reference substances were selected so that the LD~50~ was as close to
the RC prediction as possible and/or to represent the range of toxicity
in each GHS category.

Only nine reference substances of the 72 selected reference substances
fit all three criteria. One reference substance was not within the RC
acceptance interval. For the most toxic category (i.e., LD~50~ ≤ 5
mg/kg), only one RC chemical, aminopterin, was within 0.699 of the RC
regression. Sodium selenate, whose fit to the RC regression was unknown
and had not been tested in the MEIC study, was included in this toxicity
category. In addition, neither of the two reference substances chosen
for the LD~50~ ≤ 5 mg/kg category, aminopterin and sodium selenate, were
MEIC chemicals.

Table 3-8 Reference Substances Tested in Phases Ib and II

+----------------+-----------+--------+-------+----------+------------+
| **Reference    | **CASRN** | **RC   | *     | **Rodent | **Observed |
| Substances**   |           | Ref    | *MEIC | Oral**   | --         |
|                |           | erence | Refe  |          | P          |
|                |           | No.**  | rence | **LD~    | redicted** |
|                |           |        | No.** | 50~^1^** |            |
|                |           |        |       |          | **log      |
|                |           |        |       | **(      | L          |
|                |           |        |       | mg/kg)** | D~50~^2^** |
+----------------+-----------+--------+-------+----------+------------+
| ***LD~50~***   |           |        |       |          |            |
| ***≤ 5         |           |        |       |          |            |
| mg/kg***       |           |        |       |          |            |
+----------------+-----------+--------+-------+----------+------------+
| Aminopterin    | 54-62-6   | 3      | NA    | 3        | -0.652     |
+----------------+-----------+--------+-------+----------+------------+
| Sodium         | 1         | NA     | NA    | 1.6^3^   | NA         |
| selenate       | 3410-01-0 |        |       |          |            |
+----------------+-----------+--------+-------+----------+------------+
| ***5 \< LD~50~ |           |        |       |          |            |
| ≤ 50 mg/kg***  |           |        |       |          |            |
+----------------+-----------+--------+-------+----------+------------+
| Colchicine     | 64-86-8   | 6      | 60    | 6^4^     | -0.593     |
+----------------+-----------+--------+-------+----------+------------+
| Arsenic III    | 1327-53-3 | 153    | 26    | 20       | -0.591     |
| trioxide       |           |        |       |          |            |
+----------------+-----------+--------+-------+----------+------------+
| ***50 \<       |           |        |       |          |            |
| LD~50~ ≤ 300   |           |        |       |          |            |
| mg/kg***       |           |        |       |          |            |
+----------------+-----------+--------+-------+----------+------------+
| Cadmium II     | 1         | 81     | NA    | 88       | -0.336     |
| chloride       | 0108-64-2 |        |       |          |            |
+----------------+-----------+--------+-------+----------+------------+
| Sodium I       | 7681-49-4 | 106    | 14    | 180      | -0.109     |
| fluoride       |           |        |       |          |            |
+----------------+-----------+--------+-------+----------+------------+
| ***300 \<      |           |        |       |          |            |
| LD~50~ ≤ 2000  |           |        |       |          |            |
| mg/kg***       |           |        |       |          |            |
+----------------+-----------+--------+-------+----------+------------+
| DL-Propranolol | 350-60-90 | 54     | 23    | 470^4^   | -0.023     |
| HCl            |           |        |       |          |            |
+----------------+-----------+--------+-------+----------+------------+
| Lithium I      | 544-13-2  | 327^4^ | 20    | 1        | -0.256^4^  |
| carbonate      |           |        |       | 187^4,5^ |            |
+----------------+-----------+--------+-------+----------+------------+
| ***2000 \<     |           |        |       |          |            |
| LD~50~ ≤ 5000  |           |        |       |          |            |
| mg/kg***       |           |        |       |          |            |
+----------------+-----------+--------+-------+----------+------------+
| Potassium I    | 7447-40-7 | 346    | 50    | 2602     | 0.085      |
| chloride       |           |        |       |          |            |
+----------------+-----------+--------+-------+----------+------------+
| C              | 56-75-7   | 91     | 45    | 3393     | 0.441      |
| hloramphenicol |           |        |       |          |            |
+----------------+-----------+--------+-------+----------+------------+
| ***LD~50~ \>   |           |        |       |          |            |
| 5000 mg/kg***  |           |        |       |          |            |
+----------------+-----------+--------+-------+----------+------------+
| 2-Propanol     | 67-63-0   | 128    | 10    | 5843     | 0.396      |
+----------------+-----------+--------+-------+----------+------------+
| Ethylene       | 107-21-1  | 360    | 7     | 8567     | 0.321      |
| glycol         |           |        |       |          |            |
+----------------+-----------+--------+-------+----------+------------+

^1^From the RC (Halle 1998) unless otherwise indicated. Data for rats
unless otherwise indicated.

^2^Available only for chemicals included in the RC. This figure
characterizes the log LD~50~ deviation from the RC regression. Outliers
are \> ± 0.699 from the regression line.

^3^RTECS^®^ (MDL Information Systems 2002).

^4^Mouse data.

^5^[Data for lithium
sulfate.](http://www.tomescps.com/DATA/RT/RTAE4200000.HTM?Top=Yes)

[Abbreviations: CASRN = Chemical Abstracts Service Registry Number; RC =
Registry of Cytotoxicity; MEIC = Multicentre Evaluation
of](http://www.tomescps.com/DATA/RT/RTAE4200000.HTM?Top=Yes) In Vitro
Cytotoxicity; NA -- not applicable; chemical not included in the RC
and/or MEIC studies.

3.3.6 [Unsuitable and Challenging Reference Substances]{.underline}

Several reference substances could not be adequately tested for
cytotoxicity in either or both of the 3T3 or NHK NRU test methods. Under
the conditions of the NRU cytotoxicity test, the following reference
substances did not produce sufficient toxicity at soluble concentrations
for calculation of an IC~50~ at the highest concentrations that could be
tested:

-   carbon tetrachloride in either the 3T3 or NHK test method in all
    three laboratories

-   xylene in either test method in two laboratories

-   methanol in the 3T3 test method in all three laboratories and in the
    NHK test method in two laboratories

-   lithium carbonate in the 3T3 test method in two laboratories

-   1,1,1-trichloroethane in the NHK test method in two laboratories

-   valproic acid in the 3T3 test method in one laboratory

Other reference substances were difficult to test, but three acceptable
tests were obtained after a number of trials.

-   Acetonitrile and 2-propanol were so volatile and nontoxic that, even
    with the use of film plate sealers, one to seven tests failed at
    each laboratory. Tests with these two reference substances often
    failed the VC and data points criteria.

-   Disulfoton failed at least one test in both test methods in two
    laboratories due to inadequate toxicity and solubility.

-   Dibutyl phthalate failed one 3T3 test at one laboratory and one NHK
    test at one laboratory due to inadequate toxicity and solubility.

-   Lindane failed one 3T3 test due to inadequate toxicity and
    solubility and one 3T3 test due to volatility.

-   Parathion failed one test due to inadequate toxicity and solubility
    in both the 3T3 and NHK test methods and one NHK test due to
    volatility.

-   Diethyl phthalate failed one NHK test due to volatility.

-   Digoxin, gibberellic acid, and strychnine failed at least one 3T3
    test in more than one laboratory due to inadequate toxicity and
    solubility.

**3.4 Reference Substance Procurement, Coding, and Distribution**

Reference substances were purchased from the suppliers in the purities
indicated in **Appendix F** and distributed by BioReliance Corporation
(Rockville, MD). BioReliance also collected information from the
suppliers on the analytical purity, composition, and stability of the
reference substances. BioReliance tested the reference substances for
solubility, packaged them into 4 g aliquots for shipment to the
cytotoxicity testing laboratories, and archived two additional samples.
All reference substances were randomly coded to conceal the identities
from the cytotoxicity testing laboratories. Each reference substance had
a code unique for each testing facility. About 100 g of the positive
control, SLS, was distributed to each laboratory and one additional
sample was archived.

Reference substances were packaged to minimize damage during transit and
shipped under appropriate storage conditions and according to proper
regulatory transportation procedures. Testing facilities were notified
upon shipment in order to prepare for receipt. With the exception of the
positive control shipment, which was shipped directly to the Study
Directors, the reference substances were shipped to the test facility
Safety Officers. Reference substances shipments were accompanied by a
sealed information packet containing the appropriate health and safety
procedures for use (i.e., MSDS or equivalent documentation with
information regarding the proper protection for handling, procedures for
dealing with accidental ingestion or contact with skin or eyes, and
procedures for containing and recovering spills) and a disclosure key
for identifying reference substances by code. Also provided was a data
sheet giving a minimum of essential information for each reference
substance, including color, odor, physical state, weight or volume of
sample, specific density for liquid reference substances, and storage
instructions. The shipment directed the Safety Officer to:

-   notify BioReliance and the SMT upon receipt of reference substances

-   retain the health and safety package and provide the reference
    substances and chemical data sheets to the Study Director without
    revealing the identities of the reference substances

-   notify the SMT if test facility personnel open the health and safety
    packet at any time during the study

-   return the unopened health and safety package to BioReliance after
    testing is complete

*Exceptions*

The Safety Officer for ECBC required the information on reference
substance codes before the substances were shipped to the Safety Office
to satisfy the facility's environmental procedures and requirements. The
reference substance codes were stored in a classified safe located in
the Safety Office, which was in a building separate from the
cytotoxicity testing laboratory. Cytotoxicity testing personnel had no
access to the reference substance codes. The ECBC Safety Officer opened
the sealed health and safety packets for lithium carbonate and ethanol
upon receipt of those substances because the code information for these
substances was not included in the list originally provided. ECBC
cytotoxicity testing personnel never had access to the reference
substance codes.

**3.5 Reference Substances Recommended by the** *Guidance Document*
(ICCVAM 2001b)

The *Guidance Document* method for evaluating basal cytotoxicity assays
for use in predicting starting doses for acute oral toxicity assays
provides the existing performance standard (ICCVAM 2001b) for the 3T3
and NHK NRU test methods. The *Guidance Document* specifically
recommends testing the following 11 chemicals to qualify candidate basal
cytotoxicity assays: sodium dichromate dihydrate, cadmium chloride,
p-phenylenediamine, DL-propranolol HCl, trichlorfon, ibuprofen,
nalidixic acid, salicylic acid, antipyrene, dimethylformamide, and
glycerol (ICCVAM 2001b). Although the 11 reference chemicals recommended
in the *Guidance Document* were considered as candidates for testing in
the NICEATM/ECVAM validation study (see **Section 3.1.2**), only sodium
dichromate dihydrate, cadmium chloride, DL-propranolol HCl,
dimethylformamide, and glycerol were chosen for testing after the
candidate chemicals were prioritized as described in **Section 3.1.3**.
The other seven were excluded based on the criterion hierarchy used to
determine the selected chemicals (e.g., were not MEIC chemicals, not
identified as high exposure risk in TESS)

6.  **Summary**

Seventy-two reference substances were selected for testing in the
NICEATM/ECVAM validation study. The reference substances were selected
to represent: (1) the complete range of *in vivo* acute oral toxicity
ranges (in terms of LD~50~ values); (2) the types of substances
regulated by various regulatory authorities; and (3) those with human
toxicity data and/or human exposure potential. To assure the complete
range of toxicity was covered, the Globally Harmonized System of
Classification and Labelling of Chemicals (UN 2005) was used to select
12 chemicals for each acute oral toxicity category and 12 unclassified
chemicals. The set of selected reference substances had the following
characteristics:

-   38% (27/72) of the substances had pharmaceutical uses, 21% (15/72)
    had pesticide uses, 11% (8/72) had solvent uses, and 7% (5/72) had
    food additive uses. The remaining substances were used for a variety
    of manufacturing and consumer products

-   relevance of the substances to human exposures was indicated by the
    fact that 58% (42/72) were included in the MEIC study, 24% (17/72)
    were included in the EDIT program, 64% (46/72) had human exposures
    reported by TESS, 71% (51/72) had been evaluated by NTP, and 25%
    (18/72) were included in EPA's HPV list

-   81% (58/72) of the substances were also included in the RC and 38%
    (22/58) of these were outliers with respect to the RC regression

-   76% (55/72) were organic compounds and 24% (17/72) were inorganic
    compounds. The most commonly represented classes of organic
    compounds were heterocyclic compounds (26%, 14/55), carboxylic acids
    (22%, 12/55), and alcohols (18%, 10/55)

-   19 substances (26%, 19/72,) were known to have active metabolites
    and three additional substances were expected to have active
    metabolites

-   many of the selected chemicals had multiple target organs. The most
    common effects were neurological (40 chemicals), liver (17
    chemicals), kidney (15 chemicals), and cardiovascular (10
    chemicals). No target organ information was available for one
    chemical (gibberellic acid)

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