Appendix L-3

**Analysis of Outliers by Halle (1998) for the RC Millimole Regression**

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**L.3 Analysis of Outliers for the RC Millimole Regression**

The RC millimole regression was constructed from the *in vitro* IC~50X~
cytotoxicity data from multiple cell lines and the *in vivo* acute
toxicity data from rats and mice (i.e., LD~50~ values) for 347 chemicals
(Halle 1998). Halle (1998) investigated the 95 (27.4%) chemicals for
which the observed log LD~50~ values were greater than 0.699 (i.e., 0.5
log) from predicted log LD~50~ values. Of the 95 outliers, 46 were
positive outliers and 49 were negative outliers. The positive outliers
have IC~50X~ values that predict a far higher *in vivo* toxicity (i.e.,
lower LD~50~) than the actual animal experiment. The negative outliers
are more important since the IC~50X~ values predict lower toxicity (i.e.
higher LD~50~) than the observed *in vivo* toxicity. It seems that Halle
(1998) was not concerned about the positive outliers since the
prediction erred in a health protective direction. Halle (1998) was much
more concerned about trying to explain the reasons for the negative
outliers since the error was in a nonconservative direction.

Halle (1998) investigated three factors that could have explained the
negative outliers.

1.  Variation in the oral LD~50~ values

> Reported oral LD~50~ values for a particular chemical might vary by
> factor 4 to 14 even when experiments were highly standardized. They
> found LD~50~ values from other sources for 23 of the 95 outliers. They
> found that the variations in the LD~50~ values (difference between the
> RTECS® value and the "new" value found for the 23 chemicals) were
> larger for the negative outliers than for the positive outliers.

2.  Species-specificity of the oral LD~50~ values.

> Halle (1998) compared an IC~50x~--LD~50~ regression using mouse LD~50~
> values (242 values) with a regression using rat LD~50~ values (285
> values) and found no significant difference between the two
> regressions. The RC millimole regression with 347 chemicals has 285
> rat values and 65 mouse values and is not statistically different from
> either the rat or mouse regressions.

3.  The cell culture(s) used may have been unsuitable for the detection
    of cytotoxic potential or it may have been unable to simulate the
    complex process of toxicity *in vivo.*

> Halle (1998) expected, *a priori*, that three classes of compounds,
> insecticides (**Table L3-1**), neurotoxins (**Table L3-2**), and those
> requiring metabolic activation for toxicity **(Table L3-3**), would
> not fit the RC millimole regression (i.e., cytotoxicity data would not
> predict *in vivo* toxicity). Sixty-two of the 347 chemicals belong to
> these three classes. Twenty-three (37.1%) of the 62 chemicals were
> negative outliers. Of the 23, 10 were insecticides, 5 were
> neurotoxins, and 8 required metabolic activation. No positive outliers
> were identified in the three classes.

Of the 49 negative outliers, 23 (46.9%) belonged to the three classes of
concern. Examination of these classes showed that the RC millimole
prediction was accurate (i.e., predicted log LD~50~ \[mmol/kg\] was
within 0.699 of observed log LD~50~ in \[mmol/kg\]) for 50% of the
insecticides (**Table L3-1**) and chemicals that required metabolic
activation (**Table L3-3**). For neurotoxins (**Table L3-2**), the
results were even better, since 21 (80.8%) fell within the prediction
interval. Halle (1998) felt that the ability to predict the acute LD~50~
for 50% of the insecticides and xenobiotics requiring metabolic
activation and for 81% of the neurotoxic xenobiotics was sufficiently
accurate for practical purposes.

Of the 49 negative outliers in the RC millimole regression, 23 (46.9%)
of these belonged to the three classes of concern that may explain the
false negative IC~50X~ values. Findings were contrary to Halle's
assumption that *in vitro* cytotoxicity would not predict *in vivo*
toxicity for these types of chemicals. The RC millimole prediction of
LD~50~ was applicable to 50% of the insecticides and chemicals that
required metabolic activation. For neurotoxic chemicals the results were
even better, since 21 (80.8%) fell within the prediction interval. Halle
felt that the ability to predict the acute LD~50~ for 50% of the
insecticides and chemicals requiring metabolic activation and for 81% of
the neurotoxic chemicals was sufficiently accurate for practical
purposes.

In separate analyses, Halle (1998) considered the physicochemical
properties of chemicals (i.e., molecular weight and the octanol/water
partition coefficient) as independent variables in a multiple regression
analysis, but they did not improve the prediction of LD~50~ by IC~50~.

**L3-1 The Error of Prediction^a^ of 20 of The Most Important
Insecticides**

> **in the RC Ordered According to Their Chemical Characteristics^b^**

  ------------------------- -------- ------------------- -----------------
  **Chemical class**        **RC     **Name**            **LD~50~ Error of
                            No**                         Prediction^a^**

  **Chlorinated                                          
  hydrocarbon**                                          

                            26       Kelthane            0.340

                            40       Chlordan            -0.046

                            43       Aldrin              **-1.074^b^**

                            61       DDT                 **-0.775**

                            167      DDD                 -0.378

                            185      Heptachlor          **-1.050**

                            195      DDA                 0.133

                            197      DDE                 0.251

                            207      Dieldrin            **-1.223**

                            223      Lindane             **-1.043**

  **Organophosphorus                                     
  compounds**                                            

                            49       Parathion           **-2.339**

                            51       Disulfoton          **-2.346**

                            67       Malathion           0.106

                            75       Trichlorfon         -0.136

                            96       Cygon               **-0.848**

  **Carbamate compounds**                                

                            73       Carbaryl            -0.279

                            186      Zineb               **1.185**

  **Other compounds**                                    

                            134      Rotenone            0.583

                            173      Pentachlorophenol   **-0.720**

                            235      Paraquat            **-1.019**
  ------------------------- -------- ------------------- -----------------

^a^ defined as observed log LD~50~ (mmol/kg) - predicted log LD~50~
(mmol/kg)

^b^ modified from Table 10 of Halle (1998)

**bold numbers**: outliers (i.e., observed log LD~50~ (mmol/kg) -
predicted log LD~50~ (mmol/kg) \> 0.699)

**Table L3-2 The Error of Prediction ^a^ of 26 Neurotoxic Xenobiotics in
the RC Ordered According to Their *In Vivo* Potency^b^**

  ---------------------------- ------- ---------------------- -----------------
  **Chemical Class**           **RC    **Name**               **LD~50~ Error of
                               No**                           Prediction^a^**

  **Sedative, hypnotic, CNS                                   
  depressants**                                               

                               69      Secobarbital sod.      -0.651

                               83      Thiopental             -0.119

                               84      Amobarbital            -0.335

                               87      Pentobarbital sodium   -0.654

                               101     Gluthetimide           -0.270

                               118     Phenobarbital          **-1.035^b^**

                               247     (+)-Thalidomide        -0.397

                               264     Chloral hydrate        -0.349

                               317     Barbital sodium        -0.591

  **Antidepressant**                                          

                               38      Imipramine HCl         -0.093

                               90      Iproniazid             -0.273

                               183     Amitriptyline          0.021

  **Antipsychotic,                                            
  anxiolytic**                                                

                               27      Chlorpromazine         -0.176

                               44      Hydroxyzine HCl        0.248

                               63      Diazepam               0.116

                               170     Thioridazine HCl       -0.013

  **Stimulants**                                              

                               112     Caffeine               **-0.815**

                               262     Amphetamine sulfate    **-1.579**

  **Anticonvulsants**                                         

                               82      Diphenylhydantoin      -0.551

  **Analgetic (general                                        
  anesthesia)**                                               

                               229     Dextropropoxyphene HCl **-1.150**

  **Anticholinergic**                                         

                               251     Scopolamine \* HBr     -0.123

                               296     Homatropine            -0.532
                                       methylbromide          

  **Other Neurotoxins (not                                    
  insecticide)**                                              

                               102     Acrylamide             -0.338

                               137     Triethyltin chloride   **-0.852**

                               142     Methylmercury chloride 0.105

                               316     Toluene                0.571
  ---------------------------- ------- ---------------------- -----------------

^a^ defined as observed log LD~50~ (mmol/kg) - predicted log LD~50~
(mmol/kg)

^b^ modified from Table 11 of Halle (1998)

**bold numbers**: outliers (i.e., observed log LD~50~ (mmol/kg) -
predicted log LD~50~ (mmol/kg) \> 0.699)

**Table L3-3 The Error of Prediction ^a^ of the 16 Xenobiotics in the RC
that Require Metabolic Activation^b^**

  -------- -------------------------------------- --------------------------
  **RC     **Name**                               **LD~50~ error of
  No**                                            prediction^a^**

  13       Cycloheximide                          **-1.370^b^**

  33       p-Chloromercuribenzoic acid            **-1.077**

  37       Aflatoxin B~1~                         **-1.783**

  68       2.4-Dinitrophenol                      **-1.128**

  97       Phenacetin                             0.292

  109      Frusemide                              0.109

  113      Acetaminophen                          0.386

  116      Cyclophosphamide \* H~2~O              **-1.310**

  123      Isoniazid                              -0.332

  125      Carbon tetrachloride                   0.229

  192      1.3-Bis(2-chloroethyl)-1-nitrosourea   **-1.176**

  260      Coumarin                               -0.427

  273      Bromobenzene                           0.374

  279      Thioacetamide                          -0.294

  281      1.2-Dibromomethane                     **-1.106**

  292      Allylalcohol                           **-0.952**
  -------- -------------------------------------- --------------------------

^a^ defined as observed log LD~50~ (mmol/kg) - predicted log LD~50~
(mmol/kg)

^b^ modified from Table 12 of Halle (1998)

**bold numbers**: outliers (i.e., observed log LD~50~ (mmol/kg) -
predicted log LD~50~ (mmol/kg) \> 0.699

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