**Appendix L-1**

**Outlier Characterization for the 3T3 and NHK NRU Test Methods with the
RC Regression**

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

**L.1 Discordant Results for the 3T3 and NHK NRU Test Methods and RC
Millimole Regression**

The RC millimole regression and each *in vitro* NRU test method were
used to identify discordant results among the reference chemicals tested
in the validation study (i.e., those for which the rodent LD~50~ was not
accurately predicted by the *in vitro* NRU IC~50~). Discordant chemicals
are also referred to as outliers. Once identified, discordant chemicals
were then evaluated for common characteristics that may assist in
determining the types of chemicals that are not suited for use in the
3T3 and NHK NRU test methods to determine starting doses for acute
systemic toxicity assays. **Sections L.1.1** and **L.1.2** identify
discordant chemicals for the RC weight regression and the combined 3T3
or the combined NHK NRU weight regressions (see **Tables L1-1 and
L1-2**). Discordant chemicals for the millimole regressions are
discussed in this appendix.

L.1.1 [Identification of Discordant Chemicals]{.underline}

For each *in vitro* NRU test method, predicted LD~50~ values for the
reference chemicals were determined using the geometric mean IC~50~ of
the three geometric mean laboratory values in the RC millimole
regression. Discordant chemicals were identified using the RC method
(Halle 1998): a difference greater than 0.699 (or log 5) for log
observed LD~50~ (in mmol/kg) minus log predicted LD~50~ identifies a
chemical as discordant (i.e., an outlier) (see **Appendix J-1** for the
3T3 NRU test method and **Appendix J-3** for the NHK NRU test method for
the predicted LD~50~ values for each chemical). **Table L1-1** lists the
discordant chemicals for the RC millimole regression

  ------------------------------------------------------------------------
  **Table L1-1 Discordant                            
  Chemicals for the 3T3 and                          
  NHK NRU Test Methods and                           
  the RC Millimole                                   
  Regression**                                       
  -------------------------- ----------------------- ---------------------
                             **3T3 NRU**             **NHK NRU**

                             **Acetaminophen (+)**   

                             *Arsenic III trioxide   *Arsenic III trioxide
                             (-)*                    (-)*

                                                     *Aminopterin (-)*

  5-Aminosalicylic acid                              5-Aminosalicylic acid
                                                     (+)

  Busulfan                   **Busulfan (-)**        **Busulfan (-)**

  Caffeine                                           Caffeine (-)

  Cycloheximide              **Cycloheximide (-)**   **Cycloheximide (-)**

  Dibutyl phthalate          ***Dibutyl phthalate    ***Dibutyl phthalate
                             (+)***                  (+)***

                             Dichlorvos (-)          Dichlorvos (-)

                             ***Diethyl phthalate    ***Diethyl phthalate
                             (+)***                  (+)***

  Digoxin                    ***Digoxin (-)***       

  Disulfoton                 ***Disulfoton (-)***    ***Disulfoton (-)***

                             *Endosulfan (-)*        Endosulfan (-)

  Epinephrine bitartrate     Epinephrine bitartrate  Epinephrine
                             (-)                     bitartrate (-)

  Ethanol                    **Ethanol (+)**         **Ethanol (+)**

                             *Fenpropathrin (-)*     *Fenpropathrin (-)*

                             Hexachlorophene (-)     

  Lindane                    *Lindane (-)*           *Lindane (-)*

  Mercury II chloride        Mercury II chloride (-) Mercury II chloride
                                                     (-)

                                                     **Methanol (+)**

  Nicotine                   Nicotine (-)            Nicotine (-)

  Paraquat                   Paraquat (-)            Paraquat (-)

  Parathion                  ***Parathion (-)***     ***Parathion (-)***

  Phenobarbital              Phenobarbital (-)       Phenobarbital (-)

  Phenylthiourea             Phenylthiourea (-)      Phenylthiourea (-)

                             Physostigmine (-)       Physostigmine (-)

  Potassium cyanide          Potassium cyanide (-)   Potassium cyanide (-)

  Propylparaben              Propylparaben (+)       Propylparaben (+)

                             Sodium hypochlorite (+) Sodium hypochlorite
                                                     (+)

                                                     *Sodium oxalate (-)*

                             Sodium selenate (-)     Sodium selenate (-)

                             *Strychnine (-)*        *Strychnine (-)*

  Thallium I sulfate         Thallium I sulfate (-)  

  Triethylenemelamine        Triethylenemelamine (-) Triethylenemelamine
                                                     (-)

  1,1,1-Trichloroethane                              

  Verapamil HCl              **Verapamil HCl (-)**   ***Verapamil HCl
                                                     (-)***
  ------------------------------------------------------------------------

^1^Log LD~50~ (mmol/kg) = 0.435 log IC~50~ (mM) + 0.625. Log LD50
(mmol/kg) for discordant chemicals are \> 0.699 from the RC regression.

(-) - toxicity underpredicted by the *in vitro* assay (i.e., the LD~50~
value predicted by the IC~50~ is higher than the *in vivo* LD~50~
value); (+) - toxicity overpredicted by the model (i.e., the LD~50~
value predicted by the IC~50~ is lower than the rodent reference LD~50~
value).

**Bolded** chemicals have active metabolites *in vivo* (see **Appendix
F-2**).

Chemicals that showed evidence of insolubility (i.e., precipitates)
during testing (see **Table 5-8**) are identified by italics.

**Table L1-1** shows a comparison of the 22 RC outlier chemicals tested
(identified in **Table 3-2**) with the outliers identified when using
the 3T3 and NHK NRU results with the RC regression. Using the RC method
of identifying discordant chemicals (Halle 1998), there were 30
discordant chemicals for the 3T3 assay and 31 discordant chemicals for
the NHK assay. For the 58 RC chemicals that were tested, both test
methods confirmed the outlier status of 19 of the 22 RC outliers tested,
but the chemicals not identified as outliers were somewhat different for
the 3T3 and NHK assays. The 3T3 NRU cytotoxicity test method did not
identify caffeine, 5-aminosalicylic acid and 1,1,1-trichloroethane as
outliers and the NHK test method did not identify digoxin, thallium
sulfate, and 1,1,1-trichloroethane as outliers. The 3T3 assay identified
four chemicals as outliers that were not identified as outliers by the
RC: arsenic trioxide; hexachlorophene; acetaminophen; and diethyl
phthalate. The NHK assay identified five chemicals as outliers that were
not identified as outliers by the RC: aminopterin; arsenic trioxide;
sodium oxalate; diethyl phthalate; and methanol.

L.1.2 [Evaluation of Discordant Chemicals]{.underline}

To determine the attributes that may be used in the future to identify
chemicals that would result in discordant predictions, a number of
physico-chemical characteristics were evaluated for their frequency of
occurrence among the discordant chemicals versus the entire set of test
chemicals. This section provides a summary of these analyses for the
discordant chemicals identified among the validation set based on the RC
millimole regression and the RC outlier criteria.

*Physical Characteristics*

A number of physical characteristics were evaluated for their frequency
of occurrence in the set of discordant chemicals versus the entire set
of reference chemicals. The characteristics chosen were those that were
assumed to be available, or relatively easy to measure, for new
chemicals that may be tested in these assays to determine starting doses
for acute systemic toxicity assays. The characteristics examined
included chemical class, molecular weight, boiling point, IC~50~, pH,
and log K~ow~ (i.e., log octanol:water partition coefficient).
Unfortunately, these attributes were not available for all chemicals.
For example, log K~ow~ was available for only 52 of 72 (72%) chemicals
and boiling point was available for only 26 of 72 (36%) chemicals. For
boiling points \> 200^o^C, 9 of 13 (69%) chemicals were outliers using
the 3T3 NRU test method results and 8 of 13 (62%) chemicals were
outliers using the NHK NRU test method results. For molecular weight \>
400 g/mole, 5 of 7 (71%) chemicals were outliers using the 3T3 NRU test
method results and 3 of 7 (43%) chemicals were outliers using the NHK
NRU test method results. For log K~ow~ \> 3, 9 of 12 (75%) chemicals
were 3T3 outliers and 8 of 12 (67%) chemicals were NHK outliers.

*Chemical Class*

Examination of outliers by chemical class for the RC regression showed
that 3 of 3 (100%) organophosphates were outliers in both *in vitro* NRU
test methods.

*Solubility*

Another attribute that may cause a chemical to be discordant is the lack
of solubility in the solvent or when added to the tissue culture media.
Since the SMT expected the toxicity of insoluble chemicals to be
underpredicted in the *in vitro* NRU cytotoxicity assays, chemical tests
with precipitates were noted and compared with the discordant chemicals.
However, insolubility was not consistently associated with the
discordant chemicals for which toxicity was underpredicted. For example,
only 5 of the 24 (21%) underpredicted chemicals identified by applying
the 3T3 NRU test method results to the RC regression exhibited signs of
insolubility in the 3T3 assay in at least one laboratory (see **Table
5-8** for chemicals that had precipitates in the NRU assays).
Additionally, evidence of insolubility was noted for dibutyl phthalate
and diethyl phthalate in both assays, but toxicity was overpredicted
rather than underpredicted.

For the 3T3 NRU, 25 chemicals showed evidence of insolubility in the 3T3
assay in at least one lab. Eleven (4%) of these chemicals were outliers.
For the NHK NRU, 24 chemicals showed evidence of insolubility in at
least one laboratory. Nine (38%) of the 24 chemicals were outliers.

*Metabolism*

The SMT expected that the toxicity of chemicals metabolized *in vivo* to
active compounds (see **Table 3-7**) would be underpredicted *in vitro*
by 3T3 and NHK cells, which have little or no metabolic capability. Of
the 72 reference chemicals used to validate the 3T3 and NHK NRU test
methods, 22 (31%) chemicals are known to have active metabolites *in
vivo* and only 10 (45%) of these chemicals were classified as outliers
for the 3T3 NRU. Of these 10 chemicals, the toxicity of six (60%)
chemicals was underpredicted, while the toxicity of four (40%) chemicals
was overpredicted. For the NHK NRU, nine (29%) of the 31 outlier
chemicals are metabolized to active metabolites. Four (44%) of nine are
negative outliers (predicted to be less toxic than they are). See
**Table 3-7** for chemicals with active metabolites. Thus, the fact that
a chemical has active metabolites does not necessarily indicate that its
toxicity will be underpredicted by the *in vitro* NRU cytotoxicity test
methods.

Halle (1998) reported similar findings for the RC database (i.e.,
approximately half of the chemicals metabolized to active metabolites
were outliers and half were not).

*Mechanism of Action*

The contribution of mechanism of action to discordant status was
assessed by developing additional regressions for the prediction of
LD~50~ values from the *in vitro* NRU IC~50~ values. Both the 3T3 and
NHK NRU test method data and the RC database were used for this
evaluation. The RC database was used because:

-   it is the largest published compilation of IC~50x~ and rodent LD~50~
    data

-   the RC IC~50x~ data were highly correlated with the 3T3 and NHK NRU
    IC~50~ data collected for the 58 chemicals in common, and especially
    for the *in vitro* 3T3 NRU IC~50~ data (see **Figures 5-3** and
    **5-4**)

    In evaluating the contribution of mechanism of action to discordant
    status based on the RC weight regression, chemicals with mouse
    LD~50~ data only were excluded from the comparison. RC rat data only
    were used because:

-   rat and mouse data should not be combined, regardless of the high
    correlation of their LD~50~ data reported in the RC publication on
    their subset of rat and mouse LD~50~ values (see **Section 4.1.2**),
    since rats and mice may not have the same sensitivity to individual
    chemicals

-   the majority of data used in the RC regression were rat data (282
    rat data points and 65 mouse data points) (Halle 1998)

-   the great majority of acute oral systemic toxicity testing is
    performed with rats.

    The linear regression developed from the 282 rat data points in the
    RC (see **Appendix K-4**) using weight units is shown in **Table
    6-****2** and **Figure 6-5**. **Table 6-2** shows that the RC rat
    weight regression was not significantly different from the weight
    regression for the complete RC database when slopes and intercepts
    were simultaneously compared (goodness of fit F test; p=0.961).

**The following boxes characterize the** **30 discordant chemicals
(i.e., outliers) for the 3T3 NRU by counting the number of outliers in
each category and comparing to the total number of chemicals in the
category.**

[Physical Form]{.underline} [Number of]{.underline} [Outliers/Total in
Category]{.underline}

Solid 23 outliers/54 solids

Liquid 7 outliers/18 liquids

[Boiling Point]{.underline}

[(in degrees C**)**]{.underline} [Outliers/Total]{.underline}

No info 14/34

\< 100 1/8

100-200 1/5

200-300 3/4

300-400 5/6

408 1/1

960 0/1

1500 0/1

others decompose, sublime, or BPs are given @ \< atmospheric pressure

[Molecular Weight]{.underline}

[(g/mol)]{.underline} [Outliers/Total in Class]{.underline}

\< 150 3/21 chemicals (but no info on MeOH or CCl4)

\> 150-200 6/14

300. 13/20

300-400 3/11

400-500 3/4

500-600 1/1

600-700 0/1

700-800 1/1

[Chemical class]{.underline} [Number of]{.underline} [Outliers/Total in
Class]{.underline}

Alcohols 3/10

Carboxylic acids 4/12

Heterocyclic 7/14

Mercury compounds 1/1

Organophosphorous 3/3 (2 were organothiophosphorous cmpds)

Polycyclic 1/2

Sulfur compound 1/2

Sodium compound 2/5

Organometallic 1/1

Amide 1/3

Amine 1/1

Arsenical 1/2

Boron compounds 0/1

Cadmium compounds 0/1

Cyclic hydrocarbon 1/2

Halogenated hydrocarbon 1/3

Hydrocarbon 1/2

Ketone 0/1

Lithium compound 0/1

Metal compound 1/1

Nitrile 0/1

Phenol 0/1

Potassium compound 1/2

Chlorine compound 1/2

Nitrogen compound 1/1

Chromium compound 0/1

Fluorine compound 0/1

Oxygen compound 1/1

Selenium compound 1/1

[3T3 IC50 (mM)]{.underline} [Outliers/Total]{.underline}

≤ 0.0001 0/2

0.0001 - 0.001 1/2

0.001 - 0.01 1/4

0.01 - 0.1 9/13

0.1 - 1 15/23

1-10 2/12

10-100 1/9

\> 100 1/5

No IC50s for 2 chemicals

[3T3 pH]{.underline} [Outliers/Total]{.underline}

\< 7.6 0/9

7.6 0/0

7.7 1/1

7.8 0/1

7.9 3/6

8 5/12

8.1 11/18

8.2 3/6

8.3 3/8

8.4 1/5

8.5 0/1

\> 8.5 3/4

[log Kow]{.underline} [Outliers/Total]{.underline}

\< -4 0/1

-4 to --1 2/7

-1 to 0 3/7

0 to 1 4/7

1 to 2 5/13

2 -- 3 1/5

3 - 4 5/8

4 - 5 2/2

6 -- 7 2/2

No info 6/20

**The following boxes characterize the 31 discordant chemicals (i.e.,
outliers) for the NHK NRU by counting the number of outliers in each
category and comparing to the total number of chemicals in the
category.**

[Boiling Point]{.underline}

[(in degrees C**)**]{.underline} [Outliers/Total]{.underline}

No info 13/34

\< 100 2/8

100-200 1/5

200-300 3/4

300-400 5/6

408 0/1

960 0/1

1500 0/1

others decompose, sublime, or BPs are given @ \< atmospheric pressure

[Molecular Weight]{.underline}

[(g/mol)]{.underline} [Outliers/Total]{.underline}

\< 150 5/21 chemicals (but no info on CCl4)

\> 150-200 7/14

200-300 13/20

300-400 3/11

400-500 3/4

500-600 0/1

600-700 0/1

700-800 0/1

[Chemical class]{.underline} [Outliers/Total]{.underline}

Alcohols 4/10

Carboxylic acids 6/12

Heterocyclic 9/14

Mercury compounds 1/1

Organophosphorous 3/3 (2 were organothiophosphorous cmpds)

Polycyclic 0/2

Sulfur compound 1/2

Sodium compound 2/5

Organometallic 0/1

Amide 0/3

Amine 1/1

Arsenical 1/2

Boron compounds 0/1

Cadmium compounds 0/1

Cyclic hydrocarbon 0/2

Halogenated hydrocarbon 1/3

Hydrocarbon 1/2

Ketone 0/1

Lithium compound 0/1

Metal compound 0/1

Nitrile 0/1

Phenol 0/1

Potassium compound 1/2

Chlorine compound 1/2

Nitrogen compound 1/1

Chromium compound 0/1

Fluorine compound 0/1

Oxygen compound 1/1

Selenium compound 1/1

[NHK IC50 (mM)]{.underline} [Outliers/Total]{.underline}

≤ 0.0001 0/2

0.0001 - 0.001 1/2

0.001 - 0.01 3/4

0.01 - 0.1 6/13

0.1 - 1 11/23

1-10 8/12

10-100 1/9

\> 100 1/5

No IC50s for 2 chemicals

[pH]{.underline} [Outliers/Total]{.underline}

\< 7.1 0/6

7.1 0/0

7.2 1/1

7.3 0/0

7.4 1/4

7.5 3/7

7.6 4/7

7.7 9/23

7.8 11/17

7.9 0/3

8 0/1

8.1 0/0

8.2 1/1

8.3 0/0

8.4 0/0

8.5 1/1

\> 8.5 0/1

[log Kow]{.underline} [Outliers/Total]{.underline}

\< -4 0/1

-4 to --1 2/7

-1 to 0 5/7

0 to 1 3/7

1 to 2 5/13

2 -- 3 1/5

3 - 4 5/8

4 - 5 2/2

6-7 1/2

No info 7/20

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