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 Assessment 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 CC
Final 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

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Final Report 

HLS Study No.: 00-4208 

3

INDEX OF TABLES AND APPENDICES 

Table Page No.

 1. Summary of P0 Generation Male Reproductive Parameters .. 11 

 2. Summary of F1 Generation Male Reproductive Parameters ...12 

 3. Summary of F1 Generation Female Ovarian 

 Follicle Evaluation ....................................13 

Appendix Page No.

A. Individual P0 Generation Caudal Epididymis Weight, 

 Sperm Motility and Sperm Count Data .....................14 

B. Individual F1 Generation Caudal Epididymis Weight, 

 Sperm Motility and Count Data ...........................16 

C. Individual P0 Generation Sperm Morphology Data............18 

D. Individual F1 Generation Sperm Morphology Data............20 

E. Individual F1 Generation Female Ovarian 

 Follicle Count Data .....................................22 

F. Sperm Analysis Historical Control Data ...................23 

Page 1432

Final 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 (P0 and F1). The ovarian follicle 

evaluation was conducted on each ovary from 10 female rats from 

the control and high-exposure group of the F1 generation. 

 A. Male Fertility Sample Collection

 All surviving male rats from each generation (P0 and F1) were 

euthanized by exsanguination following anesthesia with inhaled 

carbon dioxide before the last F1 and F2 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 -70o

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 μm deep cannula. The cannula was immediately loaded 

into the prewarmed stage of the Hamilton Thorne IVOS (version 

10.9i for the P0 generation and version 12.1c for the F1

generation) automated sperm analyzer. The analyzer automatically 

Page 1434
Final 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 μl 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 μm 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 F1 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 μm 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 1435
Final 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 

IBMTM 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 -70o

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 1436
Final 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 P0

or F1 male sperm motility data. Group mean values were 91%, or 

higher, in the control and high exposure P0 and F1 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/m3

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

in the P0 generation (753.6 M/g). The apparent increase was 

attributed to a slightly lower P0 control group value. No 

biologically meaningful differences were observed when the 

epididymal sperm count of the 20,000 mg/m3

 exposure group (891.2 

M/g) was compared to the F1 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/m3 exposure group of the P0 generation 

(2.0% vs. 1.3% in the control group). The increase was minimal 

and was within Pathology Associates Historical Control range (0-

Page 1437
Final 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 P0 generation and 

0.25-0.26 grams for the F1 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 F1 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/m3

. A statistically significant increase 

in the number of sperm/gram of epididymis was observed in the 

20,000 mg/m3

 exposure group of the P0 generation. The mean value 

was comparable to the control group in the F1 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/m3

 exposure group of the P0

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 P0

generation were not considered treatment-related. 

No evidence of reproductive toxicity, as characterized by 

effects on sperm motility, epididymal and testicular sperm 

Page 1438

11

HLS STUDY NO.: 00-4208 

TABLE 1 

GASOLINE MTBE VAPOR CONDENSATE 

A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS 

SUMMARY OF P0 GENERATION MALE REPRODUCTIVE PARAMETERS 

Group: I IV 

Exposure Level: GMTBEVCa (mg/m3) 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 MORPHOLOGYb

 (% 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 1440
12

HLS STUDY NO.: 00-4208 

TABLE 2 

GASOLINE MTBE VAPOR CONDENSATE 

A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS 

SUMMARY OF F1 GENERATION MALE REPRODUCTIVE PARAMETERS 

Group: I IV 

Exposure Level: GMTBEVCa (mg/m3) 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 MORPHOLOGYb

 (% 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 1441
13

HLS STUDY NO.: 00-4208 

TABLE 3 

GASOLINE MTBE VAPOR CONDENSATE 

A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS 

SUMMARY OF F1 GENERATION FEMALE OVARIAN FOLLICLE EVALUATION 

PRIMORDIAL FOLLICLE COUNT 

Group: I IV 

Exposure Level: GMTBEVCa

 (mg/m3) 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: GMTBEVCa

 (mg/m3) 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 1442
14

HLS STUDY NO.: 00-4208 

APPENDIX A 

GASOLINE MTBE VAPOR CONDENSATE 

A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS 

INDIVIDUAL P0 GENERATION CAUDAL EPIDIDYMIS WEIGHT, 

SPERM MOTILITY AND SPERM COUNT DATA 

Group I: 0 mg/m3 (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 1443
15

HLS STUDY NO.: 00-4208 

APPENDIX A 

GASOLINE MTBE VAPOR CONDENSATE 

A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS 

INDIVIDUAL P0 GENERATION CAUDAL EPIDIDYMIS WEIGHT, 

SPERM MOTILITY AND SPERM COUNT DATA 

Group IV: 20,000 mg/m3 (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 1444
16

HLS STUDY NO.: 00-4208 

APPENDIX B 

GASOLINE MTBE VAPOR CONDENSATE 

A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS 

INDIVIDUAL F1 GENERATION CAUDAL EPIDIDYMIS WEIGHT, 

SPERM MOTILITY AND SPERM COUNT DATA 

Group I: 0 mg/m3 (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 1445
17

HLS STUDY NO.: 00-4208 

APPENDIX B 

GASOLINE MTBE VAPOR CONDENSATE 

A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS 

INDIVIDUAL F1 GENERATION CAUDAL EPIDIDYMIS WEIGHT, 

SPERM MOTILITY AND SPERM COUNT DATA 

Group IV: 20,000 mg/m3 (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 1446
HLS STUDY NO.: 00-4208 

APPENDIX C 

GASOLINE MTBE VAPOR CONDENSATE 

A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS 

INDIVIDUAL P0 GENERATION SPERM MORPHOLOGY DATA 

18

Group 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 

1051 199 1 0 0 0 0 0 0 1052 196 3 1 0 0 0 0 0 1053 196 4 0 0 0 0 0 0 1054 198 1 1 0 0 0 0 0 1055 200 0 0 0 0 0 0 0 1056 199 1 0 0 0 0 0 0 1057 197 3 0 0 0 0 0 0 1058 197 3 0 0 0 0 0 0 1059 198 2 0 0 0 0 0 0 1060 197 3 0 0 0 0 0 0 1061 197 2 1 0 0 0 0 0 1062 196 4 0 0 0 0 0 0 1063 196 3 1 0 0 0 0 0 1064 197 2 1 0 0 0 0 0 1065 196 4 0 0 0 0 0 0 1066 198 2 0 0 0 0 0 0 1067 197 3 0 0 0 0 0 0 1068 198 2 0 0 0 0 0 0 1069 198 1 1 0 0 0 0 0 1070 197 3 0 0 0 0 0 0 1071 196 4 0 0 0 0 0 0 1072 196 2 2 0 0 0 0 0 1073 200 0 0 0 0 0 0 0 1074 199 1 0 0 0 0 0 0 1075 197 3 0 0 0 0 0 0 1076 195 2 0 3 0 0 0 0 
HLS STUDY NO.: 00-4208 

APPENDIX C 

GASOLINE MTBE VAPOR CONDENSATE 

A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS 

INDIVIDUAL P0 GENERATION SPERM MORPHOLOGY DATA 

19

Group 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 

4051 200 0 0 0 0 0 0 0 4052 196 2 2 0 0 0 0 0 4053 195 2 0 3 0 0 0 0 4054 197 2 1 0 0 0 0 0 4055 196 4 0 0 0 0 0 0 4056 196 3 0 0 0 0 1 0 4057 197 2 0 0 0 1 0 0 4058 195 5 0 0 0 0 0 0 4059 196 3 1 0 0 0 0 0 4060 196 4 0 0 0 0 0 0 4061 194 5 1 0 0 0 0 0 4062 195 3 2 0 0 0 0 0 4063 196 3 1 0 0 0 0 0 4064 196 4 0 0 0 0 0 0 4065 197 2 1 0 0 0 0 0 4066 196 2 2 0 0 0 0 0 4067 197 3 0 0 0 0 0 0 4068 196 3 1 0 0 0 0 0 4069 195 3 2 0 0 0 0 0 4070 197 3 0 0 0 0 0 0 4071 198 2 0 0 0 0 0 0 4072 196 3 1 0 0 0 0 0 4073 191 8 1 0 0 0 0 0 4074 197 3 0 0 0 0 0 0 4075 196 4 0 0 0 0 0 0 4076 196 4 0 0 0 0 0 0 
HLS STUDY NO.: 00-4208 

APPENDIX D 

GASOLINE MTBE VAPOR CONDENSATE 

A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS 

INDIVIDUAL F1 GENERATION SPERM MORPHOLOGY DATA 

20

Group 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 

1001 200 0 0 0 0 0 0 0 1002 194 6 0 0 0 0 0 0 1003 196 4 0 0 0 0 0 0 1004 199 0 0 0 0 0 1 0 1005 199 1 0 0 0 0 0 0 1006 198 2 0 0 0 0 0 0 1007 196 4 0 0 0 0 0 0 1008 197 3 0 0 0 0 0 0 1009 200 0 0 0 0 0 0 0 1010 198 2 0 0 0 0 0 0 1011 200 0 0 0 0 0 0 0 1012 199 1 0 0 0 0 0 0 1013 200 0 0 0 0 0 0 0 1014 200 0 0 0 0 0 0 0 1015 199 1 0 0 0 0 0 0 1016 198 1 0 0 0 1 0 0 1017 200 0 0 0 0 0 0 0 1018 198 2 0 0 0 0 0 0 1019 199 1 0 0 0 0 0 0 1020 200 0 0 0 0 0 0 0 1021 200 0 0 0 0 0 0 0 1022 198 1 0 0 0 1 0 0 1023 195 5 0 0 0 0 0 0 1024 200 0 0 0 0 0 0 0 1026 200 0 0 0 0 0 0 0 
HLS STUDY NO.: 00-4208 

APPENDIX D 

GASOLINE MTBE VAPOR CONDENSATE 

A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS 

INDIVIDUAL F1 GENERATION SPERM MORPHOLOGY DATA 

21

Group 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 

4001 199 1 0 0 0 0 0 0 4003 200 0 0 0 0 0 0 0 4004 198 2 0 0 0 0 0 0 4006 200 0 0 0 0 0 0 0 4007 198 2 0 0 0 0 0 0 4008 197 3 0 0 0 0 0 0 4009 199 1 0 0 0 0 0 0 4010 196 4 0 0 0 0 0 0 4011 194 5 0 1 0 0 0 0 4012 198 2 0 0 0 0 0 0 4013 200 0 0 0 0 0 0 0 4014 200 0 0 0 0 0 0 0 4015 200 0 0 0 0 0 0 0 4016 200 0 0 0 0 0 0 0 4017 198 2 0 0 0 0 0 0 4018 199 1 0 0 0 0 0 0 4019 200 0 0 0 0 0 0 0 4020 200 0 0 0 0 0 0 0 4021 200 0 0 0 0 0 0 0 4022 198 2 0 0 0 0 0 0 4023 199 1 0 0 0 0 0 0 4024 200 0 0 0 0 0 0 0 4025 199 1 0 0 0 0 0 0 4026 197 3 0 0 0 0 0 0 
22

HLS STUDY NO.: 00-4208 

APPENDIX E 

GASOLINE MTBE VAPOR CONDENSATE 

A TWO-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATS 

INDIVIDUAL F1 GENERATION FEMALE OVARIAN FOLLICLE COUNT DATA 

Group I: 0 mg/m3 (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/m3 (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 1451
23

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 1452
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 

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 1453
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 

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 1454
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 

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 1455
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 

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 1456
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 

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 1457
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 

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 1458
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 

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 1459
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 

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 

 

Page 1460
Appendix DD

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Page 1489
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 Monitoring Results ...................................................................................1491 

B. Individual Pup Clinical Observations ......................................................................1516 

C. Individual Pup Body Weights (grams) ....................................................................1518 

D. Individual Pup Termination 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 Concentration 
























Huntingdon Life Sciences 00-4208 

 211-MTBE-2G 

MALE PUPS GFAP Report T

Individual Pup Clinical Observations 

WNL= Within Normal Limits 

Exposure Day 0 4 7 14 21 27

 Exposure Day 

0 4 7 

Group I – 0 mg/m3 

 Group III – 

10,000 mg/m3 

1566-4 WNL WNL WNL WNL WNL WNL 3551-3 WNL WNL WNL W1567-3 WNL WNL WNL WNL WNL WNL 3554-5 WNL WNL WNL W1555-3 WNL WNL WNL WNL WNL WNL 3568-1 WNL WNL WNL W1568-6 WNL WNL WNL WNL WNL WNL 3563-7 WNL WNL WNL W1564-3 WNL WNL WNL WNL WNL WNL 3560-4 WNL WNL WNL WGroup II – 2,000 mg/m3

 Group IV – 

20,000 mg/m3

2574-6 WNL WNL WNL WNL WNL WNL 4573-2 WNL WNL WNL W2561-7 WNL WNL WNL WNL WNL WNL 4565-2 WNL WNL WNL W2558-6 WNL WNL WNL WNL WNL WNL 4564-2 WNL WNL WNL W2565-2 WNL WNL WNL WNL WNL WNL 4552-4 WNL WNL WNL W2557-3 WNL WNL WNL WNL WNL WNL 4553-7 WNL WNL WNL W
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 211-MTBE-2G 

FEMALE PUPS GFAP Report T

Individual Pup Clinical Observations 

WNL= Within Normal Limits RE = Red Exudate (General) 

Exposure Day 0 4 7 14 21 27

 Exposure Day 

0 4 7 

Group I – 0 mg/m3 

 Group III – 

10,000 mg/m3 

1566-12 WNL WNL WNL WNL WNL WNL 3551-12 WNL WNL WNL W1567-11 WNL WNL WNL WNL WNL WNL 3554-12 WNL WNL WNL W1555-8 WNL WNL WNL WNL WNL WNL 3568-12 WNL WNL WNL W1568-15 WNL WNL WNL WNL WNL WNL 3563-8 WNL WNL WNL W1564-10 WNL WNL WNL WNL WNL WNL 3560-10 WNL WNL WNL WGroup II – 2,000 mg/m3

 Group IV – 

20,000 mg/m3

2574-11 WNL WNL WNL WNL WNL WNL 4573-12 WNL WNL WNL W2561-8 WNL WNL WNL WNL WNL WNL 4565-14 WNL WNL WNL W2558-13 WNL WNL WNL RE WNL WNL 4564-12 WNL WNL WNL W2565-13 WNL WNL WNL WNL WNL WNL 4552-8 WNL WNL WNL W2557-9 WNL WNL WNL WNL WNL WNL 4553-14 WNL WNL WNL W


Huntingdon Life Sciences 00-4208 Page 1520 

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Individual Pup Termination History Table D 

a

T = Terminal Sacrifice. 

b

LD = Lactation Day. 

Animal 

Number 

Date of 

Death 

Type of

Deatha

Study 

Intervalb

Animal

Number

Date of 

Death 

Type of 

Deatha

 

Study 

Intervalb

 

Group I – 0 mg/m3 (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/m3

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/m3

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/m3

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 
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Certificates of Analysis Appendix EE 

1. Feed Certificates of Analysis ..................................................................... 1522 

2. Water Certificates of Analysis ................................................................... 1568 

3. Bedding Certificates of Analysis ................................................................ 1582 
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Appendix FF

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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) 

 
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Report Amendments Appendix HH 

There are no amendments for this report at this time.