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Re: The 211(b) Working Group Responses to the Independent 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 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 reviewers have now provided 

their comments on the draft reports for studies with baseline gasoline + TBA vapor condensate 

(BG + TBA 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 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 weeks; 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 recommended 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 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 appear in final 

reports of all studies.

13 week Subchronic Inhalation Study [Huntingdon, USA]

General Comments:

Both reviewers accepted a NOEL = 2000mg/m3

Dr. Schlesinger pointed out inconsistency in numbering of exposure 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 computer 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 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 satellite 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. 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 additional 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/m3and control animals were examined histopathologically. It 

will also be stated that the presence of alpha 2-microglobulin was not verified with MalloryHeidenhain 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 programmed 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 

statistical 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 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.

Section 2.3 [p. 14] Experimental Outline

Dr Goldsworthy pointed out that an error in this table under the heading Recovery Control Group, 

Microscopic Pathology; 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 concentrations slightly exceeded target concentrations and 

were very close to nominal values, providing a reliable determination of exposure. Variations 

between measured and nominal concentrations were less than 0.1% at 2000 and 10000mg/m3

and less than 5% at 20000mg/m3. 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 (m3) 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 substance 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 these 
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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 room 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 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 the order of 10-3 mg/m3

which 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/m3females 

and the other 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/m3exposed 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/m3animals and only in 1-2 female rats at 10000mg/m3 toward the end 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 weight gain of 20000mg/m3females 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, weekly body weight changes 

were only statistically significantly lower for 20000mg/m3females 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/m3females for 

the initial four 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 justification 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 or 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 describe 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) 

to 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 counts (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 the 

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 revised 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 Weights

Dr. Goldsworthy requested that the report state that the kidneys of male rats exposed to 

10000mg/m3 were not examined histopathologically and thus the increased kidney weight 

reported in this group can only be suggested to correlate with alpha 2-microglobulin 

neuropathy.

Response: Text on p. 53 will be amended as “Although only kidneys from male rats exposed to 

20000mg/m3 were examined histopathologically, the kidney weight increases seen in 

10000mg/m3 males as well as those in 20000mg/m3 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 staining 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 employing gasoline condensate vapor 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 Conclusions 

(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/m3of vapor. Increases in kidney weight 

were also observed in male rats exposed to 10000mg/m3”.
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Section 4.0 [p. 56] Conclusions

As requested by Dr. Goldsworthy the increased kidney weights reported for 10000mg/m3and 

20000mg/m3 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 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 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 statistics) 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/m3groups and 1 female in the 

2000mg/m3group. No pituitary data was available in the 20000mg/m3 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 methods. 

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 resulted 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 should “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 noted 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: The WG acknowledges 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 hypothalamus 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 reliably 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 not 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 and 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 different 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 the statistics recommended by EPA. 

Dr. Goldsworthy suggested that it be reported in the Summary that the positive control fulfilled 

the requirements 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 chromosome 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 assays 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.