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Final Report: Gasoline Ethanol Vapor Condensate: A 13-Week Whole Body Inhalation 

Toxicity Study in Rats with Neurotoxicity Assessments and a 4-Week In Vivo Genotoxicity 

and Immunotoxicity Assessments (HLS Study No. 00-6127) 

211(b) Research Group (RG) response to EPA Second Draft report Comments 

Dated April 9, 2008 

EPA April 8, 2008 Comment #1 

In regard to immunotoxicity outcomes, the responses to comments are, with one exception, 

satisfactory. The exception is the lack of a discussion of the reported immunotoxicity results. No 

context is presented for interpreting the results. It would be helpful to include estimated blood 

alcohol levels (BAL) to put the reported suppression in context. For example, does exposure to 

20,000mg EtOH/m3

 cause extreme intoxication? The report should provide a discussion of the 

biological effects, the doses that caused the effects and the likely human effects at similar BALs. 

RG Response to EPA April 8, 2008 Comment 1: 

EPA comments to draft 1 of this report [June 9, 2003] were similar to those submitted with draft 2 

requesting BAL levels either measured or estimated and a discussion of biological mechanisms. 

The RG response to these original comments [see reviewer checklist submitted with draft 2, Nov 

12, 2007] indicated that BAL levels were not measured and that an extensive discussion of possible 

mechanisms was beyond the scope of this study. The study was performed as part of a series of 

plaque-forming assays on baseline gasoline [BG] and 6 gasoline/oxygenate blends to identify and 

compare immunotoxic potential of these materials. It was not designed as a definitive assay for 

BG+ETOH immunotoxicity. A discussion such as that recommended by the EPA reviewers in the 

absence of measured BAL data is speculative and beyond the scope of this study, a consideration 

acknowledged by the initial EPA reviewer. Dr. Kimber White, the investigator on this study 

appreciated the initial EPA reviewer’s comments but considered it more appropriate to address 

these issues in a peer-reviewed publication of the immunotoxicity results of all seven materials 

rather than in this single study. The RG continues to concur with this position. Also, the RG points 

out that the test material contained approximately 10% ethanol and 90% gasoline vapor. The 

exposure to ethanol from this mixture would never reach 20,000mg EtOH/m3

. 

Appendix Y: In Vivo-In Vitro Rat Peripheral Blood Lymphocyte Sister Chromatid Exchange Assay 

(SCE). On page 12, the report incorrectly states that in the statistical analyses of male rat 

lymphocytes there was a statistically significant increase in SCE frequency at 2000mg/m3

. An 

examination of the data in the table shows that this was instead a statistically significant decrease. 

In addition, according to the report, a one-tailed test was used. A one-tailed test is appropriate for 

detection of increases only. Consequently, there is an inconsistency between the method and the 

reported results that needs to be resolved. 

RG Response to EPA April 8, 2008 Comment 2: 
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The RG appreciates being notified of this error and the text will be corrected. The RG also 

acknowledges the appropriate application of one-tailed tests. The purpose of performing an SCE 

assay was to determine whether the test material was able to induce perturbation of genetic material 

as evidence by an increase in SCE in peripheral blood lymphocytes and a one-tailed test was 

appropriate for this purpose. The results of this assay were negative and the EPA reviewer of the 

first draft considered the data evaluation adequate to support a conclusion of “no effect”. No 

revisions of the statistics methods will be made. 

EPA April 8, 2008 Comment #3 

Appendix X: Micronucleus assay. The discussion of the results of the micronucleus (MN) assay is 

problematic. A small but statistically significant increase in the MN frequency is reported in the 

polychromatic (immature) erythocytes (PCE). The report states that this finding was biologically 

irrelevant due to the fact that the negative control was abnormally low, thus enhancing the 

statistical significance of the highest concentration examined. The report states that is the 

statistically significant high dose is removed there were no significant differences when the 2 lower 

doses were compared to the control. However, we disagree with this approach and the resulting 

conclusion. 

By examining the data presented in the Appendix, it is apparent that this study, for reasons 

unknown, was relatively insensitive for detecting MN induction. An examination of the historical 

positive controls with cyclophosphamide (assumed to be 40mg/kg) showed a mean frequency of 48 

to 49 micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes examined 

(MN PCE/2000). In the present study, where the authors dismiss the positive findings for ethanol 

due to due to abnormally low negative control (0.0 and 0.2 for males and females respectively), the 

cyclophosphamide positive controls range only between 14 and 8 MN PCEs/2000 for the male and 

female immature erythrocytes, respectively. This is roughly 6- to 4-fold lower than their usual 

historical positive control. In addition, the historical negative control data are shown to be 

between 0.71 and 0.94MN PCEs/2000. Thus, the negative controls for the current ethanol study are 

approximately 8-fold lower than normal. Since the historical positive were very low dismissing the 

positive ethanol findings due to a low negative control is misleading. This suggests that overall the 

study had low sensitivity for detecting MN, and that the findings for ethanol may in fact be higher 

and more significant than indicated if the study were run with its usual sensitivity for detecting MN. 

Therefore, it is not valid at this time to dismiss the positive findings for MN induction by ethanol, 

and a definitive results (either positive or negative) was not conclusively shown. 

RG Response to EPA April 8, 2008 Comment 3: 

The RG acknowledges the concerns raised by the EPA reviewer of draft 2 of this study. The study 

was reviewed carefully by the RG for the very reasons cited above. Our conclusion to submit the 

study to EPA as final was determined as follows. 

• The ratio of immature to mature erythrocytes did not indicate any test material induced cell 

toxicity. 
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• The low average values for negative control animals resulted from the absence of MN-PCE in 

any of the 5 male animals and 4/5 female rats [one female with MN-PCE]. The historical data 

for this laboratory gave an average of 0.71 for individuals and 0.94 for group means. However, 

the distribution figures indicated that for individual occurrences, 50% cells had no micronuclei 

and approximately 22% contained 0-0.2 occurrences for group averages. In addition, group 

occurrences for negative control values demonstrated 23% 0.6-0.8 micronuclei, suggesting that 

the incidence seen at BG+ETOH doses could have been spontaneous. 

• The incidence of micronucleated polychromatic erythrocytes in the negative control or 

BG+ETOH treated groups has no more than 1-2 MN-PCE per 2000 cells examined with the 

exception of one 20,000mg/m3

 rat with 3. In contrast, the cyclophosphamide positive control, 

although the overall average was lower than the historical positive control mean, had MN-PCE 

in every animal examined with an incidence range of 5-22 MN-PCE in 2000 cells examined. 

• The study was performed using rats exposed for 28 days in whole body inhalation chambers as 

part of a 90-day study, which had been terminated by the time these data were submitted. 

• Results from the micronucleus assay performed with unleaded baseline gasoline alone indicated 

that baseline gasoline did not induce increased incidence of MN-PCE. This study has already 

been reviewed and accepted by EPA. 

• Review of the published literature on the genetic toxicity of ethanol indicated that overall 

ethanol is not considered mutagen or clastogenic and has sometimes been used a solvent control. 

(see review by Phillips, BJ and Jenkinson, P. 2001. Mutagenesis 16(2): 91-101). 

Review of all this information suggested that, despite the low values reported for negative and 

positive controls in this study, BG+ETOH condensate is unlikely to induce biologically significant 

increases in micronucleated polychromatic erythrocytes or to be considered clastogenic. Both the 

scientific peer reviewers and the EPA reviewer of draft 1 apparently considered this conclusion to 

be adequate as well. The EPA review of the first draft (June 9, 2003) stated “The micronucleus 

assay and the SCE assay appear to have been conducted according to recognized and appropriate 

protocols. The conclusions of no effects of gas + ETOH are supported by the data. The reviewers’ 

minor comments have been adequately addressed”. This is the entirety of their comments on the 

genetic toxicity studies. Therefore the study is submitted to EPA as final.