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Re: The 211(b) Working Group Response s to the Inde pendent Peer Reviewers Comments on a

13

- week Whole Body Inhalation Study of Baseline Gasoline + Tertiary Butyl Alcohol Vapor

Condensate and Satellite Studies

In the 211(b) testing program, the draft report s 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 th e draft reports for studies with baseline gasoline + TBA vapor condensate (BG + TBA VC). The studies include a 13

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

- micronucleus and sister chromatid exchange (SC E), immunotoxicity and glial fib ri llary acidic protein ( GFAP ) analysis. All studies were performed with animals exposed to BG + TBA VC under the same chamber conditions. For the genetic toxicology and immunotoxicity studies, animals were exposed for 4 we eks; for GFAP analysis, animals were exposed 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 recom mended by EPA.

The reviewers concur that, 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 recommenda tions 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 performi ng laboratory’ s study d irector s will appear in final reports of all studies.

13 week Subchronic Inhalation Study [Huntingdon, USA]

General Comments : Both reviewers accepted a NOEL = 2000mg/m 3

Dr. Schlesinger pointed out inconsistency in numbering of ex posure groups in different sections of the report

– Roman numerals in some tables, 1 2, 3, 4 in others. Response: This inconsistency will be brought to the attention of the study director and, where practical, adjustments can be made. However, in compute r generated tables and compilation of raw data the number format is predetermined. This observation does not affect the validity

of the data.

Dr. Schlesinger recommended that consistent criteria for data evaluation be employed, especially levels of signi ficance 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 satellite studies were performed in different contract laboratories with differe nt 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 va lues are often identified in standardized statistical packages for computers.

Summary [p. 5

- 7 ] Hematology [p. 6 ]: In accordance with comments by both peer reviewers, an expanded discussion of the hematological changes will be requested to reflect additi onal information in the body of the report.

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Kidneys [p. 7 ]: In response to Dr. Goldsworthy’s request, this section will be clarified to indicate that only kidneys of the 20000mg/m 3 and control animals were examined histopathologically. It

will also be s tated that the presence of alpha 2

- microglobulin was not verified with Mallory

Heidenhain stain.

Section 2.20 [pp 42

- 44] 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. Response: A single computerized statistical testing package was employed for all parameters listed on p.42

- 43 of report. In this package, analyses by individual sex or sexes combined

were prog rammed to 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

s tatistical method employed routinely for repeat

- dose studies throughout this testing facility was not needed. Revisions of statistical methods for neurobehavioral parameters were requested by EPA for previous 13

- week studies and are outlined in this secti on.

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

Section 2.3 [p. 14] Experimental Outline Dr Goldsworthy pointed out that an error in this table under the heading Recovery Control Group, Microscopic Pat hology; the number of control males should be 9. Response: Dr. Goldsworthy is correct. The correct number will be inserted.

Section 3.1 [p. 48] Chamber Monitoring

. Dr. Schlesinger Cited the slight difference from expected 1:1 ratio between measured and nominal concentration, attributed by the study director to slight inaccuracies in calibration of chamber airflows and calibration of IR monitor and inquired why any calibration inaccuracies of IR should have

occurred? Response: In this study, measured co ncentrations slightly exceeded target concentrations and were very close to nominal values, providing a reliable determination of exposure. Variations

between measured and nominal co ncentrations were less than 0.1 % at 2000 and 10000mg/m 3

and less than 5% at 20000mg/m 3 . Nominal concentrations are calculated by measuring the weight differences in the 5 gal. cylinder from which the test sample is dispensed, divided by the total vol. of air (m 3 ) passing through the chamber. Large differences between nominal to

measured concentrations, if they occur, are useful in identifying possible leakage in sample delivery or chamber distribution, and could suggest disposition of test material on chamber walls, reducing the amount of material in the breathing atmosphere. Small differences have minimal significance. Calculation of nominal concentration can be most affected by the accuracy of calibration of the airflow rate, making calculation of nominal concentrations a less

accurate measure of chamber concentrations than analytical determinations. The calibration

of the IR monitor is the more accurate calibration than the chamber airflow calibration because the IR monitor calibration involves the precise injection (using gastight syringes) of fixed

volumes of test subst ance into a known and fixed volume of air. By comparison, the chamber airflow calibration involves the measurement of large volumes of air (~200 LPM) using a mass flow meter, a procedure that is inherently less accurate. Small variation (<5%) between the se

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measurements is not uncommon. This explanation will be added to the report if desired by

EPA.

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 r o om the surrounding exposure room to dilute the evaporated test substance. The surrounding room air is conditioned for temperature and relative humidity but is not filtered to any substantial ex tent. 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 t ypically find s room air to contain background particles on the order of 10 - 3 mg/m 3

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

Section 3.3.2 [p. 49, Table 2] Weekly Clinical Observations Dr. Schlesinger requested clarification about “yellow ano

- genital staining in 20000mg/m 3 females

and the oth er test animals”. He inquired what other test animals were involved. Response: This comment seems to reflect a misinterpretation of the sentence in the report which states: “An exposure

- related increase in the frequency of yellow ano

- genital staining was seen in the 20000mg/m 3 exposed female animals compared to Air Control and the other test substance exposed animals . Yellow ano

- genital staining was not observed at all in control or 2000mg/m 3 animals and only in 1

- 2 female rats at 10000mg/m 3 toward the e nd of the study. Only high dose females showed an exposure related response. The WG will ask the study director to rewrite this sentence to better clarify the observation.

Section 3.5 [p. 50, Fig. 1] Body Weight Dr. Schlesinger pointed out that the mean body weight of females in the figure seemed to decrease in an exposure related manner compared to controls, especially in Group IV and that this is inconsistent with the text. He also inquired why the statistically significant decrease in relative body w eight gain of 20000mg/m 3 females over weeks 0

- 4 was considered transient. Response: Dr. Schlesinger is correct that the mean body weight of treated females was slightly lower than controls, especially in the high dose group. However, w eekly body weight c hanges were only statistically significantly lower for 20000mg/m 3 females during week 0

- 4 interval. Thus, in the absence of statistically significant lower body weight in BG+TBA vapor condensate treated females, results were not considered toxicologically significant but were indicators of exposure. The statement that decreases in relative body weight of 20000mg/m 3 females for

the initial f our weeks of exposure was a “transient difference” also reflected the absence of any other statistically significant effect on this parameter. The WG will discuss revisions of the text for this section with the study director to more accurately describe the data and explain the rationale for the conclusions. Dr. Goldsworthy was satisfied with the conclusions.

Section 3.8 [p. 51] Clinical Pathology Both reviewers requested more detailed discussions of statistical changes observed in hematology parameters. Dr. Schlesinger commented that the absence of immunotoxicity or histopathologic effects should not serve as justif ication to discount clinical pathology effects. 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 physical effects o r histologically observed tissue changes. The study duration is comparatively short and individual animal variability within a small sample size may produce wide standard

deviations. The WG will ask the study director to revise the text to more fully des cribe the

effects observed.

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Section 3.8.1 [p. 51, Table 10] Hematology Both Dr. Schlesinger and Dr. Goldsworthy requested text in this section be revised. They considered changes in hematology parameters (e.g. lymphocyte counts and basophil counts)

t o be attributable to BG

- TBA exposure. Response: The concerns raised by both reviewers will be addressed by the WG in discussion with the study director. Statistically significant lower total leukocyte counts (WBC) accompanied by lower differential counts of lymphocytes and basophiles compared to controls were seen in high dose males at both wk 4 and wk 13 and total counts were slightly but not statistically significantly lower in recovery high dose males. As contrast, in high dose females, erythrocyte co unts (RBC) were elevated at wk 4 and wk 13 (p<0.01) although the WBC counts were lower only at wk 13 in all dose groups; in recovery females, all of these parameters were comparable to controls. Considering the differences in response between sexes and th e absence of histological variations in blood forming organs or other systems affected by hematological changes, it is difficult to assess the biological significance of the events as related to BG+TBA exposure. However, the text of this section will be r evised to more fully describe the effects observed, and the Conclusions and Summary sections adjusted accordingly.

Section 3.8.3 [p. 53] Clinical Chemistry Dr. Schlesinger inquired of the basis for the statement that “serum enzyme deceases are generally not considered indicative of test substance induced toxicity...”. Response: The WG agrees that this statement requires substantiation. The study director will be requested to supply appropriate reference for this statement.

Section 3.9 [p. 53

- 54] Organ W eights Dr. Goldsworthy requested that the report state that the kidneys of male rats exposed to 10000mg/m 3 were not examined histopathologically and thus the increased kidney weight reported in this group can only be suggested to correlate with alpha 2

- mic roglobulin neuropathy. Response: Text on p. 53 will be amended as “Although only kidneys from male rats exposed to 20000mg/m 3 were examined histopathologically, the kidney weight increases seen in 10000mg/m 3 males as well as those in 20000mg/m 3 males are likely correlated with microscopic findings discussed in the following section.” Text in Conclusion and the Summary will be revised accordingly.

Section 3.10.2 [p. 54

- 55] Pathology

– Microscopic Dr. Goldsworthy recommended that Mallory

- Heidenhain staini ng be performed on sections of at least some kidneys of high dose males to confirm the statement of the presence of alpha 2

- microglobulin neuropathy. Response: Alpha 2

- microglobulin neuropathy has been demonstrated previously in this series of studies emp loying gasoline condensate va por alone and gasoline+MTBE vapor condensates. Male rat kidney sections were stained with Mallory

- Heidenhain stain to confirm the presence of alpha 2

- microglobulin from animals exposed to gasoline vapor condensate and gasoline +MTBE vapor condensate. The WG decided that, since this neuropathy had been confirmed and was not related to human health issues (US EPA, 1991), special staining for the other gasoline+oxygenate blend studies could be omitted. The WG will have the Conclu sions (Sect 4.0) and the Summary revised to state that “Exposure to gasoline TBA vapor condensate resulted in kidney weight increases and histopathological results consistent with hydrocarbon nephropathy in male animals exposed to 20000mg/m 3 of vapor. Inc reases in kidney weight were also observed in male rats exposed t o 10000mg/m 3 ”.

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Section 4.0 [p. 56] Conclusions As requested by Dr. Goldsworthy the increased kidney weights reported for 10000mg/m 3 and 20000mg/m 3 males will be added to text.

GFAP Assay [Dr. J. O’Callaghan] Both reviewers agreed that the study was properly conducted and agreed with the conclusion that BG+TBA vapor condensate did not cause gliosis in the brain regions examined. Dr. Schlesinger considered this a well written report and r equested 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 stu dy specifics and setting of criteria prior to study performance. Response: The WG agrees with these comments. Dr. O’Callaghan will expand on specific criteria to identify positive or suspect materials. However, the use of this assay is not widespread 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 integrity of the study methodology (including s tatistics) and

its conclusions. Further, the WG has discussed 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 series of

studies is one of the first in which GFAP levels in pituitary have been measured. This study had usable data only for 1 male in Control and 10000mg/m 3 groups and 1 female in the 2000mg/m 3 group. No pituitary data was available in the 20000mg/m 3 dose group. To include

this limited data in s ummary 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

a ssay metho ds.

Dr. Goldsworthy also noted that in the absence of adequate pituitary data only 8 brain regions should be cited in the Summary. Response: Entry will be revised.

Results and Conclusions [p.17] Dr. Goldsworthy suggested that the decrease in hypothalamus GFAP values appeared “real” since it was observed mainly at the highest dose and was statistically significant in males and also showed a similar exposure

- related trend in females. However, he acknowledged that the significance might have res ulted from comparison to control values higher than seen in historical data. He agreed with Dr. O’Callaghan’s conclusion that BG

- TBA vapor condensate did not appear to induce significant brain gliosis as measured by enhanced GFAP but requested that text s hould “restate and clarify the potential hypothalamus alterations”. Response: The WG accepts Dr. O’Callaghan’s assessment that the neurotoxicological significance of decreases in GFAP is unknown and thus cannot be interpreted as adverse. Dr. O’Callaghan will insert into the report on R/D page 17: “Given that the decreased levels of GFAP in the hypothalamus occurred at only the high concentration, we cannot rule out the possibility that such an effect represents an adverse effect. Nevertheless, it is not ed that the 2 significant effects on GFAP occurred in brain regions that are difficult to reliably dissect and this may have been a factor in the outcome of the results”.

Dr. Schlesinger pointed out that “there appears to be the inference that the minimal changes observed in this study may, in fact, represent real effects of exposure if more samples had

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been analyzed. Thus, the reviewer is unclear as to whether or not there should be some concern regarding the test material”. Response: T he WG acknowledg es this comment and thinks that Dr. O’Callaghan has included such a caveat because the assay is relatively new and criteria are not fully set. Dr. O’Callaghan will insert into the report on R/D page 17: “Given that the decreased levels of GFAP in the hyp othalamus occurred at only the high concentration, we cannot rule out the possibility that such an effect represents an adverse effect. Nevertheless, it is noted that the 2 significant effects on GFAP occurred in brain regions that are difficult to reliab ly dissect and this may have been a factor in the outcome of the results”.

Immunotoxicology Assay [ Immunotox, Inc. ] Both reviewers agreed that the study was properly conducted and agreed with the conclusion that exposure to BG+TBA vapor condensate did no t adversely affect the functional ability of the humoral immune component of the immune system under conditions of this assay. No other

comments were made.

Micronucleus Assay [Huntingdon, UK] Both reviewers agreed that the study was properly conducted an d agreed with the conclusion that exposure to BG+TBA vapor condensate did not cause an increased frequency of micronucleated immature erythrocytes in bone marrow of treated rats.

Dr. Schlesinger has a few comments relating to syntax. On p.11, he noted that “?” in the first paragraph should be replaced by appropriate dates, and that either “negative control’ or air control” should be used in the text and not applied interchangeably. Response: The study director will supply the appropriate dates for the exposure portion of this study. The study investigator will be directed to use either “negative control” or “air control” in

the text but not both.

On p. 13, Dr Schlesinger inquired whether the items listed under Deviation from Protocol are really differ ent from protocol specification or just additional information and perhaps should be headed differently. Response: The WG believes that the protocol stated that the statistical evaluation would be performed at HRC and the change of location was therefore a protocol deviation. Similarly the change of duration of the slide hydrolysis was a deviation. The additional GLP regulations are not strictly a deviation from protocol and may be removed from this section. However it is not inappropriate to leave them as a deviation as well. The WG will request the study director to clarify these entries.

Sister Chromatid Exchange Assay [BioReliance, Inc.] Both reviewers agreed that the study was properly conducted and agreed with the conclusion that exposure to BG+ TBA vapor condensate did not induce sister chromatid exchanges (SCE) in rat peripheral blood. This study employed th e statistics recommended by EPA.

Dr. Goldsworthy suggested that it be reported in the Summary that the positive control fulfilled the req uirements for a valid test. Response: The WG will request the study director to include the statement about the positive control in the Summary.

Dr. Schlesinger requested that the study director identify which primary statistical test (p. 11) was

used to determine whether a post hoc test such as Dunnett’s was needed. Response: The WG will request the study director to revise the report to address this question.

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Dr. Schlesinger considered “the last sentence in the Evaluation of Test Results section ( p. 11) is somewhat unclear and seemed to indicate a large degree of potential subjectivity in the

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

other in vivo genetic ass ays and to strengthen predictability and reliability. The designation of “suspect” when statistical significance was observed only at the highest dose, and “equivocal” 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.