10.0 ANIMAL WELFARE CONSIDERATIONS (REFINEMENT, REDUCTION,\
AND REPLACEMENT) 10-3

10.1 Use of 3T3 and NHK NRU Test Methods to Predict Starting Doses for
Acute Systemic Toxicity Assays 10-4

10.2 Reduction and Refinement of Animal Use for the UDP 10-5

> 10.2.1 Procedure for *In Vivo* Testing Using the UDP 10-5
>
> 10.2.2 Procedure for Computer Simulation Modeling of the UDP 10-7
>
> 10.2.3 Animal Savings for the UDP When Using 3T3 and NHK
>
> NRU-Based Starting Doses 10-9
>
> 10.2.3.1 *The Effect of Dose-Response Slope on Animal Use* 10-9
>
> 10.2.3.2 *Mean Animal Use from UDP Simulations for Testing the
> NICEATM/ECVAM Reference Substances -- Comparison*
>
> *of Regressions and 3T3 and NHK NRU Test Methods* 10-11
>
> 10.2.3.3 *Animal Savings for the UDP by Toxicity Category Using*
>
> *3T3 and NHK NRU-Based Starting Doses* 10-13
>
> 10.2.4 Refinement of Animal Use for the UDP When Using 3T3 and
>
> NHK NRU-Based Starting Doses 10-23
>
> 10.3 Reduction and Refinement of Animal Use for the ATC 10-25
>
> 10.3.1 Procedure for *In Vivo* Testing Using the ATC 10-25
>
> 10.3.2 Procedure for Computer Simulation Modeling of the ATC 10-26
>
> 10.3.3 Animal Savings for the ATC When Using 3T3 and NHK
>
> NRU-Based Starting Doses 10-28
>
> 10.3.3.1 *The Effect of Dose-Response Slope on Animal Use* 10-28
>
> 10.3.3.2 *Mean Animal Use for ATC Simulations of Testing the
> NICEATM/ECVAM Reference Substances -- Comparison*
>
> *of Regressions and 3T3 and NHK NRU Test Methods* 10-29
>
> 10.3.3.3 *Animal Savings for the ATC by Toxicity Category Using*
>
> *3T3 and NHK NRU-Based Starting Doses* 10-29
>
> 10.3.4 Refinement of Animal Use for the ATC When Using 3T3
>
> and NHK NRU-Based Starting Doses 10-40

10.4 Summary 10-41

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**10.0 ANIMAL WELFARE CONSIDERATIONS (REFINEMENT, REDUCTION, AND
REPLACEMENT)**

As demonstrated in **Section 6***, in vitro* NRU basal cytotoxicity test
methods cannot be used as replacement assays[^1] for rodent acute oral
toxicity test methods for hazard classification. However, as described
in this section, such test methods can be evaluated for their ability to
reduce[^2] and refine[^3] animal use in the UDP or ATC acute oral
toxicity assays. A similar analysis cannot be conducted for the FDP as
this test method uses evident toxicity rather than death as the endpoint
of interest. The current UDP and ATC test guidelines recommend using
information on structurally-related substances and the results of any
other toxicity tests (EPA 2002b) to select a starting dose (OECD 2001a;
EPA 2002a; OECD 2001d). However, for the purposes of the reduction and
refinement evaluation conducted in this section, it was assumed that no
information other than 3T3 and NHK NRU test data would be available upon
which to base the selection of a starting dose. To determine the extent
of animal reduction or refinement that would occur in the UDP and the
ATC when using a starting dose based on 3T3 or NHK NRU IC~50~ results
rather than the default starting dose, computer models were used to
simulate the *in vivo* testing of the reference substances used in the
NICEATM/ECVAM validation study.

**Section 10.1** lists the regressions that were used with IC~50~ data
from the 3T3 and NHK NRU test methods to determine starting doses for
the UDP and ATC test methods. **Sections 10.2.1** and **10.3.1**
summarize the animal testing procedures described in the current test
guidelines for the UDP and the ATC method, respectively. The procedures
for using computer software to simulate animal testing of the
NICEATM/ECVAM reference substances are then detailed in **Sections
10.2.2** and **10.3.2**. The computer simulations were used to determine
the number of animals used and the number of animals that died for each
simulated test. The computer simulation modelling was performed using
five different dose-mortality (i.e., dose-response) slopes since no
information on dose-mortality slope was available for the substances
tested. To simplify the presentation of results, animal use figures
provided in **Sections 10.2.3**, **10.2.4**, **10.3.3**, and **10.3.4**
include two of the dose-response slopes. The results for the other three
dose-response slopes are provided in **Appendices N** and **Q**. The
number of animals used is summarized to show the mean number of animals
tested when the default starting dose is used and the mean number of
animals used when the NRU-determined starting dose (i.e., from the 3T3
or NHK NRU IC~50~ values used in the indicated regressions) is used. The
difference in animal use between the default starting doses and the
NRU-based starting doses is referred to as the animal savings.
Differences were tested for statistical significance (i.e., p \< 0.05)
using a one-sided Wilcoxon signed ranked test based on the number of
substances evaluated. **Sections 10.2** and **10.3** summarize mean
animal use by the total number of substances tested and then by the
number of substances in each GHS acute oral toxicity category.
**Sections 10.2.4** and **10.3.4** provide the mean number of animal
deaths compared to the mean number of animals used for each starting
dose (i.e., default and NRU-based) to determine whether the NRU-based
starting doses result in the refinement of animal use (i.e., reduction
in the number of animals that die).

**10.1 Use of 3T3 and NHK NRU Test Methods to Predict Starting Doses for
Acute Systemic Toxicity Assays**

The IC~50~ data from the 3T3 and NHK NRU test methods were used to
predict starting doses for acute oral systemic toxicity tests using the
following linear regressions of IC~50~-LD~50~ values presented in
**Section** **6.2** (see **Table 6-2**):

-   the RC millimole regression \[Note: The RC millimole regression was
    developed from the Registry of Cytotoxicity, a database of rat and
    mouse oral LD~50~ values from RTECS^®^ and IC~50~ values from *in
    vitro* cytotoxicity assays using multiple cell lines and
    cytotoxicity endpoints for 347 chemicals with known molecular
    weights (Halle 1998).\]

-   the RC rat-only weight regression

-   the RC rat-only weight regression excluding substances with specific
    mechanisms of toxicity other than basal cytotoxicity

    Data for the same reference substances were evaluated for each
    regression and simulated acute systemic toxicity test method.
    Forty-six substances were evaluated for the 3T3 NRU test method and
    47 substances were evaluated for the NHK NRU test method. Of the 72
    substances tested, epinephrine bitartrate, colchicine, and
    propylparaben were excluded because they were removed from the
    calculation of the RC rat-only weight regression due to the lack of
    rat oral reference LD~50~ data. The 21 substances with specific
    mechanisms of toxicity in **Table 6-3** were excluded from all
    analyses to be consistent with those removed from the RC rat-only
    weight regression excluding substances with specific mechanisms of
    toxicity. These substances have known mechanisms of toxicity that
    are not expected to be active in the 3T3 and NHK cell cultures.
    Carbon tetrachloride and methanol were excluded from the 3T3 NRU
    evaluations because no laboratory attained sufficient toxicity in
    any test for the calculation of an IC~50~. Carbon tetrachloride was
    also excluded from the NHK NRU evaluations because no laboratory
    attained sufficient toxicity in any test for the calculation of an
    IC~50.~

**10.2 Reduction and Refinement of Animal Use for the UDP**

10.2.1 [Procedure for *In Vivo* Testing Using the UDP]{.underline}

This section describes the general dosing procedure for the UDP assay
(OECD 2001a; EPA 2002a). Although doses, time between doses, and dose
progression may be adjusted as necessary, the procedures described
reflect the default guidance. Guidance on the type of animals to use,
animal housing, clinical observations, etc., are outside the scope of
the current discussion and are provided in the test guidelines (see
**Appendix M**).

*Main Test*

The UDP is based on a staircase design in which single animals are dosed
in sequence at 48-hour intervals. The outcome of the first animal
determines the dose of the next animal. If the first animal dies or is
in a moribund state, the dose administered to the next animal is lowered
by dividing the original dose by one-half log (i.e., 3.2, which is the
default dose progression). If the first animal survives, the dose
administered to the next animal is increased by one-half log times the
original dose. A dose progression of one-half log unit corresponds to a
dose-mortality (also referred to as "dose-response) slope of 2. The
default dose progression can be adjusted if the analyst has prior
information upon which to estimate a slope.

The current test guidelines recommend using information on
structurally-related substances and the results of any other toxicity
tests (EPA 2002b) for the test substance, including *in vitro*
cytotoxicity results, to approximate the LD~50~ and the slope of the
dose-response curve (OECD 2001a; EPA 2002a). The starting dose is one
dose progression step below the analyst's best estimate of the LD~50~,
since the UDP test method has a bias toward the starting dose (i.e.,
LD~50~ estimate tends to move toward the starting dose). The default
starting dose of 175 mg/kg is used if there is no information on which
to base a starting dose. The entire default dosing scheme generally uses
a dose progression of 3.2, is 1.75, 5.5, 17.5, 55, 175, 550, 1750, and
5000 mg/kg (EPA 2002a) or 1.75, 5.5, 17.5, 55, 175, 550, and 2000 mg/kg
(OECD 2001a). Dosing single animals in sequence proceeds until the first
of three conditions, referred to as stopping rules, is met:

-   three consecutive animals survive at the upper limit (2000 or 5000
    mg/kg)

-   five reversals occur in any six consecutive animals tested

-   four or more animals have followed the first reversal and the
    specified likelihood-ratios exceed the critical value. For a wide
    variety of LD~50~ values and dose-mortality slopes, this is
    satisfied with four to six animals after the first reversal. Three
    likelihood values are calculated: a likelihood at an LD~50~ point
    estimate (called the rough estimate or dose-averaging estimate); a
    likelihood at a value below the point estimate (the point estimate
    divided by 2.5); and a likelihood at a value above the point
    estimate (the point estimate multiplied by 2.5). The ratios of the
    likelihoods are examined to determine whether they exceed a critical
    value.

If none of these conditions is met, dosing stops after 15 animals have
been used.

*Limit Test*

The UDP test method guidelines include a limit test using three to five
animals dosed sequentially at 2000 mg/kg or 5000 mg/kg (OECD 2001a; EPA
2002a). The EPA guideline for testing at a limit dose of 5000 mg/kg
calls for proceeding to the main test if the first animal dosed at 5000
mg/kg dies (EPA 2002a). If the first animal lives, however, two more
animals are dosed at 5000 mg/kg. If both animals live, then testing is
terminated with

LD~50~ \> 5000 mg/kg. If one or both animals die, then two more animals
are dosed in sequence. As soon as three animals survive, the test is
terminated with the conclusion that LD~50~ \> 5000 mg/kg. However, as
soon as three animals die, the main test is conducted.

The OECD guideline for testing at a limit dose of 2000 mg/kg calls for
proceeding to the main test if the first animal dosed at 2000 mg/kg dies
(OECD 2001a). If the animal lives, however, four more animals are
sequentially dosed. Whenever three animals die, the main test is
performed. If three or more animals survive, testing is terminated with
the conclusion that the LD~50~ \> 2000 mg/kg.

10.2.2 [Procedure for Computer Simulation Modeling of the
UDP]{.underline}

Two thousand simulations of UDP testing were run for each substance, *in
vitro* NRU test method, and dose-mortality slope. Because the analysis
assumed there was no information upon which to estimate a dose-response
slope, the simulation modeling used the default dose progression factor
of 3.2. The simulations used 5000 mg/kg as the upper limit dose since
this upper limit is commonly used in the United States. If the NRU-based
starting dose was

4000 mg/mg or greater, then testing proceeded per the limit test rather
than the main test. If, during the dose progression, the next highest
dose to be administered was within 4000 mg/kg or greater, then the limit
dose of 5000 mg/kg was administered. In the case where a dose one step
below the NRU-estimated LD~50~ was used as the starting dose, the other
doses administered corresponded to the default doses specified in the
test method guidelines (OECD 2001a; EPA 2002a). The simulation modeling
procedures also used a lower limit of 1 mg/kg. Thus, if the dose
progression fell below 1 mg/kg, then a dose of 1 mg/kg was administered.
To estimate animal use by the default method, a starting dose of 175
mg/kg was used; the other doses administered after the default starting
dose corresponded to the default doses specified in the test method
guidelines (OECD 2001a; EPA 2002a).

The simulation process was performed using SAS version 8 (SAS 1999) and
implements the distributional assumptions underlying the dose-mortality
relationship. The lowest dose at which an animal dies in response to the
administration of a toxic substance varies from animal to animal. For an
entire population of animals, mortality is assumed to have a log-normal
distribution with the mean equal to the log of the true LD~50~. Sigma
(), the variability of the simulated population, is the inverse of the
slope of the dose-mortality curve. Due to a lack of information for the
real dose-mortality curves, the simulations assumed several different
values of the slope, but no corresponding changes were made in the dose
progression. Dose-mortality slopes of 0.5, 0.8, 2, 4, and 8.3 were
chosen since these were used in the simulation modeling studies that
evaluated the current version of the UDP guidelines (ICCVAM 2001c).

To model the variability of the NRU IC~50~ values within and between
laboratories, the values were log-transformed to normalize the
distribution of values for each substance. The mean and variance of
these log-transformed values were used to generate a log-normal
distribution from which to randomly select an IC~50~ value. The selected
NRU IC~50~ value was used with the regressions in two different ways to
determine starting doses. One method used the LD~50~ estimated from the
IC~50~ and the regression as the starting dose while the other method
used the closest default dose lower than the estimated LD~50~ as the
starting dose. The results from the latter method are presented in
**Section 10.2** since it is the method recommended by the EPA and OECD
test guidelines (EPA 2002a; OECD 2001a). Moreover, the UDP is only
usable for regulatory purposes if the starting dose is set below the
expected LD~50~. The results obtained when the LD~50~ estimated by the
IC~50~ and the regression was used as the starting dose are presented in
**Appendix Q.**

The simulation procedure used the following steps for each substance:

1.  The LD~50~ value (in mg/kg) from **Table 4-2** was entered as the
    true LD~50~ value and the choices of assumed slope were entered as
    the true slope for the dose-mortality curve.

2.  An IC~50~ value was selected from a distribution identified by the
    mean and variance of the IC~50~ values computed from the data to
    reflect that different laboratories produce different IC~50~ values
    in different situations (see **Table 5-3** for mean IC~50~ values
    and standard deviations).

3.  The IC~50~ value from Step 2 was used in the regression model being
    evaluated to compute a predicted LD~50~ value to use as the starting
    dose.

4.  The dosing simulation was run three times: once with the default
    starting dose of 175 mg/kg, once at the next default dose below the
    LD~50~ estimated by the regression being evaluated, and once at a
    dose equal to that of the LD~50~ estimated by the regression being
    evaluated.

5.  For each simulated trial (each substance and starting dose), the
    dosing simulation works similarly. In each trial, the animals are
    dosed sequentially; therefore for each animal(i) there is a
    corresponding dose(i) that is administered to the animal. For the
    first animal in each trial, it is the starting dose for that trial.
    For each subsequent animal, the dose is dependent on the previous
    dose and the previous animal's response as described in **Section
    10.2.1**. For animal(i), the probability of response is computed
    with the cumulative log-normal distribution at the dose
    administered. That is,
    $P(\text{response}) = P(x < \text{log}\lbrack\text{dose}(i)\rbrack)$
    where $x\text{\textasciitilde}N(\mu,\sigma)$ and $\mu$ is the log of
    the true LD~50~ value and $\sigma$ is the inverse of the assumed
    slope of the dose-mortality curve. This probability is used to
    sample one observation from a binomial distribution with this
    probability of success.

6.  Dosing simulation is stopped once one of the stopping rules is
    satisfied.

Steps 2-6 were repeated 2000 times in order to compute an average animal
use for each method evaluated.

10.2.3 [Animal Savings for the UDP When Using 3T3 and NHK NRU-Based
Starting Doses]{.underline}

10.2.3.1 *The Effect of Dose-Response Slope on Animal Use*

As described in **Section 10.2.2**, the simulation modeling of animal
use for the UDP assumed five different dose-mortality slopes to assess
animal use under various conditions of population variability. **Table
10-1** shows that the number of animals used for the UDP decreases with
increasing slope for both the default starting dose and the
NRU-determined starting dose based on the RC millimole regression. The
NRU-determined starting dose was the next default dose lower than the
regression-estimated LD~50~. For example, since the LD~50~ predicted for
cadmium chloride by the 3T3 NRU IC~50~ with the RC millimole regression
was 16 mg/kg, the starting dose was 1.75 mg/kg (i.e., the next default
dose below the predicted LD~50~). This approach is consistent with the
UDP test method guidelines (OECD 2001a; EPA 2002a) as a means for
reducing the number of animals that might experience pain and suffering
from treatment (i.e., as a test method refinement). The approach also
overcomes the nonconservative bias of the UDP, which tends to yield an
LD~50~ close to the starting dose.

**Table 10-1 Change in Animal Use^1^ with Dose-Response Slope for the
UDP^2^**

  ----------------- ----------------- ------------------ --------------------
  **Dose-Response   **With Default    **With NRU-Based   **Animals Saved^5^**
  Slope**           Starting          Starting           
                    Dose^1,3^**       Dose^1,4^**        

  **3T3 NRU Test                                         
  Method**                                               

  0.5               10.30 ± 0.13      9.43 ± 0.15        0.88\* (8.5%)

  0.8               10.34 ± 0.17      9.36 ± 0.18        0.98\* (9.4%)

  2.0               9.77 ± 0.21       8.79 ± 0.22        0.97\* (10.0%)

  4.0               8.96 ± 0.25       8.03 ± 0.27        0.93\* (10.4%)

  8.3               8.11 ± 0.26       7.20 ± 0.30        0.91\* (11.2%)

  **NHK NRU Test                                         
  Method**                                               

  0.5               10.31 ± 0.12      9.57 ± 0.17        0.74\* (7.1%)

  0.8               10.38 ± 0.16      9.47 ± 0.19        0.91\* (8.8%)

  2.0               9.75 ± 0.20       8.93 ± 0.23        0.82\* (8.4%)

  4.0               8.94 ± 0.24       8.14 ± 0.28        0.80\* (9.0%)

  8.3               8.12 ± 0.25       7.33 ± 0.30        0.79\* (9.7%)
  ----------------- ----------------- ------------------ --------------------

^1^Numbers are mean numbers of animals with standard errors for 2000
simulations for 46 substances for the 3T3 NRU test method and 47
substances for the NHK NRU test method. Although the simulations used
whole animals, averaging the results produced fractional numbers of
animals. The slight differences in the number of animals used for the
default starting dose at the same dose-response slope reflect different
simulation runs. Limit dose = 5000 mg/kg.

^2^OECD (2001a); EPA (2002a).

^3^Default starting dose = 175 mg/kg.

^4^Starting dose = next lower default dose to NRU-predicted LD~50~,
which was calculated using the geometric mean of the laboratory
geometric mean NRU IC~50~ values in the RC millimole regression: log
LD~50~ (mmol/kg) = 0.435 log IC~50~ (mM) + 0.625.

^5^Difference between mean animal use with default starting dose and
mean animal use with NRU-based starting dose. All differences denoted by
\* were statistically significant (i.e., p \< 0.05) by a one-sided
Wilcoxon signed rank test. Percentage difference is shown in
parentheses.

**Table 10-1** shows that, for each dose-response slope, the mean number
of animals saved was statistically significant (i.e., p \< 0.05) when
compared to mean animal use for the default starting dose. When
expressed as a percentage of the default animal use, animal savings also
generally increased with increasing slope.

To simplify the presentation of animal savings and comparison of the
various regressions and starting doses, the results of subsequent
analyses presented in **Section 10.2.3** will be limited to slopes of 2
and 8.3. The slope of 2 is the default slope used for the calculation of
LD~50~ by the UDP method (OECD 2001a; EPA 2002a). Animal savings results
for the other dose-mortality slopes are presented in **Appendices
N1-N3.** Although using the next lower default dose to the
NRU-determined LD~50~ value overcomes the bias of the UDP toward the
starting dose (OECD 2001a, EPA 2002a) and is the appropriate approach
for regulatory use, animal savings results using the estimated LD~50~ as
the starting dose were also calculated (see **Appendix Q**).

10.2.3.2 *Mean Animal Use from UDP Simulations for Testing the
NICEATM/ECVAM Reference Substances -- Comparison of Regressions and 3T3
and NHK NRU Test Methods*

**Table 10-2** shows the mean animal use for simulated UDP of the
testing the set of NICEATM/ECVAM reference substances described in
**Section 10.1**. Mean animal use is shown for default starting dose and
for starting doses that were one default dose lower than the LD~50~
predicted from the *in vitro* NRU test methods and the regressions
(shown in **Table 6-2**) evaluated in **Section 6.3** for prediction of
GHS acute oral toxicity category. The difference in animal use between
the two starting doses is the mean animal savings produced by using the
starting dose based on the *in vitro* NRU test methods. All differences
(i.e., mean animal savings) were statistically significant (i.e., p \<
0.05) by a one-sided Wilcoxon signed rank test. Mean animal savings
ranged from 0.79 to 1.16 (8.4 to 12.7%) animals depending upon the NRU
test method, regression, and dose-response slope. The lowest mean animal
savings were obtained for the RC millimole regression (0.82 \[8.4%\] to
0.97 \[10.0%\] animals for the various test methods and dose-response
slopes) and the highest mean animal savings were obtained with the RC
rat-only regression excluding substances with specific mechanisms of
toxicity other than basal cytotoxicity (1.00 \[12.2%\] to 1.16 \[11.8%\]
animals).

**Table 10-2 Mean Animal Use^1^ for the UDP^2^** Using Starting Doses
Based on the 3T3 and NHK NRU Test Methods with Various Regressions

  ---------------------- ----------------- ----------- ------------ ----------------- ----------- ------------ -----------------
  **Assay/Regression**   **With Default    **With      **Animals    **With Default    **With      **Animals    **Accuracy^6^**
                         Starting          NRU-Based   Saved^5^**   Starting          NRU-Based   Saved^5^**   
                         Dose^3^**         Starting                 Dose^3^**         Starting                 
                                           Dose^4^**                                  Dose^5^**                

  **3T3 NRU Test         **Dose-Response                            **Dose-Response                            
  Method**               Slope = 2**                                Slope = 8.3**                              

  RC millimole^6^        9.77 ± 0.21       8.79 ± 0.22 0.97\*       8.11 ± 0.26       7.20 ± 0.30 0.91\*       26%
                                                       (10.0%)                                    (11.2%)      

  RC rat-only weight^7^  9.79 ± 0.21       8.66 ± 0.22 1.13\*       8.14 ± 0.25       7.11 ± 0.29 1.03\*       35%
                                                       (11.6%)                                    (12.7%)      

  RC rat-only weight     9.80 ± 0.20       8.64 ± 0.23 1.16\*       8.16 ± 0.25       7.08 ± 0.31 1.08\*       46%
  excluding substances                                 (11.8%)                                    (13.3%)      
  with specific                                                                                                
  mechanisms of                                                                                                
  toxicity^8^                                                                                                  

  **NHK NRU Test         **Dose-Response                            **Dose-Response                            
  Method**               Slope = 2**                                Slope = 8.3**                              

  RC millimole ^6^       9.75 ± 0.20       8.93 ± 0.23 0.82\*       8.12 ± 0.25       7.33 ± 0.30 0.79\*       28%
                                                       (8.4%)                                     (9.7%)       

  RC rat-only weight^7^  9.77± 0.20        8.83 ± 0.23 0.94\*       8.13 ± 0.25       7.25 ± 0.30 0.88\*       30%
                                                       (9.6%)                                     (10.9%)      

  RC rat-only weight     9.78 ± 0.20       8.73 ± 0.24 1.05\*       8.15 ± 0.25       7.15 ± 0.32 1.00\*       38%
  excluding substances                                 (10.7%)                                    (12.2%)      
  with specific                                                                                                
  mechanisms of                                                                                                
  toxicity^8^                                                                                                  
  ---------------------- ----------------- ----------- ------------ ----------------- ----------- ------------ -----------------

^1^Numbers are mean numbers of animals and standard errors for 2000
simulations for each of 46 substances for the 3T3 NRU test method and 47
substances for the NHK NRU test method. Although the simulations used
whole animals, averaging the results produced fractional numbers of
animals. The slight differences in the number of animals used for the
default starting dose at the same dose-response slope reflect different
simulation runs.

^2^OECD (2001a); EPA (2002a).

^3^Default starting dose = 175 mg/kg.

^4^Starting dose = one default dose lower than the NRU-predicted LD~50~
calculated using the geometric mean of the laboratory geometric mean NRU
IC~50~ values in the specified regression.

^5^Difference between mean animal use with default starting dose and
mean animal use with NRU-based LD~50~. Differences denoted by \* were
statistically significant (i.e., p \< 0.05) by a one-sided Wilcoxon
signed rank test. Percentage difference is shown in parentheses.

^6^Proportion of substances for which the GHS acute oral toxicity
category (UN 2005) predicted by the *in vitro* NRU test methods matched
the *in vivo* category (from **Tables 6-4** to **6-6**).

^7^log LD~50~ (mmol/kg) = 0.435 log IC~50~ (mM) + 0.625.

^8^log LD~50~ (mg/kg) = 0.372 log IC~50~ (g/mL) + 2.024.

^9^log LD~50~ (mg/kg) = 0.357 log IC~50~ (g/mL) + 2.194.

**Table 10-2** also shows that animal savings increased with the
accuracy of the GHS acute oral toxicity category predictions (see
**Section 6.3**).

10.2.3.3 *Animal Savings for the UDP by Toxicity Category Using 3T3 and
NHK NRU-Based Starting Doses*

**Tables 10-3** through **10-5** show mean animal use and mean animal
savings for the UDP for the default starting dose and the NRU-determined
starting dose with the test substances grouped by GHS acute oral
toxicity category (UN 2005). The data come from the same analyses as the
data provided in **Table 10-2**. NRU-determined starting doses were
based on the:

-   RC millimole regression (**Table 10-3**).

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-   RC rat-only weight regression (**Table 10-4)**

-   RC rat-only weight regression excluding substances with specific
    mechanisms of toxicity other than basal cytotoxicity (**Table
    10-5)**

Consistencies noted in the mean animal savings data provided in the
tables included:

-   For each *in vitro* NRU cytotoxicity test method and regression,
    animal savings were statistically significant for substances in the
    2000 \< LD~50~ ≤ 5000 mg/kg and LD~50~ \> 5000 mg/kg toxicity
    categories.

-   For substances with LD~50~ ≤ 5 mg/kg, the NHK NRU test method with
    each regression used slightly more animals than the default method
    (i.e., mean animal savings were negative). The 3T3 NRU test method
    produced nonsignificant animal savings of 0.31 (2.9%) to 0.95 (8.1%)
    animal for these substances.

For substances with 50 \< LD~50~ ≤ 300 mg/kg, all test methods and
regressions produced little to no animal savings.

*[Animal Savings for the UDP by Toxicity Category Using 3T3 and NHK
NRU-Based Starting Doses with the RC Millimole Regression]{.underline}*

**Table 10-3** shows the animal savings by GHS toxicity category for the
*in vitro* NRU cytotoxicity test methods used with the RC millimole
regression. Mean animal savings were statistically significant (i.e., p
\< 0.05) by a one-tailed Wilcoxon signed rank test for the following GHS
toxicity categories, test methods, and dose-response slopes:

-   5 \< LD~50~ ≤ 50 mg/kg for the NHK NRU at dose-response slope = 2
    (0.86 \[9.2%\] animals)

-   2000 \< LD~50~ ≤ 5000 mg/kg for both NRU test methods and both
    dose-response slopes (1.25 \[13.7%\] to 1.52 \[14.1%\] animals)

-   LD~50~ \> 5000 mg/kg for both NRU test methods and both
    dose-response slopes (1.35 \[14.2%\] to 1.70 \[25.4%\] animals)

For the 3T3 NRU and NHK NRU test methods, mean animal savings were
similar for most toxicity categories at both dose-response slopes, with
the mean savings for the 3T3 NRU slightly higher than that for the NHK
NRU. For the dose-response slope of 2, mean animal savings for the 3T3
NRU test method (for the various toxicity categories) ranged from -0.09
(-1.0%) to 1.54 (16.1%) animals while mean animal savings for the NHK
NRU test method ranged from -0.25 (-2.2%) to 1.45 (13.5%) animals. For
the dose-response slope of 8.3, animal savings for the 3T3 NRU test
method ranged from 0.004 (0.05%) to 1.70 (25.4%) animals while mean
animal savings for the NHK NRU test method ranged from

-0.11 (-1.5%) to 1.45 (21.8%) animals.

For both *in vitro* NRU cytotoxicity test methods, no mean animal
savings (≤ 0.09 animal) were observed for substances with 50 \< LD~50~ ≤
300 mg/kg. This category includes the default starting dose of 175
mg/kg. Animal savings were not expected for this category since savings
were determined by comparing animal use with the NRU-based starting dose
with animal use at the default starting dose. For the 3T3 NRU, no animal
savings (-0.9 to 0.004 animals) were also observed for substances with 5
\< LD~50~ ≤ 50 mg/kg. For the NHK NRU test method, animal use actually
increased slightly compared to the default starting dose (‑0.25 to -0.09
animals) for substances with LD~50~ ≤ 5 mg/kg. Animal savings for
relatively high toxicity substances were noted for those in the LD~50~ ≤
5 mg/kg category for the 3T3 NRU (0.78 \[7.3%\] to 0.95 \[8.1%\]
animals) and in the 5 \< LD~50~ ≤ 50 mg/kg category for the NHK NRU
(0.86 \[9.2%\] to 0.87 \[10.5%\] animals). Only the 0.86 (9.2%) animal
savings for the dose-response slope of 2 (NHK NRU) were statistically
significant.

**Table 10-3 Animal Use^1^ for the UDP^2^ by GHS Toxicity Category^3^
Using Starting Doses Based on the 3T3 and NHK NRU Test Methods with the
RC Millimole Regression^4^**

+------------+-----+------+------+-------+------+------+------+----+
|            |     | **D  |      |       | **D  |      |      | *  |
|            |     | ose- |      |       | ose- |      |      | *A |
|            |     | Resp |      |       | Resp |      |      | cc |
|            |     | onse |      |       | onse |      |      | ur |
|            |     | S    |      |       | S    |      |      | ac |
|            |     | lope |      |       | lope |      |      | y^ |
|            |     | =    |      |       | =    |      |      | 8^ |
|            |     | 2**  |      |       | 8    |      |      | ** |
|            |     |      |      |       | .3** |      |      |    |
+------------+-----+------+------+-------+------+------+------+----+
| **Toxicity | **  | **   | **   | *     | **   | **   | *    |    |
| Cat        | Num | With | With | *Anim | With | With | *Ani |    |
| egory^3^** | ber | Def  | N    | als** | Def  | N    | mals |    |
|            | of  | ault | RU-B |       | ault | RU-B | Sa   |    |
|            | Ref | Star | ased | **    | St   | ased | ved^ |    |
|            | ere | ting | Star | Saved | arti | Star | 7^** |    |
|            | nce | D    | ting | ^7^** | ng** | ting |      |    |
|            | Sub | ose^ | D    |       |      | D    |      |    |
|            | sta | 5^** | ose^ |       | **D  | ose^ |      |    |
|            | nce |      | 6^** |       | ose^ | 6^** |      |    |
|            | s** |      |      |       | 5^** |      |      |    |
+------------+-----+------+------+-------+------+------+------+----+
|            |     | *    |      |       |      |      |      | *  |
|            |     | *3T3 |      |       |      |      |      | *  |
|            |     | NRU  |      |       |      |      |      | ** |
|            |     | Test |      |       |      |      |      |    |
|            |     | Meth |      |       |      |      |      |    |
|            |     | od** |      |       |      |      |      |    |
+------------+-----+------+------+-------+------+------+------+----+
| LD~50~ ≤ 5 | 7   | 1    | 10.8 | 0.95  | 1    | 9.87 | 0.78 | 0% |
| mg/kg      |     | 1.76 | ±    | (     | 0.65 | ±    | (7   |    |
|            |     | ±    | 0.64 | 8.1%) | ±    | 0.74 | .3%) |    |
|            |     | 0.16 |      |       | 0.48 |      |      |    |
+------------+-----+------+------+-------+------+------+------+----+
| 5 \<       | 6   | 9.06 | 9.15 | -0.09 | 8.04 | 8.04 | 0    | 1  |
| LD~50~ ≤   |     | ±    | ±    | (-    | ±    | ±    | .004 | 7% |
| 50 mg/kg   |     | 0.18 | 0.72 | 1.0%) | 0.24 | 0.78 | (0.  |    |
|            |     |      |      |       |      |      | 05%) |    |
+------------+-----+------+------+-------+------+------+------+----+
| 50 \<      | 6   | 7.70 | 7.61 | 0.09  | 6.63 | 6.59 | 0.03 | 6  |
| LD~50~ ≤   |     | ±    | ±    | (     | ±    | ±    | (0   | 7% |
| 300 mg/kg  |     | 0.23 | 0.18 | 1.2%) | 0.35 | 0.26 | .5%) |    |
+------------+-----+------+------+-------+------+------+------+----+
| 300 \<     | 6   | 8.76 | 7.91 | 0.84  | 7.30 | 6.69 | 0.61 | 10 |
| LD~50~ ≤   |     | ±    | ±    | (     | ±    | ±    | (8   | 0% |
| 2000 mg/kg |     | 0.34 | 0.06 | 9.6%) | 0.35 | 0.20 | .3%) |    |
+------------+-----+------+------+-------+------+------+------+----+
| 2000 \<    | 11  | 1    | 9.23 | 1     | 9.16 | 7.81 | 1.   | 0% |
| LD~50~ ≤   |     | 0.75 | ±    | .52\* | ±    | ±    | 36\* |    |
| 5000 mg/kg |     | ±    | 0.20 | (1    | 0.26 | 0.34 | (14  |    |
|            |     | 0.08 |      | 4.1%) |      |      | .8%) |    |
+------------+-----+------+------+-------+------+------+------+----+
| LD~50~ \>  | 10  | 9.59 | 8.05 | 1     | 6.69 | 4.99 | 1.   | 1  |
| 5000 mg/kg |     | ±    | ±    | .54\* | ±    | ±    | 70\* | 0% |
|            |     | 0.27 | 0.39 | (1    | 0.37 | 0.45 | (25  |    |
|            |     |      |      | 6.1%) |      |      | .4%) |    |
+------------+-----+------+------+-------+------+------+------+----+
|            |     | *    |      |       |      |      |      |    |
|            |     | *NHK |      |       |      |      |      |    |
|            |     | NRU  |      |       |      |      |      |    |
|            |     | Test |      |       |      |      |      |    |
|            |     | Meth |      |       |      |      |      |    |
|            |     | od** |      |       |      |      |      |    |
+------------+-----+------+------+-------+------+------+------+----+
| LD~50~ ≤ 5 | 7   | 1    | 1    | -0.25 | 1    | 1    | -    | 0  |
| mg/kg      |     | 1.54 | 1.79 | (-    | 0.63 | 0.72 | 0.09 |    |
|            |     | ±    | ±    | 2.2%) | ±    | ±    | (-0  |    |
|            |     | 0.25 | 0.50 |       | 0.49 | 0.54 | .8%) |    |
+------------+-----+------+------+-------+------+------+------+----+
| 5 \<       | 6   | 9.34 | 8.48 | 0     | 8.22 | 7.35 | 0.87 | 5  |
| LD~50~ ≤   |     | ±    | ± 0. | .86\* | ±    | ±    | (10  | 0% |
| 50 mg/kg   |     | 0.24 | 24   | (     | 0.31 | 0.36 | .5%) |    |
|            |     |      |      | 9.2%) |      |      |      |    |
+------------+-----+------+------+-------+------+------+------+----+
| 50 \<      | 6   | 7.82 | 7.88 | -0.06 | 6.92 | 7.02 | -    | 5  |
| LD~50~ ≤   |     | ±    | ±    | (-    | ±    | ±    | 0.11 | 0% |
| 300 mg/kg  |     | 0.22 | 0.26 | 0.7%) | 0.38 | 0.43 | (-1  |    |
|            |     |      |      |       |      |      | .5%) |    |
+------------+-----+------+------+-------+------+------+------+----+
| 300 \<     | 6   | 8.74 | 7.93 | 0.81  | 7.31 | 6.71 | 0.60 | 10 |
| LD~50~ ≤   |     | ±    | ±    | (     | ±    | ±    | (8   | 0% |
| 2000 mg/kg |     | 0.34 | 0.06 | 9.3%) | 0.34 | 0.23 | .2%) |    |
+------------+-----+------+------+-------+------+------+------+----+
| 2000 \<    | 11  | 1    | 9.29 | 1     | 9.13 | 7.88 | 1.   | 9% |
| LD~50~ ≤   |     | 0.73 | ±    | .45\* | ±    | ±    | 25\* |    |
| 5000 mg/kg |     | ±    | 0.20 | (1    | 0.25 | 0.33 | (13  |    |
|            |     | 0.08 |      | 3.5%) |      |      | .7%) |    |
+------------+-----+------+------+-------+------+------+------+----+
| LD~50~ \>  | 11  | 9.52 | 8.17 | 1     | 6.64 | 5.19 | 1.   | 0% |
| 5000 mg/kg |     | ±    | ±    | .35\* | ±    | ±    | 45\* |    |
|            |     | 0.28 | 0.41 | (1    | 0.35 | 0.44 | (21  |    |
|            |     |      |      | 4.2%) |      |      | .8%) |    |
+------------+-----+------+------+-------+------+------+------+----+

^1^Numbers are mean numbers of animals used and standard errors for 2000
simulations for each substance with a limit dose of 5000 mg/kg. Although
the simulations used whole animals, averaging the results produced
fractional numbers of animals. Results are provided for 46 substances in
the 3T3 NRU test method and 47 substances in the NHK NRU test method
categorized using the initial LD~50~ values from **Table 3-2**. The
slight differences in the number of animals used for the default
starting dose at the same dose-response slope reflect different
simulation runs.

^2^OECD (2001a); EPA (2002a).

^3^GHS-Globally Harmonized System of Classification and Labelling of
Chemicals with LD~50~ in mg/kg (UN 2005).

^4^RC millimole regression is log LD~50~ (mmol/kg) = 0.435 log IC~50~
(mM) + 0.625.

^5^Default starting dose = 175 mg/kg.

^6^Starting dose was one default dose lower than the predicted LD~50~
calculated using the geometric mean of the laboratory geometric mean NRU
IC~50~ values in the RC millimole regression.

^7^Difference between mean animal use with default starting dose and
mean animal use with NRU predicted LD~50~. Differences marked by \* are
statistically significant (p \< 0.05) by a one-sided Wilcoxon signed
rank test. Percentage difference shown in parentheses

^8^Proportion of substances for which the GHS acute oral toxicity
category (UN 2005) predicted by the *in vitro* NRU test methods matched
the *in vivo* category (from **Table 6-4**).

**Table 10-3** also shows that mean animal savings did not correlate
with the accuracy of the GHS acute oral toxicity category predictions.
Substances in categories with the lowest accuracy produced the highest
animal savings. Accuracy was the lowest (0 - 10%) for GHS acute oral
toxicity category prediction for substances with LD~50~ \> 5000 mg/kg,
but animal savings (1.35 - 1.70) were the highest. Animal savings
(0.60 - 0.84 animals) for substances with 300 ≤ LD~50~ ≤ 2000 mg/kg,
which had 100% accuracy for GHS acute oral toxicity category prediction,
were similar to animal savings (0.78 - 0.95 animals) for substances in
the LD~50~ \< 5 mg/kg category (for the 3T3 NRU), which had 0% accuracy.
Perhaps the difference between the predicted starting dose and the true
LD~50~ vs. the difference between the default starting dose and the true
LD~50~ has more influence on animal savings that the accuracy of the
LD~50~ prediction.

*[Animal Savings for the UDP by Toxicity Category Using 3T3 and NHK
NRU-Based Starting Doses with the RC Rat-Only Weight
Regression]{.underline}*

**Table 10-4** shows the mean animal savings by GHS toxicity category
for the *in vitro* NRU cytotoxicity test methods used with the RC
rat-only weight regression. A comparison of mean animal savings,
category for category, with the RC millimole regression, indicates that,
in most cases, animal savings were slightly higher for the RC rat-only
weight regression. For the RC rat-only weight regression, the mean
differences between animal use for the default starting dose and mean
animal use with the NRU-determined starting dose were statistically
significant (i.e., p \< 0.05) by a one-sided Wilcoxon signed rank test
for the following GHS toxicity categories, test methods, and
dose-response slopes:

-   300 \< LD~50~ ≤ 2000 mg/kg for the NHK NRU at dose-response slope =
    2 (0.86 \[9.8%\] animals)

-   2000 \< LD~50~ ≤ 5000 mg/kg for both NRU test methods and both
    dose-response slopes (1.50 \[16.4%\] to 1.91 \[17.7%\] animals)

-   LD~50~ \> 5000 mg/kg for both NRU test methods and both
    dose-response slopes (1.45 \[15.2%\] to 1.73 \[25.9%\] animals)

**Table 10-4 Animal Use^1^ for the UDP^2^ by GHS Toxicity Category^3^
Using Starting Doses Based on the NRU Test Methods with the RC Rat-Only
Weight Regression^4^**

  --------------- -------------- ----------------- ----------- --------------- ----------------- ----------- ------------ -----------------
                                 **Dose-Response                               **Dose-Response                            **Accuracy^8^**
                                 Slope = 2**                                   Slope = 8.3**                              

  **Toxicity      **Number of    **With Default**  **With      **Animals       **With Default    **With      **Animals    
  Category^3^**   Reference                        NRU-Based   Saved^7^**      Starting          NRU-Based   Saved^7^**   
                  Substances**                     Starting                    Dose^5^**         Starting                 
                                                   Dose**                                        Dose**                   

                                 **Starting                                                                               
                                 Dose^5^**                                                                                

                                 **3T3 NRU Test                                                                           
                                 Method**                                                                                 

  LD~50~ ≤ 5      4              11.75 ± 0. 16     10.85 ±     0.89 (7.6%)     10.66 ± 0.48      9.93 ± 0.71 0.73 (6.8%)  0%
  mg/kg                                            0.61                                                                   

  \> 5 \< LD~50~  7              9.14 ± 0.17       8.80 ± 0.54 0.34 (3.7%)     8.12 ± 0.27       7.76 ± 0.59 0.36 (4.5%)  17%
  ≤ 50 mg/kg                                                                                                              

  \> 50 \< LD~50~ 5              7.75 ± 0.22       7.60 ± 0.10 0.15 (1.9%)     6.71 ± 0.32       6.66 ± 0.23 0.05 (0.8%)  67%
  ≤ 300 mg/kg                                                                                                             

  \> 300 \<       9              8.75 ± 0.33       7.89 ± 0.07 0.86\* (9.8%)   7.29 ± 0.35       6.68 ± 0.21 0.61 (8.4%)  100%
  LD~50~ ≤ 2000                                                                                                           
  mg/kg                                                                                                                   

  \> 2000 \<      9              10.81 ± 0.08      8.90 ± 0.28 1.91\* (17.7%)  9.18 ± 0.26       7.48 ± 0.42 1.70\*       0%
  LD~50~ ≤ 5000                                                                                              (18.5%)      
  mg/kg                                                                                                                   

  \> 5000 mg/kg   12             9.59 ± 0.27       7.96 ± 0.40 1.63\* (17.0%)  6.69 ± 0.37       4.96 ± 0.45 1.73\*       10%
                                                                                                             (25.9%)      

                                 **NHK NRU Test                                                                           
                                 Method**                                                                                 

  LD~50~ ≤ 5      4              11.58 ± 0.23      11.66 ±     -0.08 (-0.7%)   10.66 ± 0.48      10.59 ±     0.07 (0.6%)  0
  mg/kg                                            0.44                                          0.53                     

  \> 5 \< LD~50~  7              9.33 ± 0.26       8.39 ± 0.27 0.94 (10.1%)    8.20 ± 0.31       7.36 ± 0.38 0.84 (10.3%) 50%
  ≤ 50 mg/kg                                                                                                              

  \> 50 \< LD~50~ 5              7.84 ± 0.21       7.93 ± 0.25 -0.09 (-1.1%)   6.94 ± 0.37       7.09 ± 0.41 -0.15        50%
  ≤ 300 mg/kg                                                                                                (-2.2%)      

  \> 300 \<       9              8.74 ± 0.34       7.92 ± 0.06 0.82 (9.3%)     7.31 ± 0.34       6.71 ± 0.23 0.60 (8.2%)  100%
  LD~50~ ≤ 2000                                                                                                           
  mg/kg                                                                                                                   

  \> 2000 \<      9              10.77 ± 0.07      9.07 ± 0.24 1.70\*(15.8%)   9.14 ± 0.25       7.64 ± 0.37 1.50\*       9%
  LD~50~ ≤ 5000                                                                                              (16.4%)      
  mg/kg                                                                                                                   

  LD~50~ \> 5000  13             9.52 ± 0.28       8.07 ± 0.40 1.45\*(15.2%)   6.64 ± 0.35       5.09 ± 0.42 1.55\*       0%
  mg/kg                                                                                                      (23.3%)      
  --------------- -------------- ----------------- ----------- --------------- ----------------- ----------- ------------ -----------------

^1^Numbers are mean number of animals used and standard errors for 2000
simulations for each substance with a limit dose of 5000 mg/kg. Although
the simulations used whole animals, averaging the results produced
fractional numbers of animals. Results are provided for 46 substances in
the 3T3 NRU test method and 47 substances in the NHK NRU test method
categorized using the reference LD~50~ values from **Table 4-2**. The
slight differences in the number of animals used for the default
starting dose at the same dose-response slope reflect different
simulation runs.

^2^OECD (2001a); EPA (2002a).

^3^GHS-Globally Harmonized System of Classification and Labelling of
Chemicals with LD~50~ in mg/kg (UN 2005).

^4^From **Table 6-2**; log LD~50~ (mg/kg) = 0.372 log IC~50~ (g/mL) +
2.024

^5^Default starting dose = 175 mg/kg.

^6^Starting dose was one default dose lower than NRU-predicted LD~50~
calculated using the geometric mean of the laboratory geometric mean NRU
IC~50~ values in the RC rat-only regression.

^7^Difference between mean animal use with default starting dose and
mean animal use with NRU predicted LD~50~. Differences marked by \* were
statistically significant (i.e., p \< 0.05) by a one-sided Wilcoxon
signed rank test. Percent difference is shown in parentheses.

^8^Proportion of substances for which the GHS acute oral toxicity
category (UN 2005) predicted by the *in vitro* NRU test methods matched
the *in vivo* category (from **Table 6-5**).

For the dose-response slope of 2, mean animal savings (for the various
toxicity categories) for the 3T3 NRU test method ranged from 0.15 (1.9%)
to 1.91 (17.7%) animals while mean animal savings for the NHK NRU test
method ranged from -0.09 (-1.1%) to 1.70 (15.8%) animals. For the
dose-response slope of 8.3, animal savings for the 3T3 NRU test method
ranged from 0.05 (0.8%) to 1.73 (25.9%) animals while animal savings for
the NHK NRU test method ranged from -0.15 (-2.2%) to 1.55 (23.3%)
animals.

For both *in vitro* NRU cytotoxicity test methods, no mean animal
savings (≤ 0.15 animal) were observed for substances with 50 \< LD~50~ ≤
300 mg/kg. This category includes the default starting dose of 175
mg/kg. Animal savings were not expected for this category since savings
were determined by comparing animal use with the NRU-based starting dose
with animal use at the default starting dose. For the NHK NRU, no animal
savings (-0.08 to 0.07 animals) were also observed for substances with
LD~50~ ≤ 5 mg/kg. Animal savings for relatively high toxicity substances
were noted in the LD~50~ ≤ 5 mg/kg category for the 3T3 NRU (0.73
\[6.8%\] to 0.89 \[7.6%\] animals) and in the 5 \< LD~50~ ≤ 50 mg/kg
category for the NHK NRU (0.84 \[10.3%\] to 0.94 \[10.1%\] animals), but
these savings were not statistically significant.

**Table 10-4** also shows that mean animal savings did not correlate
with the accuracy of the GHS acute oral toxicity category predictions
(see **Section 6.3**). The toxicity categories with the highest animal
savings had low accuracy. Substances in the 2000 \< LD~50~ ≤ 5000 mg/kg
and LD~50~ \> 5000 mg/kg categories had very low accuracy (0 - 10%) for
GHS acute oral toxicity category prediction, but the animal savings were
higher than for the other categories (1.45-1.91). Additionally, animal
savings (0.61 - 0.86 animals) for substances with

300 ≤ LD~50~ ≤ 2000 mg/kg, which had 100% accuracy for GHS acute oral
toxicity category prediction, were similar to animal savings (0.73 -
0.89 animals) for substances in the LD~50~ \< 5 mg/kg category (for the
3T3 NRU), which had 0% accuracy. Perhaps the difference between the
predicted starting dose and the true LD~50~ vs. the difference between
the default starting dose and the true LD~50~ has more influence on
animal savings than the accuracy of the LD~50~ prediction.

*[Animal Savings for the UDP by Toxicity Category Using 3T3 and NHK
NRU-Based Starting Doses with the RC Rat-Only Weight Regression
Excluding Substances with Specific Mechanisms of Action]{.underline}*

**Table 10-5** shows the mean animal savings by GHS toxicity category
for the *in vitro* NRU cytotoxicity test methods used with the RC
rat-only weight regression excluding substances with specific mechanisms
of toxicity other than basal cytotoxicity. For substances in the
categories for LD~50~ \> 2000 mg/kg, mean animal savings for the RC
rat-only weight regression excluding substances with specific mechanisms
of toxicity other than basal cytotoxicity were slightly higher than
those for the RC rat-only weight regression and those for the RC
millimole regression. Mean differences between animal use for the
default starting dose and mean animal use with the NRU-determined
starting dose were statistically significant (i.e., p \< 0.05) by a
one-sided Wilcoxon signed rank test for the following GHS toxicity
categories, test methods, and dose-response slopes:

-   5 \< LD~50~ ≤ 50 mg/kg for the NHK NRU at dose-response slope = 2
    (0.98 \[10.6%\] animals)

-   300 \< LD~50~ ≤ 2000 mg/kg for both NRU test methods and at
    dose-response = 2 (1.00 \[11.4%\] animals for the 3T3 NRU and 0.90
    \[10.3%\] animals for the NHK NRU)

-   2000 \< LD~50~ ≤ 5000 mg/kg for both NRU test methods and both
    dose-response slopes (1.75 \[19.1%\] to 2.22 \[20.5%\] animals)

-   LD~50~ \> 5000 mg/kg for both NRU test methods and both
    dose-response slopes (1.77 \[18.6%\] to 2.01 \[30.1%\] animals)

Mean animal savings for the 3T3 NRU and NHK NRU test methods were
similar for each toxicity category and dose-response slope, with the 3T3
NRU test method producing slightly higher mean animal savings in most
cases. For the dose-response slope of 2, mean animal savings across the
various toxicity categories for the 3T3 NRU ranged from -0.02 (-0.2%) to
2.22 (20.5%) animals while mean animal savings for the NHK NRU ranged
from -0.35

(-3.0%) to 1.98 (18.3%) animals.

**Table 10-5 Animal Use^1^ for the UDP^2^ By GHS Toxicity Category^3^
Using Starting Doses Based on the 3T3 and NHK**

> **^NRU\ Test\ Methods\ with\ the\ RC\ Rat-Only\ Weight\ Regression\ Excluding\ Substances\ with\ Specific\ Mechanisms\ of\ Toxicity^**4

  --------------- -------------- ----------------- ----------- -------------- ----------------- ----------- ------------ -----------------
                                 **Dose-Response                              **Dose-Response                            **Accuracy^8^**
                                 Slope = 2**                                  Slope = 8.3**                              

  **Toxicity      **Number of    **With Default**  **With      **Animals      **With Default    **With      **Animals    
  Category^3^**   Reference                        NRU-Based   Saved^7^**     Starting          NRU-Based   Saved^7^**   
                  Substances**                     Starting                   Dose^5^**         Starting                 
                                                   Dose**                                       Dose**                   

                                 **Starting                                                                              
                                 Dose^5^**                                                                               

                                 **3T3 NRU Test                                                                          
                                 Method**                                                                                

  LD~50~ ≤ 5      4              11.68 ± 0.17      11.26 ±     0.42 (3.6%)    10.62 ± 0.48      10.31 ±     0.31 (2.9%)  0%
  mg/kg                                            0.55                                         0.67                     

  \> 5 \< LD~50~  7              9.05 ± 0.13       9.03 ± 0.55 0.02 (0.3%)    8.07 ± 0.25       7.92 ± 0.59 0.15 (1.9%)  14%
  ≤ 50 mg/kg                                                                                                             

  \> 50 \< LD~50~ 5              7.82 ± 0.18       7.84 ± 0.15 -0.02 (-0.2%)  6.93 ± 0.31       6.99 ± 0.29 -0.06        80%
  ≤ 300 mg/kg                                                                                               (-0.9%)      

  \> 300 \<       9              8.81 ± 0.35       7.81 ± 0.06 1.00\* (11.4%) 7.31 ± 0.37       6.58 ± 0.18 0.73 (10.0%) 78%
  LD~50~ ≤ 2000                                                                                                          
  mg/kg                                                                                                                  

  \> 2000 \<      9              10.84 ± 0.07      8.62 ± 0.23 2.22\* (20.5%) 9.18 ± 0.26       7.19 ± 0.37 2.00\*       67%
  LD~50~ ≤ 5000                                                                                             (21.8%)      
  mg/kg                                                                                                                  

  \> 5000 mg/kg   12             9.59 ± 0.27       7.71 ± 0.40 1.88\* (19.6)% 6.69 ± 0.37       4.68 ± 0.46 2.01\*       25%
                                                                                                            (30.1%)      

                                 **NHK NRU Test                                                                          
                                 Method**                                                                                

  LD~50~ ≤ 5      4              11.55 ± 0.23      11.90 ±     -0.35(-3.0%)   10.66 ± 0.48      10.83 ±     -0.18        0
  mg/kg                                            0.32                                         0.45        (-1.6%)      

  \> 5 \< LD~50~  7              9.28 ± 0.25       8.30 ± 0.28 0.98\* (10.6%) 8.19 ± 0.32       7.30 ± 0.36 0.89 (10.9%) 14%
  ≤ 50 mg/kg                                                                                                             

  \> 50 \< LD~50~ 5              7.87 ± 0.20       8.03 ± 0.24 -0.16 (-2.0%)  7.08 ± 0.34       7.26 ± 0.40 -0.19        60%
  ≤ 300 mg/kg                                                                                               (-2.6%)      

  \> 300 \<       9              8.76 ± 0.33       7.86 ± 0.06 0.90\* (10.3%) 7.31 ± 0.34       6.61 ± 0.22 0.69 (9.5%)  89%
  LD~50~ ≤ 2000                                                                                                          
  mg/kg                                                                                                                  

  \> 2000 \<      9              10.82 ± 0.07      8.84 ± 0.26 1.98\* (18.3%) 9.15 ± 0.25       7.41 ± 0.39 1.75\*       44%
  LD~50~ ≤ 5000                                                                                             (19.1%)      
  mg/kg                                                                                                                  

  LD~50~ \> 5000  13             9.52 ± 0.28       7.75 ± 0.43 1.77\* (18.6%) 6.64 ± 0.35       4.76 ± 0.44 1.88\*       15%
  mg/kg                                                                                                     (28.4%)      
  --------------- -------------- ----------------- ----------- -------------- ----------------- ----------- ------------ -----------------

^1^Numbers are mean number of animals used and standard errors for 2000
simulations for each substance with a limit dose of 5000 mg/kg. Although
the simulations used whole animals, averaging the results produced
fractional numbers of animals. Results are provided for 46 substances in
the 3T3 NRU test method and 47 substances in the NHK NRU test method
categorized using the reference LD~50~ values from **Table 4-2**. The
slight differences in the number of animals used for the default
starting dose at the same dose-response slope reflect different
simulation runs.

^2^OECD (2001a); EPA (2002a).

^3^GHS-Globally Harmonized System of Classification and Labelling of
Chemicals with LD~50~ in mg/kg (UN 2005).

^4^From **Table 6-2**; log LD~50~ (mg/kg) = 0.357 log IC~50~ (g/mL) +
2.194.

^5^Default starting dose = 175 mg/kg.

^6^Starting dose = One default dose lower than NRU-predicted LD~50~
calculated using the geometric mean of laboratory mean IC~50~ values in
the RC rat-only weight regression excluding substances with specific
mechanisms of toxicity.

^7^Difference between mean animal use with default starting dose and
mean animal use with NRU-based LD~50~. Differences denoted by \* were
statistically significant (i.e., p \< 0.05) by a one-sided Wilcoxon
signed rank test. Percent difference is shown in parentheses.

^8^Proportion of substances for which the GHS acute oral toxicity
category (UN 2005) predicted by the *in vitro* NRU test methods matched
the *in vivo* category (from **Table 6-6**).

For the dose-response slope of 8.3, mean animal savings for the 3T3 NRU
ranged from -0.06 (-0.9%) to 2.01 (30.1%) while mean animal savings for
the NHK NRU ranged from -0.19

(-2.6%) to 1.88 (28.4%).

For both *in vitro* NRU cytotoxicity test methods, no mean animal
savings were observed for substances with 50 \< LD~50~ ≤ 300 mg/kg. In
fact, slightly more animals were used than when using the default
starting dose (i.e., animal savings were negative; -0.02 to -0.16
animal). Since this category includes the default starting dose of 175
mg/kg, animal savings were not expected for this category since savings
were determined by comparing animal use with the NRU-based starting dose
with animal use at the default starting dose. For the NHK NRU test
method, more animals were also used for substances with LD~50~ ≤ 5 mg/kg
(i.e. animal savings were -0.18 to -0.35 animals). The exceptions for
having little to no animal savings for the high toxicity substances was
for the substances in the 5 \< LD~50~ ≤ 50 mg/kg category for the NHK
NRU (0.89 \[10.9%\] to 0.98 \[10.6%\] animals), but only the 0.98
animals at dose-response = 2 was statistically significant.

**Table 10-5** also shows that mean animal savings did not correlate
with the accuracy of the GHS acute oral toxicity category predictions
(see **Section 6.3**). The toxicity categories with the highest animal
savings had low accuracy. Substances with LD~50~ \> 5000 mg/kg had
relatively low accuracy (15 - 25%) for GHS acute oral toxicity category
prediction, but the animal savings were relatively high (1.88 - 2.01
animals). For the NHK NRU, substances in the 5 \< LD~50~ ≤ 50 mg/kg
category had very low accuracy (14%) for GHS acute oral toxicity
category prediction, but the animal savings were statistically
significant (0.98 animals at dose-response = 2). Possibly the difference
between the predicted starting dose and the true LD~50~ vs. the
difference between the default starting dose and the true LD~50~ has
more influence on animal savings than the accuracy of the LD~50~
prediction. The RC rat-only weight regression excluding substances with
specific mechanisms of toxicity improved accuracy (compared with the RC
millimole regression) and animal savings for the GHS toxicity categories
for substances in the 2000 \< LD~50~ ≤ 5000 mg/kg and LD~50~ \> 5000
mg/kg categories. For substances in the 2000 \< LD~50~ ≤ 5000 mg/kg
category, accuracy increased from 0 - 9% (both *in vitro* test methods
and dose-response slopes) to 44 - 67% and animal savings increased from
1.25 -1.52 animals to 1.75 - 2.22 animals. For substances with

LD~50~ \> 5000 mg/kg, accuracy increased from 0 - 10% (both *in vitro*
NRU test methods and dose-response slopes) to 15 - 25% and animal
savings increased from 1.35 - 1.70 animals to 1.77 - 2.01 animals. The
RC rat-only weight regression excluding substances with specific
mechanisms of toxicity, however, also improved animal savings for
substances in the

300 \< LD~50~ ≤ 2000 mg/kg toxicity category while which accuracy was
decreased compared with the RC millimole regression. Animal savings for
substances in the

300 \< LD~50~ ≤ 2000 mg/kg toxicity category improved from 0.60 - 0.84
animals (for both *in vitro* NRU test methods and dose-response slopes)
to 0.69 - 1.00 animals while accuracy decreased from 100% to 78 - 89%.

10.2.4 [Refinement of Animal Use for the UDP When Using 3T3 and NHK
NRU-Based Starting Doses]{.underline}

A test method refines animal use when it lessens or eliminates pain or
distress in animals or enhances animal well-being (ICCVAM 2003). This
section evaluates whether the use of 3T3 and NHK NRU-based starting
doses refines animal use by reducing the number of animals that die
(i.e., experience pain and distress) during UDP testing compared to the
number of animals that die when using the default starting dose of 175
mg/kg. **Table 10-6** reports the refinement results for the UDP
simulation modeling using the 5000 mg/kg limit dose. For every
regression evaluated, the mean number of deaths when using the NRU-based
starting doses was slightly lower than the mean number of deaths when
using the default starting dose by approximately 0.1 to 0.2 deaths. The
percentage of deaths, however, was slightly higher for the NRU-based
starting doses than for the default starting dose since the total number
of animals used was lower for the NRU-based starting doses. In general,
fewer animals were used and fewer animals died when using an NRU-based
starting dose compared with use of the default starting dose.

**Table 10-6 Animal Deaths^1^ for the UDP^2^ Using Starting Doses Based
on the 3T3 and NHK NRU Test Methods**

  ---------------------- ----------------- ---------- ---------- ----------- ---------- ----------
  **Assay/Regression**   **With Default                          **With                 
                         Starting                                NRU-Based              
                         Dose^3^**                               Starting               
                                                                 Dose^4^**              

                         **Used**          **Dead**   **%        **Used**    **Dead**   **%
                                                      Deaths**                          Deaths**

  **3T3 NRU Test         **Dose-Response                                                
  Method**               Slope = 2**                                                    

  RC millimole^5^        9.77              4.16       42.6%      8.79        3.95       44.9%

  RC rat-only weight^6^  9.79              4.18       42.6%      8.66        3.91       45.2%

  RC rat-only weight     9.80              4.18       42.7%      8.64        4.03       46.6%
  excluding substances                                                                  
  with specific                                                                         
  mechanisms of                                                                         
  toxicity^7^                                                                           

                         **Dose-Response                                                
                         Slope = 8**                                                    

  RC millimole^5^        8.11              3.43       42.3%      7.20        3.26       45.3%

  RC rat-only weight^6^  8.14              3.44       42.3%      7.11        3.24       45.6%

  RC rat-only weight     8.16              3.45       42.3%      7.08        3.34       47.2%
  excluding substances                                                                  
  with specific                                                                         
  mechanisms of                                                                         
  toxicity^7^                                                                           

  **NHK NRU Test         **Dose-Response                                                
  Method**               Slope = 2**                                                    

  RC millimole ^5^       9.75              4.10       42.0%      8.93        3.96       44.3%

  RC rat-only weight^6^  9.77              4.11       42.0%      8.83        3.93       44.5%

  RC rat-only weight     9.78              4.12       42.1%      8.73        3.99       45.8%
  excluding substances                                                                  
  with specific                                                                         
  mechanisms of                                                                         
  toxicity^7^                                                                           

                         **Dose-Response                                                
                         Slope = 8**                                                    

  RC millimole ^5^       8.12              3.38       41.7%      7.33        3.26       44.5%

  RC rat-only weight~6~  8.14              3.39       41.7%      7.25        3.24       44.7%

  RC rat-only weight     8.15              3.40       41.7%      7.15        3.29       46.1%
  excluding substances                                                                  
  with specific                                                                         
  mechanisms of                                                                         
  action^7^                                                                             
  ---------------------- ----------------- ---------- ---------- ----------- ---------- ----------

^1^Numbers are mean numbers of animals used for 2000 simulations for
each substance. Although the simulations used whole animals, averaging
the results produced fractional numbers of animals. Upper limit dose =
5000 mg/kg. Results are provided for 46 substances in the 3T3 NRU and 47
substances in the NHK NRU test methods.

^2^OECD (2001a); EPA (2002a).

^3^Default starting dose = 175 mg/kg.

^4^Starting dose was one default dose lower than NRU-predicted LD~50~
calculated using the geometric mean of laboratory mean IC~50~ values in
the regression specified.

^5^log LD~50~ (mmol/kg) = 0.435 log IC~50~ (mM) + 0.625

^6^log LD~50~ (mg/kg) = 0.372 log IC~50~ (g/mL) + 2.024

^7^log LD~50~ (mg/kg) = 0.357 log IC~50~ (g/mL) + 2.194

**10.3 Reduction and Refinement of Animal Use for the ATC**

10.3.1 [Procedure for *In Vivo* Testing Using the ATC]{.underline}

This section describes the general dosing procedure for the conduct of
the ATC assay (OECD 2001d). The purpose of the ATC is to classify a test
substance into the appropriate GHS category for acute oral toxicity for
classification and labeling. This is done by estimating the range of the
LD~50~ values for a test substance rather than calculating a point
estimate of the LD~50~. The time between doses is determined by the
onset, duration, and severity of toxic signs. Guidance on the type of
animals to use, animal housing, clinical observations, etc., which are
outside the scope of the current discussion, are provided in the test
guidelines (See **Appendix M**).

*Main Test*

The ATC is based on the stepwise administration of test substances to
three animals at a time at one of a number of fixed doses: 5, 50, 300,
and 2000 mg/kg (and 5000 mg/kg, if necessary). The starting dose is
selected so that at least some of the animals die at that dose. If no
information on which to base a starting dose is available, the default
starting dose of 300 mg/kg is used. The next step, which may be to stop
testing, test at the same dose, test at the next higher dose, or test at
the next lower dose, is determined by the starting dose and the outcome
of the three animals tested at the starting dose. For example, if the
starting dose is 300 mg/kg and two to three animals die or are in a
moribund state, the next step is to administer 50 mg/kg to three more
animals. However, if zero to one animal dies at 300 mg/kg, three more
animals are tested at 300 mg/kg. Most substances required two to four
dose steps for substance classification. See **Appendix M** for the
outcome-based testing sequence for each starting dose.

*Limit Test*

For test substances that are likely to be nontoxic, the ATC test method
guideline includes a limit test in which six animals (three animals per
step) are tested at the limit dose of

2000 mg/kg or 5000 mg/kg (OECD 2001d).

10.3.2 [Procedure for Computer Simulation Modeling of the
ATC]{.underline}

The simulation process for the ATC was performed using MATLAB^®^ (The
MathWorks, Inc. 1996-2004) computational software, which is functionally
comparable to SAS^®^ version 8. Two thousand simulations of ATC testing
were run for each substance, NRU test method, and dose-mortality slope
using an upper limit dose of 2000 mg/kg. The simulation process
implements the distributional assumptions underlying the dose-mortality
response. The lowest dose at which an animal dies in response to the
administration of a toxic substance varies from animal to animal. For an
entire population of animals, mortality is assumed to have a log-normal
distribution with the mean equal to the log of the true LD~50~. Sigma
(), the variability of the simulated population, is the inverse of the
slope of the dose-mortality curve. For any given dose, the probability
that an animal will die is computed by the cumulative log-normal
distribution:

Probability (death) =
$\frac{1}{s\sqrt{2p}}\int_{\text{-¥}}^{\text{log}\text{dose}}{e^{\frac{- (t - \text{log}\text{trueLD}_{\text{50}})^{2}}{2s^{2}}}\text{dt}}$

Due to a lack of information for the real dose-mortality curves, the
simulations assumed several different values of the slope (i.e., the
inverse of ). Dose-mortality slopes of 0.5, 0.8, 2, 4, and 8.3 were
chosen to be comparable to those chosen for simulation modeling of the
UDP (see **Section 10.2.2**).

To model the variability of the NRU IC~50~ values within and between
laboratories, the values were log-transformed to normalize the
distribution of values for each substance. The mean and variance of
these log-transformed values were used to generate a log-normal
distribution from which to randomly select an IC~50~ value.

The simulation procedure used the following steps for each substance:

1.  The LD~50~ value (in mg/kg) from **Table 4-2** was entered as the
    true LD~50~ value and the choices of assumed slope were entered as
    the true slope for the dose-mortality curve.

2.  An IC~50~ value was selected from a distribution identified by the
    mean and variance of the IC~50~ values computed from the data to
    reflect that different laboratories produce different IC~50~ values
    in different situations (see **Table 5-3** for mean IC~50~ values
    and standard deviations).

3.  The IC~50~ value from Step 2 was used in the regression model being
    evaluated to compute a predicted LD~50~ value to use as the starting
    dose.

4.  The dosing simulation (of 2000 iterations) was run twice: once with
    the default starting dose of 300 mg/kg and once with a starting dose
    equal to the next fixed dose below the LD~50~ estimated by the
    regression being evaluated (i.e., the NRU-based starting dose). If
    the NRU-based starting dose was greater than the 2000 mg/kg limit
    dose, then testing proceeded using the 2000 mg/kg limit test rather
    than the main test.

5.  For every dose group of three animals, one observation was sampled
    from a binomial distribution with the probability of death
    calculated by the probability equation for a population of three.
    The sampled value, referred to as N1, indicates the number of
    animals, 0, 1, 2, or 3, in the dosing group that die.

6.  If N1 ≤ 1, step 4 is repeated with the same dose. Now the sampled
    value from the binomial distribution is referred to as N2.

7.  If N2 ≤ 1 and the dose is the highest dose tested, or the dose has
    already been decreased, the toxicity category is assigned and
    testing is terminated. If the dose is not the highest dose tested,
    or if the dose has not been decreased, the dose is increased to the
    next fixed dose and step 4 is repeated.

8.  If N1 \> 1 or N2 \> 2, and the dose is the lowest dose tested, or if
    the dose has already been increased, the toxicity category is
    assigned and testing is terminated. If the dose is not the lowest
    dose tested, or if the dose has not already been increased, the dose
    is decreased to the next fixed dose and step 4 is repeated.

10.3.3 [Animal Savings for the ATC When Using 3T3 and NHK NRU-Based
Starting Doses]{.underline}

10.3.3.1 *The Effect of Dose-Response Slope on Animal Use*

As described in **Section 10.3.2**, the simulation modeling of animal
use for the ATC used five different dose-mortality slopes to assess
animal use under various conditions of population variability. **Table
10-7** shows how animal use for the simulated ATC changes with
dose-response slope and mean animal use for ATC simulations when using
the default starting dose of 300 mg/kg and when using a starting dose
that was one fixed dose lower than that predicted by the 3T3 and NHK NRU
IC~50~ values with the RC millimole regression. The mean number of
animals used for the ATC decreased slightly with increasing slope for
both the default starting dose and the NRU-determined starting dose.

**Table 10-7 Change in Animal Use^1^ with Dose-Response Slope for the
ATC^2^**

  ----------------- ------------------ ------------------ ------------------
  **Dose-Response   **Default Starting **NRU-Based        **Animals
  Slope**           Dose^1,3^**        Starting           Saved^5^**
                                       Dose^1,4^**        

  **3T3 NRU Test                                          
  Method**                                                

  0.5               11.10 ± 0.07       10.11 ± 0.24       0.99\* (8.9%)

  0.8               10.98 ± 0.10       9.95 ± 0.27        1.03\* (9.4%)

  2.0               10.90 ± 0.16       9.76 ± 0.33        1.13\* (10.4%)

  4.0               10.84 ± 0.19       9.66 ± 0.35        1.17\* (10.8%)

  8.3               10.81 ± 0.21       9.64 ± 0.36        1.17\* (10.8%)

  **NHK NRU Test                                          
  Method**                                                

  0.5               11.10 ± 0.07       10.07 ± 0.22       1.03\* (9.3%)

  0.8               11.00 ± 0.09       9.90 ± 0.24        1.10\* (10.0%)

  2.0               10.93 ± 0.16       9.72 ± 0.30        1.21\* (11.1%)

  4.0               10.87 ± 0.19       9.61 ± 0.32        1.26\* (11.6%)

  8.3               10.84 ± 0.21       9.57 ± 0.34        1.27\* (11.7%)
  ----------------- ------------------ ------------------ ------------------

> ^1^Numbers are mean numbers of animals used and standard errors for
> 2000 simulations for 46 substances for the 3T3 NRU test method and 47
> substances for the NHK NRU test method. Although the simulations used
> whole animals, averaging the results produced fractional numbers of
> animals. Limit dose = 2000 mg/kg.
>
> ^2^OECD (2001d).
>
> ^3^Default starting dose = 300 mg/kg.
>
> ^4^Next fixed dose lower than the predicted LD~50~ calculated using
> the geometric mean of laboratory mean IC~50~ values in the RC
> millimole regression: log LD~50~ (mmol/kg) = 0.435 log IC~50~ (mM) +
> 0.625.
>
> ^5^Difference between mean animal use with default starting dose and
> mean animal use with NRU-based starting dose. Differences that were
> statistically significant (i.e., p \< 0.05) by a one-sided Wilcoxon
> rank test are noted by \*. Percent difference is shown in parentheses.

The mean numbers of animals saved, which was statistically significant
(i.e., p \< 0.05 by one-sided Wilcoxon signed rank tests) when compared
with mean animal use for the default dose, generally increased with
increasing slope. To simplify the presentation of animal savings and
comparison of the various regressions and starting doses, future results
in **Section 10.3.3** will be shown only for dose-response slopes of 2
and 8.3. Results for the other dose-mortality slopes are presented in
**Appendices N4-N6.**

10.3.3.2 *Mean Animal Use for ATC Simulations of Testing the
NICEATM/ECVAM Reference Substances -- Comparison of Regressions and 3T3
and NHK NRU Test Methods*

**Table 10-8** shows the mean animal use for testing the NICEATM/ECVAM
reference substances using the simulated ATC method when the starting
dose was the default starting dose and when the starting dose was one
fixed dose lower than that determined by the LD~50~ predicted from the
3T3 and NHK NRU test methods and the regressions (shown in **Table
6-2**) evaluated in **Section 6.3** for prediction of GHS acute oral
toxicity category. The mean difference in animal use between the two
starting doses is the mean animal savings. All mean differences (i.e.,
mean animal savings) were statistically significant (i.e., p \< 0.05
using one-sided Wilcoxon signed rank tests). Mean animal savings ranged
from 1.13 (10.4%) to 2.28 (21.1%) animals depending upon the test
method, regression, and dose-response slope. The lowest mean animal
savings were obtained for the RC millimole regression (1.13 \[10.4%\] to
1.27 \[11.7%\] animals) and the highest mean animal savings were
obtained with the RC rat-only regression excluding substances with
specific mechanisms of toxicity (1.68 \[15.4%\] to 2.28 \[21.1%\]
animals).

10.3.3.3 *Animal Savings for the ATC by Toxicity Category Using* *3T3
and NHK NRU-Based Starting Doses*

**Tables 10-9** through **10-11** show mean animal use and mean animal
savings for the ATC when used with the *in vitro* NRU cytotoxicity test
methods, organized by GHS toxicity category (UN 2005), and when based on
the:

-   RC millimole regression (**Table 10-9**)

-   RC rat-only weight regression (**Table 10-10)**

-   RC rat-only weight regression excluding substances with specific
    mechanisms of toxicity (**Table 10-11)**

**Table 10-8 Animal Use^1^ for the ATC^2^** Using Starting Doses Based
on NRU Test Methods with Various Regressions

  ---------------------- ----------------- ----------- ------------ ----------------- ----------- ------------ -----------------
  **Assay/Regression**   **With Default    **With      **Animals    **With Default    **With      **Animals    **Accuracy^6^**
                         Starting          NRU-Based   Saved^5^**   Starting          NRU-Based   Saved^5^**   
                         Dose^3^**         Starting                 Dose^3^**         Starting                 
                                           Dose^4^**                                  Dose^5^**                

  **3T3 NRU Test         **Dose-Response                            **Dose-Response                            
  Method**               Slope = 2**                                Slope = 8.3**                              

  RC millimole^7^        10.90 ± 0.16      9.76 ± 0.33 1.13\*       10.81 ± 0.21      9.64 ± 0.36 1.17\*       26%
                                                       (10.4%)                                    (10.8%)      

  RC rat-only weight^8^  10.90 ± 0.16      9.21 ± 0.31 1.68\*       10.81 ± 0.21      8.84 ± 0.36 1.97\*       35%
                                                       (15.5%)                                    (18.2%)      

  RC rat-only weight     10.90 ± 0.16      9.00 ± 0.29 1.90\*       10.81 ± 0.21      8.53 ± 0.33 2.28\*       46%
  excluding substances                                 (17.4%)                                    (21.1%)      
  with specific                                                                                                
  mechanisms of                                                                                                
  toxicity^9^                                                                                                  

  **NHK NRU Test         **Dose-Response                            **Dose-Response                            
  Method**               Slope = 2**                                Slope = 8.3**                              

  RC millimole^7^        10.93 ± 0.16      9.72 ± 0.30 1.21\*       10.84 ± 0.21      9.57± 0.34  1.27\*       28%
                                                       (11.1%)                                    (11.7%)      

  RC rat-only weight^8^  10.93 ± 0.16      9.45 ± 0.30 1.49\*       10.84 ± 0.21      9.22 ± 0.34 1.62\*       30%
                                                       (13.6%)                                    (14.9%)      

  RC rat-only weight     10.93 ± 0.16      9.25 ± 0.26 1.68\*       10.84 ± 0.21      8.91 ± 0.31 1.94\*       38%
  excluding substances                                 (15.4%)                                    (17.9%)      
  with specific                                                                                                
  mechanisms of                                                                                                
  toxicity^9^                                                                                                  
  ---------------------- ----------------- ----------- ------------ ----------------- ----------- ------------ -----------------

^1^Numbers are mean numbers of animals used and standard errors for 2000
ATC simulations each for 46 substances for the 3T3 NRU test method and
47 substances for the NHK NRU test method. Limit dose = 2000 mg/kg

^2^OECD (2001d).

^3^Default starting dose = 300 mg/kg.

^4^Starting dose was one fixed dose lower than NRU-predicted LD~50~
calculated using the geometric mean of laboratory mean IC~50~ values in
the regression specified.

^5^Difference between mean animal use with default starting dose and
mean animal use with NRU-based LD~50~. Percentage difference is shown in
parentheses. Differences marked by \* were statistically significant
(i.e., p \< 0.05) using a one-sided Wilcoxon signed rank test.

^6^Proportion of substances for which the GHS acute oral toxicity
category (UN 2005) predicted by the *in vitro* NRU test methods matched
the *in vivo* category (from **Tables 6-4** to **6-6**).

^7^log LD~50~ (mmol/kg) = 0.435 log IC~50~ (mM) + 0.625.

^8^log LD~50~ (mg/kg) = 0.372 log IC~50~ (g/mL) + 2.024.

^9^log LD~50~ (mg/kg) = 0.357 log IC~50~ (g/mL) + 2.194.

The summarized data come from the same analyses as the data provided in
**Table 10-8**.

Consistencies noted in the mean animal savings data provided in the
tables included:

-   For each test method and regression, the highest mean animal savings
    were generally in the LD~50~ ≤ 5 mg/kg and LD~50~ \> 5000 mg/kg
    toxicity categories.

-   For each test method and regression, the lowest mean animal savings
    were in the 50 \< LD~50~ ≤ 300 mg/kg and 300 \< LD~50~ ≤ 2000 mg/kg
    toxicity categories.

*[Animal Savings for the ATC by Toxicity Category Using 3T3 and NHK
NRU-Based Starting Doses with the RC Millimole Regression]{.underline}*

**Table 10-9** shows the mean animal savings for the ATC by GHS toxicity
category for the *in vitro* NRU test methods used with the RC millimole
regression. Mean differences between animal use for the default starting
dose and animal use with the NRU-determined starting dose were
statistically significant (i.e., p \< 0.05) by a one-sided Wilcoxon
signed rank test for the following GHS toxicity categories, test
methods, and dose-response slopes:

-   LD~50~ ≤ 5 mg/kg for the 3T3 NRU at both dose-response slopes (2.75
    \[29.5%\] to 2.80 \[31.1%\] animals)

-   2000 \< LD~50~ ≤ 5000 mg/kg for the 3T3 NRU at dose-response slope =
    8 (0.35 \[2.9%\] animals) and for the NHK NRU at dose-response slope
    = 2 (0.38 \[3.4%\] animals)

-   LD~50~ \> 5000 mg/kg for the 3T3 NRU at both dose-response slopes
    (2.32 \[29.6%\] and 2.46 \[20.5%\] animals) and for the NHK NRU at
    dose-response slope = 2 (2.34 \[19.7%\] animals)

For the dose-response slope of 2, mean animal savings for the 3T3 NRU
test method ranged from -0.24 (-2.5%) to 2.75 (29.5%) animals while
animal savings for the NHK NRU test method ranged from -0.02 (-0.2%) to
2.43 (19.9%) animals. For the dose-response slope of 8.3, mean animal
savings for the 3T3 NRU test method ranged from -0.47 (-5.1%) to 2.80
(31.1%) animals while mean animal savings for the NHK NRU test method
ranged from -0.23 (-2.4%) to 2.79 (23.0%) animals.

**Table 10-9 Animal Savings^1^ for the ATC^2^ by GHS Toxicity
Category^3^ Using Starting Doses Based on the 3T3 and NHK NRU Test
Methods with the RC Millimole Regression^4^**

  --------------- -------------- ----------------- ----------- ------------ ----------------- ----------- ------------ -----------------
                                 **Dose-Response                            **Dose-Response                            **Accuracy^8^**
                                 Slope = 2**                                Slope = 8.3**                              

  **Toxicity      **Number of    **With Default    **With      **Animals    **With Default    **With      **Animals    
  Category^3^**   Reference      Starting          NRU-Based   Saved^7^**   Starting          NRU-Based   Saved^7^**   
                  Substances**   Dose^5^**         Starting                 Dose^5^**         Starting                 
                                                   Dose^6^**                                  Dose^6^**                

                                 **3T3 NRU Test                                                                        ** **
                                 Method**                                                                              

  LD~50~ ≤ 5      7              9.35 ± 0.11       6.60 ± 0.87 2.75\*       9.00 ± 0.001      6.20 ± 0.88 2.80\*       0%
  mg/kg                                                        (29.5%)                                    (31.1%)      

  5 \< LD~50~ ≤   6              12.22 ± 0.05      11.12 ±     1.10 (9.0%)  12.13 ± 0.08      10.71 ±     1.42 (11.7%) 17%
  50 mg/kg                                         0.94                                       1.00                     

  50 \< LD~50~ ≤  6              10.70 ± 0.37      10.01 ±     0.69 (6.5%)  9.72 ± 0.48       9.39 ± 0.16 0.32 (3.3%)  67%
  300 mg/kg                                        0.08                                                                

  300 \< LD~50~ ≤ 6              9.79 ± 0.08       10.04 ±     -0.24        9.20 ± 0.11       9.67 ± 0.27 -0.47        100%
  2000 mg/kg                                       0.14        (-2.5%)                                    (-5.1%)      

  2000 \< LD~50~  11             11.18 ± 0.08      11.02 ±     0.16 (1.4%)  11.90 ± 0.04      11.55 ±     0.35\*       0%
  ≤ 5000 mg/kg                                     0.13                                       0.20        (2.9%)       

  LD~50~ \> 5000  10             11.90 ± 0.03      9.58 ± 0.91 2.32\*       12.00 ± 0.000     9.54 ± 0.97 2.46\*       10%
  mg/kg                                                        (19.5%)                                    (20.5%)      

                                 **NHK NRU Test                                                                        
                                 Method**                                                                              

  LD~50~ ≤ 5      7              9.37 ± 0.12       7.62 ± 1.12 1.76 (18.7%) 9.00 ± 0.002      7.25 ± 1.04 1.75 (19.5%) 0%
  mg/kg                                                                                                                

  5 \< LD~50~ ≤   6              12.2 ± 0.04       9.77 ± 0.34 2.43 (19.9%) 12.14 ± 0.09      9.35 ± 0.18 2.79 (23.0%) 50%
  50 mg/kg                                                                                                             

  50 \< LD~50~ ≤  6              10.75 ± 0.39      10.32 ±     0.43 (4.0%)  9.74 ± 0.49       9.97 ± 0.78 -0.23        50%
  300 mg/kg                                        0.36                                                   (-2.4%)      

  300 \< LD~50~ ≤ 6              9.79 ± 0.08       9.81 ± 0.08 -0.02        9.21 ± 0.13       9.28 ± 0.13 -0.06        100%
  2000 mg/kg                                                   (-0.2%)                                    (-0.7%)      

  2000 \< LD~50~  11             11.19 ± 0.09      10.81 ±     0.38\*       11.90 ± 0.04      11.17 ±     0.73 (6.2%)  9%
  ≤ 5000 mg/kg                                     0.27        (3.4%)                         0.73                     

  LD~50~ \> 5000  11             11.92 ± 0.02      9.58 ± 0.85 2.34\*       12.00 ± 0.000     9.52 ± 0.90 2.48 (20.6%) 0%
  mg/kg                                                        (19.7%)                                                 
  --------------- -------------- ----------------- ----------- ------------ ----------------- ----------- ------------ -----------------

^1^Numbers are mean number of animals used and standard errors for 2000
simulations for each substance with a limit dose of 2000 mg/kg. Results
are provided for 46 substances in the 3T3 NRU test method and 47
substances in the NHK NRU test method categorized using the initial
LD~50~ values from **Table 3-2**. Although the simulations used whole
animals, averaging the results produced fractional numbers of animals.

^2^OECD (2001d).

^3^GHS-Globally Harmonized System of Classification and Labelling of
Chemicals with LD~50~ in mg/kg (UN 2005).

^4^RC millimole regression is log LD~50~ (mmol/kg) = 0.435 log IC~50~
(mM) + 0.625.

^5^Default starting dose = 300 mg/kg.

^6^Starting dose was the next fixed dose lower than the predicted LD~50~
from using the NRU IC~50~ in the RC millimole regression.

^7^Difference between mean animal use with default starting dose and
mean animal use with NRU-based starting dose. Statistically significant
differences

(i.e., p \< 0.05) by a one-sided Wilcoxon signed rank test are noted by
\*. Percentage difference is shown in parentheses.

^8^Proportion of substances for which the GHS acute oral toxicity
category (UN 2005) predicted by the *in vitro* NRU test methods matched
the *in vivo* category (from **Table 6-4**).

.

For both dose-response slopes, the mean animal savings using the 3T3 NRU
test method was lower than the mean animal savings using the NHK NRU
test method for substances in four of the six toxicity categories: 5 \<
LD~50~ ≤ 50 mg/kg; 3000 \< LD~50~ ≤ 2000;

2000 \< LD~50~ ≤ 5000 mg/kg; and LD~50~ \> 5000 mg/kg. Mean animal
savings using the 3T3 NRU test method was higher than the mean animal
savings using the NHK NRU test method for substances in the other two
toxicity categories: LD~50~ ≤ 5 mg/kg and

50 \< LD~50~ ≤ 300 mg/kg. For the 3T3 NRU test method, the highest mean
animal savings occurred for substances in the category for LD~50~ ≤ 5
mg/kg (23.2 \[19.5%\] animals at dose-response slope = 2 and 2.46
\[20.5%\] animals at dose-response slope = 8.3). For the NHK NRU test
method, the highest mean animal savings occurred for substances in the
category for 5 \< LD~50~ ≤ 50 mg/kg (2.43 \[19.9%\] animals at
dose-response slope = 2 and 2.79 \[23.0%\] animals at dose-response
slope = 8.3); however, the animal savings were not statistically
significant.

For both test methods, the smallest mean animal savings (≤ 0.69) were
observed for substances with LD~50~ values between 50 and 2000 mg/kg.
Since the default starting dose was 300 mg/kg, little change in mean
animal use was expected for substances in the

50 \< LD~50~ ≤ 300 mg/kg and 300 \< LD~50~ ≤ 2000 mg/kg categories. For
both test methods and dose-response slopes, mean animal savings for the
substances in the 50 \< LD~50~ ≤ 300 mg/kg category were -0.23 to 0.69
animals. For both test methods and dose-response slopes, there were no
animal savings for substances in the 300 \< LD~50~ ≤ 2000 mg/kg
category. In fact, slight more animals were used for the NRU-based
starting doses than for the default starting dose (-0.02 to -0.47
animals).

**Table 10-9** also shows that mean animal savings did not correlate
with the accuracy of the GHS acute oral toxicity category predictions
(see **Section 6.3**). The toxicity categories with the highest animal
savings had low accuracy. The 3T3 NRU test method produced the highest
animal savings (2.75 - 2.80) for substances with LD~50~ ≤ 5 mg/kg, which
had 0% accuracy for GHS acute oral toxicity category prediction.
Substances in the

300 \< LD~50~ ≤ 2000 mg/kg category had 100% accuracy for GHS acute oral
toxicity category prediction, but had no animal savings (≤ 0.2 animals).
Possibly the difference between the predicted starting dose and the true
LD~50~ vs. the difference between the default starting dose and the true
LD~50~ has more influence on animal savings than the accuracy of the
LD~50~ prediction.

*[Animal Savings for the ATC by Toxicity Category Using 3T3 and NHK
NRU-Based Starting Doses with the RC Rat-Only Weight
Regression]{.underline}*

**Table 10-10** shows the animal savings for the simulation ATC method
by GHS toxicity category for the *in vitro* NRU cytotoxicity test
methods used with the RC rat-only weight regression. Mean animal savings
were statistically significant (i.e., p \< 0.05) by a one-tailed
Wilcoxon signed rank test for the following GHS toxicity categories,
test methods, and dose-response slopes:

-   LD~50~ ≤ 5 mg/kg for both NRU test methods and dose-response slopes
    (2.03 \[21.9%\] to 2.57 \[28.5%\] animals)

-   2000 \< LD~50~ ≤ 5000 mg/kg for the 3T3 NRU test method at both
    dose-response slopes (1.39 \[12.4%\] to 2.56 \[21.5%\] animals)

-   LD~50~ \> 5000 mg/kg for both NRU test methods and dose-response
    slopes (2.92 \[24.5%\] to 3.5 \[29.2%\] animals)

For the 3T3 NRU and NHK NRU test methods, mean animal savings were
similar for most toxicity categories at both dose-response slopes, with
the mean savings for the 3T3 NRU slightly higher than that for the NHK
NRU for most toxicity categories. For the dose-response slope of 2, mean
animal savings for the 3T3 NRU test method (for the various toxicity
categories) ranged from -0.32 (-3.3%) to 32.8 (27.5%) animals while mean
animal savings for the NHK NRU test method ranged from 0.03 (0.3%) to
2.92 (24.5%) animals. For the dose-response slope of 8.3, animal savings
for the 3T3 NRU test method ranged from -0.63 (-6.8%) to 3.50 (29.2%)
animals while mean animal savings for the NHK NRU test method ranged
from -0.23 (-2.4%) to 3.12 (26.0%) animals.

For both test methods, there were no mean animal savings (≤ 0.03
animals) for substances with LD~50~ values between 300 and 2000 mg/kg.
For both test methods and dose-response slopes, mean animal savings for
the substances in the 50 \< LD~50~ ≤ 300 mg/kg category were

**Table 10-10 Animal Savings^1^ for the ATC^2^ by GHS Toxicity
Category^3^ Using Starting Doses Based on the 3T3 and NHK NRU Test
Methods with the RC Rat-Only Weight Regression^4^**

  --------------- -------------- ----------------- ----------- ------------ ----------------- ----------- ------------ -----------------
                                 **Dose-Response                            **Dose-Response                            **Accuracy^8^**
                                 Slope = 2**                                Slope = 8.3**                              

  **Toxicity      **Number of    **With Default**  **With      **Animals    **With Default    **With      **Animals    
  Category^3^**   Reference                        NRU-Based   Saved^7^**   Starting          NRU-Based   Saved^7^**   
                  Substances**                     Starting                 Dose^5^**         Starting                 
                                                   Dose^6^**                                  Dose^6^**                

                                 **Starting                                                                            
                                 Dose^5^**                                                                             

                                 **3T3 NRU Test                                                                        
                                 Method**                                                                              

  LD~50~ ≤ 5      4              9.35 ± 0.11       6.83 ± 0.84 2.52\*       9.00 ± 0.001      6.43 ± 0.85 2.57\*       0%
  mg/kg                                                        (27.0%)                                    (28.5%)      

  \> 5 \< LD~50~  7              12.22 ± 0.05      10.33 ±     1.88 (15.4%) 12.13 ± 0.08      9.94 ± 0.54 2.20 (18.1%) 14%
  ≤ 50 mg/kg                                       0.52                                                                

  \> 50 \< LD~50~ 5              10.70 ± 0.37      9.94 ± 0.10 0.76 (7.1%)  9.72 ± 0.48       9.23 ± 0.12 0.48 (5.0%)  80%
  ≤ 300 mg/kg                                                                                                          

  \> 300 \<       9              9.79 ± 0.08       10.11 ±     -0.32        9.20 ± 0.11       9.83 ± 0.55 -0.63        78%
  LD~50~ ≤ 2000                                    0.29        (-3.3%)                                    (-6.8%)      
  mg/kg                                                                                                                

  \> 2000 \<      9              11.18 ± 0.08      9.79 ± 0.47 1.39\*       11.9 ± 0.04       9.34 ± 0.82 2.56\*       44%
  LD~50~ ≤ 5000                                                (12.4%)                                    (21.5%)      
  mg/kg                                                                                                                

  \> 5000 mg/kg   12             11.90 ± 0.03      8.62 ± 0.94 3.28\*       12.00 ± 0.00      8.50 ± 0.99 3.50\*       0%
                                                               (27.5%)                                    (29.2%)      

                                 **NHK NRU Test                                                                        
                                 Method**                                                                              

  LD~50~ ≤ 5      4              9.37 ± 0.12       7.32 ± 0.88 2.05\*       9.00 ± 0.002      6.97 ± 0.81 2.03\*       0%
  mg/kg                                                        (21.9%)                                    (22.6%)      

  \> 5 \< LD~50~  7              12.20 ± 0.04      9.72 ± 0.30 2.48 (20.3%) 12.14 ± 0.08      9.35 ± 0.17 2.79 (23.0%) 14%
  ≤ 50 mg/kg                                                                                                           

  \> 50 \< LD~50~ 5              10.75 ± 0.39      10.30 ±     0.45 (4.2%)  9.74 ± 0.49       9.97 ± 0.78 -0.23        60%
  ≤ 300 mg/kg                                      0.34                                                   (-2.4%)      

  \> 300 \<       9              9.79 ± 0.08       9.76 ± 0.08 0.03 (0.3%)  9.21 ± 0.13       9.20 ± 0.11 0.02 (0.2%)  89%
  LD~50~ ≤ 2000                                                                                                        
  mg/kg                                                                                                                

  \> 2000 \<      9              11.19 ± 0.09      10.45 ±     0.73 (6.6%)  11.90 ± 0.04      10.55 ±     1.35 (11.3%) 11%
  LD~50~ ≤ 5000                                    0.40                                       0.69                     
  mg/kg                                                                                                                

  LD~50~ \> 5000  13             11.92 ± 0.02      9.00 ± 0.88 2.92\*       12.00 ± 0.00      8.88 ± 0.93 3.12\*       8%
  mg/kg                                                        (24.5%)                                    (26.0%)      
  --------------- -------------- ----------------- ----------- ------------ ----------------- ----------- ------------ -----------------

^1^Numbers are mean number of animals used and standard errors for 2000
simulations for each substance with a limit dose of 5000 mg/kg. Although
the simulations used whole animals, averaging the results produced
fractional numbers of animals. Results are provided for 46 substances in
the 3T3 NRU test method and 47 substances in the NHK NRU test method
categorized using the reference LD~50~ values from **Table 4-2**.

^2^OECD (2001d).

^3^GHS-Globally Harmonized System of Classification and Labelling of
Chemicals with LD~50~ in mg/kg (UN 2005).

^4^From **Table 6-2;** log LD~50~ (mg/kg) = 0.372 log IC~50~ (g/mL) +
2.024

^5^Default starting dose = 300 mg/kg.

^6^Starting dose was one fixed dose lower than the NRU-predicted LD~50~
calculated using the NRU IC~50~ in the RC rat-only weight regression.

^7^Difference between mean animal use with default starting dose and
mean animal use with NRU-based LD~50~. Differences marked by \* were
statistically significant (i.e., p \< 0.05) by a one-sided Wilcoxon
signed rank test. Percentage difference is shown in parentheses.

^8^Proportion of substances for which the GHS acute oral toxicity
category (UN 2005) predicted by the *in vitro* NRU test methods matched
the *in vivo* category (from **Table 6-5**).

also relatively small (-0.23 to 0.76) animals. Since the default
starting dose was 300 mg/kg, little change in mean animal use was
expected for substances in the 50 \< LD~50~ ≤ 300 mg/kg and 300 \<
LD~50~ ≤ 2000 mg/kg categories.

**Table 10-10** also shows that mean animal savings did not correlate
with the accuracy of the GHS acute oral toxicity category predictions
(see **Section 6.3**). The toxicity categories with the highest animal
savings had low accuracy. For example, animal savings for substances in
the LD~50~ \> 5000 mg/kg category were 2.92 - 3.50 animals (for both *in
vitro* NRU test methods and dose-response slopes) and accuracy was 0 -
8%. In addition, substances in toxicity categories with the lowest
animal savings had the highest accuracy for GHS acute oral toxicity
category prediction. Substances in the 300 \< LD~50~ ≤ 2000 mg/kg
category had relatively high accuracy for GHS acute oral toxicity
category prediction (i.e., 78% for the 3T3 NRU and 89% for the NHK NRU),
but had the lowest animal savings (≤ 0.45 animals). Possibly the
difference between the predicted starting dose and the true LD~50~ vs.
the difference between the default starting dose and the true LD~50~ has
more influence on animal savings than the accuracy of the LD~50~
prediction.

*[Animal Savings for the ATC by Toxicity Category Using 3T3 and NHK
NRU-Based Starting Doses with the RC Rat-Only Weight Regression
Excluding Substances with Specific Mechanisms of Toxicity]{.underline}*

**Table 10-11** shows the animal savings by GHS toxicity category for
simulated ATC testing using the *in vitro* NRU cytotoxicity test methods
with the RC rat-only weight regression excluding substances with
specific mechanisms of toxicity. Mean animal savings were statistically
significant (i.e., p \< 0.05) by a one-tailed Wilcoxon signed rank test
for the following GHS toxicity categories, test methods, and
dose-response slopes:

-   LD~50~ ≤ 5 mg/kg for the 3T3 NRU test method at dose-response slope
    = 8.3 (2.16 \[24.0%\] animals) and for the NHK NRU test method at
    dose-response slope = 2 (1.27 \[13.5%\] animals)

-   2000 \< LD~50~ ≤ 5000 mg/kg for both NRU test methods and both
    dose-response slopes (1.23 \[11.0%\] to 3.07 \[25.8%\] animals)

-   LD~50~ \> 5000 mg/kg for both NRU test methods and both
    dose-response slopes (3.79 \[31.8%\] to 4.04 \[33.7%\] animals)

For the 3T3 NRU and NHK NRU test methods, mean animal savings were
similar for most toxicity categories at both dose-response slopes, with
the mean savings for the 3T3 NRU slightly higher than that for the NHK
NRU. For the dose-response slope of 2, mean animal savings for the 3T3
NRU test method (for the various toxicity categories) ranged from 0.02
(0.2%) to 4.08 (34.3%) animals while mean animal savings for the NHK NRU
test method ranged from 0.00 (0.0%) to 3.79 (31.8%) animals. For the
dose-response slope of 8.3, animal savings for the 3T3 NRU test method
ranged from -0.03 (-0.4%) to 4.38 (36.5%) animals while mean animal
savings for the NHK NRU test method ranged from -0.06 (-0.6%) to 4.04
(33.7%) animals.

[]{.mark}

For both test methods, there were no mean animal savings (≤ 0.02
animals) for substances with LD~50~ values between 300 and 2000 mg/kg.
For both test methods and dose-response slopes, mean animal savings for
the substances in the 50 \< LD~50~ ≤ 300 mg/kg category were also
relatively small (-0.06 to 0.79) animals. Since the default starting
dose was 300 mg/kg, little change in mean animal use was expected for
substances in the 50 \< LD~50~ ≤ 300 mg/kg and 300 \< LD~50~ ≤ 2000
mg/kg categories.

**Table 10-11** also shows that mean animal savings did not correlate
with the accuracy of the GHS acute oral toxicity category predictions
(see **Section 6.3**). The toxicity category with the highest animal
savings (LD~50~ \> 5000 mg/kg) had low accuracy (15 - 25%). Substances
in the 300 \< LD~50~ ≤ 2000 mg/kg category had very high accuracy,
78-89%, but no animal savings. Perhaps the difference between the
predicted starting dose and the true LD~50~ vs. the difference between
the default starting dose and the true LD~50~ has more influence on
animal savings than the accuracy of the LD~50~ prediction.

**^Table\ 10-11\ Animal\ Savings1\ for\ the\ ATC2\ By\ GHS\ Toxicity\ Category3\ Using\ Starting\ Doses\ Based\ on\ the\ 3T3\ and\ NHK\ NRU\ Test\ Methods\ with\ the\ RC\ Rat-Only\ Weight\ Regression\ Excluding\ Substances\ with\ Specific\ Mechanisms\ of\ Toxicity^**4

  --------------- -------------- ----------------- --------------- ------------ ----------------- ----------- ------------ -----------------
                                 **Dose-Response                                **Dose-Response                            **Accuracy^8^**
                                 Slope = 2**                                    Slope = 8.3**                              

  **Toxicity      **Number of    **With Default**  **With          **Animals    **With Default    **With      **Animals    
  Category^3^**   Reference                        NRU-Based       Saved^7^**   Starting          NRU-Based   Saved^7^**   
                  Substances**                     Starting                     Dose^5^**         Starting                 
                                                   Dose****^6^**                                  Dose^6^**                

                                 **Starting                                                                                
                                 Dose^5^**                                                                                 

                                 **3T3 NRU Test                                                                            
                                 Method**                                                                                  

  LD~50~ ≤ 5      4              9.35 ± 0.11       7.23 ± 0.83     2.12 (22.6%) 9.00 ± 0.001      6.84 ± 0.86 2.16\*       0%
  mg/kg                                                                                                       (24.0%)      

  \> 5 \< LD~50~  7              12.22 ± 0.05      10.52 ± 0.50    1.70 (13.9%) 12.13 ± 0.08      10.18 ±     1.96 (16.1%) 14%
  ≤ 50 mg/kg                                                                                      0.54                     

  \> 50 \< LD~50~ 5              10.70 ± 0.37      9.92 ± 0.09     0.79 (7.3%)  9.72 ± 0.48       9.24 ± 0.13 0.48 (4.9%)  80%
  ≤ 300 mg/kg                                                                                                              

  \> 300 \<       9              9.79 ± 0.08       9.77 ± 0.07     0.02 (0.2%)  9.20 ± 0.11       9.24 ± 0.13 -0.03        78%
  LD~50~ ≤ 2000                                                                                               (-0.4%)      
  mg/kg                                                                                                                    

  \> 2000 \<      9              11.18 ± 0.08      9.50 ± 0.47     1.67\*       11.90 ± 0.04      8.83 ± 0.82 3.07\*       67%
  LD~50~ ≤ 5000                                                    (15.0%)                                    (25.8%)      
  mg/kg                                                                                                                    

  \> 5000 mg/kg   12             11.90 ± 0.03      7.82 ± 0.77     4.08\*       12.00 ± 0.00      7.62 ± 0.82 4.38\*       25%
                                                                   (34.3%)                                    (36.5%)      

                                 **NHK NRU Test                                                                            
                                 Method**                                                                                  

  LD~50~ ≤ 5      4              9.37 ± 0.12       8.11 ± 0.65     1.27\*       9.00 ± 0.002      7.76 ± 0.58 1.24 (13.8%) 0%
  mg/kg                                                            (13.5%)                                                 

  \> 5 \< LD~50~  7              12.20 ± 0.04      9.87 ± 0.33     2.33 (19.1%) 12.14 ± 0.09      9.52 ± 0.27 2.62 (21.6%) 14%
  ≤ 50 mg/kg                                                                                                               

  \> 50 \< LD~50~ 5              10.75 ± 0.39      10.19 ± 0.26    0.55 (5.2%)  9.74 ± 0.49       9.80 ± 0.61 -0.06        60%
  ≤ 300 mg/kg                                                                                                 (-0.6%)      

  \> 300 \<       9              9.79 ± 0.08       9.79 ± 0.08     0.00 (0.0%)  9.21 ± 0.13       9.21 ± 0.12 0.01 (0.1%)  89%
  LD~50~ ≤ 2000                                                                                                            
  mg/kg                                                                                                                    

  \> 2000 \<      9              11.19 ± 0.09      9.96 ± 0.45     1.23\*       11.90 ± 0.04      9.62 ± 0.80 2.28\*       44%
  LD~50~ ≤ 5000                                                    (11.0%)                                    (19.2%)      
  mg/kg                                                                                                                    

  LD~50~ \> 5000  13             11.92 ± 0.02      8.13 ± 0.76     3.79\*       12.00 ± 0.000     7.96 ± 0.81 4.04\*       15%
  mg/kg                                                            (31.8%)                                    (33.7%)      
  --------------- -------------- ----------------- --------------- ------------ ----------------- ----------- ------------ -----------------

^1^Numbers are mean number of animals used and standard errors for 2000
simulations for each substance with a limit dose of 2000 mg/kg. Although
the simulations used whole animals, averaging the results produced
fractional numbers of animals. Results are provided for 46 substances in
the 3T3 NRU test method and 47 substances in the NHK NRU test method
categorized using the reference LD~50~ values from **Table 4-2**.

^2^OECD (2001d).

^3^GHS-Globally Harmonized System of Classification and Labelling of
Chemicals with LD~50~ in mg/kg (UN 2005).

^4^From **Table 6-2**; log LD~50~ (mg/kg) = 0.357 log IC~50~ (g/mL) +
2.194.

^5^Default starting dose = 300 mg/kg.

^6^Starting dose was one fixed dose lower than the NRU-predicted LD~50~
calculated using the NRU IC~50~ in the RC rat-only weight regression
excluding substances with specific mechanisms of toxicity.

^7^Difference between mean animal use with default starting dose and
mean animal use with NRU-based LD~50~. Statistically significant
differences (i.e., p \< 0.05) by a one-sided Wilcoxon signed rank test
are noted by \*. Percentage difference is shown in parentheses.

^8^Proportion of substances for which the GHS acute oral toxicity
category (UN 2005) predicted by the *in vitro* NRU test methods matched
the *in vivo* category (from **Table 6-5**).

The RC rat-only weight regression excluding substances with specific
mechanisms of toxicity improved accuracy (compared with the RC millimole
regression) and animal savings for the GHS toxicity categories for
substances in the 2000 \< LD~50~ ≤ 5000 mg/kg and LD~50~ \> 5000 mg/kg
categories. For the 2000 \< LD~50~ ≤ 5000 mg/kg category, accuracy
improved from 0 - 9% (both *in vitro* NRU test methods) to 44 - 67% and
animal savings improved from 0.16 - 0.73 animals to 1.23 - 3.07 animals.
For substances with LD~50~ \> 5000 mg/kg, accuracy improved from 0 - 10%
(both *in vitro* NRU test methods) to 15 - 25% and animal savings
improved from 2.32 - 2.48 animals to 3.79 - 4.38 animals. Although the
RC rat-only weight regression excluding substances with specific
mechanisms of toxicity had no animal savings for substances in the 300
\< LD~50~ ≤ 2000 mg/kg toxicity category (≤ 0.02 animals), it produced a
small improvement over the RC millimole regression since as high as 0.47
more animals were used (compared with the default starting dose).

10.3.4 [Refinement of Animal Use for the ATC when using 3T3 and NHK
NRU-Based Starting Doses]{.underline}

A test method refines animal use when it lessens or eliminates pain or
distress in animals or enhances animal well-being (ICCVAM 2003). This
section evaluates whether the use of 3T3 and NHK NRU-based starting
doses refines animal use by reducing the number of animals that die
during ATC testing compared to the number of animals that die when using
the default starting dose of 300 mg/kg. **Table 10-12** reports the
refinement results for the ATC simulation modeling using the 2000 mg/kg
limit dose. For every regression evaluated, the mean number of deaths
when using the 3T3 and NHK NRU-based starting doses was less than the
mean number of deaths when using the default starting dose by
approximately 0.6 to 0.7 deaths. For the RC millimole regression and the
RC rat-only weight regression, the percentage of deaths (compared with
the number of animals used) was also slightly lower for the NRU-based
starting dose compared with the default starting dose. For the RC
rat-only weight regression excluding substances with specific mechanisms
of action, the percentage of deaths (compared to the total number of
animals used) when using the 3T3 and NHK NRU-based starting doses was
about the same as the percentage of deaths when using the default
starting dose. In general, fewer animals were used with the NRU-based
starting dose and fewer animals died.

**Table 10-12 Animal Deaths^1^ for the ATC^2^ Using Starting Doses Based
on the 3T3 and NHK NRU Test Methods**

  ------------------ ----------------- ---------- ---------- ------------- ---------- ----------
  **Assay/           **Default                               **NRU-Based              
  Regression**       Starting                                Starting                 
                     Dose^3^**                               Dose^4^**                

                     **Used**          **Dead**   **%        **Used**      **Dead**   **%
                                                  Deaths**                            Deaths**

  **3T3 NRU**        **Dose-Response                                                  
                     Slope = 2**                                                      

  RC millimole^5^    10.90             3.55       32.6%      9.76          2.87       29.4%

  RC rat-only^6^     10.90             3.55       32.6%      9.21          2.82       30.6%

  RC rat-only        10.90             3.55       32.6%      9.00          2.92       32.4%
  excluding                                                                           
  substances with                                                                     
  specific                                                                            
  mechanisms of                                                                       
  toxicity^7^                                                                         

                     **Dose-Response                                                  
                     Slope = 8.3**                                                    

  RC millimole ^5^   10.81             3.03       28.0%      9.64          2.38       24.7%

  RC rat-only^6^     10.81             3.03       28.0%      8.84          2.33       26.3%

  RC rat-only        10.81             3.03       28.0%      8.53          2.42       28.3%
  excluding                                                                           
  substances with                                                                     
  specific                                                                            
  mechanisms of                                                                       
  toxicity^7^                                                                         

  **NHK NRU**        **Dose-Response                                                  
                     Slope = 2**                                                      

  RC millimole ^5^   10.93             3.47       31.8%      9.72          2.82       29.0%

  RC rat-only^6^     10.93             3.47       31.8%      9.45          2.78       29.4%

  RC rat-only        10.93             3.47       31.8%      9.25          2.91       31.5%
  excluding                                                                           
  substances                                                                          
  specific                                                                            
  mechanisms of                                                                       
  toxicity^7^                                                                         

                     **Dose-Response                                                  
                     Slope = 8.3**                                                    

  RC millimole ^5^   10.84             2.97       27.4%      9.57          2.34       24.4%

  RC rat-only^6^     10.84             2.97       27.4%      9.22          2.30       24.9%

  RC rat-only        10.84             2.97       27.4%      8.91          2.43       27.3%
  excluding                                                                           
  substances with                                                                     
  specific                                                                            
  mechanisms of                                                                       
  toxicity^7^                                                                         
  ------------------ ----------------- ---------- ---------- ------------- ---------- ----------

^1^Numbers are mean numbers of animals used for 2000 simulations for
each substance (46 substances in the 3T3 NRU test method and 47
substances in the NHK NRU test method). Although the simulations used
whole animals, averaging the results produced fractional numbers of
animals. Upper limit dose = 2000 mg/kg.

^2^OECD (2001d).

^3^Default starting dose = 300 mg/kg.

^4^Starting dose was one fixed dose lower than the NRU-predicted LD~50~.

^5^log LD~50~ (mmol/kg) = 0.435 log IC~50~ (mM) + 0.625.

^6^log LD~50~ (mg/kg) = 0.372 log IC~50~ (g/mL) + 2.024.

^7^log LD~50~ (mmol/kg) = 0.357 log IC~50~ (mM) + 2.194.

**10.4 Summary**

Computer simulation modeling of UDP testing using the default dose
progression shows that, for the subset of NICEATM/ECVAM reference
substances evaluated, the prediction of starting doses using the 3T3 and
NHK NRU test methods with the RC millimole regression resulted in the
use of statistically (p \< 0.05) fewer animals for UDP testing by an
average of 0.79 - 0.97 animals (8.4 - 11.2%) depending upon the *in
vitro* NRU cytotoxicity test method and the dose-response slope (of 2 or
8.3) used. Mean animal savings improved to 1.00 to 1.16 animals (10.7 -
13.3%) for the RC rat-only weight regression excluding substances with
specific mechanisms of toxicity.

When reference substances were grouped by GHS toxicity category, there
were no mean animal savings for simulated UDP testing for substances
with 50 \< LD~50~ ≤ 300 mg/kg. Statistically significant animal savings
were for observed for substances with 2000 \< LD~50~ ≤ 5000 mg/kg and
LD~50~ \> 5000 mg/kg for both NRU test methods. When using the RC
millimole regression, animal savings for these categories ranged from
1.25 to 1.70 animals (13.5 to 25.4%). Use of the RC rat-only weight
regression excluding substances with specific mechanisms of toxicity
improved animal savings for substances in these toxicity categories to
1.75 to 2.22 animals (18.3 to 30.1%). Using the 3T3 and NHK NRU IC~50~
values to estimate starting doses for the simulated UDP also resulted
approximately 0.1 to 0.2 fewer mean deaths compared with the use of the
default starting dose.

Computer simulation modeling of ATC testing with GHS cut points shows
that, for the reference substances tested in this validation study, the
prediction of starting doses using the 3T3 and NHK NRU test methods with
the RC millimole regression resulted in the use of statistically (p \<
0.05) fewer animals for ATC testing by an average of 1.13 to 1.27
animals (10.4 - 11.7%) depending upon the *in vitro* NRU cytotoxicity
test method and the dose-response slope (of 2 or 8.3) used. Animal
savings improved to a mean of 1.68 to 2.28 animals (15.4 - 21.1%) for
the RC rat-only weight regression excluding substances with specific
mechanisms of toxicity.

When test substances were grouped by GHS toxicity category, mean animal
savings for ATC testing using the RC millimole regression were
statistically significant for the 3T3 NRU at both dose-response slopes
for substances with LD~50~ ≤ 5 mg/kg (2.75 - 2.80 animals \[29.5 -
31.1%\]) and for substances with LD~50~ \> 5000 mg/kg (2.32 \[19.5%\] -
2.46 \[20.5%\] animals). Mean ATC animal savings with the RC millimole
regression were statistically significant with the NHK NRU at
dose-response = 2 for substances with 2000 \< LD~50~ ≤ 5000 mg/kg (0.38
\[3.4%\] animals) and for substances with LD~50~ \> 5000 mg/kg (2.34
animals \[19.7%\]). Using the RC rat-only weight regression excluding
substances with specific mechanisms of toxicity, statistically
significant animal savings were observed for both test methods and dose
response slopes for substances with 2000 \< LD~50~ ≤ 5000 mg/kg (1.23
\[11.0%\] - 3.07 \[25.8%\] animals) and substances with LD~50~ \> 5000
mg/kg (3.79 \[31.8%\] - 4.38 \[36.5%\] animals). Animal savings were
also statistically significant for substances with LD~50~ ≤ 5 mg/kg
using the 3T3 NRU at dose-response slope = 8.3 (2.16 \[24.0%\]) and
using the NHK NRU at dose-response slope = 2 (1.27 \[13.5%)\]. Using the
NRU IC~50~ values to estimate starting doses for the ATC refined animal
use by producing approximately 0.6 to 0.7 fewer mean animal deaths than
when the default starting dose of 300 mg/kg was used.

Spielmann et al. (1999) indicated that 76% (845/1115) of the industrial
substances submitted to the Federal Institute for Health Protection of
Consumers and Veterinary Medicine in Berlin, Germany, since 1982 had
LD~50~ \> 2000 mg/kg. Thus, the selection of starting doses using the
*in vitro* NRU methods may save a considerable number of animals since
animal savings are highest for the least toxic substances. However, the
extent to which these substances represent the world of substances in
commerce is not known.

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[^1]: **Replacement alternative:** A new or modified test method that
    replaces animals with nonanimal systems or one animal species with a
    phylogenetically lower one (e.g., a mammal with an invertebrate).

[^2]: **Reduction alternative:** A new or modified test method that
    reduces the number of animals required.

[^3]: **Refinement alternative:** A new or modified test method that
    refines procedures to lessen or eliminate pain or distress in
    animals or enhances animal well-being.