### STUDY NO. 00-4208
SPONSOR STUDY NO. 211-MTBE-2G

GASOLINE MTBE VAPOR CONDENSATE

A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION

TOXICITY STUDY IN RATS

Final Report

Volume IV of IV

Submitted to: American Petroleum Institute (API) 1220 L Street, Northwest Washington, D.C. 20005-4070

Attn: Thomas M. Gray, M.S., D.A.B.T.

Date: 14 June 2011

Page 1428 of 1650

### Huntingdon Life Sciences 00-4208 Page 1429 211-MTBE-2G Final Report

TABLE OF CONTENTS

Volume IV

COVER PAGE ...............................................................................................................1428

TABLE OF CONTENTS ...............................................................................................1429

APPENDICES

CC. Reproductive Assessm ent Report ............................................................................. 1430

DD. GFAP Report ............................................................................................................. 1461

EE. Certificates of Analysis – (Feed, Bedding and Water) ............................................. 1521

FF. Protocol and Protocol Amendments ......................................................................... 1591

GG. Testing Facility Personnel ......................................................................................... 1649

HH. Report Amendments .................................................................................................. 1650

### AUDITED FINAL REPORT

Study Phase: Reproductive Assessment

Test Site Phase Reference Number 3075-127

Testing Facility Study Number 00-4208

Sponsor Reference Number 211-MTBE-2G



# Gasoline MTBE Vapor Condensate: A Two-Generation Whole-Body Inhalation Reproduction

Toxicity Study in Rats

TEST SITE: Charles River Laboratories, Pathology Associates - Maryland

15 Worman’s Mill Court, Suite I Frederick, MD 21701

TESTING FACILITY: Huntington Life Sciences (HLS)
Mettlers Road
P.O. Box 2360 East Millstone, NJ 08875-2360

SPONSOR: American Petroleum Institute (API) 1220 L Street, Northwest Washington, D.C. 20005-4070

09-APR-2009

Page 1 of 31 Page 1430 Appendix CCFinal Report HLS Study No.: 00-4208

2 

### TABLE OF CONTENTS

Section Page No.

I. QUALITY ASSURANCE STATEMENT ................................4

II. MATERIALS AND METHODS ......................................5 A. Male Fertility Sample Collection ........................5

B. Sperm Motility Evaluation ...............................5

C. Caudal Epididymis Weight and Total Sperm Count
Determination .........................................6

D. Sperm Morphology Evaluation .............................6

E. Ovary Sample Collection and Processing ..................6

F. Ovarian Follicle Evaluation .............................6

G. Statistical Analyses ....................................7

H. GLP Compliance Statement ................................7

I. Sample and Data Archiving ...............................7

III. RESULTS ....................................................8 A. Sperm Motility ..........................................8

B. Total Sperm Count .......................................8

C. Sperm Morphology ........................................8

D. Caudal Epididymis Weight ................................9

E. Ovarian Follicle Evaluation .............................9

IV. DISCUSSION AND CONCLUSIONS .................................9

V. REFERENCES ................................................10 Page 1431Final Report HLS Study No.: 00-4208

3 

### INDEX OF TABLES AND APPENDICES

Table Page No.

1. Summary of P

0 

### Generation Male Reproductive Parameters .. 11

2. Summary of F

1 

### Generation Male Reproductive Parameters ...12

3. Summary of F

1 

### Generation Female Ovarian Follicle Evaluation ....................................13

Appendix Page No.

A. Individual P

0 

### Generation Caudal Epididymis Weight, Sperm Motility and Sperm Count Data .....................14

B. Individual F

1 

### Generation Caudal Epididymis Weight, Sperm Motility and Count Data ...........................16

C. Individual P

0 

### Generation Sperm Morphology Data............18

D. Individual F

1 

### Generation Sperm Morphology Data............20

E. Individual F

1 

### Generation Female Ovarian Follicle Count Data .....................................22

F. Sperm Analysis Historical Control Data ...................23 Page 1432Page 1433Final Report HLS Study No.: 00-4208

5 

### Gasoline MTBE Vapor Condensate A Two-Generation Whole-Body Inhalation Reproduction Toxicity Study in Rats

II. MATERIALS AND METHODS

Male fertility assessments, including sperm motility, homogenization-resistant testicular and epididymal sperm count, sperm morphology and caudal epididymis weight were conducted by PAI on all surviving male rats from control and high-exposure groups of each generation (P

0 

### and F

1 

### ). The ovarian follicle evaluation was conducted on each ovary from 10 female rats from the control and high-exposure group of the F

1 

### generation.

A. Male Fertility Sample Collection

All surviving male rats from each generation (P

0 

### and F

1 

### ) were euthanized by exsanguination following anesthesia with inhaled carbon dioxide before the last F

1 

### and F

2 

### litters, respectively, had been weaned. For all animals, the abdominal cavity was opened and the reproductive organs exposed. For motility assessment, the left vas deferens was dissected away from the testis and immediately placed in a petri dish containing 10 ml of a solution consisting of 1% Bovine Serum Albumin dissolved in Phosphate Buffered Saline. The solution was prewarmed to a temperature of approximately 38 ° C. A minimum 3 minute period was allowed for the sperm to swim out.

For total sperm count assessment, the left epididymis and left testis were then removed and placed on dry ice. The frozen epididymides and testes were then transferred to Pathology Associates - A Charles River Company (formerly known as Pathology Associates International until January 8, 2001), Frederick, MD. and stored frozen at -70 o 

### C until evaluation for caudal epididymal sperm count and homogenization-resistant testicular sperm count.

Only the control and high exposure animals were examined for percent motility, total epididymal and testicular sperm count and sperm morphology.

B. Sperm Motility Evaluation

Following the swim out period, a sperm sample was obtained using a 100 μ m deep cannula. The cannula was immediately loaded into the prewarmed stage of the Hamilton Thorne IVOS (version 10.9i for the P

0 

### generation and version 12.1c for the F

1 

### generation) automated sperm analyzer. The analyzer automatically Page 1434Final Report HLS Study No.: 00-4208

6 

### selected five fields and each motion image was digitally saved and permanently stored on optical media. The images were subsequently analyzed and the percent motility determined for each animal.

C. Caudal Epididymis Weight and Total Sperm Count Determination

Each frozen epididymis was removed from the freezer, thawed and the caudal section was trimmed and weighed. Each frozen testis was removed from the freezer, thawed, the tunica removed and the testicular parenchyma weighed. The cauda epididymis and testicular parenchyma were homogenized in deionized water and the suspensions were transferred to plastic test tubes and vortexed. A

100 μ l sample was transferred to a violet reaction vial containing a Hoechst dye (H33342) which uniquely stains the head of the sperm. A sample of the stained sperm was placed into a 20 μ m deep Cell-Vu glass slide which was loaded into the analyzer. Twenty fields were automatically selected by the analyzer for each animal and the number of cauda epididymal or homogenization-resistant testicular sperm determined. The counts reported were adjusted for caudal epididymal or testicular weight.

D. Sperm Morphology Evaluation

Two Eosin stained slides were prepared for each animal from the caudal epididymis total count preparation. The slides were evaluated and a minimum of 200 sperm cells/animal was examined for morphological development.

E. Ovary Sample Collection and Processing

The ovaries from 10 control and high-exposure F

1 

### female rats were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and evaluated microscopically for the numbers of primordial and growing follicles and the presence of corpora lutea

.

Five ovarian sections were taken at least 100 μ m apart from the inner third of each ovary. The ovarian sections were prepared by HLS personnel and evaluated by PAI personnel.

F. Ovarian Follicle Evaluation

Each ovarian section was methodically evaluated and the number of primordial and growing follicles counted and recorded. The counts are reported, per ovary and total, for primordial and growing follicles. The sections were also examined to confirm the Page 1435Final Report HLS Study No.: 00-4208

7 

### presence or absence of corpora lutea . The corpora lutea data was not summarized.

G. Statistical Analyses

The means and standard deviations for the sperm motility, caudal epididymal sperm count, testicular homogenization-resistant sperm count, sperm morphology and numbers of primordial and growing follicles by ovary and total were calculated and compared across groups using the Kruskal-Wallis nonparametric ANOVA test. If a significant effect occurred (p<0.05), the Mann-Whitney U test was used for pair-wise comparisons of each treated group to the control group. Animals with fewer than 25 sperm cells present in the motility analysis were excluded from the group mean calculation and subsequent statistical analysis. Animals with at least 25 sperm cells were included in the group mean calculation subsequent and statistical analysis; however, animals with fewer than 50 cells were identified to have a low count. The mean caudal epididymis weight for each group was calculated and compared using one-way analysis of variance (ANOVA) technique. If ANOVA is significant, Dunnett's test was used for pair-wise comparisons of each treated group to the control group at the 5% and 1% risk levels. Statistical analyses were performed using an

IBM TM 

### compatible computer with SAS computer programs (SAS/STAT User's Guide, 1989).

H. GLP Compliance Statement

The portion of this study performed by PAI was conducted in compliance with the 79.60, CFR Vol. 59, No. 122, 27 June 1994.

I. Sample and Data Archiving

Frozen epididymis and testis samples not processed for sperm count determination were transferred to and stored frozen at -70 o 

### C in PAI's long-term archive facility. The optical media used for permanent storage of the sperm motility images, raw data printouts from the sperm motility, epididymal and testicular sperm count determination, sperm morphology analysis, ovarian follicle count enumeration, and any supporting documentation, as well as the sperm morphology and ovarian histology slides will be maintained at PAI until the study has been completed. Upon finalizing the report, all non-frozen study material will be returned to HLS for archiving. Disposition of the frozen tissue samples will be directed by correspondence from HLS. Page 1436Final Report HLS Study No.: 00-4208

8 

### III. RESULTS

A. Sperm Motility

Table 1-2 (Summary Data) Appendix A-B (Individual Data)

No apparent treatment-related effects were observed in the P

0 

### or F

1 

### male sperm motility data. Group mean values were 91%, or higher, in the control and high exposure P

0 

### and F

1 

### generation males. No evidence of reduced motility due to treatment with gasoline MTBE vapor condensate (GMTBEVC) was observed.

B. Total Sperm Count

Table 1-2 (Summary Data) Appendix A-B (Individual Data) Appendix F (Historical Control)

No apparent treatment-related effects were observed in the homogenization-resistant testicular or caudal epididymal sperm counts in either generation. The sperm counts were reported as million sperm/gram of tissue or M/g. There was a statistically significant increase in the mean number of epididymal sperm for the 20,000 mg/m 3 

### exposure group (891.2 M/g) compared to the control in the P

0 

### generation (753.6 M/g). The apparent increase was attributed to a slightly lower P

0 

### control group value. No biologically meaningful differences were observed when the epididymal sperm count of the 20,000 mg/m 3 

### exposure group (891.2 M/g) was compared to the F

1 

### generation control group (887.5 M/g) or the mean reported in Pathology Associates Historical Control (869.9 M/g).

C. Sperm Morphology

Table 1-2 (Summary Data) Appendix C-D (Individual Data) Appendix F (Historical Control)

No apparent treatment-related effects were observed in the sperm morphology data of either generation. A statistically significant increase in the percent of abnormal sperm was observed in the 20,000 mg/m 3 

### exposure group of the P

0 

### generation (2.0% vs. 1.3% in the control group). The increase was minimal and was within Pathology Associates Historical Control range (0- Page 1437Final Report HLS Study No.: 00-4208

9 

### 6.7% abnormal sperm) and was not considered biologically meaningful.

D. Caudal Epididymis Weight

Table 1-2 (Summary Data) Appendix A-B (Individual Data)

No apparent treatment-related effects were observed in the caudal epididymis weight data for either generation. Group mean values ranged from 0.28-0.29 grams for the P

0 

### generation and 0.25-0.26 grams for the F

1 

### generation. No biologically meaningful differences were observed.

E. Ovarian Follicle Evaluation

Table 3 (Summary Data) Appendix E (Individual Data)

No apparent treatment-related effects were observed in the ovarian follicle count data for the F

1 

### animals evaluated. Group mean values were comparable between the control and high exposure group for the left, right and total primordial and growing follicle counts. Corpora lutea were present for all animals.

IV. DISCUSSION AND CONCLUSIONS

The percent sperm motility, caudal epididymal and homogenization-resistant testicular sperm counts, sperm morphology, caudal epididymis weight and primordial and growing follicle counts, as individual ovaries and total per animal, were not affected by exposure to gasoline MTBE vapor condensate at a dosage level of 20,000 mg/m 3 

### . A statistically significant increase in the number of sperm/gram of epididymis was observed in the 20,000 mg/m 3 

### exposure group of the P

0 

### generation. The mean value was comparable to the control group in the F

1 

### generation and to the mean reported in PAI's Historical Control. Additionally, a statistically significant increase in the percent of abnormal sperm was observed in the 20,000 mg/m 3 

### exposure group of the P

0 

### generation. The mean value of abnormal sperm is within the range of abnormal sperm reported by PAI's Historical control. As a result, the increase in the number of sperm/gram of epididymis and the increase in the percentage of abnormal sperm in the P

0 

### generation were not considered treatment-related.

No evidence of reproductive toxicity, as characterized by effects on sperm motility, epididymal and testicular sperm Page 1438Page 143911 HLS STUDY NO.: 00-4208

TABLE 1

GASOLINE MTBE VAPOR CONDENSATE
A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS

SUMMARY OF P

0 GENERATION MALE REPRODUCTIVE PARAMETERS

Group: I IV Exposure Level: GMTBEVC a (mg/m 3 ) 0 20,000

MOTILITY (%)

Mean 91 91
SD 9 8
N 25 26

EPIDIDYMAL COUNT (MILLION SPERM/GRAM)

Mean 753.6 891.2 *

SD 178.4 232.8
N 26 26

TESTICULAR COUNT (MILLION SPERM/GRAM)

Mean 99.3 108.5
SD 18.8 27.7
N 26 26

SPERM MORPHOLOGY b (% ABNORMAL)

Mean 1.3 2.0 *

SD 0.7 0.8
N 26 26

CAUDAL EPIDIDYMIS WEIGHTS (GRAM)

Mean 0.29 0.28
SD 0.04 0.03
N 26 26

a GASOLINE MTBE VAPOR CONDENSATE.

b MEAN AND STANDARD DEVIATIONS WERE CALCULATED USING THE TOTAL NUMBER OF ABNORMAL SPERM
AS A PERCENTAGE OF THE NUMBER OF SPERM EXAMINED.

* SIGNIFICANTLY DIFFERENT FROM CONTROL (p < 0.05). 

### Page 144012 HLS STUDY NO.: 00-4208

TABLE 2

GASOLINE MTBE VAPOR CONDENSATE
A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS

SUMMARY OF F

1 GENERATION MALE REPRODUCTIVE PARAMETERS

Group: I IV Exposure Level: GMTBEVC a (mg/m 3 ) 0 20,000

MOTILITY (%)

Mean 93 91
SD 7 12
N 25 24

EPIDIDYMAL COUNT (MILLION SPERM/GRAM)

Mean 887.5 933.1
SD 138.1 246.9
N 25 24

TESTICULAR COUNT (MILLION SPERM/GRAM)

Mean 96.7 102.2
SD 12.3 16.5
N 25 24

SPERM MORPHOLOGY b (% ABNORMAL)

Mean 0.7 0.6
SD 0.9 0.8
N 25 24

CAUDAL EPIDIDYMIS WEIGHTS (GRAM)

Mean 0.26 0.25
SD 0.03 0.04
N 25 24

a GASOLINE MTBE VAPOR CONDENSATE.

b MEAN AND STANDARD DEVIATIONS WERE CALCULATED USING THE TOTAL NUMBER OF ABNORMAL SPERM
AS A PERCENTAGE OF THE NUMBER OF SPERM EXAMINED.

NONE SIGNIFICANTLY DIFFERENT FROM CONTROL. 

### Page 144113 HLS STUDY NO.: 00-4208

TABLE 3

GASOLINE MTBE VAPOR CONDENSATE
A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS

SUMMARY OF F

1 GENERATION FEMALE OVARIAN FOLLICLE EVALUATION

PRIMORDIAL FOLLICLE COUNT

Group: I IV Exposure Level: GMTBEVC a (mg/m 3 ) 0 20,000

LEFT Mean 49.1 61.4
SD 30.1 28.6
N 10 10

RIGHT Mean 52.0 65.8
SD 19.4 28.2
N 10 10

TOTAL Mean 101.1 127.2
SD 47.8 51.9
N 10 10

NONE SIGNIFICANTLY DIFFERENT FROM CONTROL.

GROWING FOLLICLE COUNT

Group: I IV Exposure Level: GMTBEVC a (mg/m 3 ) 0 20,000

LEFT Mean 1.6 2.5
SD 1.8 2.2
N 10 10

RIGHT Mean 1.4 2.4
SD 1.1 1.8
N 10 10

TOTAL Mean 3.0 4.9
SD 2.1 3.5
N 10 10

NONE SIGNIFICANTLY DIFFERENT FROM CONTROL.

a GASOLINE MTBE VAPOR CONDENSATE. 

### Page 144214 HLS STUDY NO.: 00-4208

APPENDIX A

GASOLINE MTBE VAPOR CONDENSATE
A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS

INDIVIDUAL P

0 GENERATION CAUDAL EPIDIDYMIS WEIGHT,
SPERM MOTILITY AND SPERM COUNT DATA

Group I: 0 mg/m 3 (GASOLINE MTBE VAPOR CONDENSATE)

S P E R M C O U N T Animal Caudal Motility Caudal Homogenization Resistant No. Epididymis Epididymal Testicular Weight (g) (%) (million sperm/gram)

1051 0.29 94 614.5 82.4 1052 0.30 98 636.4 134.7 1053 0.31 86 1164.5 75.6 1054 0.33 99 936.2 121.6 1055 0.31 95 798.8 109.3 1056 0.36 89 961.0 90.5 1057 0.27 91 831.4 86.1 1058 0.28 100 743.8 88.6 1059 0.26 90 801.1 92.4 1060 0.36 94 945.0 82.3 1061 0.26 72 988.0 99.5 1062 0.24 94 602.6 99.6 1063 0.28 79 669.5 91.1 1064 0.28 78 429.8 84.4 1065 0.27 a 535.7 132.7 1066 0.26 97 427.2 120.2 1067 0.28 87 706.6 120.7 1068 0.27 99 677.1 109.5 1069 0.28 92 743.8 115.7 1070 0.34 97 690.8 102.2 1071 0.32 99 900.4 82.1 1072 0.24 89 573.7 124.9 1073 0.32 68 784.6 70.5 1074 0.33 97 992.3 75.2 1075 0.30 95 725.1 82.5 1076 0.23 99 714.4 107.7

a FEWER THAN 25 SPERM CELLS COUNTED IN 5 FIELDS ANALYZED; EXCLUDED FROM
STATISTICAL ANALYSIS. 

### Page 144315 HLS STUDY NO.: 00-4208

APPENDIX A

GASOLINE MTBE VAPOR CONDENSATE
A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS

INDIVIDUAL P

0 GENERATION CAUDAL EPIDIDYMIS WEIGHT,
SPERM MOTILITY AND SPERM COUNT DATA

Group IV: 20,000 mg/m 3 (GASOLINE MTBE VAPOR CONDENSATE)

S P E R M C O U N T Animal Caudal Motility Caudal Homogenization Resistant No. Epididymis Epididymal Testicular Weight (g) (%) (million sperm/gram)

4051 0.30 96 875.5 78.6 4052 0.28 88 1400.9 116.4 4053 0.29 99 857.8 102.8 4054 0.29 99 813.9 100.8 4055 0.27 84 908.5 108.9 4056 0.29 99 1201.0 132.0 4057 0.31 85 548.7 154.0 4058 0.28 89 913.3 77.5 4059 0.23 99 633.9 141.8 4060 0.27 86 1058.5 158.1 4061 0.35 93 558.7 108.6 4062 0.33 70 838.0 156.7 4063 0.30 99 725.1 147.0 4064 0.24 95 747.3 136.1 4065 0.28 95 789.3 70.7 4066 0.26 87 858.9 111.6 4067 0.32 85 922.1 104.7 4068 0.27 79 887.1 126.6 4069 0.28 88 578.5 94.7 4070 0.27 98 1058.5 81.9 4071 0.26 89 974.6 83.6 4072 0.27 94 1401.4 95.3 4073 0.27 76 784.2 70.1 4074 0.27 98 587.1 83.0 4075 0.31 100 1142.2 82.9 4076 0.27 95 1105.7 95.3 

### Page 144416 HLS STUDY NO.: 00-4208

APPENDIX B

GASOLINE MTBE VAPOR CONDENSATE
A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS

INDIVIDUAL F

1 GENERATION CAUDAL EPIDIDYMIS WEIGHT,
SPERM MOTILITY AND SPERM COUNT DATA

Group I: 0 mg/m 3 (GASOLINE MTBE VAPOR CONDENSATE)

S P E R M C O U N T Animal Caudal Motility Caudal Homogenization Resistant No. Epididymis Epididymal Testicular Weight (g) (%) (million sperm/gram)

1001 0.26 91 526.8 106.6 1002 0.22 90 922.1 77.0 1003 0.27 99 1018.5 89.8 1004 0.24 96 749.4 131.5 1005 0.32 96 797.3 98.8 1006 0.32 71 957.4 109.9 1007 0.27 96 863.6 106.5 1008 0.23 96 763.8 85.1 1009 0.32 93 777.7 105.1 1010 0.30 98 1021.2 92.1 1011 0.25 97 798.9 100.4 1012 0.30 98 644.8 101.6 1013 0.25 98 928.5 77.8 1014 0.24 96 1089.2 100.7 1015 0.28 75 914.9 103.2 1016 0.27 99 852.0 102.7 1017 0.27 93 739.7 101.6 1018 0.26 94 1009.4 102.0 1019 0.19 92 902.5 80.7 1020 0.25 97 890.9 88.1 1021 0.28 92 974.7 89.8 1022 0.28 89 926.1 77.3 1023 0.27 95 979.8 105.2 1024 0.22 93 1055.1 90.7 1025 a

1026 0.20 93 1082.2 93.8

a ANIMAL DIED PRIOR TO SCHEDULED SACRIFICE. 

### Page 144517 HLS STUDY NO.: 00-4208

APPENDIX B

GASOLINE MTBE VAPOR CONDENSATE
A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS

INDIVIDUAL F

1 GENERATION CAUDAL EPIDIDYMIS WEIGHT,
SPERM MOTILITY AND SPERM COUNT DATA

Group IV: 20,000 mg/m 3 (GASOLINE MTBE VAPOR CONDENSATE)

S P E R M C O U N T Animal Caudal Motility Caudal Homogenization Resistant No. Epididymis Epididymal Testicular Weight (g) (%) (million sperm/gram)

4001 0.27 99 762.9 102.5 4002 a

4003 0.24 97 422.6 107.8 4004 0.29 96 580.5 95.0 4005 a

4006 0.25 100 782.1 116.0 4007 0.25 71 1049.8 86.3 4008 0.25 99 656.7 102.6 4009 0.29 98 1002.4 106.3 4010 0.24 95 631.7 112.8 4011 0.28 96 944.8 74.3 4012 0.27 93 1096.0 98.8 4013 0.24 93 1289.6 111.2 4014 0.19 97 1353.8 109.7 4015 0.24 95 888.8 140.4 4016 0.19 94 599.9 61.8 4017 0.26 99 1158.2 115.4 4018 0.20 94 653.5 109.5 4019 0.32 48 1212.3 86.4 4020 0.20 95 1155.4 118.8 4021 0.25 87 949.4 94.8 4022 0.27 96 972.1 93.8 4023 0.23 90 1059.3 103.2 4024 0.20 96 1024.7 122.3 4025 0.29 65 1157.4 86.3 4026 0.28 97 989.6 97.5

a ANIMAL DIED PRIOR TO SCHEDULED SACRIFICE. 

### Page 1446HLS STUDY NO.: 00-4208APPENDIX CGASOLINE MTBE VAPOR CONDENSATEA TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATSINDIVIDUAL P0GENERATION SPERM MORPHOLOGY DATA18Group I: 0 mg/m3(GASOLINE MTBE VAPOR CONDENSATE)Animal ----------------H e a d---------------- --------T a i l--------No. Normal Amorphous Small Enlarged Double Coiled Bent Double Other1051 199 1 0 0 0 0 0 0 01052 196 3 1 0 0 0 0 0 01053 196 4 0 0 0 0 0 0 01054 198 1 1 0 0 0 0 0 01055 200 0 0 0 0 0 0 0 01056 199 1 0 0 0 0 0 0 01057 197 3 0 0 0 0 0 0 01058 197 3 0 0 0 0 0 0 01059 198 2 0 0 0 0 0 0 01060 197 3 0 0 0 0 0 0 01061 197 2 1 0 0 0 0 0 01062 196 4 0 0 0 0 0 0 01063 196 3 1 0 0 0 0 0 01064 197 2 1 0 0 0 0 0 01065 196 4 0 0 0 0 0 0 01066 198 2 0 0 0 0 0 0 01067 197 3 0 0 0 0 0 0 01068 198 2 0 0 0 0 0 0 01069 198 1 1 0 0 0 0 0 01070 197 3 0 0 0 0 0 0 01071 196 4 0 0 0 0 0 0 01072 196 2 2 0 0 0 0 0 01073 200 0 0 0 0 0 0 0 01074 199 1 0 0 0 0 0 0 01075 197 3 0 0 0 0 0 0 01076 195 2 0 3 0 0 0 0 0Page 1447HLS STUDY NO.: 00-4208APPENDIX CGASOLINE MTBE VAPOR CONDENSATEA TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATSINDIVIDUAL P0GENERATION SPERM MORPHOLOGY DATA19Group IV: 20,000 mg/m3(GASOLINE MTBE VAPOR CONDENSATE)Animal ----------------H e a d---------------- --------T a i l--------No. Normal Amorphous Small Enlarged Double Coiled Bent Double Other4051 200 0 0 0 0 0 0 0 04052 196 2 2 0 0 0 0 0 04053 195 2 0 3 0 0 0 0 04054 197 2 1 0 0 0 0 0 04055 196 4 0 0 0 0 0 0 04056 196 3 0 0 0 0 1 0 04057 197 2 0 0 0 1 0 0 04058 195 5 0 0 0 0 0 0 04059 196 3 1 0 0 0 0 0 04060 196 4 0 0 0 0 0 0 04061 194 5 1 0 0 0 0 0 04062 195 3 2 0 0 0 0 0 04063 196 3 1 0 0 0 0 0 04064 196 4 0 0 0 0 0 0 04065 197 2 1 0 0 0 0 0 04066 196 2 2 0 0 0 0 0 04067 197 3 0 0 0 0 0 0 04068 196 3 1 0 0 0 0 0 04069 195 3 2 0 0 0 0 0 04070 197 3 0 0 0 0 0 0 04071 198 2 0 0 0 0 0 0 04072 196 3 1 0 0 0 0 0 04073 191 8 1 0 0 0 0 0 04074 197 3 0 0 0 0 0 0 04075 196 4 0 0 0 0 0 0 04076 196 4 0 0 0 0 0 0 0Page 1448HLS STUDY NO.: 00-4208APPENDIX DGASOLINE MTBE VAPOR CONDENSATEA TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATSINDIVIDUAL F1GENERATION SPERM MORPHOLOGY DATA20Group I: 0 mg/m3(GASOLINE MTBE VAPOR CONDENSATE)Animal ----------------H e a d---------------- --------T a i l--------No. Normal Amorphous Small Enlarged Double Coiled Bent Double Other1001 200 0 0 0 0 0 0 0 01002 194 6 0 0 0 0 0 0 01003 196 4 0 0 0 0 0 0 01004 199 0 0 0 0 0 1 0 01005 199 1 0 0 0 0 0 0 01006 198 2 0 0 0 0 0 0 01007 196 4 0 0 0 0 0 0 01008 197 3 0 0 0 0 0 0 01009 200 0 0 0 0 0 0 0 01010 198 2 0 0 0 0 0 0 01011 200 0 0 0 0 0 0 0 01012 199 1 0 0 0 0 0 0 01013 200 0 0 0 0 0 0 0 01014 200 0 0 0 0 0 0 0 01015 199 1 0 0 0 0 0 0 01016 198 1 0 0 0 1 0 0 01017 200 0 0 0 0 0 0 0 01018 198 2 0 0 0 0 0 0 01019 199 1 0 0 0 0 0 0 01020 200 0 0 0 0 0 0 0 01021 200 0 0 0 0 0 0 0 01022 198 1 0 0 0 1 0 0 01023 195 5 0 0 0 0 0 0 01024 200 0 0 0 0 0 0 0 01026 200 0 0 0 0 0 0 0 0Page 1449HLS STUDY NO.: 00-4208APPENDIX DGASOLINE MTBE VAPOR CONDENSATEA TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATSINDIVIDUAL F1GENERATION SPERM MORPHOLOGY DATA21Group IV: 20,000 mg/m3(GASOLINE MTBE VAPOR CONDENSATE)Animal ----------------H e a d---------------- --------T a i l--------No. Normal Amorphous Small Enlarged Double Coiled Bent Double Other4001 199 1 0 0 0 0 0 0 04003 200 0 0 0 0 0 0 0 04004 198 2 0 0 0 0 0 0 04006 200 0 0 0 0 0 0 0 04007 198 2 0 0 0 0 0 0 04008 197 3 0 0 0 0 0 0 04009 199 1 0 0 0 0 0 0 04010 196 4 0 0 0 0 0 0 04011 194 5 0 1 0 0 0 0 04012 198 2 0 0 0 0 0 0 04013 200 0 0 0 0 0 0 0 04014 200 0 0 0 0 0 0 0 04015 200 0 0 0 0 0 0 0 04016 200 0 0 0 0 0 0 0 04017 198 2 0 0 0 0 0 0 04018 199 1 0 0 0 0 0 0 04019 200 0 0 0 0 0 0 0 04020 200 0 0 0 0 0 0 0 04021 200 0 0 0 0 0 0 0 04022 198 2 0 0 0 0 0 0 04023 199 1 0 0 0 0 0 0 04024 200 0 0 0 0 0 0 0 04025 199 1 0 0 0 0 0 0 04026 197 3 0 0 0 0 0 0 0Page 145022 HLS STUDY NO.: 00-4208

APPENDIX E

GASOLINE MTBE VAPOR CONDENSATE
A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS

INDIVIDUAL F

1 GENERATION FEMALE OVARIAN FOLLICLE COUNT DATA

Group I: 0 mg/m 3 (GASOLINE MTBE VAPOR CONDENSATE)

PRIMORDIAL COUNT GROWING COUNT C.L.'s Animal No. LEFT RIGHT TOTAL LEFT RIGHT TOTAL PRESENT

1501 52 60 112 5 0 5 Y 1502 121 99 220 1 3 4 Y 1503 72 59 131 3 1 4 Y 1504 28 41 69 0 1 1 Y 1516 50 38 88 3 3 6 Y 1517 37 57 94 3 2 5 Y 1521 34 28 62 0 1 1 Y 1523 11 44 55 0 2 2 Y 1525 51 51 102 1 1 2 Y 1526 35 43 78 0 0 0 Y

C.L.=CORPORA LUTEA

Group IV: 20,000 mg/m 3 (GASOLINE MTBE VAPOR CONDENSATE)

PRIMORDIAL COUNT GROWING COUNT C.L.'s Animal No. LEFT RIGHT TOTAL LEFT RIGHT TOTAL PRESENT

4503 92 52 144 7 3 10 Y 4504 59 100 159 2 3 5 Y 4512 70 92 162 2 1 3 Y 4515 82 70 152 5 7 12 Y 4517 25 52 77 2 1 3 Y 4518 7 9 16 0 3 3 Y 4519 83 96 179 1 2 3 Y 4521 89 83 172 4 2 6 Y 4524 42 46 88 1 1 2 Y 4526 65 58 123 1 1 2 Y

C.L.=CORPORA LUTEA 

### Page 145123 APPENDIX F

HLS STUDY NO.: 00-4208

PATHOLOGY ASSOCIATES

A CHARLES RIVER COMPANY FREDERICK, MD 21701

SPERM ANALYSIS HISTORICAL CONTROL DATA

Species: RAT Strain: SPRAGUE-DAWLEY Source: CHARLES RIVER LABORATORIES, INC.

November 1995 to June 2001

CUMULATIVE STUDY INFORMATION

MEAN RANGE NUMBER OF

STUDIES

MOTILITY 95 77 - 100 78 (%)

TOTAL SPERM COUNT

EPIDIDYMAL 869.9 361.1 - 1279.1 81 (million sperm/gram)

TESTICULAR 105.2 82.1 - 141.6 25 (million sperm/gram)

MORPHOLOGY 1.0 0.0 – 6.7 61 (% abnormal) 

### Page 1452APPENDIX F

HLS STUDY NO.: 00-4208

PATHOLOGY ASSOCIATES

A CHARLES RIVER COMPANY FREDERICK, MD 21701

SPERM ANALYSIS HISTORICAL CONTROL DATA

Species: RAT Strain: SPRAGUE-DAWLEY Source: CHARLES RIVER LABORATORIES, INC.

November, 1995 to June, 2001

INDIVIDUAL STUDY INFORMATION

TOTAL SPERM COUNT

HISTORICAL NUMBER MOTILITY EPIDIDYMAL TESTICULAR MORPHOLOGY DATA OF percent M/G % abnormal STUDY NO. ANIMALS (range) N (range) N (range) N

24 1 30 97 (82 - 100) 30 742.5 (291.1 - 1499.6) 29 NP/NA 2.0 (0.0 - 7.5) 30

2 25 94 (0 - 100) 25 876.3 (190.6 - 2132.9) 25 NP/NA 1.0 (0.0 - 4.5) 25

3 10 99 (98 - 100) 10 1131.5 (759.9 - 1498.4) 10 NP/NA 1.6 (0.0 - 7.0) 10

4 10 99 (97 - 100) 10 1158.0 (628.1 - 1541.4) 10 NP/NA NP/NA

5 15 92 (48 - 100) 15 1092.4 (450.9 - 1749.7) 15 NP/NA 1.0 (0.0 - 8.5) 15

6 25 99 (92 - 100) 25 1117.3 (819.2 - 1504.2) 25 NP/NA 0.7 (0.0 - 5.0) 25

7 10 99 (98 - 100) 10 1243.3 (791.9 - 2063.5) 10 NP/NA 0.3 (0.0 - 2.5) 10

8 10 100 (98 - 100) 8 1279.1 (642.8 - 1843.1) 10 NP/NA 0.7 (0.0 - 3.0) 10

9 10 100 (99 - 100) 10 1116.8 (689.8 - 1615.6) 10 NP/NA 0.8 (0.0 - 2.0) 10

11 10 100 (99 - 100) 10 1141.4 (418.8 - 1807.9) 10 NP/NA 0.3 (0.0 - 1.5) 10

12 25 98 (81 - 100) 25 873.2 (476.4 - 1533.1) 25 NP/NA 0.3 (0.0 - 1.5) 25

13 10 96 (86 - 100) 9 552.7 (293.1 - 763.7) 10 NP/NA NP/NA 

### Page 1453APPENDIX F

HLS STUDY NO.: 00-4208

PATHOLOGY ASSOCIATES

A CHARLES RIVER COMPANY FREDERICK, MD 21701

SPERM ANALYSIS HISTORICAL CONTROL DATA

Species: RAT Strain: SPRAGUE-DAWLEY Source: CHARLES RIVER LABORATORIES, INC.

November, 1995 to June, 2001

INDIVIDUAL STUDY INFORMATION

TOTAL SPERM COUNT

HISTORICAL NUMBER MOTILITY EPIDIDYMAL TESTICULAR MORPHOLOGY DATA OF percent M/G % abnormal STUDY NO. ANIMALS (range) N (range) N (range) N

25 14 24 99 (97 - 100) 24 942.6 (413.9 - 1424.1) 24 NP/NA 0.1 (0.0 - 1.0) 24

15 10 99 (98 - 100) 10 1034.9 (482.1 - 1541.4) 10 NP/NA NP/NA

16 12 99 (98 - 100) 12 1170.1 (686.8 - 1944.8) 12 NP/NA 0.2 (0.0 - 1.0) 12

18 15 91 (57 - 99) 15 826.5 (479.4 - 1235.4) 15 NP/NA 0.0 (0.0 - 0.5) 15

19 10 92 (69 - 98) 10 693.3 (422.2 - 1360.5) 10 NP/NA NP/NA

20 10 87 (73 - 96) 8 1151.3 (543.8 - 1731.3) 10 NP/NA NP/NA

21 24 94 (25 - 100) 24 1171.3 (745.2 - 1801.7) 24 NP/NA 0.2 (0.0 - 1.0) 24

22 20 95 (48 - 100) 20 941.3 (316.7 - 1755.6) 20 NP/NA 0.1 (0.0 - 1.0) 20

24 26 94 (77 - 100) 19 556.3 (0.0 - 1376.9) 26 NP/NA 0.0 (0.0 - 0.5) 20

26 10 92 (83 - 99) 10 764.3 (272.7 - 1326.8) 10 NP/NA 0.1 (0.0 - 0.5) 10

27 26 96 (75 - 100) 26 1030.2 (686.4 - 1484.6) 26 NP/NA 0.1 (0.0 - 1.0) 26

28 10 97 1045.9 NP/NA 0.3 

### Page 1454APPENDIX F

HLS STUDY NO.: 00-4208

PATHOLOGY ASSOCIATES

A CHARLES RIVER COMPANY FREDERICK, MD 21701

SPERM ANALYSIS HISTORICAL CONTROL DATA

Species: RAT Strain: SPRAGUE-DAWLEY Source: CHARLES RIVER LABORATORIES, INC.

November, 1995 to June, 2001

INDIVIDUAL STUDY INFORMATION

TOTAL SPERM COUNT

HISTORICAL NUMBER MOTILITY EPIDIDYMAL TESTICULAR MORPHOLOGY DATA OF percent M/G % abnormal STUDY NO. ANIMALS (range) N (range) N (range) N

26 (91 - 100) 10 (836.7 - 1539.8) 10 (0.0 - 2.5) 10

29 10 94 (79 - 100) 7 732.0 (507.8 - 885.9) 10 NP/NA NP/NA

30 15 96 (92 - 100) 13 1063.9 (400.5 - 1360.7) 15 NP/NA 0.1 (0.0 - 0.5) 14

31 5 99 (97 - 100) 5 825.4 (578.5 - 1256.3) 5 NP/NA 0.0 (0.0) 5

32 25 90 (13 – 100) 25 994.7 (484.9 - 1444.2) 25 141.6 (72.3 - 233.6) 25 0.1 (0.0 - 0.5) 25

33 26 94 (59 – 100) 25 808.1 (231.4 - 1328.7) 26 105.1 (9.6 - 222.8) 26 0.1 (0.0 - 0.5) 25

34 10 NP/NA 921.3 (729.0 - 1295.9) 10 133.2 (88.4 - 203.8) 10 NP/NA

35 10 NP/NA 755.7 (585.2 - 909.1) 10 98.0 (68.8 - 141.2) 10 NP/NA

36 30 97 (79 – 99) 29 757.8 (214.1 – 1116.2) 30 102.4 (50.1 – 172.1) 29 0.0 (0.0 – 0.5) 30

37 29 89 (56 – 100) 29 843.2 (373.4 – 1231.5) 29 89.9 (64.4 – 124.4) 29 0.1 (0.0 – 1.0) 29

38 29 96 (84 - 100) 27 924.0 (389.2 - 1476.0) 29 82.1 (49.9 - 130.2) 29 0.1 (0.0 - 1.0) 29

39 30 91 (10 - 100) 29 906.3 (472.5 - 1198.6) 30 98.0 (70.7 - 134.7) 30 0.4 (0.0 - 5.0) 30 

### Page 1455APPENDIX F

HLS STUDY NO.: 00-4208

PATHOLOGY ASSOCIATES

A CHARLES RIVER COMPANY FREDERICK, MD 21701

SPERM ANALYSIS HISTORICAL CONTROL DATA

Species: RAT Strain: SPRAGUE-DAWLEY Source: CHARLES RIVER LABORATORIES, INC.

November, 1995 to June, 2001

INDIVIDUAL STUDY INFORMATION

TOTAL SPERM COUNT

HISTORICAL NUMBER MOTILITY EPIDIDYMAL TESTICULAR MORPHOLOGY DATA OF percent M/G % abnormal STUDY NO. ANIMALS (range) N (range) N (range) N

27 40 26 96 (81 - 99) 26 806.6 (410.2 - 1200.5) 26 119.4 (79.7 - 185.7) 26 0.3 (0.0 - 1.0) 26

41 25 93 (69 - 98) 23 909.8 (585.3 - 1179.3) 25 104.5 (58.4 - 142.2) 25 NP/NA

42 26 98 (90 – 100) 26 841.6 (264.5 – 1294.2) 26 98.7 (71.2 – 140.4) 26 0.2 (0.0 – 1.0) 26

43 26 97 (89 – 100) 26 1070.7 (704.3 – 1495.3) 26 106.5 (49.8 – 150.6) 26 0.1 (0.0 – 1.0) 26

44 10 98 (97 - 100) 10 682.5 (342.5 - 1103.7) 10 NP/NA NP/NA

45 15 82 (63 - 96) 13 1148.3 (621.4 - 1552.4) 15 NP/NA 0.3 (0.0 - 2.5) 15

46 10 88 (19 - 98)10 980.8 (642.0 - 1611.0) 10 NP/NA NP/NA

49 15 96 (84 – 99) 15 715.6 (441.4 – 1106.2) 15 NP/NA NP/NA

50 25 93 (20 – 100) 25 773.7 (356.0 – 1189.5) 25 NP/NA 0.1 (0.0 – 0.5) 25

51 10 97 (94 – 99) 10 784.2 (518.2 – 1177.0) 10 NP/NA NP/NA

52 10 95 (89 – 100) 10 845.7 (347.1 – 1045.0) 10 NP/NA 0.1 (0.0 – 0.5) 10

53 10 95 1064.0 NP/NA 0.1 

### Page 1456APPENDIX F

HLS STUDY NO.: 00-4208

PATHOLOGY ASSOCIATES

A CHARLES RIVER COMPANY FREDERICK, MD 21701

SPERM ANALYSIS HISTORICAL CONTROL DATA

Species: RAT Strain: SPRAGUE-DAWLEY Source: CHARLES RIVER LABORATORIES, INC.

November, 1995 to June, 2001

INDIVIDUAL STUDY INFORMATION

TOTAL SPERM COUNT

HISTORICAL NUMBER MOTILITY EPIDIDYMAL TESTICULAR MORPHOLOGY DATA OF percent M/G % abnormal STUDY NO. ANIMALS (range) N (range) N (range) N

28 (80 – 99) 10 (818.9 – 1152.8) 10 (0.0 – 0.5) 10

54 12 89 (70 – 97) 12 958.8 (557.5 – 1314.9) 12 107.8 (78.8 – 134.9) 12 NP/NA

57 26 NP/NA 1010.1 (636.4 – 1475.3) 26 115.5 (91.8 – 167.2) 26 0.1 (0.0 – 1.0) 26

59 10 99 (96 – 100) 10 935.8 (568.9 – 1198.1) 10 NP/NA NP/NA

62 10 97 (93 – 100) 8 945.5 (257.7 – 1371.0) 10 NP/NA NP/NA

63 26 98 (89 – 100) 23 966.8 (582.4 – 1428.4) 26 105.8 (67.6 – 147.5) 26 0.2 (0.0 – 1.0) 26

64 12 90 (77 – 97) 12 927.8 (714.4 – 1224.9) 12 102.6 (77.9 – 127.1) 12 NP/NA

65 10 97 (95 – 99) 9 1035.8 (624.8 – 1356.0) 10 NP/NA 0.1 (0.0 – 0.5) 10

66 10 95 (83 – 100) 9 938.0 (177.0 – 1332.4) 10 NP/NA 0.3 (0.0 – 1.5) 10

67 26 91 (46 – 100) 26 903.9 (185.1 – 1941.7) 26 115.7 (79.5 – 152.0) 26 0.3 (0.0 – 1.5) 26

69 26 91 (63 – 100) 26 957.1 (405.0 – 1874.5) 26 111.9 (35.0 – 154.3) 26 0.2 (0.0 – 1.0) 24

71 25 93 (74 – 100) 25 777.4 (184.1 – 1211.5) 25 NP/NA 0.2 (0.0 – 1.0) 25 

### Page 1457APPENDIX F

HLS STUDY NO.: 00-4208

PATHOLOGY ASSOCIATES

A CHARLES RIVER COMPANY FREDERICK, MD 21701

SPERM ANALYSIS HISTORICAL CONTROL DATA

Species: RAT Strain: SPRAGUE-DAWLEY Source: CHARLES RIVER LABORATORIES, INC.

November, 1995 to June, 2001

INDIVIDUAL STUDY INFORMATION

TOTAL SPERM COUNT

HISTORICAL NUMBER MOTILITY EPIDIDYMAL TESTICULAR MORPHOLOGY DATA OF percent M/G % abnormal STUDY NO. ANIMALS (range) N (range) N (range) N

29 72 25 94 (75 – 100) 25 887.2 (366.4 – 1276.4) 25 104.3 (80.6 – 123.7) 25 6.7 (0.0 – 13.0) 25

75 15 96 (89 – 98) 15 1012.5 (652.6 – 1418.7) 15 NP/NA 0.1 (0.0 – 0.5) 15

77 15 95 (88 – 99) 13 715.9 (42.4 – 1208.2) 13 NP/NA 0.2 (0.0 – 1.0) 11

78 25 98 (69 – 100) 25 728.3 (270.0 – 1322.4) 25 NP/NA 5.0 (1.1 – 9.0) 25

79 10 95 (80 – 99) 9 787.8 (311.9 – 1300.3) 10 NP/NA 5.0 (2.0 – 8.0) 10

80 10 94 (56 – 99) 10 700.2 (389.8 – 1117.2) 10 NP/NA 0.3 (0.0 – 1.0) 10

81 10 98 (93 – 100) 10 627.5 (419.4 – 796.6) 10 NP/NA 0.3 (0.0 – 1.0) 10

82 10 98 (88 – 100) 10 718.6 (411.8 – 1125.2) 10 NP/NA 3.1 (0.0 – 11.5) 10

84 26 98 (85 – 100) 26 696.9 (231.4 – 1384.9) 26 92.7 (25.9 – 123.2) 26 1.6 (0.0 – 6.0) 26

85 15 97 (92 – 100) 15 895.0 (459.2 – 1276.9) 15 NP/NA 1.7 (0.0 – 5.5) 15

86 12 86 (58 – 97) 12 875.8 (495.2 – 1223.2) 12 94.8 (68.5 – 119.2) 12 NP/NA 

### Page 1458APPENDIX F

HLS STUDY NO.: 00-4208

PATHOLOGY ASSOCIATES

A CHARLES RIVER COMPANY FREDERICK, MD 21701

SPERM ANALYSIS HISTORICAL CONTROL DATA

Species: RAT Strain: SPRAGUE-DAWLEY Source: CHARLES RIVER LABORATORIES, INC.

November, 1995 to June, 2001

INDIVIDUAL STUDY INFORMATION

TOTAL SPERM COUNT

HISTORICAL NUMBER MOTILITY EPIDIDYMAL TESTICULAR MORPHOLOGY DATA OF percent M/G % abnormal STUDY NO. ANIMALS (range) N (range) N (range) N

30 87 10 99 (97 – 100) 10 664.7 (518.9 – 896.7) 10 NP/NA 1.8 (0.5 – 3.5) 10

89 12 94 (84 – 99) 11 450.9 (19.3 – 949.8) 12 105.9 (72.9 – 150.1) 11 5.7 (0.0 – 10.4) 12

90 15 96 (86 – 100) 14 713.3 (82.6 – 1123.5) 15 NP/NA 1.4 (0.0 – 2.5) 15

91 12 93 (84 – 100) 12 620.2 (298.6 – 794.0) 12 NP/NA 1.8 (0.0 – 4.5) 12

92 12 94 (80 – 100) 12 361.1 (182.7 – 564.1) 12 87.2 (58.8 – 125.9) 12 3.9 (1.0 – 16.1) 12

93 12 95 (80 – 100) 11 622.3 (316.9 – 973.9) 11 109.7 (72.8 – 133.5) 11 1.8 (0.5 – 4.6) 11

94 6 77 (2 – 98) 6 609.0 (363.7 – 1160.9) 6 NP/NA 1.7 (0.5 – 3.5) 6

95 12 94 (77 – 100) 12 654.7 (440.8 – 844.7) 12 97.6 (61.2 – 127.8) 12 4.2 (1.1 – 9.4) 12

96 10 100 (99 – 100) 10 719.7 (511.0 – 1053.3) 10 NP/NA 0.8 (0.0 – 2.0) 10

97 10 99 (97 – 100) 10 867.2 (540.0 – 1836.2) 10 NP/NA NP/NA

98 5 94 (84 – 97) 5 755.2 (450.4 – 997.5) 5 NP/NA 1.0 (0.0 – 2.0) 5

99 10 98 615.3 NP/NA NP/NA 

### Page 1459APPENDIX F

HLS STUDY NO.: 00-4208

PATHOLOGY ASSOCIATES

A CHARLES RIVER COMPANY FREDERICK, MD 21701

SPERM ANALYSIS HISTORICAL CONTROL DATA

Species: RAT Strain: SPRAGUE-DAWLEY Source: CHARLES RIVER LABORATORIES, INC.

November, 1995 to June, 2001

INDIVIDUAL STUDY INFORMATION

TOTAL SPERM COUNT

HISTORICAL NUMBER MOTILITY EPIDIDYMAL TESTICULAR MORPHOLOGY DATA OF percent M/G % abnormal STUDY NO. ANIMALS (range) N (range) N (range) N

31 (93 – 100) 10 (489.8 – 838.9) 10 

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### Huntingdon Life Sciences 00-4208 Page 1490 211-MTBE-2G Final Report

Animal Exposure and Animal Data
Preface Appendix A

INTRODUCTION: The following is data generated at Huntingdon Life Sciences, East Millstone, NJ. The separately issued main study report should be referenced for details of the procedures used for test atmosphere generation/characterization and animal
evaluations.

STUDY DATES: Date of Parental Animal Receipt: 29 May 2001

Experimental Initiation Date: 12 June 2001 (in-life)

Experimental Completion Date: 10 October 2001 (in-life)

Draft Report Date: 26 August 2002

EXPOSURES AND IN-LIFE SUMMARY: The actual measured results during the exposures of the parental animals (before, during and after gestation, until the day before pups were sacrificed) were comparable to the targeted exposure levels. There were no exposure-related effects seen in the pups with regards to clinical observations or body weights.

TABLE OF CONTENTS

TABLES

A. Chamber Monitori ng Results ...................................................................................1491

B. Individual Pup Clinical Observations ......................................................................1516
C. Individual Pup Body We ights (grams) ....................................................................1518

D. Individual Pup Termin ation History ........................................................................1520

### Huntingdon Life Sciences 00-4208 Page 1491 211-MTBE-2G Final Report

GFAP Report Table A

Chamber Monitoring Results Preface Key to Abbreviations

MMAD = Mass Median Aerodynamic Diameter GSD = Geometric Standard Deviation TMC = Total Mass ConcentrationHuntingdon Life Sciences 00-4208 Page 1516211-MTBE-2G Final ReportMALE PUPSGFAP ReportTable BIndividual Pup Clinical ObservationsWNL= Within Normal LimitsExposureDay0 4 7 142127Exposure Day047142127Group I –0 mg/m3Group III –10,000 mg/m31566-4 WNL WNL WNL WNL WNL WNL 3551-3 WNL WNL WNL WNL WNL WNL1567-3 WNL WNL WNL WNL WNL WNL 3554-5 WNL WNL WNL WNL WNL WNL1555-3 WNL WNL WNL WNL WNL WNL3568-1 WNL WNL WNL WNL WNL WNL1568-6 WNL WNL WNL WNL WNL WNL3563-7 WNL WNL WNL WNL WNL WNL1564-3 WNL WNL WNL WNL WNL WNL3560-4 WNL WNL WNL WNL WNL WNLGroup II –2,000 mg/m3Group IV –20,000 mg/m32574-6 WNL WNL WNL WNL WNL WNL 4573-2 WNL WNL WNL WNL WNL WNL2561-7 WNL WNL WNL WNL WNL WNL 4565-2 WNL WNL WNL WNL WNL WNL2558-6 WNL WNL WNL WNL WNL WNL4564-2 WNL WNL WNL WNL WNL WNL2565-2 WNL WNL WNL WNL WNL WNL4552-4 WNL WNL WNL WNL WNL WNL2557-3 WNL WNL WNL WNL WNL WNL4553-7 WNL WNL WNL WNL WNL WNLHuntingdon Life Sciences 00-4208 Page 1517211-MTBE-2G Final ReportFEMALE PUPSGFAP ReportTable BIndividual Pup Clinical ObservationsWNL= Within Normal LimitsRE = Red Exudate (General)ExposureDay0 4 7 142127Exposure Day047142127Group I –0 mg/m3Group III –10,000 mg/m31566-12 WNL WNL WNL WNL WNL WNL 3551-12 WNL WNL WNL WNL WNL WNL1567-11 WNL WNL WNL WNL WNL WNL 3554-12 WNL WNL WNL WNL WNL WNL1555-8 WNL WNL WNL WNL WNL WNL3568-12 WNL WNL WNL WNL WNL WNL1568-15 WNL WNL WNL WNL WNL WNL3563-8 WNL WNL WNL WNL WNL WNL1564-10 WNL WNL WNL WNL WNL WNL3560-10 WNL WNL WNL WNL WNL WNLGroup II –2,000 mg/m3Group IV –20,000 mg/m32574-11 WNL WNL WNL WNL WNL WNL 4573-12 WNL WNL WNL WNL WNL WNL2561-8 WNL WNL WNL WNL WNL WNL 4565-14 WNL WNL WNL WNL WNL WNL2558-13 WNL WNL WNL RE WNL WNL4564-12 WNL WNL WNL WNL WNL WNL2565-13 WNL WNL WNL WNL WNL WNL4552-8 WNL WNL WNL WNL WNL WNL2557-9 WNL WNL WNL WNL WNL WNL4553-14 WNL WNL WNL WNL WNL WNL

### Huntingdon Life Sciences 00-4208 Page 1520 211-MTBE-2G Final Report

Individual Pup Termination History Table D

a 

### T = Terminal Sacrifice.

b 

### LD = Lactation Day. Animal
Number Date of
Death Type of
Death a Study
Interval b Animal
Number Date of
Death Type of
Death a Study
Interval b

Group I – 0 mg/m 3 (air only)

1566-4 10 Oct 01 T LD 27 1566-12 10 Oct 01 T LD 27

1567-3 10 Oct 01 T LD27 1567-11 10 Oct 01 T LD 27

1555-3 10 Oct 01 T LD 27 1555-8 10 Oct 01 T LD 27

1568-6 10 Oct 01 T LD 27 1568-15 10 Oct 01 T LD 27

1564-3 10 Oct 01 T LD 27 1564-10 10 Oct 01 T LD 27

Group II – 2,000 mg/m 3

2574-6 10 Oct 01 T LD 27 2574-11 10 Oct 01 T LD 27

2561-7 10 Oct 01 T LD 27 2561-8 10 Oct 01 T LD 27

2558-6 10 Oct 01 T LD 27 2558-13 10 Oct 01 T LD 27

2565-2 10 Oct 01 T LD 27 2565-13 10 Oct 01 T LD 27

2557-3 10 Oct 01 T LD 27 2557-9 10 Oct 01 T LD 27

Group III – 10,000 mg/m 3

3551-3 10 Oct 01 T LD 27 3551-12 10 Oct 01 T LD 27

3554-5 10 Oct 01 T LD 27 3554-12 10 Oct 01 T LD 27

3568-1 10 Oct 01 T LD 27 3568-12 10 Oct 01 T LD 27

3563-7 10 Oct 01 T LD 27 3563-8 10 Oct 01 T LD 27

3560-4 10 Oct 01 T LD 27 3560-10 10 Oct 01 T LD 27

Group IV – 20,000 mg/m 3

4573-2 10 Oct 01 T LD 27 4573-12 10 Oct 01 T LD 27

4565-2 10 Oct 01 T LD 27 4565-14 10 Oct 01 T LD 27

4564-2 10 Oct 01 T LD 27 4564-12 10 Oct 01 T LD 27

4552-4 10 Oct 01 T LD 27 4552-8 10 Oct 01 T LD 27

4553-7 10 Oct 01 T LD 27 4553-14 10 Oct 01 T LD 27

### Huntingdon Life Sciences 00-4208 Page 1521 211-MTBE-2G Final Report

Certificates of Analysis Appendix EE

1. Feed Certificates of Anal ysis ..................................................................... 1522 2. Water Certificat es of A nalysis ................................................................... 1568 3. Bedding Certificat es of A nalysis ................................................................ 1582

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### Huntingdon Life Sciences 00-4208 Page 1649 211-MTBE-2G Final Report

Testing Facility Personnel Appendix GG

TITLE/DEPARTMENT NAME/DEGREE

VICE PRESIDENT, SAFETY ASSESSMENT

VICE PRESIDENT, OPERATIONS

VICE PRESIDENT, ANALYTICAL SERVICES Sylvie J. Gosselin, D.V.M., Ph.D., Diplomate A.C.V.P.

Michael Caulfield

Dari Dadgar, Ph.D.

SENIOR DIRECTOR, TOXICOLOGY Carol S. Auletta, M.B.A., D.A.B.T., R.A.C.

DIRECTOR, QUALITY ASSURANCE Nicki S. Iacono, B.S.

STUDY DIRECTOR Gary M. Hoffman, B.A., D.A.B.T.

PATHOLOGIST Katharine M. Whitney, D.V.M., Ph.D., Diplomate A.C.V.P.

STUDY MONITOR Brian P. Colfer, B.S.

VETERINARIAN Teresa S. Kusznir, V.M.D.

MANAGER/SUPERVISOR

Inhalation/Pharmacy Reproductive Toxicology Analytical Services Pathology Services Ellen H. Whiting, AALAS LAT Robert Faust, B.S. Kay Saladdin, B.S. Barbara A. Litzenberger, B.S., M.T. (ASCP)

### Huntingdon Life Sciences 00-4208 Page 1650 211-MTBE-2G Final Report

Report Amendments Appendix HH

There are no amendments for this report at this time.