4.0 *IN VIVO* RODENT TOXICITY REFERENCE VALUES USED TO ASSESS THE
ACCURACY OF THE 3T3 AND NHK NRU TEST METHODS 4-3

4.1 Methods Used to Determine *In Vivo* Rodent Toxicity Reference Values
4-3

> 4.1.1 Identification of Candidate *In Vivo* Rodent Toxicity Reference
> Data 4-3
>
> 4.1.2 Criteria Used to Select Candidate *In Vivo* Rodent Toxicity Data
> for Determination of Reference Values 4-6

4.2 Final *In Vivo* Rodent Toxicity Reference Values 4-10

4.3 Relevant Toxicity Information for Humans 4-16

4.4 Accuracy and Reliability of the *In Vivo* Rodent Toxicity Reference

> Values 4-16

4.5 Summary 4-20

***\[This Page Intentionally Left Blank\]***

**4.0 *IN VIVO* RODENT TOXICITY REFERENCE VALUES USED TO ASSESS THE
ACCURACY OF THE 3T3 AND NHK NRU TEST METHODS**

The aim of the procedures and analyses presented in this section is to
identify the most appropriate *in vivo* rodent toxicity data with which
to compare the *in vitro* cytotoxicity data. The *in vitro* NRU
cytotoxicity test methods are intended to be used in a weight of
evidence approach to determine the starting dose for the *in vivo* acute
oral systemic toxicity test methods using rodents. Thus, rodent LD~50~
values from acute oral systemic toxicity tests are the most appropriate
reference data for the *in vitro* NRU IC~50~ values. This section
describes the methods for identifying and evaluating the most
appropriate rodent LD~50~ data to use for determining reference LD~50~
values for the 72 reference substances tested in the NICEATM/ECVAM
validation study. These *in vivo* rodent toxicity reference values will
be used in **Section 6** to establish the accuracy of the *in vitro*
IC~50~ data from the 3T3 and NHK NRU test methods for predicting LD~50~
values from rodent acute oral systemic toxicity tests.

**4.1 Methods Used to Determine *In Vivo* Rodent Toxicity Reference
Values**

4.1.1 [Identification of Candidate *In Vivo* Rodent Toxicity Reference
Data]{.underline}

No animal experiments were performed to obtain *in vivo* reference data
for acute oral systemic toxicity. To identify LD~50~ reference data for
the 72 reference substances, rat oral LD~50~ data were located through
literature searches, secondary references, and electronic database
searches. PubMed and ISI Web of Science^®^ searches were conducted using
each chemical name and "lethal dose 50." Secondary sources included NTP
technical reports, Toxicological Profiles from the Agency for Toxic
Substances and Disease Registry (ATSDR), Cosmetic Ingredient Reviews by
the Cosmetics Industry Council, pesticide handbooks, Merck Index, and
various other summary sources. **Table 4-1** lists databases searched
via the Internet to locate references for rat oral LD~50~ values. Rat
LD~50~ data were preferred because the current oral acute toxicity test
guidelines recommend using rats (OECD 2001a, c, d; EPA 2002a). Taking
the same approach used for the Registry of Cytotoxicity (RC), mouse
LD~50~ data were sought for a particular chemical if rat LD~50~ values
could not be located. \[The RC is a database of acute oral LD~50~ values
for rats and mice, obtained from RTECS^®^ and IC~50~ values from *in
vitro* cytotoxicity assays using multiple cell lines and cytotoxicity
endpoints for chemicals with known molecular weights (Halle 1998).\]

+--------------------------------+-------------------------------------+
| **Table 4-1 Internet           |                                     |
| Accessible Databases with      |                                     |
| LD~50~ Information**           |                                     |
+================================+=====================================+
| **Database**                   | **Sponsor**                         |
+--------------------------------+-------------------------------------+
| Agency for Toxic Substances    | U.S. Department of Health and Human |
| and Disease Registry (ATSDR)   | Services (DHHS)                     |
+--------------------------------+-------------------------------------+
| Center for Drug Evaluation and | U.S. Food and Drug Administration   |
| Research (CDER)                | (FDA)                               |
+--------------------------------+-------------------------------------+
| CHEMFINDER                     | CambridgeSoft Corporation           |
+--------------------------------+-------------------------------------+
| Chemical Carcinogenesis        | NCI; National Institutes of Health  |
| Research Information System    | (NIH); DHHS                         |
| (CCRIS)                        |                                     |
|                                |                                     |
| National Cancer Institute      |                                     |
| (NCI) Website                  |                                     |
+--------------------------------+-------------------------------------+
| Chemical Evaluation Search and | Michigan Department of Natural      |
| Retrieval System (CESARS)      | Resources; Ontario Ministry of the  |
|                                | Environment; CCOHS CHEMpendium     |
+--------------------------------+-------------------------------------+
| Chemical Hazard Response       | U.S. Coast Guard                    |
| (CHRIS)                        |                                     |
+--------------------------------+-------------------------------------+
| Chemical Ingredients Database  | U.S. Environmental Protection       |
|                                | Agency (EPA) Office of Pesticide    |
|                                | Programs (OPP); California EPA      |
|                                | Department of Pesticide Regulation  |
+--------------------------------+-------------------------------------+
| CHEMINDEX                      | # Ca                                |
|                                | nadian Centre for Occupational Heal |
| CHEMINFO                       | th and Safety (CCOHS) CHEMpendium  |
+--------------------------------+-------------------------------------+
| ChemRTK High Production Volume | EPA Office of Pollution Prevention  |
| (HPV) Challenge Program        | and Toxics (OPPT)                   |
|                                |                                     |
| OPPT Chemical Fact Sheets      |                                     |
|                                |                                     |
| Chemical Information           |                                     |
| Collection and Data            |                                     |
| Development                    |                                     |
+--------------------------------+-------------------------------------+
| CIS Chemical Information       | World Health Organization (WHO)     |
|                                | International Programme on Chemical |
|                                | Safety (IPCS); CCOHS; International |
|                                | Labour Organisation (ILO)           |
|                                | Occupational Safety and Health      |
|                                | Information Centre (CIS)            |
+--------------------------------+-------------------------------------+
| Concise International Chemical | WHO IPCS; CCOHS; ILO; United        |
| Assessment Documents (CICADS)  | Nations Environment Programme       |
|                                | (UNEP)                              |
+--------------------------------+-------------------------------------+
| Consumer Product Safety        | U.S. Consumer Product Safety        |
| Commission Website             | Commission (CPSC)                   |
+--------------------------------+-------------------------------------+
| Deutsches Institut fur         | Zentralstelle zur Erfassung und     |
| Medizinische Dokumentation und | Bewertungvon Ersatz- und            |
| Information (DIMDI) \[The      | Erganzungsmethoden zum Tierversuch  |
| German Institute for Medical   | (ZEBET) \[German Centre for the     |
| Documentation and              | Documentation and Validation of     |
| Information\]                  | Alternative Methods\]               |
|                                |                                     |
| Registry of Cytotoxicity (RC)  |                                     |
+--------------------------------+-------------------------------------+
| Developmental and Reproductive | EPA; The National Library of        |
| Toxicology/Environmental       | Medicine (NLM); The National        |
| Teratology Information Center  | Institute of Environmental Health   |
| (DART^®^/ETIC)                 | Sciences (NIEHS); National Center   |
|                                | for Toxicological Research (NCTR)   |
+--------------------------------+-------------------------------------+
| Emergency Response Guidebook   | Transport Canada; U.S. Department   |
| (ERG 2000)                     | of Transportation (DOT);            |
|                                | Secretariat of Communications and   |
|                                | Transportation of Mexico            |
+--------------------------------+-------------------------------------+
| Environmental Health Criteria  | WHO IPCS; CCOHS                     |
| (EHC) monographs               |                                     |
|                                |                                     |
| Health and Safety Guides (HSG) |                                     |
|                                |                                     |
| International Agency for       |                                     |
| Research on Cancer (IARC)      |                                     |
+--------------------------------+-------------------------------------+
| European Centre for the        | European Commission Joint Research  |
| Validation of Alternative      | Centre                              |
| Methods (ECVAM) Scientific     |                                     |
| Information Service (ECVAM     |                                     |
| SIS)                           |                                     |
+--------------------------------+-------------------------------------+
| HAZARDTEXT^®^; MEDITEXT^®^;    | TOMES Plus^®^, MICROMEDEX,          |
| INFOTEXT^®^; SARATEXT^®^;      | Greenwood Village, CO               |
| REPROTEXT^®^; REPROTOX^®^      |                                     |
+--------------------------------+-------------------------------------+
| Integrated Risk Information    | EPA Office of Research and          |
| System (IRIS)                  | Development (ORD)                   |
+--------------------------------+-------------------------------------+
| International Chemical Safety  | WHO IPCS; CCOHS; Commission of the  |
| Cards (ICSC)                   | European Union                      |
|                                |                                     |
| IPCS/EC Evaluation of          |                                     |
| Antidotes Series               |                                     |
+--------------------------------+-------------------------------------+
| International Uniform Chemical | European Chemicals Bureau           |
| Information Database (IUCLID)  |                                     |
+--------------------------------+-------------------------------------+
| Joint Expert Committee on Food | WHO IPCS; CCOHS; Food and           |
| Additives (JECFA)              | Agriculture Organization (FAO) of   |
|                                | the United Nations                  |
| Joint Meeting on Pesticide     |                                     |
| Residues (JMPR)                |                                     |
|                                |                                     |
| Pesticide Data Sheets (PDSs)   |                                     |
+--------------------------------+-------------------------------------+
| Material Safety Data Sheets    | Interactive Living Paradigms,       |
| (MSDS)                         | Incorporated                        |
+--------------------------------+-------------------------------------+
| Multicentre Evaluation of In   | Scandinavian Society for Cell       |
| Vitro Cytotoxicity (MEIC)      | Toxicology                          |
+--------------------------------+-------------------------------------+
| National Toxicology Program    | NIEHS                               |
| (NTP) Chemical Health and      |                                     |
| Safety Database                |                                     |
+--------------------------------+-------------------------------------+
| National Transportation        | DOT                                 |
| Library                        |                                     |
+--------------------------------+-------------------------------------+
| New Jersey Hazardous Substance | New Jersey Department of Health and |
| Fact Sheets                    | Senior Services                     |
+--------------------------------+-------------------------------------+
| Oil and Hazardous              | EPA Office of Waste and Water       |
| Materials/Technical Assistance | Management                          |
| Data System (OHM/TADS)         |                                     |
+--------------------------------+-------------------------------------+
| Organisation for Economic      | IPCS; CCOHS; International Register |
| Co-operation and Development   | of Potentially Toxic Chemicals      |
| (OECD) Screening Information   | (IRPTC); UNEP                       |
| Data Sets (SIDS)               |                                     |
+--------------------------------+-------------------------------------+
| Pesticide Action Network       | Pesticide Action Network North      |
| Pesticide Database             | America                             |
+--------------------------------+-------------------------------------+
| Pesticide Product Information  | EPA Office of Pesticide Programs    |
| System (PPIS)                  | (OPP)                               |
+--------------------------------+-------------------------------------+
| Poisons Information Monographs | IPCS; CCOHS                         |
| (PIMs)                         |                                     |
+--------------------------------+-------------------------------------+
| Registry of Toxic Effects of   | National Institute for Occupational |
| Chemical Substances (RTECS^®^) | Safety and Health (NIOSH)           |
|                                |                                     |
| # NIOSH Po                     |                                     |
| cket Guide to Chemical Hazards |                                     |
+--------------------------------+-------------------------------------+
| SCORECARD                      | Environmental Defense               |
+--------------------------------+-------------------------------------+
| The EXtension TOXicology       | University of California, Davis;    |
| NETwork (EXTOXNET)             | Oregon State University; Michigan   |
|                                | State University; Cornell           |
|                                | University; University of Idaho     |
+--------------------------------+-------------------------------------+
| The Right-to-Know Network (RTK | Office of Management and Budget     |
| NET)                           | Watch; Center for Public Data       |
|                                | access                              |
+--------------------------------+-------------------------------------+
| Toxic Chemical Release         | The National Library of Medicine    |
| Inventory (TRI)                | (NLM)                               |
|                                |                                     |
| GENE-TOX                       |                                     |
+--------------------------------+-------------------------------------+
| Toxic Substances Control Act   | EPA OPPT                            |
| Test Submissions (TSCATS)      |                                     |
+--------------------------------+-------------------------------------+
| TOXLINE^®^                     | NLM (TOXNET)                        |
|                                |                                     |
| Hazardous Substances Data Bank |                                     |
| (HSDB)                         |                                     |
|                                |                                     |
| ChemIDplus                     |                                     |
+--------------------------------+-------------------------------------+

A total of 195 LD~50~ references retrieved through these searches were
reviewed and evaluated. Information regarding the materials and methods
used to derive the 491 LD~50~ values reported by these references were
compiled and are provided in **Appendix H-1** in a spreadsheet format.
**Appendix H-2** provides a narrative characterization and evaluation of
the values.

4.1.2 [Criteria Used to Select Candidate *In Vivo* Rodent Toxicity Data
for Determination of Reference Values]{.underline}

From the data retrieved, the goal was to derive a set of high quality
reference LD~50~ values (i.e., data that were collected using
standardized protocols, accompanied by documentation showing that
established testing procedures were followed in compliance with national
and international GLP guidelines \[OECD 1998; FDA 2003; EPA 2003a,b\]).
After a review of the collected data, the SMT determined that a
requirement for GLP compliance would eliminate 99% (452 of the 459
values remaining after exclusion of 30 duplicate values and two
erroneous values) of the oral LD~50~ values, since only seven had been
obtained in compliance with GLP guidelines. GLP-compliant studies were
found for only four of the 72 (6%) reference substances.

The SMT then considered limiting the selection of LD~50~ values to those
from studies that used the type of animals recommended by the current
oral acute toxicity test guidelines, since these guidelines will be used
for future acute systemic toxicity testing. The current guidelines
recommend using young adult rats, 8-12 weeks of age, of a common
laboratory strain and the most sensitive sex (OECD 2001a, c, d; EPA
2002a). Female animals are suggested if there is no information on which
to determine the most sensitive sex. Selecting LD~50~ values from
animals that fit this description yielded a limited number of values.
Only 3% (14/459) of the oral LD~50~ values were determined using 8-12
week old female laboratory rats. Another 15 LD~50~ values were obtained
with female rats in an appropriate weight range (\~ 176-250 g according
to Charles River \[[http://www.criver.com](http://www.criver.com/)\],
Harlan \[<http://www.harlan.com/us/index.htm>\], and Taconic Farms
\[<http://www.taconic.com/anmodels/spragued.htm>\] websites) for that
age. Thus, only 6% (29/459) of the LD~50~ values in the database,
covering 21 of the 72 reference substances (29 %), were obtained from
studies that used the strain, sex, and age of rats recommended by
current test guidelines (OECD 2001a; EPA 2002a).

*Final Exclusion Criteria*

Since so few studies met the initial criteria (i.e., GLP compliance and
use of animals recommended by current acute oral toxicity test
guidelines), the database was reviewed and evaluated to derive
alternative criteria for the development of reference LD~50~ values. For
this evaluation, the SMT looked for commonalities among the data records
that, when selected, provided a comparable data set for each chemical.
Review of the available data indicated that the majority of acute oral
toxicity tests were conducted with unanesthetized young adult laboratory
rats of both genders, to which chemicals were administered by gavage.
Thus, to compile a homogenous set of reference LD~50~ values for each
chemical, the selection process was revised to exclude studies that
reflected the following, less typical, materials and methods:

-   feral rats

-   rats \< 4 weeks of age

-   anesthetized rats

-   test chemical administered in food or capsule

-   LD~50~ reported as a range or inequality

Data from feral rats were excluded, since the health status of these
animals was uncertain. All laboratory rat strains/stocks were deemed
acceptable, since they were expected to be healthy and provided with
adequate care and housing during testing. Data from neonates or
weanlings were excluded since their sensitivity to chemical toxicity may
differ from that of adults. Four weeks was considered the minimum
acceptable age, since rats are weaned at about 3 weeks of age (Barrow
2000). Data from feeding experiments or experiments that involved
administration of the chemical in capsules were also excluded, since
gavage is the most common mode of administration for acute oral studies
and the rate of gastrointestinal absorption for these methods is likely
to be different (Nebendahl 2000). Since LD~50~ point estimates are
required for the prediction model, LD~50~ values reported as ranges or
inequalities were considered unacceptable.

*Assumptions*

The level of detail for materials and methods for the LD~50~ studies
varied greatly. Some studies reported only the use of white rats while
other acute oral toxicity studies provided complete information on
stock/strain, gender, and age of animals; the number of animals tested
per dosing group, method of administration, doses administered, clinical
signs, and times of death. To use as much of the available data as
possible, the following assumptions were made if a study report did not
declare otherwise.

-   The rats were assumed to have been young adults of a common
    laboratory strain.

-   The rats were assumed to have been unanesthetized.

-   The oral route of administration was by gavage.

*Calculation of Reference Values*

If there were multiple acceptable LD~50~ values for a chemical after the
application of the exclusionary criteria, outliers at the 99% level
(Dixon and Massey 1981) were excluded. A geometric mean and 95%
confidence limits were calculated from the remaining values to serve as
the reference LD~50~. A geometric mean is the antilog of the mean of the
logarithm of the values and it is less affected by extreme values than
the arithmetic mean. Use of a geometric mean corresponds with the
approach used for the RC regression to obtain a single IC~50~ value from
multiple IC~50~ values (Halle 1998) and with the approach used to derive
the IC~50~ value for each chemical for the *in vitro* - *in vivo*
regressions for the NICEATM/ECVAM validation study (see **Section 6**).

In addition to the statistical evaluation of outliers, an extreme value,
which was not a statistical outlier, for trichloroacetic acid was also
evaluated based on biological plausibility. There were five LD~50~
values that ranged from 400-8900 mg/kg after applying the exclusionary
criteria for trichloroacetic acid. The lowest value of 400 mg/kg was
rejected as biologically implausible since up to 1000 mg/kg/day has been
used in chronic rodent carcinogenicity studies (EPA 1996).

*Use of Rat and Mouse Data*

If no rat oral LD~50~ values could be found for a chemical, mouse oral
LD~50~ values were located, retrieved, and evaluated by the same method
as that used for rat oral values. Although a model using entirely rat
data or entirely mouse data would be preferable, the use of mouse values
was considered to be justified by a significant correlation of rat and
mouse oral LD~50~ values reported by Ekwall et al. (1998a) for the 50
chemicals tested in the MEIC study. Using values from RTECS^®^, Ekwall
et al. (1998a) reported a coefficient of determination, R^2^, of 0.85
for a linear regression analysis of rat LD~50~ - mouse LD~50~.
Furthermore, Halle (1998) compared IC~50~ - LD~50~ linear regressions
with 285 rat values and 242 mice values and found no significant
difference in the intercepts or slopes.

A correlation of the 173 chemicals in the RC that had both rat and mouse
LD~50~ values is shown in **Figure 4-1**. A Spearman correlation
analysis of the log transformed rat and mouse data yielded a significant
correlation (p\< 0.0001) with r~s~ = 0.88.

**Figure 4-1 Correlation of Rat and Mouse LD~50~ Values for 173 RC
Chemicals**

![](media/image1.pct){width="5.346527777777778in"
height="4.929861111111111in"}

The diagonal line shows the 1:1 relationship.

**4.2 Final *In Vivo* Rodent Toxicity Reference Values**

After the application of the exclusionary criteria, there were 385
acceptable LD~50~ values with which to calculate reference values.
**Table 4-2** shows the reference LD~50~ value for each reference
substance in ascending order. The reference values are the geometric
means of the acceptable LD~50~ values. Also shown for each substance are
the 95% confidence limits around the mean, the ratio of the maximum to
the minimum acceptable value, the number of LD~50~ values used to
calculate the reference value, the number of LD~50~ values available
(not including duplicate values or the erroneous values for
acetylsalicylic acid and sodium oxalate), and the LD~50~ initially used
for hazard category (often referred to as "toxicity" or "LD~50~"
category) classification of the substance (see **Table 3-2**). Ratios
for the maximum to minimum LD~50~ values ranged from 1.0 to 25.9. The
average ratio was 4.1. Six of the 62 reference substances for which
ratios were calculated had ratios greater than one order of magnitude:
triethylenemelamine, parathion, busulfan, triphenyltin hydroxide,
phenol, and trichloroacetic acid. Three of these substances,
triethylenemelamine, parathion, and busulfan, were in the two highest
toxicity categories (i.e., LD~50~ ≤ 50 mg/kg).

**Table 4-2** shows the reference substances grouped by GHS acute oral
toxicity category (UN 2005) using the reference LD~50~ values. The
initial categorization for this study, which used the LD~50~ values in
the far right column of **Table 4-2** (i.e., values reported in **Table
3-2**, which come from the RC unless otherwise specified), placed 12
substances in each toxicity category. **Table 4-3** compares the number
of substances in each GHS toxicity category based on the reference
LD~50~ values with the number of substances in each category based on
the initial LD~50~ values. **Table 4-3** shows that the initial and
reference LD~50~ values placed 74% of the substances in the same GHS
category. Compared with the initial LD~50~, the reference LD~50~ was
higher for 25% of the substances and lower for 1% of the substances.

+-------------+-------+-------+------+--------+-------+------+-------+
| **Table 4-2 |       |       |      |        |       |      |       |
| Reference   |       |       |      |        |       |      |       |
| LD~50~      |       |       |      |        |       |      |       |
| Values by   |       |       |      |        |       |      |       |
| GHS         |       |       |      |        |       |      |       |
| Ca          |       |       |      |        |       |      |       |
| tegory^1^** |       |       |      |        |       |      |       |
+=============+=======+=======+======+========+=======+======+=======+
| **GHS       | *     | **95% | **R  | **95%  | Max   | **N  | **In  |
| Category^1^ | *Refe | Confi | efer | Conf   | imum: | A    | itial |
| /Chemical** | rence | dence | ence | idence | Mi    | vera | R     |
|             | Oral  | I     | Oral | Inter  | nimum | ged^ | odent |
|             | LD~5  | nterv | L    | val^3^ |       | 5^** | Oral  |
|             | 0~^2^ | al^3^ | D~50 | (mmol  | **    |      | LD~5  |
|             | (mg/  | (mg/  | ~^2^ | /kg)** | Value |      | 0~^6^ |
|             | kg)** | kg)** | (mm  |        | ^3^** |      | (mg/  |
|             |       |       | ol/k |        |       |      | kg)** |
|             |       |       | g)** |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| *           |       |       |      |        |       |      |       |
| **LD~50~*** |       |       |      |        |       |      |       |
| ***≤ 5      |       |       |      |        |       |      |       |
| mg/kg (N =  |       |       |      |        |       |      |       |
| 7)***       |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Cy          | 2     | NC    | 0.0  | NC     | 2.5   | 3    | 2     |
| cloheximide |       |       | 0711 |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Phe         | 3     | NC    | 0.   | NC     | NC    | 1    | 3     |
| nylthiourea |       |       | 0197 |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Sodium      | 3     | NC    | 0.   | NC     | 3.7   | 2    | 2^7^  |
| selenate    |       |       | 0159 |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Epinephrine | 4     | NC    | 0.   | NC     | NC    | 1    | 4     |
| bitartrate  | (m    |       | 0196 |        |       |      | (m    |
|             | ouse) |       |      |        |       |      | ouse) |
+-------------+-------+-------+------+--------+-------+------+-------+
| Triethyl    | 4     | 1-25  | 0.   | 0.003  | 13.0  | 4    | 1     |
| enemelamine |       |       | 0120 | 7-0.12 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Ph          | 5     | NC    | 0.   | NC     | NC    | 1    | 5^7^  |
| ysostigmine |       |       | 0182 |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Disulfoton  | 5     | 2-10  | 0.   | 0.009  | 5.5   | 6    | 2     |
|             |       |       | 0182 | -0.036 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| ***5 \<     |       |       |      |        |       |      |       |
| LD~50~ ≤ 50 |       |       |      |        |       |      |       |
| mg/kg (N =  |       |       |      |        |       |      |       |
| 12)***      |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Parathion   | 6     | 3-12  | 0.   | 0.010  | 16.7  | 10   | 2     |
|             |       |       | 0209 | -0.041 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Strychnine  | 6     | NC    | 0.   | NC     | 6.9   | 3    | 2^7^  |
|             |       |       | 0188 |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Aminopterin | 7     | NC    | 0    | NC     | NC    | 1    | 3     |
|             |       |       | .016 |        |       |      | (m    |
|             |       |       |      |        |       |      | ouse) |
+-------------+-------+-------+------+--------+-------+------+-------+
| Potassium   | 7     | 5-10  | 0    | 0.07   | 2.0   | 7    | 10    |
| cyanide     |       |       | .111 | 7-0.15 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Busulfan    | 12    | NC    | 0    | 0.00   | 15.3  | 4    | 2     |
|             |       |       | .049 | 8-0.38 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Colchicine  | 15    | NC    | 0.   | NC     | 4.9   | 3    | 6     |
|             | (m    |       | 0375 |        |       |      | (m    |
|             | ouse) |       |      |        |       |      | ouse) |
+-------------+-------+-------+------+--------+-------+------+-------+
| Thallium I  | 25    | NC    | 0.   | NC     | NC    | 1    | 29    |
| sulfate     |       |       | 0495 |        |       |      | (m    |
|             |       |       |      |        |       |      | ouse) |
+-------------+-------+-------+------+--------+-------+------+-------+
| Arsenic III | 25    | 10-64 | 0    | 0.05   | 6.3   | 5    | 20    |
| trioxide    |       |       | .127 | 0-0.32 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Endosulfan  | 28    | NC    | 0    | NC     | 2.4   | 2    | 18^7^ |
|             |       |       | .068 |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Digoxin     | 28    | NC    | 0.   | NC     | NC    | 1    | 18    |
|             |       |       | 0362 |        |       |      | (m    |
|             |       |       |      |        |       |      | ouse) |
+-------------+-------+-------+------+--------+-------+------+-------+
| Mercury II  | 40    | 27-60 | 0    | 0.01   | 7.7   | 10   | 1     |
| chloride    |       |       | .148 | 0-0.22 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Sodium      | 44    | 36-53 | 0    | 0.2    | 1.5   | 5    | 41^7^ |
| arsenite    |       |       | .336 | 8-0.40 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| ***50 \<    |       |       |      |        |       |      |       |
| LD~50~ ≤    |       |       |      |        |       |      |       |
| 300 mg/kg   |       |       |      |        |       |      |       |
| (N =12)***  |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Sodium      | 51    | 44-58 | 0    | 0.1    | 1.9   | 11   | 50    |
| dichromate  |       |       | .193 | 7-0.22 |       |      |       |
| dihydrate   |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Dichlorvos  | 59    | 40-88 | 0    | 0.1    | 5.7   | 9    | 17^7^ |
|             |       |       | .266 | 8-0.40 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Nicotine    | 70    | 68-72 | 0    | 0.4    | 1.0   | 4    | 50    |
|             |       |       | .430 | 2-0.44 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Fe          | 76    | 5     | 0    | 0.1    | 3.4   | 9    | 18^7^ |
| npropathrin |       | 7-100 | .217 | 6-0.29 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Hexa        | 82    | 68-98 | 0    | 0.1    | 3.8   | 19   | 61    |
| chlorophene |       |       | .202 | 7-0.24 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Paraquat    | 93    | 6     | 0    | 0.3    | 2.0   | 5    | 58    |
|             |       | 5-132 | .498 | 5-0.71 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Lindane     | 100   | 7     | 0    | 0.2    | 1.4   | 4    | 76    |
|             |       | 8-129 | .344 | 7-0.44 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Verapamil   | 111   | NC    | 0    | NC     | 1.1   | 2    | 108   |
| HCl         |       |       | .226 |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Sodium I    | 127   | 9     | 3    | 2.1    | 4.4   | 12   | 180   |
| fluoride    |       | 2-175 | .020 | 9-4.16 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Cadmium II  | 135   | 8     | 0    | 0.4    | 2.4   | 5    | 88    |
| chloride    |       | 8-208 | .738 | 8-1.14 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Diquat      | 160   | NC    | 0    | NC     | 1.9   | 3    | 231   |
| dibromide   |       |       | .466 |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Ph          | 224   | NC    | 0    | NC     | 2.0   | 3    | 163   |
| enobarbital |       |       | .966 |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| ***300 \<   |       |       |      |        |       |      |       |
| LD~50~ ≤    |       |       |      |        |       |      |       |
| 2000 mg/kg  |       |       |      |        |       |      |       |
| (N = 16)*** |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Caffeine    | 310   | 25    | 1.59 | 1.3    | 2.5   | 10   | 192   |
|             |       | 6-374 |      | 2-1.93 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| T           | 329   | 20    | 0    | 0.5    | 25.9  | 15   | 44    |
| riphenyltin |       | 8-520 | .896 | 7-1.42 |       |      |       |
| hydroxide   |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Haloperidol | 330   | NC    | 0    | NC     | 6.6   | 2    | 1     |
|             |       |       | .877 |        |       |      | 28^7^ |
+-------------+-------+-------+------+--------+-------+------+-------+
| Am          | 348   | NC    | 1.18 | NC     | 1.2   | 2    | 319   |
| itriptyline |       |       |      |        |       |      |       |
| HCl         |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Propranolol | 466   | NC    | 1    | NC     | NC    | 1    | 470   |
| HCl         |       |       | .575 |        |       |      | (m    |
|             |       |       |      |        |       |      | ouse) |
+-------------+-------+-------+------+--------+-------+------+-------+
| Cupric      | 474   | 26    | 1.90 | 1.0    | 4.1   | 6    | 300   |
| sulfate \*  |       | 9-836 |      | 8-3.35 |       |      |       |
| 5 H2O       |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Phenol      | 548   | 43    | 5.82 | 4.8    | 4.7   | 14   | 414   |
|             |       | 4-692 |      | 2-7.68 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Lithium     | 590   | 47    | 7.98 | 6      | 1.4   | 4    | 1187  |
| carbonate   |       | 9-728 |      | .5-9.9 |       |      | (m    |
|             |       |       |      |        |       |      | ouse; |
|             |       |       |      |        |       |      | su    |
|             |       |       |      |        |       |      | lfate |
|             |       |       |      |        |       |      | salt) |
+-------------+-------+-------+------+--------+-------+------+-------+
| G           | 600   | NC    | 2.76 | NC     | NC    | 1    | 600   |
| lutethimide |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Sodium      | 633   | NC    | 4    | NC     | 1.3   | 2    | 155   |
| oxalate     |       |       | .724 |        |       |      | (mous |
|             |       |       |      |        |       |      | e)^8^ |
+-------------+-------+-------+------+--------+-------+------+-------+
| Chloral     | 638   | 391   | 3.86 | 2.3    | 1.8   | 4    | 479   |
| hydrate     |       | -1040 |      | 6-6.29 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Atropine    | 819   | 641   | 1.21 | 0.9    | 1.9   | 7    | 623   |
| sulfate     |       | -1045 |      | 5-1.54 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Valproic    | 995   | NC    | 6.91 | NC     | 2.2   | 2    | 6     |
| acid        |       |       |      |        |       |      | 70^7^ |
+-------------+-------+-------+------+--------+-------+------+-------+
| Meprobamate | 1387  | 1291  | 6.35 | 5.9    | 1.2   | 6    | 7     |
|             |       | -1489 |      | 2-6.82 |       |      | 94^7^ |
+-------------+-------+-------+------+--------+-------+------+-------+
| Acet        | 1506  | 1224  | 8.36 | 6.     | 4.6   | 14   | 1000  |
| ylsalicylic |       | -1854 |      | 8-10.3 |       |      |       |
| acid        |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| P           | 1950  | NC    | 8    | NC     | NC    | 1    | 19    |
| rocainamide |       |       | .286 |        |       |      | 50^7^ |
| HCl         |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| ***2000 \<  |       |       |      |        |       |      |       |
| LD~50~ ≤    |       |       |      |        |       |      |       |
| 5000 mg/kg  |       |       |      |        |       |      |       |
| (N = 11)*** |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Ac          | 2163  | NC    | 14.3 | NC     | 1.2   | 2    | 2404  |
| etaminophen |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Potassium I | 2799  | NC    | 37.6 | NC     | 1.2   | 2    | 2602  |
| chloride    |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Ca          | 2805  | NC    | 11.9 | NC     | 2.1   | 2    | 19    |
| rbamazepine |       |       |      |        |       |      | 57^7^ |
+-------------+-------+-------+------+--------+-------+------+-------+
| Boric aid   | 3426  | 2617  | 55.4 | 42.    | 1.9   | 6    | 26    |
|             |       | -4486 |      | 3-72.6 |       |      | 60^7^ |
+-------------+-------+-------+------+--------+-------+------+-------+
| 5-Ami       | 3429  | NC    | 22.4 | NC     | 1.5   | 2    | 7749  |
| nosalicylic |       |       |      |        |       |      | (m    |
| acid        |       |       |      |        |       |      | ouse) |
+-------------+-------+-------+------+--------+-------+------+-------+
| Chlo        | 3491  | NC    | 10.8 | NC     | 2.0   | 3    | 3393  |
| ramphenicol |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| A           | 3598  | 2951  | 87.6 | 71     | 6.2   | 26   | 3798  |
| cetonitrile |       | -4375 |      | .9-107 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Lactic acid | 3639  | NC    | 40.3 | NC     | 1.1   | 2    | 3730  |
+-------------+-------+-------+------+--------+-------+------+-------+
| Carbon      | 3783  | 3024  | 24.6 | 20-31  | 4.3   | 15   | 2799  |
| te          |       | -4732 |      |        |       |      |       |
| trachloride |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Sodium      | 4046  | 2917  | 69.3 | 50-96  | 2.0   | 5    | 2998  |
| chloride    |       | -5623 |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Xylene      | 4667  | 1294- | 43.9 | 12-158 | 5.6   | 4    | 4300  |
|             |       | 16827 |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| ***LD~50~   |       |       |      |        |       |      |       |
| \> 5000     |       |       |      |        |       |      |       |
| mg/kg (N =  |       |       |      |        |       |      |       |
| 14)***      |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| 2-Propanol  | 5105  | 4624  | 84.9 | 77-94  | 1.3   | 6    | 5843  |
|             |       | -5636 |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Tric        | 5229  | 2745  | 32.0 | 16.    | 2.7   | 4    | 4999  |
| hloroacetic |       | -9961 |      | 8-61.0 |       |      |       |
| acid        |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Dimeth      | 5309  | 3548  | 72.6 | 49-108 | 2.6   | 6    | 2800  |
| ylformamide |       | -7925 |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Citric Acid | 5929  | NC    | 30.9 | NC     | 3.9   | 2    | 30    |
|             |       |       |      |        |       |      | 00^7^ |
+-------------+-------+-------+------+--------+-------+------+-------+
| Gibberellic | 6040  | NC    | 17.4 | NC     | 1.1   | 2    | 6305  |
| acid        |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Pr          | 6332  | NC    | 35.1 | NC     | NC    | 1    | 6326  |
| opylparaben | (m    |       |      |        |       |      | (m    |
|             | ouse) |       |      |        |       |      | ouse) |
+-------------+-------+-------+------+--------+-------+------+-------+
| Ethylene    | 7161  | 6266  | 1    | 1      | 2.5   | 16   | 8567  |
| glycol      |       | -8204 | 15.4 | 01-132 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Methanol    | 8710  | 6223- | 272  | 1      | 2.3   | 6    | 13012 |
|             |       | 12218 |      | 94-381 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Dibut       | 8892  | 6180- | 31.9 | 22-46  | 1.7   | 4    | 11998 |
| ylphthalate |       | 12794 |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Dieth       | 9311  | NC    | 41.9 | NC     | 1.2   | 2    | 8602  |
| ylphthalate |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Sodium      | 10328 | NC    | 62.8 | NC     | 1.6   | 2    | 89    |
| h           |       |       |      |        |       |      | 10^9^ |
| ypochlorite |       |       |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Ethanol     | 11324 | 8610- | 2    | 1      | 2.5   | 8    | 14008 |
|             |       | 14894 | 45.7 | 87-323 |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| 1,1,1-Tric  | 12078 | 1     | 90.5 | 75-109 | 1.7   | 6    | 10298 |
| hloroethane |       | 0000- |      |        |       |      |       |
|             |       | 14588 |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+
| Glycerol    | 19770 | 1     | 215  | 1      | 2.2   | 4    | 12691 |
|             |       | 0495- |      | 14-403 |       |      |       |
|             |       | 37154 |      |        |       |      |       |
+-------------+-------+-------+------+--------+-------+------+-------+

^1^GHS- Globally Harmonized System of Classification and Labelling of
Chemicals (UN 2005). Chemicals categorized using reference oral LD~50.~

^2^Based on a geometric mean of acceptable LD~50~ values from laboratory
rats unless otherwise specified.

^3^For the geometric mean of the acceptable LD~50~ values.

^4^Ratio of minimum acceptable LD~50~ to maximum acceptable LD~50~

^5^Number of values used for geometric mean.

^6^Values rounded to the nearest one; from the RC unless otherwise
specified; rat data unless otherwise specified.

^7^RTECS^®^ (MDL Information Systems 2002).

^8^RC reference for rat oral LD~50~ of 155 mg/kg is Shrivastava et al.
(1992), which references Klinger and Kersten (1961). Klinger and Kersten
(1961) indicates the value was determined by intraperitoneal
administration to mice.

^9^NLM (2002).

Abbreviations: NC -- Not calculated. N was three or less and considered
too small for a meaningful result.

The reference LD~50~ values caused the reclassification of 19 reference
substances (i.e., the sum of the numbers in the mismatching cells in
**Table 4-3)**. Seven substances remain in the lowest LD~50~ category
(i.e., LD~50~ ≤ 5 mg/kg). Five substances originally in this category
(aminopterin, mercury chloride, busulfan, parathion, and strychnine)
moved to the next higher category (5 \< LD~50~ ≤ 50 mg/kg) due the
change in the reference LD~50~ values. In the 5 \< LD~50~ ≤ 50 mg/kg
category, four substances (dichlorvos, fenpropathrin, sodium dichromate
dihydrate, and nicotine) moved to the next higher LD~50~ category (50 \<
LD~50~ ≤ 300 mg/kg) and one substance (triphenyltin hydroxide) moved up
two categories to 300 \< LD~50~ ≤ 2000 mg/kg. In the 50 \< LD~50~ ≤ 300
category, four substances (haloperidol, caffeine, copper sulfate
pentahydrate, and sodium oxalate) moved up to the next toxicity category
(300 \< LD~50~ ≤ 2000 mg/kg). In the 300 \< LD~50~ ≤ 2000 mg/kg
category, only carbamazepine moved up to the next toxicity category
(2000 \< LD~50~ ≤ 5000 mg/kg). In the 2000 \< LD~50~ ≤ 5000 mg/kg
category, citric acid, trichloroacetic acid and dimethylformamide moved
up to the next higher LD~50~ category (LD~50~ \> 5000 mg/kg). In the
LD~50~ \> 5000 mg/kg category, 5-aminosalicylic acid moved down into the
2000 \< LD~50~ ≤ 5000 mg/kg category. 5-Aminosalicylic acid was the only
substance that moved to a lower LD~50~ (i.e., more toxic) category.

**Table 4-3 GHS^1^ Toxicity Category Matches for the Initial and
Reference LD~50~ Values^2^**

<table>
<colgroup>
<col style="width: 9%" />
<col style="width: 6%" />
<col style="width: 6%" />
<col style="width: 7%" />
<col style="width: 9%" />
<col style="width: 10%" />
<col style="width: 7%" />
<col style="width: 6%" />
<col style="width: 9%" />
<col style="width: 12%" />
<col style="width: 12%" />
</colgroup>
<tbody>
<tr class="odd">
<td rowspan="2"><p><strong>Initial LD<sub>50</sub></strong></p>
<p><strong>(mg/kg)</strong></p></td>
<td colspan="6"><strong>Reference LD<sub>50</sub></strong></td>
<td rowspan="2"><strong>Total</strong></td>
<td rowspan="2"><p><strong>Category</strong></p>
<p><strong>Match</strong></p></td>
<td rowspan="2"><strong>Reference LD<sub>50</sub> Lower</strong></td>
<td rowspan="2"><strong>Reference LD<sub>50</sub> Higher</strong></td>
</tr>
<tr class="even">
<td>≤ <strong>5</strong></td>
<td><strong>5-50</strong></td>
<td><strong>50 - 300</strong></td>
<td><strong>300 - 2000</strong></td>
<td><strong>2000 - 5000</strong></td>
<td><strong>&gt; 5000</strong></td>
</tr>
<tr class="odd">
<td>≤ 5</td>
<td>7</td>
<td>5</td>
<td>0</td>
<td>0</td>
<td>0</td>
<td>0</td>
<td>12</td>
<td>58%</td>
<td>0%</td>
<td>42%</td>
</tr>
<tr class="even">
<td>5-50</td>
<td>0</td>
<td>7</td>
<td>4</td>
<td>1</td>
<td>0</td>
<td>0</td>
<td>12</td>
<td>58%</td>
<td>0%</td>
<td>42%</td>
</tr>
<tr class="odd">
<td>50 - 300</td>
<td>0</td>
<td>0</td>
<td>8</td>
<td>4</td>
<td>0</td>
<td>0</td>
<td>12</td>
<td>67%</td>
<td>0%</td>
<td>33%</td>
</tr>
<tr class="even">
<td>300 - 2000</td>
<td>0</td>
<td>0</td>
<td>0</td>
<td>11</td>
<td>1</td>
<td>0</td>
<td>12</td>
<td>92%</td>
<td>0%</td>
<td>8%</td>
</tr>
<tr class="odd">
<td>2000 - 5000</td>
<td>0</td>
<td>0</td>
<td>0</td>
<td>0</td>
<td>9</td>
<td>3</td>
<td>12</td>
<td>75%</td>
<td>0%</td>
<td>25%</td>
</tr>
<tr class="even">
<td>&gt; 5000</td>
<td>0</td>
<td>0</td>
<td>0</td>
<td>0</td>
<td>1</td>
<td>11</td>
<td>12</td>
<td>92%</td>
<td>8%</td>
<td>0%</td>
</tr>
<tr class="odd">
<td>Total</td>
<td>7</td>
<td>12</td>
<td>12</td>
<td>16</td>
<td>11</td>
<td>14</td>
<td>72</td>
<td>74%</td>
<td>1%</td>
<td>25%</td>
</tr>
</tbody>
</table>

^1^Globally Harmonized System of Classification and Labelling of
Chemicals (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^Number of chemicals. Darkened cells show the number of chemicals for
which the categories match.

**4.3 Relevant Toxicity Information for Humans**

The relevance of rodent acute systemic toxicity data to human lethality
was assessed by the MEIC program as a comparison to the evaluation of
*in vitro* predictions of human acute toxicity (Ekwall et al. 1998b).
The MEIC program collected mouse and rat oral LD~50~ data from RTECS^®^
(Ekwall et al. 1998a). Mean lethal doses in humans were collected mainly
from handbooks containing human clinical toxicity information (Ekwall et
al. 1998a). Data from the handbooks were supplemented, when necessary,
by an in-house compendium from the Swedish Poisons Information Centre.
Ekwall et al. (1998b) calculated least squares linear regressions for
the prediction of the mean human lethal doses by rat oral LD~50~ data
and by mouse oral LD~50~ data for the 50 MEIC chemicals using units of
log mol/kg. Ekwall et al. (1998b) reported R^2^ = 0.607 for the rat
LD~50~ prediction of mean human lethal doses and R^2^ = 0.653 for the
mouse LD~50~ prediction of mean human lethal doses.

The relevance of the NRU data collected in the NICEATM/ECVAM study to
the prediction of human acute toxicity will be addressed elsewhere by
ECVAM.

**4.4 Accuracy and Reliability of the *In Vivo* Rodent Toxicity
Reference Values**

Accuracy is the closeness of agreement between the results of an
alternative test method and an accepted reference test method (ICCVAM
2003). Since there is no accepted reference test for the rodent acute
oral toxicity test, the accuracy of the reference LD~50~ values for
predicting the oral LD~50~ in humans cannot be determined. Acute
toxicity testing in rodents leads to a relative ranking of the toxicity
of chemicals for regulatory purposes. The reliability of the reference
LD~50~ values determined in this section may be judged by evaluating the
range of acceptable LD~50~ values for each chemical and by comparing the
values (and their variability) with other LD~50~ values.

*Variability Among the Acceptable LD~50~ Values*

The variability of the acceptable LD~50~ values used to calculate the
reference value for each reference substance was assessed by calculating
the ratio of the maximum to the minimum value (see **Table 4-2**). For
the 62 reference substances with more than one acceptable LD~50~ value,
the average maximum:minimum ratio ranged from 1.1 to 25.9 with a mean of
4.3 and a median of 2.2. The maximum:minimum ratios were greater than 10
for four substances: triethylenemelamine, parathion, busulfan, and
triphenyltin hydroxide.

The low LD~50~ values for triethylenemelamine, busulfan, and parathion
may have contributed to the high maximum:minimum ratios for these
substances, since the range of values did not seem to be extremely wide.
The four LD~50~ values for triethylenemelamine ranged from 1 to 13
mg/kg, the four LD~50~ values for busulfan ranged from 1.9 to 29 mg/kg,
and the 10 LD~50~ values for parathion ranged from 1.8 to 30 mg/kg.
**Table 4-4** shows the maximum:minimum ratios by toxicity category. The
substances in the higher toxicity categories (i.e., LD~50~ ≤ 50 mg/kg)
tended to have higher maximum:minimum LD~50~ ratios than substances in
the lower toxicity categories (i.e., LD~50~ \> 50 mg/kg); however, there
were also fewer substances in the higher toxicity categories.

**Table 4-4 Maximum:Minimum LD~50~ Ratios by GHS^1^ Toxicity Category**

+---------------+---------------+---------------+---------------+-----+
| **GHS         | **Mean        | **Median      | **Range of    | **  |
| Category^1^** | Ma            | Ma            | Ma            | N** |
|               | ximum:Minimum | ximum:Minimum | ximum:Minimum |     |
| **(LD~50~ in  | LD~50~        | LD~50~        | LD~50~        |     |
| mg/kg)**      | Ratio**       | Ratio**       | Ratio**       |     |
+---------------+---------------+---------------+---------------+-----+
| LD~50~ ≤ 5    | 6.2           | 4.6           | 2.5 -- 13.0   | 4   |
+---------------+---------------+---------------+---------------+-----+
| 5 \< LD~50~ ≤ | 7.1           | 6.3           | 2.0 - 16.7    | 9   |
| 50            |               |               |               |     |
+---------------+---------------+---------------+---------------+-----+
| 50 \< LD~50~  | 2.4           | 1.9           | 1.1 - 5.7     | 12  |
| ≤ 300         |               |               |               |     |
+---------------+---------------+---------------+---------------+-----+
| 300 \< LD~50~ | 4.6           | 2.2           | 1.2 - 25.9    | 13  |
| ≤ 2000        |               |               |               |     |
+---------------+---------------+---------------+---------------+-----+
| 2000 \<       | 2.6           | 2.0           | 1.2- 22.3     | 11  |
| LD~50~ ≤ 5000 |               |               |               |     |
+---------------+---------------+---------------+---------------+-----+
| LD~50~ \>     | 2.3           | 2.3           | 1.1 - 3.9     | 13  |
| 5000          |               |               |               |     |
+---------------+---------------+---------------+---------------+-----+

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

N = number of chemicals with more than one acceptable LD~50~ value after
application of the exclusion criteria in

**Section 4.1.2**.

*Comparison of Reference Values with RC Values*

The correspondence of the reference LD~50~ values with the LD~50~ values
for the 58 validation study reference substances in common with the RC
are shown on a log scale in **Figure 4-2**. A Spearman correlation
analysis for the two sets of log transformed values yielded a
significant correlation (p \< 0.0001) with a correlation coefficient,
r~s~, of 0.97. **Figure 4-2** shows that the reference values tended to
be higher than the RC LD~50~ values. The LD~50~ values used in the RC
were largely from the 1983/84 RTECS**^®^**, which publishes the lowest
LD~50~ value found for a particular chemical without regard to the
source (i.e., from a primary publication or a review) and without
scientific review before publication. Thus, since the reference LD~50~
values are based on the geometric mean from multiple studies, it is not
surprising that these values tended to be higher than those included in
the RC database.

When comparing the reference LD~50~ values to the RC values, the
substances with the largest differences in LD~50~ were busulfan,
triphenyltin hydroxide, and mercury chloride **(**see **Figure 4-2)**.

-   The reference LD~50~ for busulfan was six times that of the RC value
    (12 mg/kg vs. 1.9 mg/kg). The RC value (i.e., the 1983/84
    RTECS**^®^** value) was from a paper by Schmahl and Osswald (1970)
    in which they cited a rat oral LD~50~ of 1.86 mg/kg. We also found
    rat oral LD~50~ values of 28 and 29 mg/kg for male and female
    Sprague-Dawley rats, respectively (Matsuno et al. 1971).

-   The reference LD~50~ for triphenyltin hydroxide was 7.5 times the RC
    LD~50~ (329 mg/kg vs. 44 mg/kg). The 15 LD~50~ values used to
    determine the reference value included the RC value and had a wide
    range, 44-1200 mg/kg. Due to the relatively large variation in the
    data, neither the highest nor the lowest values were statistical
    outliers.

-   The reference LD~50~ for mercury chloride was 40 mg/kg, while the RC
    value was 1 mg/kg. The RC value was from a summary document that
    reported the rat oral LD~50~ as a range of 1-5 mg/kg (Worthing and
    Walker 1991). Since it was reported as a range, it was excluded from
    the calculation of the reference value. The remaining 11 LD~50~
    values ranged from 12 to 160 mg/kg. As previously stated, 160 mg/kg
    was an outlier compared to the other 10 values and therefore
    excluded from the calculation of the reference value.

**Figure 4-2 Correlation of LD~50~ Values for the 58 RC Chemicals**

![](media/image2.pct){width="5.570138888888889in"
height="5.236805555555556in"}

The diagonal line shows the 1:1 relationship.

*Comparison of the Variability Among Acceptable LD~50~ Values to Other
Studies*

When compared to other studies on the variation of acute oral LD~50~
values, the variation determined for 61 reference substances with
multiple LD~50~ values was not unusual. Weil and Wright (1967) showed
that even LD~50~ values from multiple laboratories using exactly the
same protocol varied by as much as five-fold for the 10 substances they
tested in eight laboratories. In addition, they showed that allowing the
laboratories to use their own protocols for LD~50~ determination
produced data somewhat more variable, but the observed differences were
not reported. Another multicenter study that did not control the LD~50~
protocols reported maximum:minimum ratios from 3.6 to 11.3 for five
substances (Hunter et al. 1979). The 65 participating laboratories in
eight countries reported LD~50~ values ranging from 44 to 5420 mg/kg for
the five substances tested:

-   Compound I/PCP 44 -- 523 mg/kg

-   Compound II/Sodium salicylate 800 - 4150 mg/kg

-   Compound III/Aniline 350 -- 1280 mg/kg

-   Compound IV/Acetanilide 805 -- 5420 mg/kg

-   Compound V/Cadmium chloride 70 -- 513 mg/kg

The results of a follow on study in which the same substances were
tested by about 100 laboratories in 13 countries showed that adhering to
a specific protocol reduced the range of maximum:minimum LD~50~ ratios
from 3.6 -- 11.3 to 2.4 -- 8.4 (Zbinden and Flury-Roversi 1981).

Although the LD~50~ data collected from the literature for the
NICEATM/ECVAM validation study used various strains, sexes, observation
durations, and calculation methods for estimating the LD~50~, the
variation in LD~50~ values for individual substances was similar to the
data by Hunter et al. (1979). The current study found six of the 61
substances with multiple LD~50~ values had maximum:minimum LD~50~ values
higher than that reported by Hunter et al. (1979). Three of the
reference substances: triethylenemelamine, parathion, and busulfan, were
in the lowest LD~50~ (i.e., highest toxicity categories). Hunter et al.
(1979) also observed that the largest variation was associated with the
most toxic substances.

**4.5 Summary**

*In vivo* reference data for comparison with the *in vitro* NRU
cytotoxicity data for the 72 substances were determined by analyzing
rodent LD~50~ values identified by literature searches and secondary
references. Rat LD~50~ values were preferred, but when rat data could
not be located for three substances, mouse LD~50~ values were used. The
491 LD~50~ values located consisted of 485 rat oral LD~50~ values and
six mouse oral LD~50~ values. Identifying a high quality data set
determined under GLP guidelines was not possible since only 3% of the
data records were in compliance. Instead, a homogenous set of LD~50~
values for each substance was identified by excluding studies that
employed the following materials and methods:

-   feral rats

-   rats \< 4 weeks of age

-   anesthetized rats

-   test chemical administered in food or capsule

-   LD~50~ reported as a range or inequality

After analyzing the remaining acceptable data for outliers, the
remaining 385 values were used to determine *in vivo* reference values
by calculating a geometric mean of the values for each reference
substance. The reference LD~50~ values for 20 substances varied enough
from the initial LD~50~ values, which came from the RC and other summary
sources, that the substances were classified into different GHS oral
toxicity categories.

Since there is no reference test for the rodent oral LD~50~, the
accuracy of the reference values for predicting the oral LD~50~ in
humans could not be determined. The reliability of the reference values
was assessed by comparison to other evaluations of the performance of
the *in vivo* acute oral toxicity tests. Although the correlation of the
reference values for the 58 RC chemicals with the RC LD~50~ was high
(r~s~ = 0.97), the reference LD~50~ values tended to be higher than the
RC values. The maximum:minimum ratio of the acceptable values for the 62
reference substances that had more than one LD~50~ value ranged from 1.1
to 25.9. The maximum:minimum ratios for four chemicals were greater than
one order of magnitude.

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