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 Gasoline 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 Condensate. A 13 Week Whole-Body Inhalation 

Toxicology Study in Rats with Neurotoxicology 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 period. In this report, 

standard toxicology and neurotoxicity parameters were presented (genotoxicity and 

immunotoxicity results were presented in separate reports; all reviews presented below). 

The report was clearly written and comprehensive. The design of the study was 

appropriate and sound and the study was properly conducted. Environmental conditions 

(temperature, humidity, noise level, illumination) 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 expected finding - for consistency 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 adequate 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-TBA in their expected proportions. Week to 

week data consistency was noted, indicating 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 absolute 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 exposure-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 potential change that was deemed not exposure 

related. An increase in motor activity during exposure and recovery periods were seen in 
the high exposure group relative to the control group. The revised statistical procedures 

undertaken (Protocol Amendments 1 and 2) appear appropriate to assess the responses 

observed. 

 Effects of G-TBA exposure were noted in hematology parameters and coagulation 

values in Table 10; report notes and suggests that these slight differences were most 

likely attributable to normal variation and supported by lack of evidence of 

immunotoxicity or histopathological changes. However, the data demonstrating 

decreased reductions in lymphocytes counts and decreased basophil counts are (a) 

different in both male and female exposed animals, (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 control values. I would consider 

these changes to be attributable to G-TBA exposure and thus Report text (Summary, 

Conclusions, and Results - page 52) may be modified to reflect this. Serum cholesterol 

values were increased in the mid and high exposed 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 brain 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 the kidneys of high dose male rats. These renal 

findings were consistent with hydrocarbon-induced nephropathy (even though the 

presence of alpha 2u-globulin was not confirmed by Mallory staining). 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/m3

 exposure 

group. This may be expected since 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 comment on nephropathy, Conclusion (as well as 

Summary and Results - page 56) should note the 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 Experimental 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 study specifics, remain. The text reports the 

statistical alterations in the cortex and hypothalamus. Given the lack of dose-response 

and 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 although not always statistically 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 hypothalamus values than usually observed in historical control 

values. Overall , I concur with the generalized conclusion the G-TBA does not appear to 

result in significant brain gliosis as measured by enhanced GFAP regions. Text should 

restate and clarify the potential hypothalamus alterations as well as modify conclusion to 

note that only 8 brain regions were examined (pituitary was not fully assessed). 

Study Title: In Vivo-In Vitro Rat Peripheral Lymphocyte Sister 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 note that the positive 

control fulfilled the requirements for a valid test. 

Study Title: Satellite Procedure 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 micronucleated 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 Evaluation 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 effect 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 assay conditions. The conclusions that, under the 
experimental conditions of this study, G-TBA 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.