Dr. Lorraine Twerdok October 3, 2004

Manager, Health Sciences

API

1220 L Street, NM Washington, DC 20005

Dr. Twerdok,

Below is my review of the various Gaso line TBA (G-TBA) studies conducted for the American Petroleum Institute. Comments are provided for each report. Please feel free to contact me if there are any questions.

Study Title: Gasoline-TBA Vapor Condensat e. A 13 Week Whole-Body Inhalation Toxicology Study in Rats with Neurotoxi cology Assessments and 4-week In Vivo Genotoxicity and Immunotoxicity Assessments

The potential inhalation toxicity of G- TBA was assessed via whole body exposures to male and female rats for 13 weeks followed by a 4-week recovery peri od. In this report, standard toxicology and neur otoxicity parameters were presented (genotoxicity and immunotoxicity results were presented in sepa rate reports; all revi ews presented below). The report was clearly written and compre hensive. The design of the study was appropriate and sound and the study was pr operly conducted. Environmental conditions (temperature, humidity, noise level, illuminatio n) were appropriate. The deviations noted had no impact on the overall study conclusions or data integrity; the lack of the Mallory Heidenhain staining to confirm the presence of alpha 2u-globulin nephropathy does not permit the report to definitely state this e xpected finding - for cons istency and to place this sole major finding within proper context, I recommend staining with Mallory’s on at least some high dose animals. Exposure techniques and procedures were ad equate to test the potential toxicity of GTBA. Exposure levels of test substance were relatively close to target levels. Animals were exposed to all major components of G-TB A in their expected proportions. Week to week data consistency was noted, indicatin g stability of test substance and proper techniques. Experimental data revealed no effect of G-TBA on survival, feed consumption or gross abnormalities at necropsy. Yellow ano-genital staining was observed in high exposed

female animals; these observations abated following the 4 week recovery period. No exposure related changes were noted in ab solute body weights or in final body weight changes; a transient decrease in body weight gain was observed for the first 4 weeks of exposure in high dosed female rats. No ex posure-related effect was observed with opthalmoscopic assessments. G-TBA exposure did not appear to be associated with FOB measurement count changes; foot splay data indicated a poten tial change that was deemed not exposure related. An increase in motor activity during ex posure and recovery pe riods were seen inthe high exposure group relative to the cont rol group. The revised statistical procedures undertaken (Protocol Am endments 1 and 2) appear appr opriate to assess the responses

observed. Effects of G-TBA exposure were noted in hematology parameters and coagulation values in Table 10; report notes and suggest s that these slight differences were most likely attributable to nor mal variation and supported by lack of evidence of immunotoxicity or histopathological change s. However, the data demonstrating decreased reductions in lymphocytes count s and decreased basophil counts are (a) different in both male and female exposed an imals, (b) at both the 4 and 13 week time points, and (c) often seen in the mid and high exposed groups. Furthermore, these changes were often statistically different, and when they did not reach statistical significance level, they were still decreased relative to cont rol values. I would consider these changes to be attributable to G-TB A exposure and thus Report text (Summary, Conclusions, and Results - page 52) may be modified to re flect this. Serum cholesterol values were increased in the mid and high ex posed male rats; this difference abated by the end of the recovery period. It is interesting that this change was only noted in male rats. No other significant alterations in clinical chemistry were attributed to G-TBA

exposure. Absolute and/or relative to body or brai n weight differences in kidney weights were observed in mid and high exposed male rats. These increases were expected, consistent with microscopic findings, and most likely due to hydrocarbon-induced nephropathy. No other exposure related differences in organ weight were noted. Microscopic findings were present in th e kidneys of high dose male rats. These renal findings were consistent with hydrocar bon-induced nephropathy (even though the presence of alpha 2u-globulin was not conf irmed by Mallory staini ng). The kidneys of the mid level exposure group in male rats were not examined microscopically and thus it could be determined if nephropathy was also present at the 10,000 mg/m 3 exposure group. This may be expected si nce renal weights were also increased at the mid exposure level. The text is written inappropriately regarding the induction of hydrocarbon induced nephropathy in that this finding is suggested to occur only in the high exposure group of

male animals. Text should be reworded (Results, Conclusions, Summary) to more accurately reflect what is known and what is not (e.g. only see high dose effect because only examined high dose). Summary: In addition to the above co mment on nephropathy, Conclusion (as well as Summary and Results - page 56) should note th e increased renal weights at the mid and high exposure groups in male rats. I concur that the 2000 mg/m3 exposure level be

considered the NOAEL.

Editorial: The Table on Page 14, Section 2.3 E xperimental Outline, lists 10 male rats in the Control Recovery Microscopic Pathology - Recovery Group; I believe this should be 9, as seen in the other recovery group listings.

Study Title: GFAP Levels in Specific Brain Areas Following a 13 Week Whole-body Inhalation Exposure to G-TBA.These studies appear to be properly conducted. Editorial comments from previous reviews regarding format, e.g. lack of st udy specifics, remain. The text reports the

statistical alterations in th e cortex and hypothalamus. Given the lack of dose-responseand the observance in only male rats, the cortex changes do not appear to be related to GTBA exposure. The decreases in hypothalamus values appear real since it is observed mainly at the highest exposure groups and a lthough not always statis tically significant, the decrease is seen in both males and females (additionally, the female data is a classic dose response and most likely positive in trend analysis). This result may possibly be due to the higher control hypothala mus values than usually obse rved in historical control

values. Overall , I concur w ith the generalized conclusion th e G-TBA does not appear to result in significant brain gliosis as meas ured by enhanced GFAP regions. Text should restate and clarify the potential hypothalamus alte rations as well as modify conclusion to note that only 8 brain regions were examin ed (pituitary was not fully assessed).

Study Title: In Vivo-In Vitr o Rat Peripheral Lymphocyte Si ster Chromatid Exchange Assay

This study was properly conducted and scientifically sound. The positive control fulfilled the requirements for a valid test. The report correctly concludes the G-TBA was negative for the induction of SCEs in male and female rat peripheral lymphocytes.

Editorial comments include suggesting that the Summary Section not e that the positive control fulfilled the requirements for a valid test.

Study Title: Satellite Procedur e G-TBA Rat Micronucleus Test

This study was properly conducted and scientifically sound. Positive and negative controls performed as expected and within historical range. G-TBA did not cause any significant increases in the incidence of micr onucleated mature erythrocytes and did not cause any decreases in the proportion of immature erythrocytes. No evidence of bone marrow toxicity was observed. I concur with the report conclusions.

Study Title: Immunological Ev aluation of G-TBA in Female Spraque Dawley Rats

This study evaluated the potential of 4 weeks of exposure to G-TBA to affect the humoral immune component of the immune system. This study was properly conducted and scientifically sound. Data demonstrated that G-TBA did not e ffect terminal body weights or spleen and thymus weights. G-TBA did not alter the spleen IgM antibodyforming cell response to sheep erythrocytes. The positive control CPS demonstrated the anticipated results, verifying the proper a ssay conditions. The conclusions that, under theexperimental conditions of this study, G-TB A did not adversely affect the functional ability of the humoral immune component of the immune system accurately reflect the study results.

Please contact me if you have any questions,

Sincerely,

Thomas L. Goldsworthy, PhD.