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Response to Independent Peer Reviewers Comments: 13

- week Whole Body Inhalation Study of Baseline Gasoline + Diisopropyl Ether Vapor Condensate and Satellite Studies

In the 211(b) testing program, the draft reports of all 13

- week inhalation and satellite studies are reviewed by two independent peer reviewers: Dr. Thomas Goldsworthy, Consultant, and Dr. Richard Schlesinger, Pace University, New York, NY. Those peer revie wers have now provided their comments on the draft reports for studies with baseline gasoline + DIPE vapor condensate (BG + DIPE VC). The studies include a 13

- week inhalation study including neurotoxicity and 4 satellite reports for genetic toxicology

- micronucleus and sister chromatid exchange (SCE), immunotoxicity and glial fibrillary a cidic protein (GFAP) analysis. All studies were performed with animals exposed to BG + DIPE VC under the same chamber conditions. For the genetic toxicology and immunotoxicity studies, animals were exposed for 4 weeks; for GFAP analysis, animals were exp osed for all 13 weeks and samples were collected at terminal sacrifice of the subchronic study. This draft report set includes the revised statistics for sister chromatid exchange and neurobehavioral endpoints recommended by EPA.

The reviewers concur tha t, overall, the studies were scientifically sound, protocols were appropriately designed and accurately followed, and results and conclusions reflect the data presented. None of the reviewers‟ comments or recommendations invalidates the studies. The 211( b) Working Group (WG) responses to specific comments are organized by study and reviewer. Where comments were similar from both reviewers, the response is addressed jointly. Recommendations accepted by the performing laboratory‟s study directors will app ear in final reports of all studies.

13

- week Subchronic Inhalation Study [Huntingdon, USA]

General Comments : Dr. Goldsworthy suggested that the titles of associated study reports be specified on pp. 34, 35,

and 36. Response: Rather than including the ext ended study titles, the WG will direct the study director to refer to these studies as Sister chromatid exchange report [BioReliance], Micronucleus report [Huntingdon, UK] and Immunotoxicology report [Immunotox, Inc].

Dr. Schlesinger recommended that cons istent criteria for data evaluation be employed, especially levels of significance for all aspects of the study, including satellite reports. Response: Levels of significance are identified variously as p=0.05 and p=0.01 in different

studies because satel lite studies were performed in different contract laboratories with different standard protocols for levels of significance which are not adjusted from client to client. To accommodate client needs, some contract laboratories [e.g.Huntingdon, US] provide both 0.05 and 0.01 levels of significance for the same data set and both values are often identified in standardized statistical packages for computers.

Summary [p.6] Dr Schlesinger noted that the correspondence between the measured and nominal

concentrat ions was quite good and that the sentence describing reasons for occurrences when the correspondence was not good does not apply and should be deleted. Response: The WG agrees with this comment. The inappropriate sentence [1 st paragraph, line 7] will be deleted.

Dr. Goldsworthy suggested that the information on motor activity [2 nd paragraph, p.6] be expanded in the summary to correspond to the body of the report. Text should indicate that

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statistically significant differences observed in low and medium exposure groups were not considered associated with BG+DIPE VC treatment because changes were not consistent among dose levels. Response: The observed changes in motor activity and the reasons for not considering them

treatment

- related are well described in the report. There was no linear relationship between exposure levels or any consistent change in activity related to exposure. The purpose of the summary is to highlight conclusions without extensive detail. The WG does not consider it
necessary to expand the Summary with additional motor activity detail.

Section 2.11.5 [p. 23] Administration of Test Substances Dr Schlesinger pointed out that the group numbers are inconsistent as I, II, III, IV in the text and then 1, 2, 3, 4 in the Tables. Response : These variations result from use of Arabic numerals in the computerized data gathering system, which can‟t be altered and the routine use of Roman numerals in the text. The study director will be requested to revise the text numbers to match the tables

.

Section 2.20 [pp42

- 43] Statistical Analysis Dr Schlesinger: Inquired why each time interval was analyzed independently and requested an explanation of why a 2

- way ANOVA with time as a parameter was not used instead. He also requested

clarification on which post

- hoc tests were used for which data sets. Response: A single computerized statistical testing package was employed for all parameters listed on p.43

- 44 of report. In this package, analyses by individual sex or sexes combined were programmed t o be performed separately by one

- way ANOVA, rather than analyzed together by two

- way ANOVA. Terminal and recovery data were also analyzed separately. After discussion with the study director, it was decided that additional justification for the

statistic al method employed routinely for repeat

- dose studies throughout this testing facility was probably not needed. Revisions of statistical methods for neurobehavioral parameters were requested by EPA and are outlined in this section.

Thought that the use of both 5% and 1% significance levels for some endpoints did not have adequate justification and that only one level should be used. Response: Contract laboratories provide both levels of significance to satisfy needs of a range of

clients and both values are often identified in standardized statistical packages for computers.

Inquired about the meaning of the statement under Motor Activity and FOB data that “separate analyses by sex may have been done to explain the nature of the effect”. Response: Addi tional analysis of neurobehavioral endpoints was requested by EPA for 211(b)

subchronic studies. That work was performed by ExxonMobil Biomedical Sciences (M. Nicolich

and G

. Bukhbinder ) and used to write the draft report. The WG will instruct the study director to

work with the contract statisticians to further clarify the study report.

Section 3.1 [p. 48] Chamber Monitoring

. Dr Schlesinger requested an explanation of why “some particles are invariably occurring in

chamber air”. He assumed that if chambers were properly filtered there should be no particles. Response: The exposure chambers draw in air room the surrounding exposure room to dilute the evaporated test substance. The surrounding room air is conditioned for temperature and

relative hum idity but is not filtered to any substantial extent. Therefore, the chamber air for all test groups is reflective of the room air during the conduct of a gas or vapor exposure. This laboratory typically finds room air to contain background particles on t he order of 10 - 3 mg/m 3

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which was the range for this study. The WG will ask the study director to clarify this issue in the report.

Section 3.4 [p. 50] Body Weight Dr. Schlesinger suggested that the weight gain seen in the 10000mg/m 3 group females might indicate that “something is different about these animals.” Response: The higher body weights in the 10000mg/m 3 female rats and the statistically significant (p<0.05) increase in overall weight gain (wk 0

- 12) originated back at the beginning of

the stud y. Although all groups were matched for body weight at 1 week prior to study initiation, the 10000mg/m 3 females already weighed 3 grams more by week 0 the start of the study. It would appear that the rats in this group grew faster than other groups inclu ding the controls and that the weight gain was unlikely to be related to exposure. Moreover, as stated in the report,” increased weight gain is not usually considered an adverse response to treatment”.

Section 3.6 [p. 50] Feed Consumption Dr. Schlesinger noted that both male and female high dose rats showed increases feed consumption beginning at week 14 and requested that this observation be included in the text. Response: Weeks 14

- 17 constitute the untreated recovery period for this study. Although sl ight increased feed consumption occurred [Males 2

- 3 g; Females 2

- 4g, 7g at wk16] compared to controls, increases were not statistically significant and were not of sufficient magnitude to be considered reportable in the text. The WG does not consider it n ecessary to amend the report.

Section 3.7.1 [p.50] Motor Activity Both peer reviewers had comments on this section. Dr. Goldsworthy recommended that the reasons for discounting the statistical significance in motor activity (MA) in the low and mid dose gr oups be added to the Summary. The WG did not consider it necessary to expand the Summary with this information (see Response to Summary on p.1)

Dr. Schlesinger : Is the response difference between low and mid groups found in the Pretest MA or in the data obtained during exposure? Response: MA changes occurred in the exposure data. As indicated in Table 8, which includes the analyses o f data report from ExxonMobil Biomedical Sciences (M. Nicolich and G

. Bukhbinder ) , the pretest response is used as a cova riate in the mixed model analysis of covariance and is not analyzed independently.

Requested clarification of the basis for the statement that “there is an increase in activity in the

females at mid

- exposure during the 4 th week”. Response: This comment a ppears inaccurate. The statement in the report says “data plots do not suggest a consistent exposure related response, but show an increase in activity in the female control group and decrease in activity in female mid exposure group only during week

4.” Additional analysis of neurobehavioral endpoints was requested by EPA for 211(b)

subchronic studies. That work was performed by ExxonMobil Biomedical Sciences (M. Nicolich

and G

. Bukhbinder ) and used to write the draft report. The WG will instruct the s tudy director to

work with the contract statisticians to review and if appropriate clarify the text in the report.

Section 3.8 [p. 51] Clinical Pathology Both reviewers requested more detailed discussions of statistical changes observed in hematology and clinical chemistry parameters. Response: The biological significance of variable statistically significant changes in clinical pathology parameters in subchronic studies is difficult to determine in the absence of overt

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physical effects or histologically observed tissue changes. The study duration is comparatively short and individual animal variability may produce wide standard deviations.

Section 3.8.1 [p. 51, Table 10] Hematology Dr. Goldsworthy: Requested revision of reticulocyte count statements h ere and in the Summary (p. 7). He recommended that the designations „very slight” and “slight” be replaced with “statistical” decreases and that concluding statements (p. 7 and 55) should clarify that the decreases in reticulocyte counts occurred at all d ose levels [in males] at week 4. Response: The WG agrees that “slight” and “very slight” should be replaced with “statistical” unless there is an established quantified definition of these terms for “% change”. Although decreases in reticulocyte counts w ere observed in all male dose groups at week 4 as reported in the text (p. 51

- 52), only the 20000mg/m 3 males showed consistent non

- reversible occurrences at termination and after the recovery period warranting inclusion as a significant finding in the conc lusions (p. 55) and Summary (p.7). No revisions are needed.

Noted that the text did not address statistically significant (p<0.01) decrease in absolute monocyte counts in 20000mg/m 3 females at week 4 and at recovery. Response: Although the statisticall y significant decrease in absolute monocyte counts in high dose females at week 4 and recovery (values at termination were lower than controls but were not statistically significant) are not likely to be biologically significant, the WG will instruct the

s tudy director to include this finding in the text with appropriate comment.

Section 3.8.2 [p. 52] Coagulation time Dr. Schlesinger suggested that the increase in mean coagulation values in all animals exposed to

BG+DIPE VC should be addressed. Response: The cited increase in coagulation values occurred only in male rats at week 4. Increased prothrombin time and activated partial prothrombin time (APPT) were elevated above controls at approximately the same value for all doses at a significance level of 0 .05 or 0.01 respectively (PT: 11.9 sec. control vs 12.5, 12.6, 12.5 seconds for low, mid and high doses and APPT: 17.7 sec.control vs 20.4, 20.4, and 21.1 seconds for low, mid and high doses, respectively). At terminal sampling, values were similar to wee k 4 but were not statistically significant. The study director will add a description of this effect to the text but the change is likely a statistical anomaly rather than biologically significant in the absence of dose

related increases and no statistica lly significant value at the terminal evaluation.

Section 3.8.3 [p. 52] Clinical Chemistry Both reviewers commented that this section does not adequately address the data and cite CK and SDH changes in males and SDH changes in females. Discussion appeare d to focus on males only. Although Dr. Goldworthy agreed that “it is appropriate to not consider these clinical chemistry effects adverse”, he maintained the effects should be included in the results and the Summary. Response: The WG agrees that addition al information should be supplied in the Clinical Chemistry section. Expanded entries will be included on statistically significant changes in lactate dehydrogenase and sorbitol dehydrogenase levels in males and female animals, and

increased levels of Na+ and K+ in female rats at all sampling intervals. The Summary section (p. 7) will be revised to include female data entries. However, the WG agrees with the study director that these changes are not consistent enough to be considered biologically signifi cant effects of BG+DIPE VC exposure.

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Dr. Schlesinger was concerned that changes at week 4 that did not also occur at termination were not regarded as significant. Response: Statistically significant changes that were observed only at week 4 in this study

, included decreased glucose and creatinine kinase levels in males at all dose levels and bile acids (all groups), glucose (high dose) and alt (high dose) in females. In the absence of a persistent pattern of effect at these dose levels at termination and /or in high dose recovery animals, these early changes are considered transient adjustments to exposure that have no biologically significant effects.

Section 3.9 [p. 53] Organ Weights Dr. Schlesinger pointed out a decrease in lung weight in males exposed at 20000mg/m 3 that

should be included in the text. Response: The text indicated that a few statistically significant differences in organ weights were observed but no details were given. The WG will advise that study dire ctor to include an entry

that “ a statistically significant decrease in absolute lung weight compared to controls was observed in 20000mg/m 3 exposed male rats but no statistical significance was found for lung weight relative to body weight or to brain weight, and no abnormal histopatholo gy was observed.”

GFAP Assay [Dr. J. O’Callaghan] Both reviewers agreed that the study was properly conducted and agreed with the conclusion that BG+DIPE VC did not cause gliosis in the brain regions examined. Dr. Schlesinger considered this a well wr itten report and requested only that the name of the
post

- hoc test used be provided in the section on Statistics (p.17) Response: The study director will request Dr. O‟Callaghan to supply this information.

Dr. Goldsworthy referred to earlier comments on the absence of study specifics and setting of criteria prior to study performance. Response: The WG agrees with these comments. Dr. O‟Callaghan has been requested to expand on specific criteria to identify positive or suspect materials. However, widespre ad use of this assay is relatively recent and firm parameters to identify responses are still being established. It is also worth noting that Dr. O'Callaghan is internationally recognized for his work on the GFAP assay, and the WG has confidence in the in tegrity of the study methodology (including statistics) and its conclusions. Further, the WG has discussd with Dr. O‟Callaghan, deletion of sparse pituitary data from Tables 5 and 6, but retention of individual values in Table 7, primarily because this se ries of studies is one of the first in which GFAP levels in pituitary have been measured. This study had usable data only for 2 males and 1 female in the control group and three males in the 20,000mg/m 3 dose group. To include this limited data in summary tables (5&6) gives an erroneous impression that sufficient animals/group responded. Keeping individual entries (Table 7) provides an idea of the level of GFAP likely to be present in this small organ, providing a starting point for improvement of assay m ethods.

Immunoto xicology Assay [Immunotox, Inc. ] Dr. Schlesinger inquired about the statement in the text that the “effect of DIPE on spleen weights and spleen cell numbers cannot be appropriately evaluated since the animals in this study were sensitize d with sheep erythrocytes”. He wondered why these data are included if it is not possible to provide any conclusion. Response: The statement in this report refers to comparison of these data with the spleen data

in the 13

- week study in which these animal s were co

- exposed but where no effects on spleen weight were reported. The overall increased relative spleen weights from animals induced with

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S

- RBC is not indicative of the effect of BG+DIPE VC on rats in general but is believed to be a useful measurement for immunologically related changes in this assay by the study director (Dr. White). The WG will not ask for changes in the report.

Dr. Goldsworthy: Requested that some historical control data be supplied to determine the significance of the

decrease i n spleen IgM response at the 10000mg/m 3 level compared to “normal vehicle controls”. The text [p. 16] had stated that vehicle control animal values were „somewhat lower than had been observed in the past.” Response: Dr. White will be requested to supply historical vehicle control data for this comparison.

Suggested that the report conclusion on p. 9 and p. 17 provide a NOAEL = 10000mg/m 3

. Response: Dr. White will be requested to consider adding a NOAEL to the report.

Micronucleus Assay [Huntingdon, UK] Both reviewers agreed that the study was properly conducted and agreed with the conclusion that BG+DIPE VC did not cause an increased frequency of micronucleated immature erythrocytes in bone marrow of treated rats. No other comments were made.

Sister C hromatid Exchange Assay [BioReliance, Inc.] Both reviewers agreed that the study was properly conducted and agreed with the conclusion that BG+DIPE VC did not induce sister chromatid exchanges (SCE) in rat peripheral blood. This study employed the statist ics recommended by EPA.

Dr. Goldsworthy suggested that it be reported in the Summary that the positive control fulfilled the requirements for a valid test and that the study title should include Gasoline

- DIPE Vapor

Condensate. Response: The WG will reque st the study director to include the statement about the positive control in the Summary. It does not seem necessary to change the title of the report since

“Gasoline

- DIPE Vapor Condensate” appears in the next line under Test Substance.

Although the resu lts of this study are clearly negative, Dr. Schlesinger was concerned in general about some criteria used to evaluate SCE data. He did not agree that “a statistically significant increase in the high exposure level without statistically significant exposu re level responses at lower doses should be considered suspect”. He suggested that this event should be considered a full positive response indicating that “some exposure threshold for response had

been reached”. Response: The SCE assay was developed as an indicator of DNA perturbation with possible application for suggesting a potential for chromosome or other genetic damage. Criteria for positive responses developed by many investigators evaluating numerous assays were designed to be sufficiently rigor ous to minimize false positive and negative results in relation to

other in vivo genetic assays and to strengthen predictability and reliability. The designation of “suspect” when statistical significance was observed only at the highest dose, and “equivo cal” when positive results are observed at two doses in the absence of an exposure response trend contributed to improving this reliability by ranking the relevance of responses. The scientific expertise of the investigator is a significant factor in the accurate assessment of SCE data.