STUDY NO. 00-4204

SPONSOR STUDY NO. 211-ETBE-1G

GASOLINE ETBE VAPOR CONDENSATE:

A ONE-GENERATION WHOLE-BODY INHALATION REPRODUCTION

TOXICITY STUDY IN RATS

Final Report

Volume II of II

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

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

Date: 18 July 2011

Page 513 of 845Huntingdon Life Sciences 00-4204 Page 514
211-ETBE-1G Final Report

TABLE OF CONTENTS

Volume II

COVER PAGE .................................................................................................................513

TABLE OF CONTENTS .................................................................................................514

APPENDICES

Z. Individual Animal Gross and Mi croscopic Observations .......................................... 515

AA. Released Pup and Histor ical Control Data ................................................................. 719

BB. Sperm Analysis Report ............................................................................................... 752

CC. Certificates of Analysis – (Feed, Bedding, and Water) .............................................. 767

DD. Protocol and Protocol Amendments ........................................................................... 808

EE. Testing Facility Personnel ........................................................................................... 844

FF. Report Amendments .................................................................................................... 845Huntingdon Life Sciences 00-4204 Page 515 211-ETBE-1G Final Report

Individual Animal Gross and Microscopic Observations

Preface Appendix Z

Key to Abbreviations

Gl = Gland LN = Lymph Node Oviducts/Fallop = Oviducts/Fallopian

Notes

1. Unless otherwise specified in a histopathology note, the organ/tissue examined was the requir ed (routine) section.

2. The dimensions of gross lesions are presented as a general range based on the actual dimensions noted during macr oscopic examinations. The actual dimensions of gross lesions can be found in the raw data.

Corresponding exposure levels for each group were as follows:

Group 1 - 0 mg/m 3
Group 2 - 2000 mg/m 3
Group 3 - 10000 mg/m 3
Group 4 - 20000 mg/m 3Page 516Page 517Page 518Page 519Page 520Page 521Page 522Page 523Page 524Page 525Page 526Page 527Page 528Page 529Page 530Page 531Page 532Page 533Page 534Page 535Page 536Page 537Page 538Page 539Page 540Page 541Page 542Page 543Page 544Page 545Page 546Page 547Page 548Page 549Page 550Page 551Page 552Page 553Page 554Page 555Page 556Page 557Page 558Page 559Page 560Page 561Page 562Page 563Page 564Page 565Page 566Page 567Page 568Page 569Page 570Page 571Page 572Page 573Page 574Page 575Page 576Page 577Page 578Page 579Page 580Page 581Page 582Page 583Page 584Page 585Page 586Page 587Page 588Page 589Page 590Page 591Page 592Page 593Page 594Page 595Page 596Page 597Page 598Page 599Page 600Page 601Page 602Page 603Page 604Page 605Page 606Page 607Page 608Page 609Page 610Page 611Page 612Page 613Page 614Page 615Page 616Page 617Page 618Page 619Page 620Page 621Page 622Page 623Page 624Page 625Page 626Page 627Page 628Page 629Page 630Page 631Page 632Page 633Page 634Page 635Page 636Page 637Page 638Page 639Page 640Page 641Page 642Page 643Page 644Page 645Page 646Page 647Page 648Page 649Page 650Page 651Page 652Page 653Page 654Page 655Page 656Page 657Page 658Page 659Page 660Page 661Page 662Page 663Page 664Page 665Page 666Page 667Page 668Page 669Page 670Page 671Page 672Page 673Page 674Page 675Page 676Page 677Page 678Page 679Page 680Page 681Page 682Page 683Page 684Page 685Page 686Page 687Page 688Page 689Page 690Page 691Page 692Page 693Page 694Page 695Page 696Page 697Page 698Page 699Page 700Page 701Page 702Page 703Page 704Page 705Page 706Page 707Page 708Page 709Page 710Page 711Page 712Page 713Page 714Page 715Page 716Page 717Page 718Huntingdon Life Sciences 00-4204 Page 719 211-ETBE-1G Final Report

Released Pup and Historical Control Data Appendix AA

Note

During Lactation Days 25-27 on 18 July 2002, a Group 1 rack containing the pups

from dam numbers 1554, 1556, 1560, 1563, 1565, 1566, 1568, 1569, 1572, 1574, 1575, and 1576 was accidentally overturned releas ing the litters so that it could not be determined which pups were from which li tters. The dams were in the exposure

chamber at the time of the incident and were unaffected. Of the 12 litters of
control-group pups, 108 pups in total were released and 105 survived. The
laboratory necropsied and collected or gan weight data on all 105 pups.

In addition, one female pup missing fr om dam number 1552 on Lactation Day 28 was apparently mis-identified as a releas ed pup. Organ weights were collected and
the pup was necropsied with the released pups.

The data for all 106 pups was separated according to sex, and the mean, standard deviation and range of values is presented separately from data of unaffected litters. The corresponding Lacta tion Day (LD) for each litter on the day of necropsy and data collection is as follows:

1554 LD27 1556 LD28 1560 LD28 1563 LD26

1565 LD26 1566 LD27 1568 LD27 1569 LD27

1572 LD27 1574 LD27 1575 LD27 1576 LD27

Table of Contents

Released Pup Data Mean Body Wei ght Val ues ............................................................................. 720 Summary of Pup Necr opsy Observat ions ....................................................... 722 Summary of Absolu te Organ We ights ............................................................ 725 Summary of Organ Weight to Body Weight Ratio ........................................... 727 Summary of Organ Weight to Brain Wei ght Rati o .......................................... 729

Individual Body Weight Va lues ....................................................................... 731 Individual Pup Necr opsy Observat ions ........................................................... 735 Individual Organ and Fi nal Body We ights ....................................................... 743

Historical Control Data Summary of Mean Pup Body Weight s/Litte r ................................................... 747 Summary of Pup Necr opsy Observat ions ....................................................... 748 Summary of Mean Pup Organ Weights:

Male Organ/Body and Organ/Br ain Weight Ra tios (%) ................................ 750 Female Organ/Body and Organ/Br ain Weight Ra tios (%) ............................ 751Page 720Page 721Page 722Page 723Page 724Page 725Page 726Page 727Page 728Page 729Page 730Page 731Page 732Page 733Page 734Page 735Page 736Page 737Page 738Page 739Page 740Page 741Page 742Page 743Page 744Page 745Page 746Huntingdon Life Sciences 00-4204 Page 747 211-ETBE-1G Final Report

Historical Control Data Summary of Mean Pup Body Weights/Litter Appendix AA

Study No. Lactation Day

1 4 7 14 21 28

00-4202 Mean 7.2 10.4 14.5 26.0 42.5 78.8

S.D. 0.52 1.03 1.23 1.68 3.87 5.12

N 23 22 22 22 22 22

00-4203 Mean 7.3 10.6 14.7 25.6 41.3 76.7

S.D. 0.48 0.98 1.50 2.25 3.97 6.16

N 25 25 25 25 25 25

00-4207 Mean 7.0 10.4 14.6 24.4 40.7 69.5

S.D. 0.75 1.36 1.70 2.31 3.99 7.40

N 24 24 24 24 24 24

00-4208 Mean 6.9 9.7 13.5 23.7 38.7 72.6

S.D. 0.67 0.98 1.13 1.78 3.47 5.60

N 23 23 23 23 23 23

Overall Mean 7.1 10.3 14.3 25.0 40.8 74.3

S.D. 0.64 1.13 1.48 2.20 4.01 7.04

N 95 94 94 94 94 94Huntingdon Life Sciences 00-4204 Page 748 211-ETBE-1G Final Report

Historical Control Data Summary of Pup Necropsy Observations Appendix AA

Study Number 00-4202 00-4203 00-4207 00-4208 Total

Rat Strain: Sprague-Dawley [Crl: CD IGS BR]

Total # male pups 114 117 117 137 485 Total # female pups 127 112 117 100 456

Total # undetermined 0 1 0 0 1

Stillborn 2 5 8 1 16 Lungs: floatation test - stillborn negative 2 5 6 1 14 Stomach: no milk present 0 4 6 1 11

Stomach: milk undetermined 2 1 2 0 5 Gross exam: autolysis 2 0 0 0 2

Gross exam: cannibalized 1 1 2 0 4

Gross exam: internal sex undetermined 1 1 0 0 2 Abdominal cavity: fluid filled 0 1 0 0 1

Skin: discolored area 0 1 0 0 1 Skin: edematous - general 0 1 0 0 1

Died prior to Cull 16 0 7 6 29

No remarkable observation 14 0 0 0 14 Lungs: floatation test - found dead, positive 0 0 2 2 4

Stomach: milk present 1 0 3 4 8 Stomach: no milk present 0 0 3 1 4 Head: skull damaged 0 0 0 0 0 Gross exam: autolysis 1 0 1 1 3

Day 4 Cull 93 75 80 97 345

No remarkable observation 93 75 80 97 345

Died prior to 28 Day Sacrifice 0 0 1 0 1 Head: skull damage 0 0 1 0 1 Stomach: milk present 0 0 1 0 1

Day 28 Sacrifice 130 150 138 133 551

No remarkable observation 127 136 130 126 519 Epididymides: small 0 0 0 1 1 External exam: left hindpaw missing 0 1 0 0 1 Eyes: damaged 0 0 1 0 1Huntingdon Life Sciences 00-4204 Page 749 211-ETBE-1G Final Report

Historical Control Data Summary of Pup Necropsy Observations Appendix AA

Study Number 00-4202 00-4203 00-4207 00-4208 Total

Rat Strain: Sprague-Dawley [Crl: CD IGS BR]

Gross exam: cannibalized 0 0 2 0 2 Kidney: cystic 3 6 3 6 18 Kidney: dilated pelvis 0 6 0 1 7

Liver: discolored foci 0 0 1 0 1 Skin: alopecia - general 0 0 1 0 1

Testes: small 0 0 0 1 1
Testes: undescended, unilateral 0 1 0 0 1Huntingdon Life Sciences 00-4204 Page 750211-ETBE-1G Final ReportMalesHistorical Control DataSummary of Mean Pup Organ WeightsAppendix AATBW=TerminalBody Weight; ORG=Organ WeightStudy TBW Brain Spleen ThymusNo. Wt. ORG/TBWWT ORG/TBWORG/BRAINWT ORG/TBWORG/BRAIN(g) (g) (%) (g) (%) (%) (g) (%) (%)00-4202 Mean 82.3 1.5825 1.9312 0.3245 0.3951 20.514 0.3586 0.4392 22.655S.D. 6.4 0.0629 0.1263 0.0500 0.0588 3.142 0.0547 0.0512 3.381N 22 22 22 22 22 22 22 22 2200-4203 Mean 81.8 1.6118 1.9785 0.3445 0.4210 21.381 0.3673 0.4499 22.754S.D. 6.8 0.0713 0.1305 0.0477 0.0472 2.845 0.0887 0.1105 5.300N 25 25 25 25 25 25 25 25 2500-4207 Mean 72.7 1.5939 2.2127 0.2792 0.3850 17.589 0.3238 0.4471 20.489S.D. 8.1 0.1572 0.2845 0.0491 0.0613 3.031 0.0788 0.1076 5.486N 23 23 23 23 23 23 23 23 2300-4208 Mean 75.6 1.5634 2.0757 0.3218 0.4258 20.582 0.3322 0.4389 21.234S.D. 5.6 0.0591 0.1267 0.0484 0.0589 3.031 0.0600 0.0718 3.687N 22 22 22 22 22 22 22 22 22Overall Mean 78.2 1.5888 2.04900.3180 0.4067 20.035 0.3459 0.4440 21.801S.D. 7.9 0.0967 0.2077 0.0537 0.0584 3.300 0.0737 0.0885 4.626N 92 92 92 92 92 92 92 92 92Huntingdon Life Sciences 00-4204 Page 751211-ETBE-1G Final ReportFemalesHistorical Control DataSummary of Mean Pup Organ WeightsAppendix AAStudy TBW Brain Spleen ThymusNo. Wt. ORG/TBWWt. ORG/TBWORG/BRAINWT ORG/TBWORG/BRAIN(g) (g) (%) (g)(%) (%) (g) (%) (%)00-4202 Mean 76.7 1.5323 2.0070 0.2884 0.3741 18.875 0.3836 0.5005 25.068S.D. 5.5 0.0671 0.1472 0.0681 0.0702 4.548 0.0468 0.0486 3.049N 22 22 22 22 22 22 22 22 2200-4203 Mean 75.1 1.5506 2.0750 0.3043 0.4041 19.597 0.3459 0.4384 22.301S.D. 6.36 0.0631 0.1576 0.0559 0.0606 3.307 0.0466 0.1045 2.830N 25 25 25 25 25 25 25 25 2500-4207 Mean 67.4 1.5015 2.2454 0.2440 0.3613 16.388 0.3253 0.4869 21.854S.D. 6.87 0.1191 0.2698 0.0473 0.0564 3.713 0.0884 0.1423 6.683N 23 23 23 23 23 23 23 23 2300-4208 Mean 69.9 1.5284 2.1971 0.2831 0.4058 18.506 0.3420 0.4891 22.349S.D. 5.90 0.0688 0.1724 0.0428 0.0573 2.541 0.0607 0.0805 3.704N 22 22 22 22 22 22 22 22 22Overall Mean 72.3 1.5287 2.13050.2803 0.3867 18.3614 0.3488 0.4775 22.862S.D. 7.1 0.0833 0.2120 0.0580 0.0633 3.7352 0.0653 0.1020 4.450N 92 92 92 92 92 92 92 92 92Huntingdon Life Sciences 00-4204 Page 752 211-ETBE-1G Final Report

Sperm Analysis Report Appendix BBAUDITED FINAL REPORT

Study Phase: Sperm Analysis

Test Site Phase Reference Number 93075219

Testing Facility Study Number 00-4204

Sponsor Reference Number 211-ETBE-1G



## Gasoline ETBE Vapor Condensate: A One-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 14 Page 753Final Report HLS Study No.: 00-4204

2 TABLE OF CONTENTS

Section Page No.

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

II. MATERIALS AND METHODS .................................... 5

A. Sample Collection................................... 5

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

C. Caudal Epididymis Weight and Total Sperm Count

Determination ...................................... 5

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

E. Statistical Analyses................................ 6

F. GLP Compliance Statement............................ 6

G. Sample and Data Archiving........................... 7

III. RESULTS .................................................. 7

A. Sperm Motility...................................... 7

B. Total Sperm Count................................... 7

C. Sperm Morphology.................................... 7

D. Caudal Epididymis Weight............................ 7

IV. DISCUSSION AND CONCLUSIONS ............................... 8

V. REFERENCES ............................................... 9 Page 754Final Report HLS Study No.: 00-4204

3 INDEX OF TABLES AND APPENDICES

Table Page No.

1. Summary of Male Reproductive Parameters .................10

Appendix Page No.

A. Individual Caudal Epididymis Weight, Sperm Motility and Sperm Count Data .....................11

B. Individual Sperm Morphology Data .........................13 Page 755Page 756Final Report HLS Study No.: 00-4204

5 Gasoline ETBE Vapor Condensate: A One-Generation Whole-Body Inhalation Reproduction Toxicity Study in Rats

II. MATERIALS AND METHODS

A. Sample Collection

All surviving male rats were euthanized by exsanguination following anesthesia with inhaled carbon dioxide after the last F 1 litters were delivered. 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 o 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, sperm morphology and caudal epididymis weight.

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 automated sperm analyzer (version 12.1.c). The analyzer automatically 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 Page 757Final Report HLS Study No.: 00-4204

6 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 homogenization-resistant sperm and cauda epididymal 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. Statistical Analyses

The means and standard deviations for the sperm motility, caudal epididymal sperm count, testicular homogenization-resistant spermatid count and sperm morphology data were calculated and compared across groups using the Kruskal-Wallis nonparametricANOVA test. If a significant effect occurred (p<0.05), the MannWhitney 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).

F. GLP Compliance Statement

The portion of this study performed by PAI was conducted in compliance with US EPA 79.60, CFR Vol. 59, No. 122, 27 June 1994. Page 758Final Report HLS Study No.: 00-4204

7 G. 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 and sperm morphology analysis, as well as any supporting documentation and the sperm morphology 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.

III. RESULTS

A. Sperm Motility

Table 1 (Summary Data) Appendix A (Individual Data)

No apparent treatment-related effects were observed in the sperm motility data. Group mean values were 96% and 94% for the control and high-exposure groups, respectively.

B. Total Sperm Count

Table 1 (Summary Data) Appendix A (Individual Data)

The number of sperm per gram of caudal epididymis and testis was comparable between the control and high-exposure groups. Group mean values were 956.7 and 1053.0 million sperm/gram for the epididymal sperm count and 105.7 and 106.5 million sperm/gram for the homogenization-resistant testicular count.

C. Sperm Morphology

Table 1 (Summary Data) Appendix B (Individual Data)

A low incidence of head/tail abnormalities was observed for animals in the control and high-exposure groups. Group mean values were 0.8 and 0.6 percent abnormal sperm. No treatment
related differences were observed.

D. Caudal Epididymis Weight

Table 1 (Summary Data) Page 759Page 760Final Report HLS Study No.: 00-4204

9 V. REFERENCES

Dunlap, W.P., Marx, M.S. and Agamy, G.G. 1981. Fortran IV functions for calculating probabilities associated with

Dunnett’s test. Behav. Res. Methods and Instrumentation 13:

363-366.

Dunnett, C. W. 1955. A multiple comparison procedure for comparing several treatments with a control. Journal of the

American Statistical Association 50:1096-1121.

Dunnett, C. W. 1964. New tables for multiple comparisons with a

control. Biometrics 20:482-491.

Kruskal, W.H. and Wallis, W.A. 1952. Use of ranks in onecriterion variance analysis. Journal of the American

Statistical Association 47: 583-621.

Kruskal, W.H. and Wallis, W.A. 1953. Errata for Kruskal-Wallis (1952). Journal of the American Statistical Association 48:

907-911.

Kruskal, W.H. 1957. Historical notes on the Wilcoxon unpaired two-sample test. Journal of the American Statistical

Association 52: 356-360.

SAS/STAT User's Guide, 1989a. SAS Institute Inc., Version 6, Fourth Edition, Cary, NC:SAS Institute Inc., 1989. Vol. 1, p. 209-244. Vol. 2 p. 1195-1210. Page 76110 HLS STUDY NO.: 00-4204

TABLE 1

GASOLINE ETBE VAPOR CONDENSATE:

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

SUMMARY OF MALE REPRODUCTIVE PARAMETERS

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

MOTILITY (%)

Mean 96 94

SD 3 5

N 25 26

EPIDIDYMAL COUNT (MILLION SPERM/GRAM)

Mean 956.7 1053.0

SD 164.8 215.1

N 26 26

TESTICULAR COUNT (MILLION SPERM/GRAM)

Mean 105.7 106.5

SD 21.3 21.0

N 26 26

SPERM MORPHOLOGY b

(% ABNORMAL)

Mean 0.8 0.6

SD 0.8 0.6

N 26 26

CAUDAL EPIDIDYMIS WEIGHTS (GRAM)

Mean 0.27 0.28

SD 0.03 0.03

N 26 26

a GASOLINE ETBE 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 76211 HLS STUDY NO.: 00-4204

APPENDIX A

GASOLINE ETBE VAPOR CONDENSATE:

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

INDIVIDUAL CAUDAL EPIDIDYMIS WEIGHT,

SPERM MOTILITY AND SPERM COUNT DATA

Group I: 0 mg/m 3 (GASOLINE ETBE 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 0.28 98 870.1 88.1

1052 0.24 91 954.1 98.3

1053 0.23 93 1227.5 91.9

1054 0.27 100 910.1 114.1

1055 0.20 97 705.8 105.3

1056 0.23 98 1036.5 146.9

1057 0.28 100 780.5 151.1

1058 0.26 96 949.1 69.7

1059 0.29 95 739.2 79.6

1060 0.29 99 1016.8 79.1

1061 0.25 93 803.0 91.1

1062 0.28 90 690.9 140.4

1063 0.34 93 925.7 75.2

1064 0.26 99 1089.9 107.3

1065 0.25 b a 949.4 b 106.9

1066 0.27 95 844.3 117.7

1067 0.27 99 1192.8 139.4

1068 0.27 97 1045.6 109.0

1069 0.32 92 1035.8 103.9

1070 0.28 87 1041.9 97.0

1071 0.28 99 1120.3 111.7

1072 0.25 97 1183.7 119.0

1073 0.24 96 1172.0 99.5

1074 0.27 99 619.6 97.1

1075 0.27 96 1014.7 115.0

1076 0.23 92 954.7 94.3

a NO SPERM PRESENT FOR ANALYSIS.

b LEFT EPIDIDYMIS AND LEFT TESTIS PRESERVED FOR POSSIBLE HISTOPATHOLOGICAL

EVALUATION; RIGHT EPIDIDYMIS AND RIGHT TESTIS USED FOR SPERM COUNT

DETERMINATION. Page 76312 HLS STUDY NO.: 00-4204

APPENDIX A

GASOLINE ETBE VAPOR CONDENSATE:

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

INDIVIDUAL CAUDAL EPIDIDYMIS WEIGHT,

SPERM MOTILITY AND SPERM COUNT DATA

Group IV: 20,000 mg/m 3 (GASOLINE ETBE 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 0.28 94 1105.4 101.0

4052 0.30 97 962.0 88.4

4053 0.29 94 1305.2 76.9

4054 0.23 97 1272.9 94.2

4055 0.19 79 627.4 77.8

4056 0.30 99 930.6 82.7

4057 0.26 88 1351.3 120.5

4058 0.25 94 819.8 97.8

4059 0.28 88 903.7 93.3

4060 0.32 98 1195.9 93.2

4061 0.28 96 1198.7 92.2

4062 0.28 97 1333.2 122.6

4063 0.23 96 836.5 144.0

4064 0.30 89 1153.7 77.0

4065 0.21 95 1050.6 110.2

4066 0.28 97 1056.8 105.3

4067 0.27 93 867.5 128.7

4068 0.32 97 1392.0 87.8

4069 0.29 96 1117.7 108.7

4070 0.25 95 1292.4 144.3

4071 0.30 98 1104.9 105.2

4072 0.30 87 1164.1 126.2

4073 0.32 98 630.6 108.4

4074 0.28 94 847.7 121.0

4075 0.29 100 1020.4 151.0

4076 0.25 88 836.5 110.1 Page 764HLS STUDY NO.: 00-4204APPENDIX BGASOLINE ETBE VAPOR CONDENSATE:A ONE-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATSINDIVIDUAL SPERM MORPHOLOGY DATA13Group I: 0 mg/m3(GASOLINE ETBE VAPOR CONDENSATE)Animal----------------H e a d---------------- --------T a i l--------No. NormalAmorphous Small Enlarged Double Coiled Bent Double Other1051 200 0 0 0 0 0 0 0 01052 200 0 0 0 0 0 0 0 01053 199 1 0 0 0 0 0 0 01054 196 4 0 0 0 0 0 0 01055 198 2 0 0 0 0 0 0 01056 198 2 0 0 0 0 0 0 01057 200 0 0 0 0 0 0 0 01058 199 1 0 0 0 0 0 0 01059 200 0 0 0 0 0 0 0 01060 197 3 0 0 0 0 0 0 01061 196 3 0 1 0 0 0 0 01062 199 1 0 0 0 0 0 0 01063 198 1 1 0 0 0 0 0 01064 197 3 0 0 0 0 0 0 01065 196 4 0 0 0 0 0 0 01066 198 2 0 0 0 0 0 0 01067 198 1 0 0 0 1 0 0 01068 200 0 0 0 0 0 0 0 01069 200 0 0 0 0 0 0 0 01070 200 0 0 0 0 0 0 0 01071 199 1 0 0 0 0 0 0 01072 200 0 0 0 0 0 0 0 01073 200 0 0 0 0 0 0 0 01074 198 2 0 0 0 0 0 0 01075 200 0 0 0 0 0 0 0 01076 195 5 0 0 0 0 0 0 0Page 765HLS STUDY NO.: 00-4204APPENDIX BGASOLINE ETBE VAPOR CONDENSATE:A ONE-GENERATION WHOLE-BODY INHALATION REPRODUCTION TOXICITY STUDY IN RATSINDIVIDUAL SPERM MORPHOLOGY DATA14Group IV: 20,000 mg/m3(GASOLINE ETBE VAPOR CONDENSATE)Animal----------------H e a d---------------- --------T a i l--------No. NormalAmorphous Small Enlarged Double Coiled Bent Double Other4051 200 0 0 0 0 0 0 0 04052 199 1 0 0 0 0 0 0 04053 198 2 0 0 0 0 0 0 04054 200 0 0 0 0 0 0 0 04055 198 2 0 0 0 0 0 0 04056 198 1 0 0 0 1 0 0 04057 200 0 0 0 0 0 0 0 04058 200 0 0 0 0 0 0 0 04059 199 1 0 0 0 0 0 0 04060 200 0 0 0 0 0 0 0 04061 198 2 0 0 0 0 0 0 04062 200 0 0 0 0 0 0 0 04063 198 2 0 0 0 0 0 0 04064 200 0 0 0 0 0 0 0 04065 195 5 0 0 0 0 0 0 04066 198 2 0 0 0 0 0 0 04067 198 2 0 0 0 0 0 0 04068 200 0 0 0 0 0 0 0 04069 200 0 0 0 0 0 0 0 04070 199 1 0 0 0 0 0 0 04071 198 2 0 0 0 0 0 0 04072 200 0 0 0 0 0 0 0 04073 197 3 0 0 0 0 0 0 04074 200 0 0 0 0 0 0 0 04075 199 1 0 0 0 0 0 0 04076 198 2 0 0 0 0 0 0 0Page 766Huntingdon Life Sciences 00-4204 Page 767 211-ETBE-1G Final Report

Certificates of Analysis Appendix CC

1. Feed Certificates of Anal ysis ....................................................................... 768

2. Water Certificat es of A nalysis ..................................................................... 794 3. Bedding Certificat es of A nalysis .................................................................. 801Page 768Page 769Page 770Page 771Page 772Page 773Page 774Page 775Page 776Page 777Page 778Page 779Page 780Page 781Page 782Page 783Page 784Page 785Page 786Page 787Page 788Page 789Page 790Page 791Page 792Page 793Page 794Page 795Page 796Page 797Page 798Page 799Page 800Page 801Page 802Page 803Page 804Page 805Page 806Page 807Huntingdon Life Sciences 00-4204 Page 808 211-ETBE-1G Final Report

Protocol and Protocol Amendments Appendix DDPage 809Page 810Page 811Page 812Page 813Page 814Page 815Page 816Page 817Page 818Page 819Page 820Page 821Page 822Page 823Page 824Page 825Page 826Page 827Page 828Page 829Page 830Page 831Page 832Page 833Page 834Page 835Page 836Page 837Page 838Page 839Page 840Page 841Page 842Page 843Huntingdon Life Sciences 00-4204 Page 844 211-ETBE-1G Final Report

Testing Facility Personnel Appendix EE

TITLE/DEPARTMENT NAME/DEGREE

SENIOR VICE PRESIDENT,

SAFETY ASSESSMENT Sylvie J. Gosselin, D.V.M., Ph.D., Diplomate A.C.V.P.

SENIOR DIRECTOR, SAFETY

ASSESSMENT Carol S. Auletta, M.B.A., D.A.B.T.,

R.A.C.

DIRECTOR, ANALYTICAL SERVICES Barbara A. Litzenberger, B.S., M.T. (ASCP)

DIRECTOR, TOXICOLOGY

OPERATIONS Ian Vanterpool, F.I.A.T.

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 Janet E. Scimone, B.A.

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

MANAGER/SUPERVISOR

Reproductive Toxicology

Inhalation Analytical Services Clinical Pathology Histology Robert Faust, B.S. Ellen H. Whiting, AALAS LAT Kay Saladdin, B.S. Lisa A. Spall, B.S., M.T. (ASCP)

Janet KusistoHuntingdon Life Sciences 00-4204 Page 845 211-ETBE-1G Final Report

Report Amendments Appendix FF

There are no amendments for this report at this time.