### STUDY NO. 00

- 6130 SPONSOR STUDY NO. 211

- DIPE -S

GASOLINE DIPE VAPOR CONDENSATE:

A 13

- WEEK WHOLE

- BODY INHALATION TOXICITY STUDY IN RA TS

WITH NEUROTOXICITY A SSESSMENTS AND 4

- WEEK IN VIVO

GENOTOXICITY AND IMMUNOTOXICITY ASSESSME NTS

Final Report

Volume III of IV

Submitted to: American Petroleum Institute (API) 1220 L Street, Northwest Washington, DC 20005

- 4070

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

Date: 31 May 2013

Page 816 of 1446

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TABLE OF CONTENTS

Volume III

COVER PAGE .................................................................................................................816 TABLE OF CONTENTS .................................................................................................817

APPENDICES N. Equipment List ...........................................................................................................818 O. Miran Calibration .......................................................................................................821 P. Chamber Distribution Records ..................................................................................824 Q. Testing Room and Chambers Environmental Monitoring .........................................826 R. Animal Termination History ......................................................................................828 S. Analytical Report .......................................................................................................844 T. Neurobehavioral Positive Control ..............................................................................869 U. Neuropathology Historical Control ............................................................................962 V. Protocol and Protocol Amendment ............................................................................968 W. Testing Facility Personnel ........................................................................................1056

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Equipment List Appendix N

Exposure Chambers 1000 Liter glass and stainless steel chamber (Wahmann)

Chamber Air Flow Gauges Dwyer ® 

### Magnehelic ® 

### gauge (Dwyer ® 

### Instruments Inc.); calibrated prestudy with a Side Trak  III, equipped with a Digital Meter, Model 831

- N2 (Sierra Instruments Inc.)

Chamber Static Pressure Gauges Dwyer ® 

### Magnehelic ® 

### gauge (Dwyer ® 

### Instruments Inc.); calibrated prestudy with a Dwyer ® 

### Mark II Manometer, Model 25 (Dwyer ® 

### Instruments Inc.)

Compound Generator Counter

- Current Volatilization Unit, coiled glass rod ins ert with nichro me wire (Crown Glass Co., Inc.) Flowmeter with built

- in valve, size 0

- 65 mm, tu be #’s 6G02R3 (Key Instruments) Variable Autotransformer, Type 3PN 1010 (Statco Energy Products Company) Minitrol Autotransformer, Type PL

- 312 (Glas

- col ® 

### A pparatu s Company) T ° Sentry Digital Alarm Module, Model 110 (Hampshire Controls Corp.) Balston ® 

### Microfibre  Disposable Filter Units, No. L9933

- 05 (Grade DQ) Quick

- disconnect fitting with toggle valve (Rego ® 

### )

Compound Reservoir 4 gallon cylinder (Manchester Tank)

Flowmeters Flowmeter, size 0 - 4, 0 - 10, 0 - 20, 0 - 40 Lpm (Dwyer ® 

### ) Top Trak  Mass Flow Meter, size 0 - 1 Lpm, Model 821

- 1, (Sierra Instruments), calibrated with Gilibrator ® 

### Bubble Generator, P/N D800286, S/N 569

- S, with flow cell assembly P/N D800268

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Equipment List Appendix N

Pressure/Vacuum Gauges Norgreen backpressure gauge, PN 9892K23 and R07

- 219RGKA Ashcrof t backpressure gauge, PN 733

- 47 Matheson backpressure gauge, PN 63

- 3161 USG backpressure gauge, PN 12672 -1 GAST ® 

### vacuum gauge, Model 169

- 01

Regulators Norg reen, PN 9892K23 and PN 219RGKA

Valves Metering Valve, Model SS

- 4L Series (Nupro ® 

### Co.)

Tubing Plastic, size 1⁄4”, 1⁄2” (Norton) Teflon ® 

### , size 1/8”, 1⁄4”, 1⁄2” Stainless steel, size 1⁄2” Stainless steel 1⁄4” “T” Swage stainless steel “T”

Air Analyzer MIRAN ® 

### 1A

- CVF Ambient Air Analyzer (Wilks) with a Cole

- Parmer strip chart recorder, Model # 201 and a Micronta ® 

### LCD Benchto p Digital Multimeter No. 22

- 195

Particle Sizer/Analyzer TSI Aerodynamic Particle Sizer, Model 331001 and a DELL computer, Model 486P/25, equipped with an Epson LQ

- 570+ Dot matrix printer, Model P630B

Absorbent Tube Sampling Charcoal Tubes , ORBO

- 32, Lot # 2000 (Supelco)

Vacuum Pumps Thomas Industries Inc., Model 707CM50 Thomas Industries Inc., Model 107CA183 Becton

- Dickinson, Model 12 97

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Equipment List Appendix N

Timers Gr alab Universal Timer, Model 171

Environmental Monitoring VWR Big

- Digit Hygrometer/Thermometer Model No. 61161

- 378 Oxygen/Gas Analyzer, Model 1214S, (Gastech) Digital Sound Level Meter, Model 840029 (Sper Scientific) Photometer 1 Light Met er (Quantum Instruments)

Miscellaneous Ball and Socket joint

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Miran Calibration Appendix O
Methodology for Gasoline DIPE Vapor Condensate
Settings: The instrument settings for the unit are summarized below:
wavelength, microns 10.2 pathlength, dial setting 5.30 slit width, mm 1 range, absorbance 1 response, seconds 1 gain High chart speed, cm/min 1 chart volts 1

Calibrations: The Miran ® 

### was turned on and allowed to warm up for approximately 10 minutes. The cell was flushed with room air for approximately one minute. The loop was closed, the unit was zeroed and the calibration ser ies was performed as shown below. The resultant data were plotted to obtain a calibration curve. Each observer used a separate syringe for calibration.
Injection Calculated Absorbance 2



### . Volume Concentration 1 

### Operator 1 Operator 2 Average (μL) (mg/m 3 

### ) (volts) (volts) (volts)

8.4 953 0.0333 0.0325 0.0313 0.0304 0.0319 17 1929 0.0684 0.0711 0.0675 0.0698 0.0692 84 9532 0.319 0.321 0.315 0.316 0.318 170 19291 0.6 48 0.580 0.618 0.640 0.622 210 23830 0.788 0.838 0.770 0.760 0.789

1 

### Calculated Conc. (mg/m 3 

### ) = Injection volume (μL) x Density (mg/μL) x 1000 L/m 3 

### 5.64 L where density = 0.64 mg/μL and 5.64 L = volume of Miran cell

2 

### Due to the variability of the initial results, two additional injections (one per operator)

were performed for each concentration. All four values per concentration were used for the regression analysis.

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Miran Calibration Appendix O

Ca libration Checks: A three

- point calibration check of the Miran ® 

### was performed for each exposure prior to sampling the chambers. The parameters are shown below:
Expected Acceptable Injection Calculated Absorbance Absorbance Volume Concentration Rea ding Range (μL) (mg/m 3 

### ) (volts) (volts)

17 1929 0.0692 0.0588 – 0.0796 84 9532 0.318 0.2703 – 0.366 170 19291 0.622 0.529 – 0.715
The absorbance was recorded after each injection. The absorbance was considered satisfactory if it was within 15% of t he original calibration series. If any of the absorbance values fell outside the 15% range, the injection was rechecked as follows. The volume for the value that was out of range was reinjected twice. The closer pair of the three injections were averaged and the results were compared to the original curve. If the average of the pair was within the 15% range, the original was accepted. If the value of the average was outside the 15% range, the Study Director decided if a new graph was to be prepared.

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Miran Calibration Appendix O

Calibration Curve for Gasoline DIPE Vapor Condensate

y = -40124x 4 + 41398x 3 - 6533.9x 2 + 29104x
R 2 = 0.9977

0 5000 10000 15000 20000 25000 30000

0.0000 0.2000 0.4000 0.6000 0.8000 1.0000 Absorbance (Volts)

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Chamber Distribution Records Appendix P

Group (target) Date Port IR Conc (mg/m 3 

### ) Ratio to H

- 1

IIA (2000 mg/m 3 

### ) 04

- Feb

- 02 H

- 1 2090 1.00 H

- 2 2100 1.00 H

- 8 1980 0.95 H

- 7 1960 0.94 H

- 1 2110 1.00 H

- 12 2090 0.99 H

- 11 2100 1.00 H

- 13 2120 1.00 H

- 14 2120 1.00
IIB (2000 mg/m 3 

### ) 04

- Feb

- 02 H

- 1 2170 1.00 H

- 2 2270 1.05 H

- 8 2290 1.06 H

- 7 2310 1.06 H

- 1 2130 1.00 H

- 14 2930 1.38 H

- 13 2160 1.01 H

- 11 2120 1.00 H

- 12 2440 1.15 07

- Feb

- 02 H

- 1 2030 1.00 H

- 14 2270 1.12
IIIA (10000 mg/m 3 

### ) 0 5

- Feb

- 02 H

- 1 10000 1.00 H

- 2 10200 1.02 H

- 8 10300 1.03 H

- 7 10200 1.02 H

- 1 10500 1.00 H

- 12 11300 1.08 H

- 11 11000 1.05 H

- 1 8970 1.00 H

- 13 9390 1.05 H

- 14 9540 1.06

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Chamber Distribution Records Appendix P

Group (target) Date Port IR Conc (mg/m 3 

### ) Ratio to H

- 1

IIIB ( 10000 mg/m 3 

### ) 05

- Feb

- 02 H

- 1 11000 1.00 H

- 2 10600 0.96 H

- 8 10700 0.97 H

- 7 11000 1.00 H

- 1 11100 1.00 H

- 12 11300 1.02 H

- 11 11500 1.04 H

- 13 11300 1.02 H

- 1 10200 1.00 H

- 14 10100 0.99
IVA (20000 mg/m 3 

### ) 06

- Feb

- 02 H

- 1 20500 1.00 H

- 2 20500 1.00 H

- 8 20500 1.00 H

- 7 20400 1.00 H

- 1 20400 1.00 H

- 14 20000 0.98 H

- 13 20800 1.02 H

- 1 20900 1.00 H

- 12 20100 0.96 H

- 11 20000 0.96
IVB (20000 mg/m 3 

### ) 06

- Feb

- 02 H

- 1 20100 1.00 H

- 2 20800 1.03 H

- 8 21100 1.05 H

- 7 21100 1.05 H

- 1 20000 1.00 H

- 14 20800 1.04 H

- 13 20000 1.00 H

- 1 19800 1.00 H

- 12 20900 1.06 H

- 11 20900 1.06

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Testing Room and Chambers Environmental Monitoring Appendix Q

Interval Location Test Substance

(mg/m 3 

### ) Light (Ft Candles) Noise (dB) Oxygen (%) Particle Sizing (mg/m 3 

### ) b



### Pretest Front Room 0 37.9 61.9 - Back Room 0 38.6 63.3 - Group IA Chamber - - - 20 2.14 x 10 -3


### Group IB Chamber - - - 20 1.61 x 10 -3


### Group IIA Chamber - - - 20 2.38 x 10 -3


### Group IIB Chamber - - - 20 2.68 x 10 -3


### Group IIIA Chamber - - - 20 3.42 x 10 -3


### Group IIIB Chamber - - - 20 6.59 x 10 -3


### Group IVA Chamber - - - 20 3.60 x 10 -3


### Group IVB Chamber - - - 20 4.84 x 10 -3



### Week 0 Front Room 0 37.9 65.0 - Back Room 0 37.9 62.3 - Group IA Chamber - - - 20 Group IB Chamber - - - 20 Group IIA Chamber - - - 20 Group IIB Chamber - - - 20 Group IIIA Chamber - - - 20 Group IIIB Chamber - - - 20 Group IVA Chamber - - - 20 Group IVB Chamber - - - 20

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Testing Room and Chambers Environmental Monitoring Appendix Q

Interval Location Test Substance

(mg/m 3 

### ) Light (Ft Candles) Noise (dB) Oxygen (%) Particle Sizing (mg/m 3 

### ) b



### Week 6 a



### Week 12 Front Room 0 38.3 61.4 - Back Room 0 39.1 59.6 - Group IA Chamber - - - 20 Group IB Chamber - - - 20 Group IIA Chamber - - - 20 Group IIB Chamber - - - 20 Group IIIA Chamber - - - 20 Group IIIB Chamber - - - 20 Group IVA Chamber - - - 20 Group IVB Chamber - - - 20

a 

### Due to oversight, all of the parameters designated for the middle of the study

were not collected.25-MAR-200416:16HuntingdonLifeSciences00-6130ToxicitySub-Group(MainandRecovery)MALESGROUP1ANIMAL#10511052105310541055105610571058105910601061106210631064106510661067106810691070APPENDIXRGASOLINEDIPEVAPORCONDENSATE:A13-WEEKWHOLE-BODYINHALATIONTOXICITYSTUDYINRATSWITHNEUROTOXICITYASSESSMENTSAND4-WEEKINVIVOGENOTOXICITYANDIMMUNOTOXICITYASSESSMENTS0MG/M3TYPEOFDEATHRECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICEANIMALTERMINATIONHISTORYDATEOFDEATH14-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0213-MAY-0213-MAY-0213-MAY-0213-MAY-0213-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-02WEEKOFSTUDY1717171717171717171712121212121313131313STUDYDAY12212212212212212212212212212290909090909191919191Page 82825-MAR-200416:16HuntingdonLifeSciences00-6130ToxicitySub-Group(MainandRecovery)MALESGROUP2ANIMAL#2051205220532054205520562057205820592060APPENDIXRGASOLINEDIPEVAPORCONDENSATE:A13-WEEKWHOLE-BODYINHALATIONTOXICITYSTUDYINRATSWITHNEUROTOXICITYASSESSMENTSAND4-WEEKINVIVOGENOTOXICITYANDIMMUNOTOXICITYASSESSMENTS2000MG/M3TYPEOFDEATHTERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICEREASSIGNEDTOSATELLITEGROUPANIMALTERMINATIONHISTORYDATEOFDEATH13-MAY-0213-MAY-0213-MAY-0213-MAY-0213-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-02WEEKOFSTUDY12121212121313131313STUDYDAY90909090909191919191Page 82925-MAR-200416:16HuntingdonLifeSciences00-6130ToxicitySub-Group(MainandRecovery)MALESGROUP3ANIMAL#3051305230533054305530563057305830593060APPENDIXRGASOLINEDIPEVAPORCONDENSATE:A13-WEEKWHOLE-BODYINHALATIONTOXICITYSTUDYINRATSWITHNEUROTOXICITYASSESSMENTSAND4-WEEKINVIVOGENOTOXICITYANDIMMUNOTOXICITYASSESSMENTS10000MG/M3TYPEOFDEATHTERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICEANIMALTERMINATIONHISTORYDATEOFDEATH13-MAY-0213-MAY-0213-MAY-0213-MAY-0213-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-02WEEKOFSTUDY12121212121313131313STUDYDAY90909090909191919191Page 83025-MAR-200416:16HuntingdonLifeSciences00-6130ToxicitySub-Group(MainandRecovery)MALESGROUP4ANIMAL#40514052405340544055405640574058405940604061406240634064406540664067406840694070APPENDIXRGASOLINEDIPEVAPORCONDENSATE:A13-WEEKWHOLE-BODYINHALATIONTOXICITYSTUDYINRATSWITHNEUROTOXICITYASSESSMENTSAND4-WEEKINVIVOGENOTOXICITYANDIMMUNOTOXICITYASSESSMENTS20000MG/M3TYPEOFDEATHRECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICEANIMALTERMINATIONHISTORYDATEOFDEATH14-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0213-MAY-0213-MAY-0213-MAY-0213-MAY-0213-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-02WEEKOFSTUDY1717171717171717171712121212121313131313STUDYDAY12212212212212212212212212212290909090909191919191Page 83125-MAR-200416:16HuntingdonLifeSciences00-6130ToxicitySub-Group(MainandRecovery)FEMALESGROUP1ANIMAL#15511552155315541555155615571558155915601561156215631564156515661567156815691570APPENDIXRGASOLINEDIPEVAPORCONDENSATE:A13-WEEKWHOLE-BODYINHALATIONTOXICITYSTUDYINRATSWITHNEUROTOXICITYASSESSMENTSAND4-WEEKINVIVOGENOTOXICITYANDIMMUNOTOXICITYASSESSMENTS0MG/M3TYPEOFDEATHRECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICEANIMALTERMINATIONHISTORYDATEOFDEATH14-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0213-MAY-0213-MAY-0213-MAY-0213-MAY-0213-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-02WEEKOFSTUDY1717171717171717171712121212121313131313STUDYDAY12212212212212212212212212212290909090909191919191Page 83225-MAR-200416:16HuntingdonLifeSciences00-6130ToxicitySub-Group(MainandRecovery)FEMALESGROUP2ANIMAL#2551255225532554255525562557255825592560APPENDIXRGASOLINEDIPEVAPORCONDENSATE:A13-WEEKWHOLE-BODYINHALATIONTOXICITYSTUDYINRATSWITHNEUROTOXICITYASSESSMENTSAND4-WEEKINVIVOGENOTOXICITYANDIMMUNOTOXICITYASSESSMENTS2000MG/M3TYPEOFDEATHTERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICEANIMALTERMINATIONHISTORYDATEOFDEATH13-MAY-0213-MAY-0213-MAY-0213-MAY-0213-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-02WEEKOFSTUDY12121212121313131313STUDYDAY90909090909191919191Page 83325-MAR-200416:16HuntingdonLifeSciences00-6130ToxicitySub-Group(MainandRecovery)FEMALESGROUP3ANIMAL#3551355235533554355535563557355835593560APPENDIXRGASOLINEDIPEVAPORCONDENSATE:A13-WEEKWHOLE-BODYINHALATIONTOXICITYSTUDYINRATSWITHNEUROTOXICITYASSESSMENTSAND4-WEEKINVIVOGENOTOXICITYANDIMMUNOTOXICITYASSESSMENTS10000MG/M3TYPEOFDEATHTERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICEANIMALTERMINATIONHISTORYDATEOFDEATH13-MAY-0213-MAY-0213-MAY-0213-MAY-0213-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-02WEEKOFSTUDY12121212121313131313STUDYDAY90909090909191919191Page 83425-MAR-200416:16HuntingdonLifeSciences00-6130ToxicitySub-Group(MainandRecovery)FEMALESGROUP4ANIMAL#45514552455345544555455645574558455945604561456245634564456545664567456845694570APPENDIXRGASOLINEDIPEVAPORCONDENSATE:A13-WEEKWHOLE-BODYINHALATIONTOXICITYSTUDYINRATSWITHNEUROTOXICITYASSESSMENTSAND4-WEEKINVIVOGENOTOXICITYANDIMMUNOTOXICITYASSESSMENTS20000MG/M3TYPEOFDEATHRECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICERECOVERYSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICEANIMALTERMINATIONHISTORYDATEOFDEATH14-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0214-JUN-0213-MAY-0213-MAY-0213-MAY-0213-MAY-0213-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-0214-MAY-02WEEKOFSTUDY1717171717171717171712121212121313131313STUDYDAY12212212212212212212212212212290909090909191919191Page 83525-MAR-200416:17HuntingdonLifeSciences00-6130NNeurotoxicitySub-GroupMALESGROUP1ANIMAL#10711072107310741075APPENDIXRGASOLINEDIPEVAPORCONDENSATE:A13-WEEKWHOLE-BODYINHALATIONTOXICITYSTUDYINRATSWITHNEUROTOXICITYASSESSMENTSAND4-WEEKINVIVOGENOTOXICITYANDIMMUNOTOXICITYASSESSMENTS0MG/M3TYPEOFDEATHTERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICEANIMALTERMINATIONHISTORYDATEOFDEATH16-MAY-0216-MAY-0216-MAY-0217-MAY-0217-MAY-02WEEKOFSTUDY1313131313STUDYDAY9393939494Page 83625-MAR-200416:17HuntingdonLifeSciences00-6130NNeurotoxicitySub-GroupMALESGROUP2ANIMAL#20612062206320642065APPENDIXRGASOLINEDIPEVAPORCONDENSATE:A13-WEEKWHOLE-BODYINHALATIONTOXICITYSTUDYINRATSWITHNEUROTOXICITYASSESSMENTSAND4-WEEKINVIVOGENOTOXICITYANDIMMUNOTOXICITYASSESSMENTS2000MG/M3TYPEOFDEATHTERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICEANIMALTERMINATIONHISTORYDATEOFDEATH16-MAY-0216-MAY-0217-MAY-0217-MAY-0217-MAY-02WEEKOFSTUDY1313131313STUDYDAY9393949494Page 83725-MAR-200416:17HuntingdonLifeSciences00-6130NNeurotoxicitySub-GroupMALESGROUP3ANIMAL#30613062306330643065APPENDIXRGASOLINEDIPEVAPORCONDENSATE:A13-WEEKWHOLE-BODYINHALATIONTOXICITYSTUDYINRATSWITHNEUROTOXICITYASSESSMENTSAND4-WEEKINVIVOGENOTOXICITYANDIMMUNOTOXICITYASSESSMENTS10000MG/M3TYPEOFDEATHTERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICEANIMALTERMINATIONHISTORYDATEOFDEATH16-MAY-0216-MAY-0216-MAY-0217-MAY-0217-MAY-02WEEKOFSTUDY1313131313STUDYDAY9393939494Page 83825-MAR-200416:17HuntingdonLifeSciences00-6130NNeurotoxicitySub-GroupMALESGROUP4ANIMAL#40714072407340744075APPENDIXRGASOLINEDIPEVAPORCONDENSATE:A13-WEEKWHOLE-BODYINHALATIONTOXICITYSTUDYINRATSWITHNEUROTOXICITYASSESSMENTSAND4-WEEKINVIVOGENOTOXICITYANDIMMUNOTOXICITYASSESSMENTS20000MG/M3TYPEOFDEATHTERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICETERMINALSACRIFICEANIMALTERMINATIONHISTORYDATEOFDEATH16-MAY-0216-MAY-0217-MAY-0217-MAY-0217-MAY-02WEEKOFSTUDY1313131313STUDYDAY9393949494Page 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Table of Contents

Functional Observational Battery Study .......................................................... 870 Motor Activity Study ......................................................................................... 935

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This is a reformat of text and tables from Study No. H-34, 00-14500

Table of Contents

Introducti on ...................................................................................................... 871 Materials and Methods .................................................................................... 871 Results and Di scussion ................................................................................... 890 Conclusi on ....................................................................................................... 895 Mean Body Weights, Grip Strength and Landing Foot Splay Values ............... 896 Incidence Summary of Functional Obse rvational Battery Evaluations ............. 907

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1. INTRODUCTION This study was designed to train tec hnicians to perform the functional observational battery evaluations for rats and to provide evidence, by use of test materials with known neurological effects, that the observational methods used at Huntingdon Life Sciences, Princeton Resear ch Center, are capable of detecting major neurotoxic endpoints. In addition, these studies were used to demonstrate interobserver reliability for neurobehavioral assessments by evaluating consistency of observations performed by different technicians.

2. MATERIALS AND METHODS 2.1. REGULATORY REFERENCES
2.1.1. TEST GUIDELINES This study was designed to provi de positive control data for neurotoxicity screening studies in accord with the US EPA Health Effects Test Guidelines, OPPTS 870.6200 Neurotoxicity Screening Battery, August 1998.

2.1.2. GOOD LABORATORY PRACTICES This study was conducted in comp liance with Part 160 of 40 CFR (EPA/FIFRA Good Laboratory Pr actice Standards) with the exception that assays to verify concentration, stability and homogeneity of the test articles in carriers were not performed.

2.1.3. ANIMAL WELFARE ACT COMPLIANCE This study complied with all appropriate parts of the Animal Welfare Act regulations: 9 CFR Parts 1 and 2 Final Rules, Federal Register, Volume 54, No. 168, August 31, 1989, pp. 36112-36163 effective October 30, 1989 and 9 CFR Part 3 Animal Welfare Standards; Final Rule, Federal Register, Volume 56, No. 32, February 15, 1991, pp. 6426-6505 effective March 18, 1991.

2.1.4. FACILITIES MANAGEMENT/ANIMAL HUSBANDRY Currently acceptable practices of good animal husbandry were followed e.g., Guide for the Care and Use of Laboratory Animals ; National Academy Press, 1996. Huntingdon Life Sciences, East Millstone, New Jersey is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

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2.2. STUDY MANAGEMENT
2.2.1. SPONSOR Huntingdon Life Sciences P.O. Box 2360 Mettlers Road East Millstone, New Jersey 08875-2360

2.2.2. TESTING FACILITY Huntingdon Life Sciences P.O. Box 2360 Mettlers Road East Millstone, New Jersey 08875-2360

2.2.3. STUDY DIRECTOR Rosemary C. Mandella, Ph.D., D.A.B.T.

2.3. EXPERIMENTAL DESIGN
2.3.1. TRAINING SESSIONS Training consisted of two parts. In the first part, the technicians were required to view a tr aining video for a functional observational battery (United States Environmental Protection Agency/American Industrial Health Council Training Video and Reference Manual for an FOB, 1996) . The second part consisted of observations of animals treated with chemicals known to produce neurological effects as indicated below:

Number of Animals

Group Route of Admin. Test Article Daily Doses a Initial Functional Observational Battery Dose (mg/kg) Volume (mL/kg) Conc. (mg/mL) M F M F 1 b i.p. Control - 5 - 3 - 3 - 2 i.p. Chlorpromazine 4 5 0.8 3 - 3 - 3 i.p. Amphetamine 2 5 0.4 3 - 3 - 4 i.p. Amphetamine 8 5 1.6 - 3 - 3 5 i.p. Physostigmine 1 5 0.2 - 3 - 3 6 Oral Ethanol 3000 10 300 - 3 - 3

a Represent doses of test article as supplied; i.e., no correction for percent active ingredient was made. b Control animals received 0.9% saline. M = Male; F = Female; i.p. = intraperitoneal; Oral, via gastric intubation; mg/kg = milligrams of test article per kilogram of body weight

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2.3.2. FOB PERFORMANCE EVALUATIONS After completion of the training session, each technician was required to perform a functional ob servational battery evaluation in which the technician was blinded as to the treatment group. Each technician evaluated 20 males or 20 females (5 animals/group).

Number of Animals

Group Route of Admin. Test Article Daily Doses a Initial Functional Observational Battery Dose (mg/kg) Volume (mL/kg) Conc. (mg/mL) M F M F
1 b i.p. Control - 5 - 5 5 5 5 2 i.p. Chlorpromazine 4 5 0.8 5 5 5 5 3 i.p. Amphetamine 2 5 0.4 5 5 5 5
4 s.c. c Physostigmine 1 5 0.2 5 5 5 5

a Represent doses of test article as supplied; i.e., no correction for percent active ingredient was made. b Control animals received 0.9% saline.

c Intraperitoneal injections were administered for the Training Sessions and for the Performance Evaluations conducted by Observer 8; subcutan eous injections were administered for the Performance Evaluations conducted by all other observers. M = Male; F = Female; i.p. = intraperitoneal; s.c. = subcutaneous injection mg/kg = milligrams of test artic le per kilogram of body weight



### 2.4. STUDY DATES
2.4.1. STUDY INITIATION 15 February 2000 (Date Study Director signed the Protocol)

2.4.2. DATE OF ANIMAL RECEIPT
3 March 2000

2.4.3. DOSING INITIATION Training Session: 23 March 2000 (Experimental Start Date) Performance Evaluations: 26 April 2000

2.4.4. DOSING TERMINATION 6 July 2000

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2.4.5. EXPERIMENTAL TERMINATION 1 August 2001

2.4.6. STUDY COMPLETION 1 August 2003 (Date Study Director signed the Final Report)

2.5. TEST ARTICLES
2.5.1. CHLORPROMAZINE
Manufacturer Sigma Chemical Company St. Louis, Missouri 63178

Lot Number

48H1403
Purity
98%
Description White/white with a yellow cast powder

Date Received
1 December 1999
Expiration Date April 2001
Storage Room temperature, protected from light

2.5.2. D-AMPHETAMINE SULFATE
Manufacturer Sigma Chemical Company St. Louis, Missouri 63178

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Lot Number

69H1239
Purity
Assume 100%
Description
Powder

Date Received 7 January 2000
Expiration Date

June 2001
Storage Room temperature

2.5.3. PHYSOSTIGMINE
Manufacturer Sigma Chemical Company St. Louis, Missouri 63178

Lot Number

48H1126
Purity
Assume 100%
Description
Powder

Date Received
1 December 1999

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Expiration Date
1 December 2004
Storage Room temperature, protected from light

2.5.4. ETHANOL
Manufacturer Spectrum Chemical Manufacturing Corporation Gardena, California 90248

Lot Number

MI0504
Purity
100%
Description Clear liquid

Date Received
16 March 1998
Expiration Date
March 2003
Storage Room temperature

2.5.5. ANALYSIS Documentation of the identity, strength, purity, composition and stability; and synthesis, fabrication, and/or derivation of the test articles was the responsibility of the Manufacturers.

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2.5.6. ARCHIVAL SAMPLE Samples from the lots of Chlorpromazine, D-Amphetamine Sulfate, Physostigmine and Ethanol are stored in the Archives of the Testing Facility under conditions specified for test article storage.

2.5.7. DISPOSITION The unused portions of Chlorpromazine, D-Amphetamine Sulfate and Physostigmine were submitted to the Archives of the Testing Facility as archival samples. The remaining portions of Ethanol were retained by the Testing Facility for possible future use.

2.6. CONTROL ARTICLE
0.9% Saline

2.6.1. MANUFACTURER Baxter Healthcare Corporation Deerfield, Illinois 60015

2.6.2. LOT NUMBER

G949941

2.6.3. PURITY
100%

2.6.4. DESCRIPTION Clear, colorless liquid

2.6.5. DATE RECEIVED
5 November 1999

2.6.6. EXPIRATION DATE
October 2000

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2.6.7. STORAGE Room Temperature

2.6.8. ANALYSIS Documentation of the identity, strength, purity, composition and stability; and synthesis, fabrication, and/or derivation of the control article was the responsibility of the Manufacturer.

2.6.9. ARCHIVAL SAMPLE A sample from each lot of control ar ticle is stored in the Archives of the Testing Facility under conditions specified for control article storage.

2.6.10. DISPOSITION The unused portion of the control article will be retained for use in future studies. Any empty control article containers will be discarded by the Testing Facility following completion of the study.

2.7. TEST ANIMALS
2.7.1. SPECIES Albino Rats (Outbred) VAF/Plus 


### CD ® 

### (Sprague-Dawley derived) [Crl:CD ® 

### (SD)IGS BR]

2.7.2. SUPPLIER
Charles River Laboratories Kingston, New York 12484

2.7.3. JUSTIFICATION FOR ANIMAL SELECTION The rat is the animal model recommended by the testing guidelines for use in neurotoxicity screening studies. In addition, a historical database is available for comparative evaluation.

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2.7.4. NUMBER OF ANIMALS Transferred from the In-house Colony: 60 total (30 males, 30 females)
Placed on test: 40 total (20 males, 20 females) Females were nulliparous and non-pregnant.

2.7.5. JUSTIFICATION FOR NUMBER OF ANIMALS The neurotoxicants used in this study were chosen because they produce transient neurological effects and do not cause permanent damage to the nervous system at the doses used in this study. Therefore, the animals were used more than once during the training program. During the training portion of the study, a total of 18 animals (3 males or 3 fema les/chemical) were used. This was considered the minimum number necessary to demonstrate the range of effects of the chemi cal based on individual animal variability. During the performance part of the study, a total of 20 males and 20 females were used (including animals used in the training portion of the study). This allowed each technician undergoing training to perform the FOB evaluation for 20 animals (5 males or 5 females/group) and allowed two separate evaluations per week. Five animals per gr oup were considered the minimum number necessary to produce scientific and statistically valid data.

2.7.6. AGE AT RECEIPT 23 days

2.7.7. AGE AT INITIATION OF TRAINING SESSION 43 days

2.7.8. AGE AT INITIATION OF PERFORMANCE EVALUATIONS 77 days

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2.7.9. WEIGHT AT INITIATION OF TRAINING SESSION (GRAMS)

Mean Range Male: 205 178 to 225 Female: 172 161 to 180

2.7.10. WEIGHT AT INITIATION OF PERFORMANCE EVALUATIONS (GRAMS)

Mean Range Male: 440 379 to 498 Female: 270 235 to 339

2.7.11. ACCLIMATION PERIOD Animals were acclimated for approximately 3 weeks. All animals were examined during the acclimation period to confirm suitability for study.

2.8. SELECTION AND GROUP ASSIGNMENT More animals than required for the study were transferred from the inhouse colony. Twenty males and twen ty females were randomly selected for use on study and randomly assigned to control or treatment groups. Individual weights of animals used on study at initiation of the training session were within ±20% of the mean weight for each sex. Disposition of all animals not utilized in the study is maintained in the study file.

2.9. ANIMAL IDENTIFICATION
Each rat was identified with a metal ear tag bearing its assigned animal number. The assigned animal number plus the study number comprised the unique animal number for each animal. In addition, each cage was provided with a cage card which was color-coded for dose level identification and contained study number and animal number information.

2.10. VETERINARY CARE Animals were monitored by the t echnical staff for any conditions requiring possible veterinary care.

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2.11. HUSBANDRY
2.11.1. HOUSING Animals were doubly housed in elevated, stainless steel, wire mesh cages during the first week of the acclimation period and individually housed thereafter.

2.11.2. FEED Certified Rodent Diet, No. 5002; (Meal) (PMI Nutrition International, St. Louis, Missouri) was available without restriction. Fresh feed was presented weekly.

2.11.3. FEED ANALYSIS Analysis of each feed lot used during this study was performed by the manufacturer. Results are maintained on file.

2.11.4. WATER Water was available without restriction via an automated watering system (Elizabethtown Water Company, Westfield, New Jersey).

2.11.5. WATER ANALYSIS Water analyses are conducted by Elizabethtown Water Company, Westfield, New Jersey (Raritan-East Millstone Plant) to ensure that water meets standards specified under the EPA Federal Safe Drinking Water Act Regulations (40 CFR Part 141). In addition, water samples are collected biannually from representative rooms in the Testing Facility; chemical and microbiological water analyses are conducted on these samples by a subcontract laboratory. Results of all water an alyses are maintained on file at the Testing Facility.

2.11.6. CONTAMINANTS
There were no known contaminants in the feed or water which were expected to interfere with the results of this study.

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2.11.7. ENVIRONMENTAL CONDITIONS Light/Dark Cycle A twelve hour light/dark cycle wa s provided and controlled via an automatic timer.

Temperature Temperature was monitored and recorded twice daily and maintained within the specified range to the maximum extent possible. Excursions outside the specified range were not considered to have affected the integrity of the study.

Desired: 18 to 26 ° C Actual: 15 to 24°C
Relative Humidity Relative humidity was monitored and recorded once daily and maintained within the specified range to the maximum extent possible. Excursions outside the specified range were not considered to have affected the integrity of the study.

Desired: 30 to 70% Actual: 28 to 68%

2.12. TEST ARTICLE PREPARATION Appropriate amounts of the test article s were mixed with 0.9% saline to achieve the desired concentrations. Dosing solutions were prepared either the day prior to or the day of dos e administration and were stored refrigerated when not in use.

2.13. ANALYSIS OF DOSE SOLUTIONS Analysis of the dosing solutions was not performed for this study.

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2.14. TEST ARTICLE ADMINISTRATION
2.14.1. ROUTE OF ADMINISTRATION Chlorpromazine, D-Amphetamine Sulfate and saline were administered by intraperitoneal injection into the abdominal cavity near the inguinal region using a sterile needle and syringe of appropriate size. Doses were calculated using the most recent body weights available. A volume of 5 mL/kg of body weight was administered.
Physostigmine was administered by intraperitoneal injection into the abdominal cavity near the inguinal region or by subcutaneous injection into an area on the back, using a sterile needle and syringe of appropriate size. In traperitoneal injections were administered for the Training Sessions and for the Performance Evaluations conducted by Observer 8; subcutaneous injections were administered for the Performance Evaluations conducted by all other observers. Doses were calculated using the most recent body weights available. A volume of 5 mL/kg of body weight was administered.
Ethanol was administered by oral intubation using a stainless steel dosing needle and syringe of appropriate size. Doses were calculated using the most recent body weights available. A volume of 10 mL/kg of body weight was administered.

2.14.2. ROUTE JUSTIFICATION The intraperitoneal, subcutaneous and oral routes provide rapid test article absorption and have been used in previous studies with the test articles.

2.14.3. DOSE LEVELS Training Sessions 4 mg Chlorpromazine/kg body weight (males) 2 mg D-Amphetamine Sulfate/kg body weight (males) 8 mg D-Amphetamine Sulfate/kg body weight (females) 1 mg Physostigmine/kg body weight (females) 3000 mg Ethanol/kg body weight (females)

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Performance Evaluations 4 mg Chlorpromazine/kg body weight 2 mg D-Amphetamine Sulfate/kg body weight 1 mg Physostigmine/kg body weight

2.14.4. DOSE VOLUME 0.9% Saline, Chlorpromazine, D-Amphetamine Sulfate, and Physostigmine were administered at a volume of 5 mL/kg of body weight. Ethanol was administer ed at a volume of 10 mL/kg of body weight.

2.14.5. FREQUENCY OF DOSING Each animal was dosed no more than once per week. There was a washout period of approximately one to five weeks between doses.

2.15. EXPERIMENTAL EVALUATIONS
2.15.1. VIABILITY CHECKS Animals were observed in their cages once daily for mortality and signs of severe toxic or pharmacologic effects.

2.15.2. PHYSICAL/NEUROBEHAVIO RAL EXAMINATIONS Animals were removed from their cages and examined once pretest to determine suitability for study. Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia, occurrence of secretions and excretions, autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait or posture, response to handling, presence of clonic or tonic movements, stereotypy (e.g., excessive grooming, repetitive circling) and bizarre behavior (e.g., self-mutilation, walking backward).

2.15.3. BODY WEIGHT Animals were removed from their cages and weighed once pretest and once prior to each dose to determine dose volumes. Body

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weights were also recorded during the study as part of the functional observational battery.

2.15.4. FUNCTIONAL OBSERVATIONAL BATTERY EVALUATIONS
Training Session Training consisted of two parts. In the first part, all technicians were required to view a tr aining video for a functional observational battery (United States Environmental Protection Agency/American Industrial Health Council Training Video and Reference Manual for an FOB, 1996) . The second part consisted of observations of animals treated with chemicals known to produce neurological effects. Two experienced technicians performed the FOB evaluations and instructed the novice technicians, under the direction of the Study Director or other senior staff member. These evaluations were not performed “blind”.

FOB Performance Evaluation After training, the experienced and novice technicians were each required to evaluate 20 animals (5 males or females/group) using the Functional Observational Battery. Observations by each technician were performed blind, i.e., the observer was unaware of the treatment of each animal. Animals were randomized and blinded according to the standard operating procedures of the Testing Facility.
The results of each FOB evaluation were tabulated and the Study Director determined whether the observations performed by each technician comprised a treatment profile consistent with known neurological effects of each test article. Interobserver reliability was considered established for technicians that accurately identified the treatment profile for each test article. The expected neurological effects were base d on published reports in the literature, a previous validation study conducted at the Testing

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Facility (Study No. 96-4516), and observations by the experienced technicians.
Technicians not accurately identifying the treatment profile for each test article were given additional training and/or conducted additional performance evaluations before being allowed to conduct FOB evaluations on a study.
In addition to being able to identify the treatment profile for each test article, each technician was required to demonstrate consistency in performing the quantitative measures of hindlimb and forelimb grip strength and landi ng foot splay. This was done by determining the mean and standard deviation for the control animals during the performance ev aluation for each technician and calculating the coefficient of vari ance [(standard deviation/mean) x 100%]. The coefficient of variance was required to be approximately 25% or lower to be considered consistent. If a technician did not achieve this leve l of consistency, further training and evaluations were performed.

Parameters Evaluated Temperature, humidity, noise level and illumination were measured and recorded to ensure that variations in environmental conditions were minimal during all evaluations.
Evaluations were performed according to the Testing Facility’s standard operating procedures that include defined scales for each function listed below:

1. Assessment of signs of autonomic function: Ranking of the degree of lacrimation and salivation Presence or absence of piloerection and exophthalmus Count of urination and defecation Pupillary function (constriction of pupil in response to light) Degree of palpebral closure

2. Description, incidence, and severity of any convulsions, tremors, or abnormal motor movements

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3. Ranking of general level of activity in the open field

4. Description and incidence of posture and gait abnormalities and ranking of gait abnormalities

5. Quantitative measures of forelimb and hindlimb grip strength

6. Quantitative measure of landing foot splay

7. Ranking of reactivity to general stimuli; sensorimotor responses to visual and auditory stimuli; and the response to a tail pinch.

2.16. POSTMORTEM
At the conclusion of all evaluations , all animals were sacrificed and discarded. No necropsy was perfor med and no tissues were collected. Animals were euthanized by carbon dioxide inhalation.

2.17. STATISTICAL ANALYSIS The following parameters were analyzed statistically:
body weight (from the functional observational battery) forelimb and hindlimb grip strength measurements landing foot splay measurements

2.17.1. METHOD OF ANALYSIS Mean values of all dose groups we re compared to the mean value for the control group at each time interval.
Evaluation of equality of group means was made by the appropriate statistical method, followed by a multiple comparison test if needed. Bartlett’s test (Bartlett, 1937) was performed to determine if groups had equal variances. If the variances were equal, parametric procedures were used; if not, nonparametric procedures were used. The parametric method was the standard one-way analysis of variance (ANOVA) using the F ratio to assess significance (Armitage, 1971). If significant differences among the means were indicated, Dunnett’s test (Dunnett, 1955, 1964) or

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Williams test (Williams, 1971, 1972) were used to determine which means were significantly diffe rent from the control. The nonparametric method was the Kruskal-Wallis test (Kruskal and Wallis, 1952, 1953) and if differences were indicated, Shirley’s test (Shirley, 1977) or Dunn’s te st (Dunn, 1964) were used to determine which means differed from control. Bartlett’s test for equality of variance was conducted at the 1% significance level; all other statistical tests were conducted at the 5% and 1% significance levels.

2.18. DATA STORAGE All raw data and retained samples, as well as the original study protocol and the original final report will be maintained in the Archives of the Testing Facility upon completion of the study.

2.19. PROTOCOL DEVIATIONS The following protocol deviations o ccurred during the study but were not considered to have compromised the validity or integrity of the study.

1. Viability checks were performed once per day instead of twice per day as specified in the Protocol.

2. Animals were approximately 3 weeks old at receipt instead of approximately 4 weeks old as specified in the protocol. In addition, the animals were approximately 11 weeks old at initiation of performance evaluations rather than 8 weeks or less as specified by the protocol.

3. Animals were not randomly assigned to study groups to try to equalize mean body weights as specified in the Protocol. Animals were arbitrarily assigned animal numbers and placed into groups by animal number (i.e. Nos. 5001 to 5005 were assigned to Group 1; Nos. 5006 to 5010 were assigned to Group 2; etc.).

4. Body weights of some animals exceed ed the Protocol specified range of 150 to 250 grams at first dose.

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5. Initial FOB performance evaluations by Observers 1 and 6 conducted on 26 April 2000 were performed on 10 animals/sex/observer instead of 20 males or 20 females/observer as specified in the Protocol. Evaluations were re-conducted by these observers using the correct number of animals. The initial evaluations are not presented in this report, but are maintained with the study data.

6. The washout period between doses was approximately one to five weeks instead of approximately one week as specified in the Protocol.

7. Body weights for the calculation of dose volumes were not recorded weekly as specified in the Protocol , but were recorded prior to each dose.

8. Fresh dosing solutions were prepared on the day prior to or on day of dose rather than once weekly as required by protocol.

9. The acceptable variability established in the report of 25-30% differed from the range specified in the protocol of 25% or less.

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3. RESULTS AND DISCUSSION

3.1. FUNCTIONAL OBSERVATIONAL BATTERY (FOB) EVALUATIONS
Nine technicians were trained or retr ained in the performance of the FOB evaluations and subsequently tested for their ability to identify the behavioral pattern produced by thr ee test articles that are known to produce neurological effects. Three of the technicians (Observers 1, 5, and 6) had been previously trained and helped to set the standard for the expected pattern of observations for each compound. Results of a previous training study conducted at the Testing Facility (Study No. 96-4516), as well as published data , were also used to set the performance standards. Results of the FOB evaluations for each test article are presented in Table 2 for all nine observers. The standard pattern of effects for each of the test articles and the comparative evaluations for each observer are pres ented in the following sections for all observers, with the exception of Observer 3 who is no longer employed at the Testing Facility.

3.2. FOB EVALUATIONS FOR RATS TREATED WITH CHLORPROMAZINE The pattern of effects observed afte r administration of chlorpromazine (Text Table 1) are consistent with the actions of the drug which is a central nervous system depressant. The pattern of effects is also similar to that observed in previous training studies. All observers noted postural effects in the home cage, with the anim al lying on its side or flattened; effects on gait and posture in the ope n field, mainly body drags or limbs splayed or dragging; decreased or no locomotion; low or very low arousal level; and no response to the approach of a blunt object. Seven of the eight observers that were compared al so noted effects on palpebral closure in the home cage, with eyelids s lightly drooping or half-closed and moderate or extreme lacrimation. Si x of the eight observers also noted no response to an auditory stimulus a nd some in coordination in the air righting reflex. In the functional evaluations, there was an increase in

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landing foot splay of 17% to 46% for the Chlorpromazine-treated animals, as compared to the saline controls, for 7/7 observers.
Text Table 1 - FOB Evaluations for Rats Treated with Chlorpromazine No. of Observations Males (n = 5)) Females (n = 5) Evaluations Obs. 1 Obs. 2 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9 Home Cage

• Posture lying on Side flattened

• Palpebral Closure sl. drooping half-closed 2 1

4 1 0 1

1 0 2 0

0 0 0 1

1 1 1 2

1 0 0 2

2 0 0 1

4 0 0 4

4 0 Handling

• Lacrimation moderate extreme 4 0 1 0 2 0 0 0 1 3 1 2 2 1 2 2 Open Field

• Gait and Posture body drags limbs splayed

• Locomotion decreased

none

• Arousal low very low 1 5

0 5

0 5 0 5

0 5

0 5 2 1

0 3

0 3 2 1

1 4

0 5 1 3

0 4

0 4 2 2

1 4

2 3 3 2

4 1

1 4 2 3

1 4

1 4 Reflex

• Approach no response

• Audition no response

• Air Righting sl. uncoordinated lands on side 3

0

3 1 3

2

2 0 2

2

0 1 4

0

3 0 3

2

2 0 5

2

0 0 2

2

0 0 5

4

1 1 Landing Foot Splay

• % increase 35% 34% 17% 21% 46% 17% a 25%

a Some measurements were performed incorrectly and not considered valid. The number of valid measurements was considered to be too small to provide a valid comparison.

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3.3. FOB EVALUATIONS FOR RATS TREATED WITH AMPHETAMINE D-amphetamine, a catecholamine analog, is a central nervous system stimulant that induces increased locomotor activity, mainly through noradrenergic receptors, and/or produces stereotypic behavior via dopaminergic mechanisms. The predom inant effects that are produced are dependent on both the dose of amphetamine and individual variability. The effects observed in the FOB after administration of D-amphetamine to rats are consistent with both actions of the drug. Stereotypic behavior, consisting of excessive repetitive actions such as sniffing, was noted by all eight observers and was seen in most of the animals. Increased locomotor activity and high or very high arousal state was noted by seven of eight observers, although the incidence was lower than that for stereotypic behavior. Decreased locomotor activity and low or very low arousal was also noted by some observers. This paradoxical effect was attributed to the predominance of the stereotypic eff ects compared to the stimulation of motor activity, which resulted in less movement and arousal in the animals.
Text Table 2 – FOB Evaluations for Rats Treated with Amphetamine No. of Observations Males (n = 5)) Females (n = 5) Evaluations Obs. 1 Obs. 2 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9 Open Field

• Locomotion increased decreased

• Arousal high, sl. excitement very high, hyperalert low very low 1 3

1

1 1 1 2 1

0

1 1 1 2 0

2

0 0 0 0 0

4

0 1 0 3 2

4

0 1 0 5 0

5

0 0 0 4 0

4

0 0 0 4 0

5

0 0 0 Stereotypic Behavior present 4 5 2 4 5 5 4 5 Reflex

• Approach no response 1 0 3 1 4 1 0 0

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3.4. FOB EVALUATIONS FOR RATS TREATED WITH PHYSOSTIGMINE Physostigmine is a cholinesterase inhibitor whose administration results in an excess of the neurotransmitter acetylcholine at cholinergic nerve endings. This results in stimulati on of cholinergic nerves in both the peripheral and central nervous system. The effects observed in the FOB are consistent with the wide range of effects produced by cholinergic stimulation (Text Table 3). Findings associated with stimulation of the parasympathetic nervous system, including increased salivation and miosis, were noted by all observers in most of the animals. Effects on muscles, as indicated by abnormal posture in the home cage, abnormal posture and gait in the open field, decreased or absent locomotion, muscle fasciculations, and incoordination in the air righting reflex, were also noted by all observers in most animals. In addition, 5/7 observers noted increases of 19% to 30% in mean landing foot splay measurements. Findings indicative of depression of the central nervous system included a low or very low arousal state, as not ed by all observers, and no reaction to the approach of a blunt object (6/8 observers) and no reaction to a pain stimulus (7/8 observers). The incide nce scores for Observer No. 8 were lower than those recorded by the other observers. Since the scores were low for all measures of cholinergic ac tivity, this suggests that the animals were not evaluated at the time of peak effect or that the animals did not receive a full dose of physostigmine and this was not considered an indication of inability to recognize these effects.

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Text Table 3 – FOB Evaluations for Rats Treated with Physostigmine No. of Observations Males (n = 5)) Females (n = 5) Evaluations Obs. 1 Obs. 2 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9 Home Cage

• Posture lying on side flattened 0 5 3 2 0 4 0 5 0 5 0 1 0 1 0 5 Handling

• Salivation slight moderate extreme 3 2 0 2 1 2 2 3 0 2 3 0 2 1 0 3 1 0 1 0 0 2 0 0 Open Field

• Gait and Posture ataxia body drags limbs splayed hunched posture

• Locomotion decreased

none

• Arousal low very low 0 3 5 0

0 5

2 3 0 5 3 0

0 5

0 5 0 5 0 0

3 2

1 4 0 5 3 0

0 5

0 5 0 3 3 0

2 3

1 4 2 1 1 1

5 0

3 2 0 1 0 0

1 0

1 0 2 3 1 0

3 2

0 5 Motor Movements

• Fasciculations slight

severe 4 1 4 1 5 0 4 1 1 4 5 0 1 0 3 2 Reflex

• Approach no response

• Pain no reaction

• Pupil Response miosis

• Air Righting sl. uncoordinated lands on side lands on back 2

4

5

3 2 0 0

4

5

5 0 0 3

5

5

1 3 1 4

5

5

2 0 1 2

1

5

2 3 0 2

1

5

2 2 0 0

0

2

0 1 0 2

4

5

2 1 0 Landing Foot Splay

• % increase 29% 22% a 23% 30% a b 19%

a No change in landing foot splay, as compared to saline- treated animals. b Some measurements were performed incorrectly and not considered valid. The number of valid measurements was considered to be too small to provide a valid comparison.

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3.5. CONSISTENCY OF GRIP STRENGTH AND LANDING FOOT SPLAY MEASUREMENTS Acceptable variability (25-30%) in the measurement of forelimb and hindlimb grip strength and landing foot splay was achieved by all observers.
Text Table 4 - Consistency of Grip Strength and Landing Foot Splay Measurements Coefficient of Variability (%)
Evaluations Obs. 1 Obs. 2 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9 Forelimb grip strength 11.1 21.1 21.3 22.0 16.0 b 8.7 8.8 21.1 b Hindlimb grip strength 15.3 25.3 32.8 a
27.2 a 13.6 18.4 20.5 16.0 20.6

Landing foot splay 23.5 19.1 25.4 22.4 15.4 23.4 9.8 c 29.1

a Original value was unacceptable. Evaluations were repeated using untreated animals (5 males and

5 females). b Original value was unacceptable. Evaluations were repeated using untreated animals (10 females).

c Measurements of landing foot splay were performed incorrectly and were rep eated with ten untreated

animals.



### 4. CONCLUSION Treatment of Sprague-Dawley rats with Chlorpromazine, D-Amphetamine Sulfate, or Physostigmine produced pattern s of behavior consistent with their expected neurological effects. These da ta provide evidence that the procedures used for the Functional Observational Batte ry evaluations in the Testing Facility are capable of detecting major neurotoxic endpoints. In addition, the neurobehavioral profiles for each co mpound were correctly identified by Observers 1-2 and 4-9, who were blinde d with respect to treatment. This demonstrates that there is interobserver reliability among the trained technicians at the Testing Facility in the conduct of the Functional Observational Battery evaluations. Acceptable variability in th e performance of forelimb and hindlimb grip strength measurements and landing f oot splay measurements were achieved by all observers. In conclusion, Observer s 1, 2, 4, 5, 6, 7, 8, and 9 were considered to be fully qualified to perform Functional Observational Battery evaluations.

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*Significantly different from control mean; p ≤ 0.05. **Significantly different from control mean; p ≤ 0.01. If no asterisks, no statistically significant differences from control mean.

%CV = (Control Group S.D./C ontrol Group Mean) x 100. Observer 1 Group 1 Group 2 Group 3 Group 4 Test Article: Saline Chlorpromazine D-Amphetamine Sulfate Physostigmine

Body Weight Mean 567.0 569.7 604.1 544.0 S.D. 27.9 25.0 39.8 45.7 N 5 5 5 5

* Forelimb Grip Strength Mean 926.0 680.5 1070.0 851.5 S.D. 102.5 142. 5 137.5 167.6 N 5 5 5 5 %CV 11.1

* Hindlimb Grip Strength Mean 781.5 635.5 831.0 622.5 S.D. 119.8 85.6 69.9 80.4 N 5 5 5 5 %CV 15.3

Landing Foot Splay Mean 4.8 6.5 5.4 6.2 S.D. 1.1 1.6 1.7 1.6 N 5 5 5 5 %CV 23.5

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*Significantly different from control mean; p ≤ 0.05. **Significantly different from control mean; p ≤ 0.01. If no asterisks, no statistically significant differences from control mean.

%CV = (Control Group S.D./C ontrol Group Mean) x 100. Observer 2 Group 1 Group 2 Group 3 Group 4 Test Article: Saline Chlorpromazine D-Amphetamine Sulfate Physostigmine

Body Weight Mean 572.4 572.0 608.2 547.6 S.D. 20.9 26.6 36.4 45.9 N 5 5 5 5

Forelimb Grip Strength Mean 858.0 1210.0 797.0 941.5 S.D. 181.2 369.8 266.0 244.6 N 5 5 5 5 %CV 21.1

Hindlimb Grip Strength Mean 908.5 876.5 773.0 807.5 S.D. 230.1 216.4 103.4 127.5 N 5 5 5 5 %CV 25.3

* Landing Foot Splay Mean 6.7 9.0 5.7 8.2 S.D. 1.3 1.8 0.9 1.0 N 5 5 5 5 %CV 19.1

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*Significantly different from control mean; p ≤ 0.05. **Significantly different from control mean; p ≤ 0.01. If no asterisks, no statistically significant differences from control mean.

%CV = (Control Group S.D./C ontrol Group Mean) x 100. Observer 3 Group 1 Group 2 Group 3 Group 4 Test Article: Saline Chlorpromazine D-Amphetamine Sulfate Physostigmine

Body Weight Mean 475.4 490.0 506.3 462.3 S.D. 24.2 30.2 28.9 37.5 N 5 5 5 5

Forelimb Grip Strength Mean 1499.5 1323.5 1909.0 1278.0 S.D. 470.4 715.3 344.9 351.5 N 5 5 5 5 %CV 31.4

Hindlimb Grip Strength Mean 955.5 837.5 1102.0 906.5 S.D. 270.5 255.7 230.2 251.5 N 5 5 5 5 %CV 28.3

Landing Foot Splay Mean 6.8 8.3 6.1 6.3 S.D. 0.9 2.5 0.8 1.4 N 5 5 5 5 %CV 13.4

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*Significantly different from control mean; p ≤ 0.05. **Significantly different from control mean; p ≤ 0.01. If no asterisks, no statistically significant differences from control mean.

%CV = (Control Group S.D./C ontrol Group Mean) x 100. Observer 4 Group 1 Group 2 Group 3 Group 4 Test Article: Saline Chlorpromazine D-Amphetamine Sulfate Physostigmine

Body Weight Mean 456.9 468.7 494.6 448.3 S.D. 21.3 30.1 26.5 37.0 N 5 5 5 5

* Forelimb Grip Strength Mean 1224.0 1161.0 1311.0 901.5 S.D. 261.3 149.0 179.6 276.0 N 5 5 5 5 %CV 21.3

Hindlimb Grip Strength Mean 622.5 577.5 601.0 572.0 S.D. 238.7 202.9 122.3 206.0 N 5 5 5 5 %CV 38.3

Landing Foot Splay Mean 7.2 8.4 7.1 6.9 S.D. 1.8 1.8 1.1 1.5 N 5 5 5 5 %CV 25.4

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*Significantly different from control mean; p ≤ 0.05. **Significantly different from control mean; p ≤ 0.01. If no asterisks, no statistically significant differences from control mean.

%CV = (Control Group S.D./C ontrol Group Mean) x 100. Observer 5 Group 1 Group 2 Group 3 Group 4 Test Article: Saline Chlorpromazine D-Amphetamine Sulfate Physostigmine

Body Weight Mean 558.9 564.5 591.7 530.9 S.D. 27.4 30.3 37.8 41.4 N 5 5 5 5

* Forelimb Grip Strength Mean 1352.5 1179.0 1458.5 980.5 S.D. 296.9 327.7 170.8 186.7 N 5 5 5 5 %CV 22.0

** Hindlimb Grip Strength Mean 1428.0 1105.0 1266.0 941.0 S.D. 193.8 312.4 162.7 300.3 N 5 5 5 5 %CV 13.6

Landing Foot Splay Mean 8.7 10.5 8.9 10.7 S.D. 1.9 1.7 1.3 1.5 N 5 5 5 5 %CV 22.4

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*Significantly different from control mean; p ≤ 0.05. **Significantly different from control mean; p ≤ 0.01. If no asterisks, no statistically significant differences from control mean.

%CV = (Control Group S.D./C ontrol Group Mean) x 100. Observer 6 Group 1 Group 2 Group 3 Group 4 Test Article: Saline Chlorpromazine D-Amphetamine Sulfate Physostigmine

Body Weight Mean 313.0 314.5 311.5 311.0 S.D. 18.4 26.3 46.9 10.5 N 5 5 5 5

Forelimb Grip Strength Mean 667.0 847.5 1005.5 708.0 S.D. 294.4 143.6 159.5 203.3 N 5 5 5 5 %CV 44.1

Hindlimb Grip Strength Mean 827.5 835.5 947.0 820.0 S.D. 152.2 161.6 50.1 136.6 N 5 5 5 5 %CV 18.4

* Landing Foot Splay Mean 5.0 7.3 4.0 6.5 S.D. 0.8 0.6 0.8 1.8 N 5 5 5 5 %CV 15.4

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*Significantly different from control mean; p ≤ 0.05. **Significantly different from control mean; p ≤ 0.01. If no asterisks, no statistically significant differences from control mean.

%CV = (Control Group S.D./C ontrol Group Mean) x 100. Observer 7 Group 1 Group 2 Group 3 Group 4 Test Article: Saline Chlorpromazine D-Amphetamine Sulfate Physostigmine

Body Weight Mean 313.0 312.3 305.5 310.1 S.D. 17.5 26.1 33.0 13.2 N 5 5 5 5

Forelimb Grip Strength Mean 815.0 869.5 975.5 766.5 S.D. 71.2 124.3 147.4 161.4 N 5 5 5 5 %CV 8.7

Hindlimb Grip Strength Mean 620.0 560.5 673.0 642.5 S.D. 127.2 116.7 105.2 174.5 N 5 5 5 5 %CV 20.5

Landing Foot Splay Mean 6.6 7.7 5.7 6.4 S.D. 1.5 0.8 0.8 1.8 N 5 5 5 5 %CV 23.4

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*Significantly different from control mean; p ≤ 0.05. **Significantly different from control mean; p ≤ 0.01. If no asterisks, no statistically significant differences from control mean.

%CV = (Control Group S.D./C ontrol Group Mean) x 100. Observer 8 Group 1 Group 2 Group 3 Group 4 Test Article: Saline Chlorpromazine D-Amphetamine Sulfate Physostigmine

Body Weight Mean 268.7 298.0 276.1 280.3 S.D. 19.5 55.7 27.3 12.4 N 5 5 5 5

Forelimb Grip Strength Mean 1006.5 799.5 912.5 829.5 S.D. 88.6 148.1 108.9 180.2 N 5 5 5 5 %CV 8.8

Hindlimb Grip Strength Mean 735.0 605.5 571.0 695.5 S.D. 117.9 158.6 101.9 238.0 N 5 5 5 5 %CV 16.0

Landing Foot Splay Mean 6.4 6.2 6.0 5.1 S.D. 1.6 0.3 - 0.6 N 3 3 1 2 %CV 25.0

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*Significantly different from control mean; p ≤ 0.05. **Significantly different from control mean; p ≤ 0.01. If no asterisks, no statistically significant differences from control mean.

%CV = (Control Group S.D./C ontrol Group Mean) x 100. Observer 9 Group 1 Group 2 Group 3 Group 4 Test Article: Saline Chlorpromazine D-Amphetamine Sulfate Physostigmine

Body Weight Mean 315.9 313.5 310.8 311.1 S.D. 18.3 25.5 48.3 11.1 N 5 5 5 5

Forelimb Grip Strength Mean 539.5 963.0 823.0 606.5 S.D. 292.8 456.9 330.6 327.3 N 5 5 5 5 %CV 54.3

Hindlimb Grip Strength Mean 632.0 588.0 637.0 647.5 S.D. 130.3 67.2 128.0 161.7 N 5 5 5 5 %CV 20.6

Landing Foot Splay Mean 6.3 7.9 4.4 7.5 S.D. 1.8 1.4 1.3 1.8 N 5 5 5 5 %CV 29.1

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%CV = (Control Group S.D./C ontrol Group Mean) x 100. Observer 4 Repeat Test Article: Untreated Untreated Males Females

Forelimb Grip Strength Mean 1373.0 1167.0 S.D. 168.3 293.8 N 5 5 %CV 12.3 25.2

Hindlimb Grip Strength Mean 864.5 645.5 S.D. 235.0 211.9 N 5 5 %CV 27.2 32.8

Observer 6 Repeat Test Article: Untreated Females

Forelimb Grip Strength Mean 970.5 S.D. 154.9 N 10 %CV 16.0

Hindlimb Grip Strength Mean 743.3 S.D. 147.0 N 10 %CV 19.8

Observer 8 Repeat Test Article: Saline
Males

Landing Foot Splay Mean 9.5 S.D. 0.9 N 10 %CV 9.8

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%CV = (Control Group S.D./C ontrol Group Mean) x 100. Observer 9 Repeat Test Article: Untreated Females

Forelimb Grip Strength Mean 882.0 S.D. 186.0 N 10 %CV 21.1

Hindlimb Grip Strength Mean 555.8 S.D. 178.3 N 10 %CV 32.1

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Corresponding test articles for each group were as follows:
Group 1 - Saline Group 2 - Chlorpromazine Group 3 - D-Amphetamine Sulfate Group 4 - Physostigmine
For summarization purposes, comments de scribing location [i.e., forelimb, hindlimb, right, unilateral, etc.] are not presented in this appendix. These data are contained in the study raw data if needed.
When an animal received two scores during Open Field Evaluations of Gait and Posture (i.e. the animal was noted to be dragging it’s body and it’s limbs were splayed or dragging), the two scores were summarized individually. Therefore, the total incidence of scores may be greater than the num ber of animals examined.

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Observer 1 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Home Cage Evaluations Posture Sitting or Standing Normally 1 2 1 0 Alert, Oriented Toward Observer 4 0 4 0 Asleep 0 0 0 0 Lying on Side 0 2 0 0 Flattened 0 1 0 5 Crouched 0 0 0 0

Vocalizations Not Present 5 5 5 5 Present 0 0 0 0

Palpebral Closure Eyelids Open 5 0 5 5 Eyelids Completely Closed 0 0 0 0 Eyelids Slightly Drooping 0 4 0 0 Eyelids Half Closed 0 1 0 0

Handling Evaluations Ease of Removal Very Easy 3 4 2 5 Easy 1 1 2 0 Moderately Difficult 1 0 1 0 Freezes 0 0 0 0 Difficult 0 0 0 0

Ease of Handling Normal (easy, alert) 4 5 4 5 Moderately Difficult 1 0 1 0 Freezes 0 0 0 0 Difficult 0 0 0 0 Totally limp 0 0 0 0

Chromodacryorrhea Not Present 5 5 5 5 Present 0 0 0 0

Lacrimation Not Present 5 1 5 5 Moderate 0 4 0 0 Extreme 0 0 0 0

Coat Normal 5 5 5 5 Slightly Soiled 0 0 0 0 Moderately Soiled 0 0 0 0 Extremely Soiled 0 0 0 0

Salivation Not Present 5 5 4 0 Slight 0 0 1 3 Moderate 0 0 0 2 Extreme 0 0 0 0

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Observer 1 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Open Field Evaluations Gait and Posture Normal 5 0 4 0 Ataxia 0 0 1 0 Body Drags 0 1 0 3 Limbs Splayed or Dragging 0 5 0 5 Hunched Posture 0 0 0 0

Locomotion Normal 5 0 1 0 Increased Movement 0 0 1 0 Decreased Movement 0 0 3 0 None 0 5 0 5

Arousal Normal 4 0 1 0 Low, Slight Stupor 1 0 1 2 Very Low, Stupor 0 5 1 3 High, Slight Excitement, Tense 0 0 1 0 Very High, Hyper-Alert 0 0 1 0

Piloerection Not Present 5 5 5 5 Present 0 0 0 0

Exophthalmia Not Present 5 5 5 5 Present 0 0 0 0

Feces Number of Boluses 6 11 0 3 Unformed Stool 0 0 0 0

Urine Number of Pools 4 3 0 5 Polyuria 0 0 0 0

Stereotypic Behavior Stereotypic Behavior Not Present 5 5 1 5 Present 0 0 4 0

Motor Movements Fasciculations Not Present 5 5 5 0 Slight 0 0 0 4 Severe 0 0 0 1

Convulsions Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

Tremors Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

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Observer 1 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Reflex Assessments Approach Response Normal: Slowly Approac hes, Sniffs and/or Turns Away 5 2 4 3 No Reaction 0 3 1 2 Freezes or Pulls Away Slightly 0 0 0 0 Jumps or Turns Abruptly to Avoi d, or attacks and/or bites 0 0 0 0

Audition Normal: Flinches or Flicks Ears 5 5 5 5 No Reaction 0 0 0 0 Exaggerated; Jumps, Flips, Bites 0 0 0 0

Pain Normal: Turns/Bites or Walks Forward/Away 5 4 3 1 No Reaction 0 1 2 4 Apparent Increased Pain 0 0 0 0

Pupil Response Pupil Constricts 5 4 5 0 No Constriction 0 0 0 0 Miosis 0 1 0 5 Mydriasis 0 0 0 0

Pinna Ear Flattens Against Head 0 0 0 0 Animal Shakes Head 5 5 5 5 No Response 0 0 0 0

Proprioception Normal: Returns Leg to Original Position 5 5 5 5 Returns Leg Only Partially to Original Position 0 0 0 0 No Response 0 0 0 0

Air Righting Reflex Normal: Lands on All Four Feet 5 1 5 0 Slightly Uncoordinated 0 3 0 3 Lands on Side 0 1 0 2 Lands on Back 0 0 0 0

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Observer 2 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Home Cage Evaluations Posture Sitting or Standing Normally 4 3 1 0 Alert, Oriented Toward Observer 1 1 4 0 Asleep 0 0 0 0 Lying on Side 0 0 0 3 Flattened 0 1 0 2 Crouched 0 0 0 0

Vocalizations Not Present 5 5 5 5 Present 0 0 0 0

Palpebral Closure Eyelids Open 5 4 5 5 Eyelids Completely Closed 0 0 0 0 Eyelids Slightly Drooping 0 1 0 0 Eyelids Half Closed 0 0 0 0

Handling Evaluations Ease of Removal Very Easy 0 1 4 4 Easy 5 4 1 1 Moderately Difficult 0 0 0 0 Freezes 0 0 0 0 Difficult 0 0 0 0

Ease of Handling Normal (easy, alert) 5 5 5 5 Moderately Difficult 0 0 0 0 Freezes 0 0 0 0 Difficult 0 0 0 0 Totally limp 0 0 0 0

Chromodacryorrhea Not Present 5 4 5 5 Present 0 1 0 0

Lacrimation Not Present 5 4 5 5 Moderate 0 1 0 0 Extreme 0 0 0 0

Coat Normal 5 5 5 5 Slightly Soiled 0 0 0 0 Moderately Soiled 0 0 0 0 Extremely Soiled 0 0 0 0

Salivation Not Present 5 5 4 0 Slight 0 0 1 2 Moderate 0 0 0 1 Extreme 0 0 0 2

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Observer 2 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Open Field Evaluations Gait and Posture Normal 5 0 4 0 Ataxia 0 0 1 0 Body Drags 0 0 0 5 Limbs Splayed or Dragging 0 5 0 3 Hunched Posture 0 0 0 0

Locomotion Normal 4 0 2 0 Increased Movement 0 0 2 0 Decreased Movement 1 0 1 0 None 0 5 0 5

Arousal Normal 4 0 2 0 Low, Slight Stupor 1 0 1 0 Very Low, Stupor 0 5 1 5 High, Slight Excitement, Tense 0 0 0 0 Very High, Hyper-Alert 0 0 1 0

Piloerection Not Present 5 5 5 5 Present 0 0 0 0

Exophthalmia Not Present 5 5 5 5 Present 0 0 0 0

Feces Number of Boluses 6 1 1 1 Unformed Stool 0 0 0 0

Urine Number of Pools 12 7 6 12 Polyuria 0 0 0 0

Stereotypic Behavior Stereotypic Behavior Not Present 5 5 0 4 Present 0 0 5 1

Motor Movements Fasciculations Not Present 5 5 5 0 Slight 0 0 0 4 Severe 0 0 0 1

Convulsions Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

Tremors Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 913 211-DIPE-S Final Report

Males Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 2 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Reflex Assessments Approach Response Normal: Slowly Approac hes, Sniffs and/or Turns Away 5 2 5 5 No Reaction 0 3 0 0 Freezes or Pulls Away Slightly 0 0 0 0 Jumps or Turns Abruptly to Avoi d, or attacks and/or bites 0 0 0 0

Audition Normal: Flinches or Flicks Ears 4 3 5 3 No Reaction 1 2 0 0 Exaggerated; Jumps, Flips, Bites 0 0 0 2

Pain Normal: Turns/Bites or Walks Forward/Away 5 5 5 1 No Reaction 0 0 0 4 Apparent Increased Pain 0 0 0 0

Pupil Response Pupil Constricts 5 3 4 0 No Constriction 0 0 0 0 Miosis 0 2 1 5 Mydriasis 0 0 0 0

Pinna Ear Flattens Against Head 0 0 1 0 Animal Shakes Head 5 5 4 5 No Response 0 0 0 0

Proprioception Normal: Returns Leg to Original Position 5 5 5 5 Returns Leg Only Partially to Original Position 0 0 0 0 No Response 0 0 0 0

Air Righting Reflex Normal: Lands on All Four Feet 5 3 5 0 Slightly Uncoordinated 0 2 0 5 Lands on Side 0 0 0 0 Lands on Back 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 914 211-DIPE-S Final Report

Males Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 3 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Home Cage Evaluations Posture Sitting or Standing Normally 2 3 0 1 Alert, Oriented Toward Observer 3 0 5 0 Asleep 0 0 0 0 Lying on Side 0 0 0 1 Flattened 0 2 0 3 Crouched 0 0 0 0

Vocalizations Not Present 5 5 5 5 Present 0 0 0 0

Palpebral Closure Eyelids Open 5 3 5 5 Eyelids Completely Closed 0 0 0 0 Eyelids Slightly Drooping 0 2 0 0 Eyelids Half Closed 0 0 0 0

Handling Evaluations Ease of Removal Very Easy 3 3 5 5 Easy 1 2 0 0 Moderately Difficult 1 0 0 0 Freezes 0 0 0 0 Difficult 0 0 0 0

Ease of Handling Normal (easy, alert) 3 4 5 5 Moderately Difficult 2 1 0 0 Freezes 0 0 0 0 Difficult 0 0 0 0 Totally limp 0 0 0 0

Chromodacryorrhea Not Present 5 5 5 4 Present 0 0 0 1

Lacrimation Not Present 5 3 5 5 Moderate 0 2 0 0 Extreme 0 0 0 0

Coat Normal 5 5 5 4 Slightly Soiled 0 0 0 1 Moderately Soiled 0 0 0 0 Extremely Soiled 0 0 0 0

Salivation Not Present 5 5 5 2 Slight 0 0 0 1 Moderate 0 0 0 2 Extreme 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 915 211-DIPE-S Final Report

Males Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 3 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Open Field Evaluations Gait and Posture Normal 5 0 5 0 Ataxia 0 1 0 1 Body Drags 0 3 0 4 Limbs Splayed or Dragging 0 3 0 3 Hunched Posture 0 0 0 0

Locomotion Normal 5 0 4 0 Increased Movement 0 0 1 1 Decreased Movement 0 2 0 1 None 0 3 0 3

Arousal Normal 5 0 1 0 Low, Slight Stupor 0 2 0 2 Very Low, Stupor 0 3 0 3 High, Slight Excitement, Tense 0 0 4 0 Very High, Hyper-Alert 0 0 0 0

Piloerection Not Present 5 5 5 5 Present 0 0 0 0

Exophthalmia Not Present 5 5 5 5 Present 0 0 0 0

Feces Number of Boluses 1 1 0 2 Unformed Stool 0 0 0 0

Urine Number of Pools 3 5 0 3 Polyuria 0 0 0 0

Stereotypic Behavior Stereotypic Behavior Not Present 5 5 2 5 Present 0 0 3 0

Motor Movements Fasciculations Not Present 5 5 5 2 Slight 0 0 0 2 Severe 0 0 0 1

Convulsions Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

Tremors Not Present 5 5 5 4 Slight 0 0 0 0 Severe 0 0 0 1

### Huntingdon Life Sciences 00-6130 Page 916 211-DIPE-S Final Report

Males Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 3 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Reflex Assessments Approach Response Normal: Slowly Approac hes, Sniffs and/or Turns Away 5 1 2 3 No Reaction 0 4 3 2 Freezes or Pulls Away Slightly 0 0 0 0 Jumps or Turns Abruptly to Avoi d, or attacks and/or bites 0 0 0 0

Audition Normal: Flinches or Flicks Ears 5 4 5 4 No Reaction 0 1 0 1 Exaggerated; Jumps, Flips, Bites 0 0 0 0

Pain Normal: Turns/Bites or Walks Forward/Away 5 5 5 3 No Reaction 0 0 0 2 Apparent Increased Pain 0 0 0 0

Pupil Response Pupil Constricts 5 5 5 2 No Constriction 0 0 0 1 Miosis 0 0 0 2 Mydriasis 0 0 0 0

Pinna Ear Flattens Against Head 0 0 1 0 Animal Shakes Head 5 5 4 5 No Response 0 0 0 0

Proprioception Normal: Returns Leg to Original Position 5 5 5 4 Returns Leg Only Partially to Original Position 0 0 0 0 No Response 0 0 0 1

Air Righting Reflex Normal: Lands on All Four Feet 5 5 5 1 Slightly Uncoordinated 0 0 0 2 Lands on Side 0 0 0 2 Lands on Back 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 917 211-DIPE-S Final Report

Males Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 4 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Home Cage Evaluations Posture Sitting or Standing Normally 5 3 3 1 Alert, Oriented Toward Observer 0 0 2 0 Asleep 0 0 0 0 Lying on Side 0 0 0 0 Flattened 0 2 0 4 Crouched 0 0 0 0

Vocalizations Not Present 5 5 5 5 Present 0 0 0 0

Palpebral Closure Eyelids Open 5 5 5 5 Eyelids Completely Closed 0 0 0 0 Eyelids Slightly Drooping 0 0 0 0 Eyelids Half Closed 0 0 0 0

Handling Evaluations Ease of Removal Very Easy 4 4 4 5 Easy 1 1 0 0 Moderately Difficult 0 0 1 0 Freezes 0 0 0 0 Difficult 0 0 0 0

Ease of Handling Normal (easy, alert) 5 5 5 5 Moderately Difficult 0 0 0 0 Freezes 0 0 0 0 Difficult 0 0 0 0 Totally limp 0 0 0 0

Chromodacryorrhea Not Present 5 5 5 5 Present 0 0 0 0

Lacrimation Not Present 5 3 5 3 Moderate 0 2 0 0 Extreme 0 0 0 2

Coat Normal 5 5 5 5 Slightly Soiled 0 0 0 0 Moderately Soiled 0 0 0 0 Extremely Soiled 0 0 0 0

Salivation Not Present 5 5 5 0 Slight 0 0 0 2 Moderate 0 0 0 3 Extreme 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 918 211-DIPE-S Final Report

Males Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 4 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Open Field Evaluations Gait and Posture Normal 5 2 5 0 Ataxia 0 0 0 0 Body Drags 0 2 0 5 Limbs Splayed or Dragging 0 1 0 0 Hunched Posture 0 0 0 0

Locomotion Normal 5 2 3 0 Increased Movement 0 0 2 0 Decreased Movement 0 0 0 3 None 0 3 0 2

Arousal Normal 4 2 3 0 Low, Slight Stupor 0 0 0 1 Very Low, Stupor 0 3 0 4 High, Slight Excitement, Tense 1 0 2 0 Very High, Hyper-Alert 0 0 0 0

Piloerection Not Present 5 5 4 5 Present 0 0 1 0

Exophthalmia Not Present 5 5 5 5 Present 0 0 0 0

Feces Number of Boluses 5 8 0 1 Unformed Stool 0 0 0 0

Urine Number of Pools 4 5 2 6 Polyuria 0 0 0 0

Stereotypic Behavior Stereotypic Behavior Not Present 5 5 3 4 Present 0 0 2 1

Motor Movements Fasciculations Not Present 5 5 5 0 Slight 0 0 0 5 Severe 0 0 0 0

Convulsions Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

Tremors Not Present 5 5 5 3 Slight 0 0 0 1 Severe 0 0 0 1

### Huntingdon Life Sciences 00-6130 Page 919 211-DIPE-S Final Report

Males Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 4 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Reflex Assessments Approach Response Normal: Slowly Approac hes, Sniffs and/or Turns Away 5 3 2 2 No Reaction 0 2 3 3 Freezes or Pulls Away Slightly 0 0 0 0 Jumps or Turns Abruptly to Avoi d, or attacks and/or bites 0 0 0 0

Audition Normal: Flinches or Flicks Ears 5 3 5 4 No Reaction 0 2 0 0 Exaggerated; Jumps, Flips, Bites 0 0 0 1

Pain Normal: Turns/Bites or Walks Forward/Away 5 5 4 0 No Reaction 0 0 1 5 Apparent Increased Pain 0 0 0 0

Pupil Response Pupil Constricts 5 5 5 0 No Constriction 0 0 0 0 Miosis 0 0 0 5 Mydriasis 0 0 0 0

Pinna Ear Flattens Against Head 0 1 1 0 Animal Shakes Head 5 4 4 5 No Response 0 0 0 0

Proprioception Normal: Returns Leg to Original Position 5 5 5 5 Returns Leg Only Partially to Original Position 0 0 0 0 No Response 0 0 0 0

Air Righting Reflex Normal: Lands on All Four Feet 5 4 5 0 Slightly Uncoordinated 0 0 0 1 Lands on Side 0 1 0 3 Lands on Back 0 0 0 1

### Huntingdon Life Sciences 00-6130 Page 920 211-DIPE-S Final Report

Males Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 5 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Home Cage Evaluations Posture Sitting or Standing Normally 3 3 0 0 Alert, Oriented Toward Observer 2 1 5 0 Asleep 0 0 0 0 Lying on Side 0 0 0 0 Flattened 0 1 0 5 Crouched 0 0 0 0

Vocalizations Not Present 5 5 5 5 Present 0 0 0 0

Palpebral Closure Eyelids Open 5 3 5 5 Eyelids Completely Closed 0 0 0 0 Eyelids Slightly Drooping 0 1 0 0 Eyelids Half Closed 0 1 0 0

Handling Evaluations Ease of Removal Very Easy 5 4 5 5 Easy 0 1 0 0 Moderately Difficult 0 0 0 0 Freezes 0 0 0 0 Difficult 0 0 0 0

Ease of Handling Normal (easy, alert) 5 4 5 5 Moderately Difficult 0 1 0 0 Freezes 0 0 0 0 Difficult 0 0 0 0 Totally limp 0 0 0 0

Chromodacryorrhea Not Present 5 5 5 5 Present 0 0 0 0

Lacrimation Not Present 5 5 5 5 Moderate 0 0 0 0 Extreme 0 0 0 0

Coat Normal 5 5 5 4 Slightly Soiled 0 0 0 1 Moderately Soiled 0 0 0 0 Extremely Soiled 0 0 0 0

Salivation Not Present 5 5 4 0 Slight 0 0 1 2 Moderate 0 0 0 3 Extreme 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 921 211-DIPE-S Final Report

Males Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

a Gait for two Group 2 animals could not be determin ed because the animals did not move during the observation period. Observer 5 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Open Field Evaluations Gait and Posture a Normal 5 1 5 0 Ataxia 0 0 0 0 Body Drags 0 2 0 5 Limbs Splayed or Dragging 0 1 0 3 Hunched Posture 0 0 0 0

Locomotion Normal 5 0 5 0 Increased Movement 0 0 0 0 Decreased Movement 0 1 0 0 None 0 4 0 5

Arousal Normal 4 0 0 0 Low, Slight Stupor 1 0 1 0 Very Low, Stupor 0 5 0 5 High, Slight Excitement, Tense 0 0 4 0 Very High, Hyper-Alert 0 0 0 0

Piloerection Not Present 5 5 5 5 Present 0 0 0 0
Exophthalmia Not Present 5 5 5 5 Present 0 0 0 0

Feces Number of Boluses 6 4 0 1 Unformed Stool 0 0 0 0

Urine Number of Pools 2 6 0 6 Polyuria 0 0 0 0
Stereotypic Behavior Stereotypic Behavior Not Present 5 5 1 5 Present 0 0 4 0

Motor Movements Fasciculations Not Present 5 5 5 0 Slight 0 0 0 4 Severe 0 0 0 1

Convulsions Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

Tremors Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 922 211-DIPE-S Final Report

Males Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 5 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Reflex Assessments Approach Response Normal: Slowly Approac hes, Sniffs and/or Turns Away 5 1 4 1 No Reaction 0 4 1 4 Freezes or Pulls Away Slightly 0 0 0 0 Jumps or Turns Abruptly to Avoi d, or attacks and/or bites 0 0 0 0

Audition Normal: Flinches or Flicks Ears 5 5 4 4 No Reaction 0 0 0 0 Exaggerated; Jumps, Flips, Bites 0 0 1 1

Pain Normal: Turns/Bites or Walks Forward/Away 5 5 4 0 No Reaction 0 0 0 5 Apparent Increased Pain 0 0 1 0

Pupil Response Pupil Constricts 5 5 5 0 No Constriction 0 0 0 0 Miosis 0 0 0 5 Mydriasis 0 0 0 0

Pinna Ear Flattens Against Head 2 1 1 0 Animal Shakes Head 3 4 4 5 No Response 0 0 0 0

Proprioception Normal: Returns Leg to Original Position 5 4 5 1 Returns Leg Only Partially to Original Position 0 0 0 1 No Response 0 1 0 3

Air Righting Reflex Normal: Lands on All Four Feet 5 2 5 2 Slightly Uncoordinated 0 3 0 2 Lands on Side 0 0 0 0 Lands on Back 0 0 0 1

### Huntingdon Life Sciences 00-6130 Page 923 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 6 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Home Cage Evaluations Posture Sitting or Standing Normally 4 1 0 0 Alert, Oriented Toward Observer 1 1 5 0 Asleep 0 0 0 0 Lying on Side 0 1 0 0 Flattened 0 2 0 5 Crouched 0 0 0 0

Vocalizations Not Present 5 5 5 5 Present 0 0 0 0

Palpebral Closure Eyelids Open 5 4 5 5 Eyelids Completely Closed 0 0 0 0 Eyelids Slightly Drooping 0 1 0 0 Eyelids Half Closed 0 0 0 0

Handling Evaluations Ease of Removal Very Easy 3 5 4 5 Easy 1 0 1 0 Moderately Difficult 0 0 0 0 Freezes 0 0 0 0 Difficult 0 0 0 0

Ease of Handling Normal (easy, alert) 5 5 5 5 Moderately Difficult 0 0 0 0 Freezes 0 0 0 0 Difficult 0 0 0 0 Totally limp 0 0 0 0

Chromodacryorrhea Not Present 5 5 5 5 Present 0 0 0 0

Lacrimation Not Present 5 1 5 3 Moderate 0 1 0 1 Extreme 0 3 0 1

Coat Normal 5 5 5 5 Slightly Soiled 0 0 0 0 Moderately Soiled 0 0 0 0 Extremely Soiled 0 0 0 0

Salivation Not Present 5 5 5 2 Slight 0 0 0 2 Moderate 0 0 0 1 Extreme 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 924 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 6 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Open Field Evaluations Gait and Posture Normal 5 1 5 0 Ataxia 0 0 0 0 Body Drags 0 1 0 3 Limbs Splayed or Dragging 0 3 0 3 Hunched Posture 0 1 0 0

Locomotion Normal 5 1 0 0 Increased Movement 0 0 3 0 Decreased Movement 0 0 2 2 None 0 4 0 3

Arousal Normal 5 1 0 0 Low, Slight Stupor 0 0 1 1 Very Low, Stupor 0 4 0 4 High, Slight Excitement, Tense 0 0 4 0 Very High, Hyper-Alert 0 0 0 0

Piloerection Not Present 5 5 5 5 Present 0 0 0 0

Exophthalmia Not Present 5 5 5 5 Present 0 0 0 0

Feces Number of Boluses 0 3 0 0 Unformed Stool 0 0 0 0

Urine Number of Pools 0 5 0 4 Polyuria 0 0 0 0

Stereotypic Behavior Stereotypic Behavior Not Present 5 5 0 3 Present 0 0 5 2

Motor Movements Fasciculations Not Present 5 5 5 0 Slight 0 0 0 1 Severe 0 0 0 4

Convulsions Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

Tremors Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 925 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 6 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Reflex Assessments Approach Response Normal: Slowly Approac hes, Sniffs and/or Turns Away 5 2 1 3 No Reaction 0 3 4 2 Freezes or Pulls Away Slightly 0 0 0 0 Jumps or Turns Abruptly to Avoi d, or attacks and/or bites 0 0 0 0

Audition Normal: Flinches or Flicks Ears 5 3 5 5 No Reaction 0 2 0 0 Exaggerated; Jumps, Flips, Bites 0 0 0 0

Pain Normal: Turns/Bites or Walks Forward/Away 5 5 5 4 No Reaction 0 0 0 1 Apparent Increased Pain 0 0 0 0

Pupil Response Pupil Constricts 5 5 5 0 No Constriction 0 0 0 0 Miosis 0 0 0 5 Mydriasis 0 0 0 0

Pinna Ear Flattens Against Head 0 0 2 0 Animal Shakes Head 5 5 3 5 No Response 0 0 0 0

Proprioception Normal: Returns Leg to Original Position 5 3 5 3 Returns Leg Only Partially to Original Position 0 1 0 1 No Response 0 1 0 1

Air Righting Reflex Normal: Lands on All Four Feet 5 3 5 0 Slightly Uncoordinated 0 2 0 2 Lands on Side 0 0 0 3 Lands on Back 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 926 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 7 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Home Cage Evaluations Posture Sitting or Standing Normally 2 3 1 3 Alert, Oriented Toward Observer 3 0 4 1 Asleep 0 0 0 0 Lying on Side 0 0 0 0 Flattened 0 2 0 1 Crouched 0 0 0 0

Vocalizations Not Present 5 5 5 5 Present 0 0 0 0

Palpebral Closure Eyelids Open 5 3 5 5 Eyelids Completely Closed 0 0 0 0 Eyelids Slightly Drooping 0 2 0 0 Eyelids Half Closed 0 0 0 0

Handling Evaluations Ease of Removal Very Easy 5 5 4 5 Easy 0 0 1 0 Moderately Difficult 0 0 0 0 Freezes 0 0 0 0 Difficult 0 0 0 0

Ease of Handling Normal (easy, alert) 5 5 4 5 Moderately Difficult 0 0 1 0 Freezes 0 0 0 0 Difficult 0 0 0 0 Totally limp 0 0 0 0

Chromodacryorrhea Not Present 5 4 5 5 Present 0 1 0 0

Lacrimation Not Present 5 2 5 5 Moderate 0 1 0 0 Extreme 0 2 0 0

Coat Normal 5 5 5 5 Slightly Soiled 0 0 0 0 Moderately Soiled 0 0 0 0 Extremely Soiled 0 0 0 0

Salivation Not Present 4 5 5 1 Slight 1 0 0 3 Moderate 0 0 0 1 Extreme 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 927 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 7 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Open Field Evaluations Gait and Posture Normal 5 1 5 0 Ataxia 0 0 0 2 Body Drags 0 2 0 1 Limbs Splayed or Dragging 0 2 0 1 Hunched Posture 0 0 0 1

Locomotion Normal 2 0 0 0 Increased Movement 1 0 5 0 Decreased Movement 2 1 0 5 None 0 4 0 0

Arousal Normal 3 0 0 0 Low, Slight Stupor 2 2 0 3 Very Low, Stupor 0 3 0 2 High, Slight Excitement, Tense 0 0 5 0 Very High, Hyper-Alert 0 0 0 0

Piloerection Not Present 5 5 5 5 Present 0 0 0 0

Exophthalmia Not Present 5 5 5 5 Present 0 0 0 0

Feces Number of Boluses 0 8 0 0 Unformed Stool 0 0 0 0

Urine Number of Pools 0 4 1 4 Polyuria 0 0 0 0

Stereotypic Behavior Stereotypic Behavior Not Present 5 5 0 1 Present 0 0 5 4

Motor Movements Fasciculations Not Present 5 5 5 0 Slight 0 0 0 5 Severe 0 0 0 0

Convulsions Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

Tremors Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 928 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 7 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Reflex Assessments Approach Response Normal: Slowly Approac hes, Sniffs and/or Turns Away 3 0 4 3 No Reaction 2 5 1 2 Freezes or Pulls Away Slightly 0 0 0 0 Jumps or Turns Abruptly to Avoi d, or attacks and/or bites 0 0 0 0

Audition Normal: Flinches or Flicks Ears 5 3 5 5 No Reaction 0 2 0 0 Exaggerated; Jumps, Flips, Bites 0 0 0 0

Pain Normal: Turns/Bites or Walks Forward/Away 5 5 5 4 No Reaction 0 0 0 1 Apparent Increased Pain 0 0 0 0

Pupil Response Pupil Constricts 5 4 5 0 No Constriction 0 1 0 0 Miosis 0 0 0 5 Mydriasis 0 0 0 0

Pinna Ear Flattens Against Head 3 0 4 0 Animal Shakes Head 2 5 1 5 No Response 0 0 0 0

Proprioception Normal: Returns Leg to Original Position 5 4 5 5 Returns Leg Only Partially to Original Position 0 1 0 0 No Response 0 0 0 0

Air Righting Reflex Normal: Lands on All Four Feet 4 5 4 1 Slightly Uncoordinated 1 0 1 2 Lands on Side 0 0 0 2 Lands on Back 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 929 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 8 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Home Cage Evaluations Posture Sitting or Standing Normally 0 4 0 3 Alert, Oriented Toward Observer 4 0 4 1 Asleep 1 0 0 0 Lying on Side 0 0 0 0 Flattened 0 1 0 1 Crouched 0 0 0 0

Vocalizations Not Present 5 5 5 5 Present 0 0 0 0

Palpebral Closure Eyelids Open 4 1 5 5 Eyelids Completely Closed 1 0 0 0 Eyelids Slightly Drooping 0 4 0 0 Eyelids Half Closed 0 0 0 0

Handling Evaluations Ease of Removal Very Easy 3 4 4 3 Easy 2 1 1 2 Moderately Difficult 0 0 0 0 Freezes 0 0 0 0 Difficult 0 0 0 0

Ease of Handling Normal (easy, alert) 4 5 5 5 Moderately Difficult 1 0 0 0 Freezes 0 0 0 0 Difficult 0 0 0 0 Totally limp 0 0 0 0

Chromodacryorrhea Not Present 5 4 5 5 Present 0 1 0 0

Lacrimation Not Present 5 2 5 5 Moderate 0 2 0 0 Extreme 0 1 0 0

Coat Normal 5 5 5 5 Slightly Soiled 0 0 0 0 Moderately Soiled 0 0 0 0 Extremely Soiled 0 0 0 0

Salivation Not Present 5 5 5 4 Slight 0 0 0 1 Moderate 0 0 0 0 Extreme 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 930 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 8 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Open Field Evaluations Gait and Posture Normal 5 1 5 4 Ataxia 0 0 0 0 Body Drags 0 3 0 1 Limbs Splayed or Dragging 0 2 0 0 Hunched Posture 0 0 0 0

Locomotion Normal 5 0 1 4 Increased Movement 0 0 4 0 Decreased Movement 0 4 0 1 None 0 1 0 0

Arousal Normal 5 0 1 4 Low, Slight Stupor 0 1 0 1 Very Low, Stupor 0 4 0 0 High, Slight Excitement, Tense 0 0 4 0 Very High, Hyper-Alert 0 0 0 0

Piloerection Not Present 5 5 5 5 Present 0 0 0 0

Exophthalmia Not Present 5 5 5 5 Present 0 0 0 0

Feces Number of Boluses 0 4 0 0 Unformed Stool 0 0 0 0

Urine Number of Pools 0 0 0 1 Polyuria 0 0 0 0

Stereotypic Behavior Stereotypic Behavior Not Present 5 5 1 4 Present 0 0 4 1

Motor Movements Fasciculations Not Present 5 5 5 4 Slight 0 0 0 1 Severe 0 0 0 0

Convulsions Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

Tremors Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 931 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 8 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Reflex Assessments Approach Response Normal: Slowly Approac hes, Sniffs and/or Turns Away 5 3 5 5 No Reaction 0 2 0 0 Freezes or Pulls Away Slightly 0 0 0 0 Jumps or Turns Abruptly to Avoi d, or attacks and/or bites 0 0 0 0

Audition Normal: Flinches or Flicks Ears 5 3 5 5 No Reaction 0 2 0 0 Exaggerated; Jumps, Flips, Bites 0 0 0 0

Pain Normal: Turns/Bites or Walks Forward/Away 5 4 5 5 No Reaction 0 1 0 0 Apparent Increased Pain 0 0 0 0

Pupil Response Pupil Constricts 5 3 4 2 No Constriction 0 2 0 1 Miosis 0 0 0 2 Mydriasis 0 0 1 0

Pinna Ear Flattens Against Head 0 1 1 0 Animal Shakes Head 5 4 4 5 No Response 0 0 0 0

Proprioception Normal: Returns Leg to Original Position 5 5 5 5 Returns Leg Only Partially to Original Position 0 0 0 0 No Response 0 0 0 0

Air Righting Reflex Normal: Lands on All Four Feet 5 5 5 4 Slightly Uncoordinated 0 0 0 0 Lands on Side 0 0 0 1 Lands on Back 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 932 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 9 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Home Cage Evaluations Posture Sitting or Standing Normally 4 1 4 0 Alert, Oriented Toward Observer 1 0 1 0 Asleep 0 0 0 0 Lying on Side 0 0 0 0 Flattened 0 4 0 5 Crouched 0 0 0 0

Vocalizations Not Present 5 5 5 5 Present 0 0 0 0

Palpebral Closure Eyelids Open 5 1 5 5 Eyelids Completely Closed 0 0 0 0 Eyelids Slightly Drooping 0 4 0 0 Eyelids Half Closed 0 0 0 0

Handling Evaluations Ease of Removal Very Easy 3 4 4 4 Easy 2 1 1 1 Moderately Difficult 0 0 0 0 Freezes 0 0 0 0 Difficult 0 0 0 0

Ease of Handling Normal (easy, alert) 5 5 5 5 Moderately Difficult 0 0 0 0 Freezes 0 0 0 0 Difficult 0 0 0 0 Totally limp 0 0 0 0

Chromodacryorrhea Not Present 5 4 5 5 Present 0 1 0 0

Lacrimation Not Present 5 1 5 3 Moderate 0 2 0 2 Extreme 0 2 0 0

Coat Normal 5 5 4 2 Slightly Soiled 0 0 1 3 Moderately Soiled 0 0 0 0 Extremely Soiled 0 0 0 0

Salivation Not Present 5 5 5 3 Slight 0 0 0 2 Moderate 0 0 0 0 Extreme 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 933 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 9 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Open Field Evaluations Gait and Posture Normal 5 1 5 0 Ataxia 0 0 0 2 Body Drags 0 2 0 3 Limbs Splayed or Dragging 0 3 0 1 Hunched Posture 0 0 0 0

Locomotion Normal 3 0 1 0 Increased Movement 0 0 4 0 Decreased Movement 2 1 0 3 None 0 4 0 2

Arousal Normal 3 0 0 0 Low, Slight Stupor 2 1 0 0 Very Low, Stupor 0 4 0 5 High, Slight Excitement, Tense 0 0 5 0 Very High, Hyper-Alert 0 0 0 0

Piloerection Not Present 5 5 5 5 Present 0 0 0 0

Exophthalmia Not Present 5 5 5 5 Present 0 0 0 0

Feces Number of Boluses 0 3 0 0 Unformed Stool 0 0 0 0

Urine Number of Pools 0 2 0 0 Polyuria 0 0 0 0

Stereotypic Behavior Stereotypic Behavior Not Present 5 5 0 0 Present 0 0 5 5

Motor Movements Fasciculations Not Present 5 5 5 0 Slight 0 0 0 3 Severe 0 0 0 2

Convulsions Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

Tremors Not Present 5 5 5 5 Slight 0 0 0 0 Severe 0 0 0 0

### Huntingdon Life Sciences 00-6130 Page 934 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Functional Observational Battery Incidence Summary of Functional Observational Battery Evaluations Appendix T

Observer 9 Group: 1 2 3 4

Number of Animals Examined 5 5 5 5

Reflex Assessments Approach Response Normal: Slowly Approac hes, Sniffs and/or Turns Away 3 0 5 3 No Reaction 2 5 0 2 Freezes or Pulls Away Slightly 0 0 0 0 Jumps or Turns Abruptly to Avoi d, or attacks and/or bites 0 0 0 0

Audition Normal: Flinches or Flicks Ears 5 1 5 3 No Reaction 0 4 0 2 Exaggerated; Jumps, Flips, Bites 0 0 0 0

Pain Normal: Flinches or Flicks Ears 5 5 5 1 No Reaction 0 0 0 4 Exaggerated; Jumps, Flips, Bites 0 0 0 0

Pupil Response Pupil Constricts 4 3 5 0 No Constriction 1 1 0 0 Miosis 0 1 0 5 Mydriasis 0 0 0 0

Pinna Ear Flattens Against Head 0 0 0 0 Animal Shakes Head 5 5 5 5 No Response 0 0 0 0

Proprioception Normal: Returns Leg to Original Position 5 5 5 5 Returns Leg Only Partially to Original Position 0 0 0 0 No Response 0 0 0 0

Air Righting Reflex Normal: Lands on All Four Feet 5 3 5 2 Slightly Uncoordinated 0 1 0 2 Lands on Side 0 1 0 1 Lands on Back 0 0 0 0

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Neurobehavioral Positive Control Data: Motor Activity Preface Appendix T

This is a reformat of text and tables from Study No. H-34, 00-14501

Table of Contents

Introducti on ...................................................................................................... 936 Materials and Methods .................................................................................... 936 Result s ............................................................................................................ 951 Discussi on ....................................................................................................... 952 Conclusi on ....................................................................................................... 952 Mean Motor Activi ty Val ues ............................................................................. 953

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1. INTRODUCTION This study was designed to assess the eff ect of Amphetamine and Chlorpromazine on the locomotor activity in young rats 13, 17, 21, and 62 days of age. This work was performed in order to provide Po sitive Control data and was conducted according to test guidelines of the U.S. Environmental Protection Agency (Office of Prevention, Pesticides and Toxic Substances, Health Effects Guidelines, OPPTS 870.6300, Developmental Neurotoxicity, August 1998).

2. MATERIALS AND METHODS

2.1. REGULATORY REFERENCES

2.1.1. TEST GUIDELINES This study was designed to provi de positive control data for neurotoxicity screening studies in accord with the US EPA Health Effects Test Guidelines, OPPTS 870.6200 Neurotoxicity Screening Battery, August 1998.

2.1.2. GOOD LABORATORY PRACTICES This study was conducted in comp liance with Part 160 of 40 CFR (EPA/FIFRA Good Laboratory Practice Regulations) with the exception that assays to verify concentration, stability and homogeneity of the test articles in carriers were not performed.

2.1.3. ANIMAL WELFARE ACT COMPLIANCE This study complied with all appropriate parts of the Animal Welfare Act regulations: 9 CFR Part s 1 and 2 final Rules, Federal Register, Volume 54, No. 168, August 31, 1989, pp. 36112-36163 effective October 30, 1989 and 9 CFR Part 3 Animal Welfare Standards; Final Rule, Federal Register, Volume 56, No. 32, February 15, 1991, pp. 6426-6505 effective March 18, 1991.

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2.2. FACILITIES MANAGEMENT/ANIMAL HUSBANDRY Currently acceptable practices of good animal husbandry were followed,
e.g., Guide for the Care and Use of Laboratory Animals ; National Academy Press, 1996. Huntingdon Life Sciences is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

2.3. STUDY MANAGEMENT

2.3.1. SPONSOR AND TESTING FACILITY Huntingdon Life Sciences Mettlers Road PO Box 2360 East Millstone, NJ 08875-2360

2.3.2. STUDY DIRECTOR 1


### Suzanne R. Thornton, Ph.D. P.J. Shem Patyna, Ph.D. George L. Brown, M.S. Edward R. Frizell, M.D., Ph.D.

1 Suzanne R. Thornton, Ph.D. served as Study Director during the testing portion of Study No. 00-14501. Effective 19 May 2000, P.J. Shem Patyna assumed the responsibilities of Study Dir ector. George Brown, M.S. assumed all Study Director responsibilities follo wing Dr. Patyna’s depart ure from Huntingdon Life Sciences, effective 22 December 2000. Effective 10 N ov 2008, Edward R. Frizell, M.D., Ph.D. assumed Study Director responsibilities for Study No. 00-14501 for the purpose of amending the Final Report only.

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Neurobehavioral Positive Control Data: Motor Activity Appendix T

2.4. EXPERIMENTAL DESIGN

Group Treatment Dosage (mg/kg) Dose Volume (mL/kg) Treatment Schedule Locomotor Activity Assessment Number of Animals

I a Control 0 5 Postnatal Day 13, 17, 21, 62 Approximately 30 minutes after injection 10/sex

II Amphetamine 2.0 5 Postnatal Day 13, 17, 21, 62 Approximately 30 minutes after injection 10/sex

III Chlorpromazine 10.0 5 Postnatal Day 13, 17, 21, 62 Approximately 30 minutes after injection 10/sex

a Control animals were administered 0.9% saline at the same dose volume as the test articles.



### 2.5. STUDY DATES

2.5.1. STUDY INITIATION 10 February 2000

2.5.2. DATE OF ANIMAL RECEIPT 11 February 2000

2.5.3. DOSE INITIATION DATES FOR LOCOMOTOR ACTIVITY ASSESSMENTS Postnatal Day 13: 14 February 2000 Postnatal Day 17: 18 February 2000 Postnatal Day 21: 22 February 2000 Postnatal Day 62: 03 April 2000

2.5.4. TRANSFER OF ANIMALS TO EXTRA RAT COLONY 04 April 2000

2.5.5. STUDY COMPLETION 13 March 2001 (Date Study Director signed the Final Report)

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Neurobehavioral Positive Control Data: Motor Activity Appendix T

2.6. TEST ARTICLES

2.6.1. AMPHETAMINE
Supplier Sigma 3050 Spruce Strret St. Louis, MO 63103

Lot Number

69H1239

Purity
Assume 100%

Date Received 07 January 2000

Expiration Date

June 2001

Description
Powder

Storage Room temperature

Archival Sample A sample of amphetamine is stored in the Archives of the Testing Facility under conditions specified for test article storage.

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Disposition of Unused Test Article Unused test article was used as the archival sample for this test article.

2.6.2. CHLORPROMAZINE
Supplier Sigma 3050 Spruce Street St. Louis, MO 63103

Lot Number

48H1403

Purity
98%

Date Received
01 December 1999

Expiration Date April 2001

Description White/White with a yellow cast, powder

Characteristics Light sensitive

Storage Room temperature, protected from light

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Archival Sample A sample of chlorpromazine is stored in the Archives of the Testing Facility under conditions specified for test article storage.

Disposition of Unused Test Article Unused test article was used as the archival sample for this test article.

2.7. CONTROL ARTICLE
0.9 % Saline

2.7.1. SUPPLIER
VWR Scientific Products 200 Center Square Rd. Bridgeport, NJ 08014

2.7.2. LOT/BATCH NUMBER
G-949941 / 173-191

2.7.3. DATE RECEIVED

04 June 1999

2.7.4. EXPIRATION DATE
October 2000

2.7.5. DESCRIPTION Clear liquid

2.7.6. STORAGE Room temperature

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2.7.7. ARCHIVAL SAMPLE A sample of the control article is stored in the Archives of the Testing Facility under conditions specified for test article storage.

2.8. TEST ARTICLE AND CONTROL ARTICLE PREPARATION

2.8.1. CONTROL ARTICLE PREPARATION Control article was used neat; as received.

2.8.2. TEST ARTICLE PREPARATION Appropriate amounts of test article were mixed with 0.9% saline to achieve the desired concentrations in a graduated cylinder, capped, inverted and shaken well prior to use. Fresh dosing solutions were prepared either 1 or 3 days prior to or the day of dose administration and were refrigerated (2-8 ° C) when not in use.

2.9. ANALYSIS OF DOSING SUSPENSIONS Analyses were not performed in this study.

2.10. TEST ANIMALS

2.10.1. SPECIES
Albino Rats [Crl: CD ® 

### (SD) IGS BR]

2.10.2. SUPPLIER
Charles River Laboratories Kingston, NY facility

2.10.3. JUSTIFICATION FOR TEST SYSTEM SELECTION The rat is a rodent animal model commonly utilized in reproduction studies as recommended in the referenced guidelines.

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2.10.4. ANIMAL RECEIPT INFORMATION Purchased 8 litters (dam and 5 pups/sex/litter) Placed on study 6 litters (dam and 5/sex/litter)

2.10.5. AGE OF PUPS AT RECEIPT Pups were received on Postnatal Day 10.

2.10.6. ACCLIMATION PERIOD All female rats and their litters were acclimated from the time they were received until locomotor activity assessment began on the
pups.

2.11. SELECTION/GROUP ASSIGNMENT More animals than required for the study were purchased and acclimated. Animals considered suitable for the study on the basis of pretest physical examinations were randomized into groups at receipt using a method which ranked the Postnatal Day 10 body weights and randomly assigned each within the block of three into groups. Animals were replaced as required based on physical examinations and/or body weight change up to Postnatal Day 13 to achieve the desired number of animals (10/sex/group). Disposition of all animals not utilized in this study is maintained in the study file.

2.12. ANIMAL IDENTIFICATION Each female was ear tagged (metal ear tag) with a unique identification number by the supplier. Each Postnatal Day 10 pup was uniquely identified by tattoo upon receipt. At weaning (Postnatal Day 21), each pup was ear-tagged with a number assigned by the Testing Facility upon receipt. These numbers plus the study number served as each animal’s unique and permanent identification fo r the duration of the study. Each cage was also provided with a cag e card which contained the study number and animal number.

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2.13. VETERINARY CARE Animals were monitored by the t echnical staff for any conditions requiring possible veterinary care.

2.14. ANIMAL HUSBANDRY

2.14.1. HOUSING Each female rat and litter, fr om the time of receipt through Postnatal Day 20 were individually housed in a covered, clear plastic “shoebox” with bedding. From Postnatal Day 21 until Postnatal Day 28, the weanling pups (two or three of the same sex) were housed in stainless steel suspended cages with wire mesh floors and fronts. After Postnatal Day 28, and for the remainder of the study, animals were individually housed in stainless steel suspended cages with wire mesh floors. A glass food jar with a stainless steel lid, and food saver, was secured in each cage. Clean food jars and fresh feed were provided at least weekly.

2.14.2. FEED Certified Rodent Diet, No. 5002; (Meal) (PMI Nutrition International, St. Louis, MO) was provided ad libitum

. Analytical certification of batches of feed, provided by the manufacturer, were maintained on file at the Testing Facility. Feed was analyzed for nutritional content (protein, fat, fiber), inorganic constituents (arsenic, cadmium, calcium, lead, mercury, phosphorus, selenium), organophosphates (diazinon, ethion, methyl parathion, thimet, trithion, disulfoton, malathion, parathion, thiodan), and pesticides (aldrin, beta-BHC, DDE, delta-BHC, endrin, heptachlor, lindane, mirex, alpha-BHC, chlordane, total DDT, dieldrin, HCB, heptachlor epoxide, methoxychlor), plus PCB, and aflatoxin.

There were no known contaminants in the feed which were considered capable of interfering with the results of this study.

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2.14.3. WATER Facility water was supplied by Elizabethtown Water Company, (Westfield, NJ); and was provided ad libitum to individual animal cages via an automated watering system.
Monthly water analyses were conducted by Elizabethtown Water Company, Westfield, New Jersey (R aritan-East Millstone Plant) to ensure that water meets standards specified under the EPA Federal Safe Drinking Water Act Regulati ons (40 CFR part 141). Water analyses, provided by the supplier, were maintained on file at the Testing Facility. In addition, water samples were collected biannually from representative rooms in the Testing Facility; chemical and microbiological wa ter analyses were conducted on these samples by a subcontract laboratory. Results were maintained on file at the Te sting Facility. There are no known contaminants in the water which are expected to interfere with this study.

2.14.4. BEDDING ARTICLE
Ground corncob bedding (Bed-O’-Cobs ® 

### 1/4 inch Irradiated, The Andersons, Maumee, Ohio) was provided for each female rat and litter from time of receipt through Postnatal Day 20. Fresh bedding was provided weekly and as needed.
Bedding was analyzed for inorganic constituents (arsenic, bismuth, cadmium, lead, magnesium, manganese, mercury, nickel, zinc), organophosphates (diazinon, ethion, methyl parathion, trithion, malthion, ethyl parathion), and pestcides (aldrin, beta-BHC, DDE, delta-BHC, endrin, heptchlor, lindane, mirex, alpha-BHC, chlordane, total DDT, dieldrin, HCB, heptachlor epoxide, methoxychlor), plus PCB, and aflatoxin. There were no known contaminants in the bedding which were expected to interfere with the results of this study. Cont aminant analyses were performed periodically. Results of the bedding analysis were maintained on file at the Testing Facility.

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2.14.5. ENVIRONMENTAL CONDITIONS
Light/Dark Cycle Twelve-hour light/dark cycle provided via automatic timer.

Temperature Temperature was monitored and recorded twice daily and maintained within the desired ranges to the maximum extent possible.

Desired: 18 - 26 ° C Actual: 21 - 24 ° C

Relative Humidity Relative humidity was monitored and recorded once daily and maintained within the desired ranges to the maximum extent possible.

Desired: 30 - 70% Actual: 20 – 68%
Relative humidity was not within the specified range on eight occasions during this study (20-28%). This was not considered to have affected the integrity of the study.

2.15. TEST ARTICLE ADMINISTRATION

2.15.1. ROUTE OF ADMINISTRATION Intraperitoneal injection

2.15.2. JUSTIFICATION FOR ROUTE OF ADMINISTRATION The intraperitoneal route provides rapid test article absorption and has been used in previous studies with the test articles.

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2.15.3. FREQUENCY AND DURATION OF ADMINISTRATION The test articles (Amphetamine, Chlorpromazine) and control article were administered to pups on Postnatal Days 13, 17, 21, and 62. Each animal was dosed mo re than once per week with a washout period of 4 to 41 days between doses.

2.15.4. ADMINISTRATION OF TEST AND/OR CONTROL ARTICLE The test articles (Amphetamine, Chlorpromazine) and the control article (saline) were administered by intraperitoneal injection into the abdominal cavity near the ingui nal region using a sterile needle and syringe of appropriate size. Doses were calculated using the most recent body weights available. A volume of 5 mL/kg of body weight was administered.

2.16. EXPERIMENTAL EVALUATION

2.16.1. VIABILITY OBSERVATIONS (CAGESIDE) Observations for mortality, morbidity and signs of severe toxic or pharmacological effects were made at least twice daily (morning and afternoon) for the female rats and pups.

2.16.2. CLINICAL OBSERVATIONS A detailed physical examination was given to all female rats and pups upon receipt. The rat pups had a detailed physical examination on the day of locomotor activity behavioral assessment and weekly during the postweaning period until they were placed in a colony.

2.16.3. BODY WEIGHTS Body weights were recorded for pups on Postnatal Days 10, 13, 17, 21, 62, and weekly during the postweaning period.

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Neurobehavioral Positive Control Data: Motor Activity Appendix T

2.17. LITTER EVALUATIONS

2.17.1. OBSERVATIONS Litters were observed and each pup was sexed at the time of receipt. Pups in the litter were c ounted on Postnatal Days 10,13,17, and 21.

2.17.2. WEANING Pups were weaned on Postnatal Da y 21 and housed two or three of the same sex per cage.

2.17.3. LOCOMOTOR ACTIVITY BEH AVIORAL ASSESSMENT The locomotor activity assessment was measured in 10 pups/sex/group on Postnatal Days 13, 17, 21 and 62. On each day of testing, 10 rat pups/sex/group were adminstered either vehicle, amphetamine, or chlorpromazine. Approximately thirty minutes after the injection they were placed in a clear plastic shoebox cage equipped with a 5-beam automated Photobeam Activity System (PAS; San Diego Instruments, Inc.). Activity was monitored during a 60-minute session composed of 12, five-minute intervals. A record was made of the total number of photobeam breaks which occurred during each of the 12, five-minute intervals. The locomotor activity was assessed using a repeated measures analysis of variance to determine if there was a test article effect. Rat pups were randomized so that there was an equal representation of the treatment groups during each locomotor activity session.

Temperature Temperature was monitored and recorded twice daily, just prior to the first evaluation and just after the last evaluation.

Desired: 18 to 26 ° C Actual: 22 to 24 ° C

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Relative Humidity Relative humidity was monitored and recorded twice daily, just prior to the first evaluation and just after the last evaluation.

Desired: 30 to 70% Actual: 29 to 56%

Noise Level Noise level was monitored using a Digital Sound Level Meter, Model 840029 (Sper Scientific, Ltd., Scottsdale, Arizona) and recorded twice daily, just prior to the first evaluation and just after the last evaluation.

Desired: 55 to 65 dB Actual: 60.1 to 61.4 dB

Illumination Illumination was monitored using a Photometer 1 (Quantum Instruments, Inc., Garden City, New York) and recorded twice daily, just prior to the first evaluation and just after the last evaluation.

Desired: <80 footcandles Actual: 36.5 to 55.3 footcandles

2.18. POSTMORTEM No postmortem examinations were performed as all animals were transferred to the in-house rat colony.

2.19. DISPOSITION OF ANIMALS After the pups were weaned, the fema le rats were euthanized via an overdose of carbon dioxide and discarde d. After the locomotor activity behavioral assessment on 62, the rats we re transferred to the in-house rat colony.

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2.20. PRESERVATION OF RECORDS AND SPECIMENS All data documenting experimental details and study procedures and observations were recorded and maintained as raw data.
At the completion of the study, a ll reports, raw data, protocol and amendments, and retained samples will be maintained in the Testing Facility’s Archives.

2.21. PROTOCOL DEVIATIONS The following protocol deviations o ccurred during the study, but were not considered to have compromised the validity or integrity of the study.

1. Litter counts were not recorded on Postnatal Days 10, 13, 17, and 21 due to technician error. Howeve r, individual body weights recorded for those days (Postnatal Days 10, 13, 17, and 21) are considered documentation for the count.

2. Documentation of pups not selected for this study was omitted from the data due to technician error.

3. As there is no documentation in th e study data indicating the age of the dams at time of receipt at the Testing Facility, it can not be determined that the protocol requirement that the dams be 75-90 days old was met.

4. Protocol specified that the was hout period between doses would be approximately 2 days even though it also specified dosing would occur on Postnatal Days 13, 17, 21 and 60 (±2) days. The washout periods in this study ranged from 4 to 41 days.

5. Fresh dosing solutions were prepared either 1 or 3 days prior to or the day of dose administration rather than once weekly as specified in the protocol.

2.22. COMPUTER SYSTEM The Toxicology Analysis System Customized - Reproduction Module (TASC-R), version 1.1.13, from Pat hologist Associates International

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Neurobehavioral Positive Control Data: Motor Activity Appendix T

(PAI), Frederick, Maryland, was the computer software system utilized during the conduct of this study for randomizations, data collection, reporting, and statistical analysis.

2.23. STATISTICAL EVALUATIONS The locomotor activity behavioral assessments were analyzed for each period using split-plot repeated measures analysis of variance with model terms for group, animal within group, interval and group by interval interaction. If the group by treatment interaction was statistically significant, indicating non-parallelism in the behavioral profile between groups, a separate one-way ANOVA for group effects was performed at each interval. If the response data passed on the parallel hypothesis, an ANOVA (using summed responses over intervals for each animal) was used to test for the overall treatment effect with respect to the level hypothesis. If any significant overa ll treatment group effect was found by any of the above ANOVAs, Dunnett’s t-test was used to find groups that differed from control. No investig ation of model fit was requested or performed. Analyses were performed for sexes separately and combined. Treatment group effects were deemed significant at the α =0.05 level. Plots, tables, listings, and analyses were generated using SAS® version 6.12 for WINDOWS. Analyses were conducted by CATO Research, 200 Westpark Corporate Center, 4364 South Alston Avenue, Durham, NC 27713-2280.

3. RESULTS Results from pups treated with 2 mg/kg Amphetamine (Group II) indicate a statistically significant increase in locomotor activity for male and female rats combined, on Postnatal Days 13, 17, 21, and 62, when compared to the control group. Statistically significant increases in locomotor activity were observed for Group II male and female rats individually, on Postnatal Day 21 and 62 only.
Results from pups treated with 10 mg/ kg Chlorpromazine (Group III) indicated a statistically significant decrease in locomo tor activity for male and female rats combined, on Postnatal Days 17, 21, and 62, when compared to the control group. Statistically significant decreases in locomotor activity were observed for Group III male and female rats individually, on Postnatal Days 17, 21, and 62 only.

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4. DISCUSSION This study evaluated the behavioral effects of Amphetamine and Chlorpromazine on locomotor activity of young male and female rats, in order to generate positive control data for future developmental neurotoxicity studies.
Rats dosed and tested on Postnatal Day 17, 21, and 62 responded to test article treatment, with increased activity for Group II (2 mg/kg amphetamine) and decreased activity for Group III (10 mg/kg chlorpromazine). Amphetamine or Chlorpromazine behavioral effects we re observed equally in both male and female rats in the treatment groups.
Rats treated with Amphetamine exhibite d locomotor activity similar to control levels at the beginning of the testing period. Unlike the control animals, the Amphetamine treated animals did not habituate, and remained in a more active state for the duration of the 60-minute testing period.
Rats treated with Chlorpromazine e xhibited decreased locomotor activity compared to control levels at the beginning of the testing period. Unlike the control animals, the Chlorpromazine treated animals did not habituate, and remained in a less active state for the duration of the 60-minute testing period.
The pups dosed and tested on Postnatal Day 13 did not respond to treatment as dramatically as in latter dosing/testing days. The lack of statistically significant locomotor activity differences in the Po stnatal Day 13 treatment groups may stem from the low basal level of activity in pups of such a young age. The presence of age-related differences in locomotor activ ity in this study suggests the importance of evaluating behavioral endpoints at seve ral different time-points, when testing for developmental neurotoxicity.

5. CONCLUSION Under the conditions of this study, 2 mg/kg of Amphetamine or 10 mg/kg of Chlorpromazine, administered to young male and female rats on Postnatal Days 13, 17, 21, and 62, via intraperit oneal injection produced statistically significant behavioral effects on locomotor activity. Based on these results, it was shown that both amphetamine and chlorpromazine are excellent model compounds for the generation of locomoto r activity alterations in young Sprague Dawley rats.

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Neurobehavioral Positive Control Data: Motor Activity Mean Motor Activity Values Preface Appendix T

Summary of Statistical Results for Motor Activity Data

Post Natal Day Sex Parallel Hypothesis

(p-value) Level Hypothesis

(p-value) Significant Pairwise Results*

13 Male 0.1824 0.3593 NA Female 0.1097 0.0723 NA Combined 0.0074 NA 1 :NS; 2 :NS; 10 :II>I; 11 :II>I; 12 :II>I 17 Male 0.0203 NA 1 :I>III; 2 :I>III; 4 :I>III Female 0.0001 NA 1 :I>III; 2 :I>III; 3 :I>III; 4 :NS; 5 :I>III Combined 0.0001 NA 1 :I>III; 2 :I>III; 3 :I>III; 4 :I>III; 5 :I>III; 8 :NS; 10 :NS; 11 :NS; 12: II>I 21 Male 0.0001 NA 1 :II>I, I>III; 2 :II>I, I>III; 3 :II>I, I>III; 4 :II>I; 5 :II>I; 6 :II>I; 7 :II>I; 8 :II>I; 9 :II>I; 10 :II>I; 11 :II>I; 12: II>I Female 0.0001 NA 1 :II>I, I>III; 2 :II>I, I>III; 3 :II>I, I>III; 4 :II>I; 5 :II>I; 6 :II>I; 7 :II>I; 8 :II>I; 9 :II>I; 10 :II>I; 11 :II>I; 12: II>I Combined 0.0001 NA 1 :II>I, I>III; 2 :II>I, I>III; 3 :II>I, I>III; 4 :II>I; 5 :II>I; 6 :II>I, I>III; 7 :II>I; 8 :II>I; 9 :II>I; 10 :II>I; 11 :II>I; 12: II>I 62 Male 0.0001 NA 1 :I>III; 2 :I>III; 3 :I>III; 4 :I>III; 5 :I>III; 6 :II>I, I>III; 7 :II>I, I>III; 8 :II>I, I>III; 9 :II>I, I>III; 10 :II>I, I>III; 11 :II>I; 12: II>I Female 0.0001 NA 1 :I>III; 2 :I>III; 3 :I>III; 4 :I>III; 5 :II>I, I>III; 6 :II>I, I>III; 7 :I>III; 8 :II>I, I>III; 9 :II>I, I>III; 10 :II>I, I>III; 11 :II>I; 12: II>I, I>III Combined 0.0001 NA 1 :I>III; 2 :I>III; 3 :I>III; 4 :I>III; 5 :II>I, I>III; 6 :II>I, I>III; 7 :II>I, I>III; 8 :II>I, I>III; 9 :II>I, I>III; 10 :II>I, I>III; 11 :II>I, I>III; 12: II>I, I>III *Only intervals with significant differences between treatments are reported. NA = Not applicable; NS = No si gnificant differences from control x: a>b indicates mean for group a significantly greater than mean for group b in interval x. I = control, II = amphetamine, III = chlorpromazine

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Males Neurobehavioral Positive Control Data: Motor Activity Mean Motor Activity Values Postnatal Day 13 Appendix T

Interval 1 2 3 4 5 6 7 8 9 10 11 12
Group I – Control Mean 27.70 12.30 18.40 23.60 10.60 14.10 12.90 7.40 7.10 9.40 8.80 9.90 S.D. 21.88 15.32 30.96 40.81 12.43 20.80 12.81 10.24 11.32 10.15 7.18 11.90 N 10 10 10 10 10 10 10 10 10 10 10 10
Group II – 2.0 mg/kg Amphetamine Mean 37.70 21.10 18.40 17.20 10.20 9.20 6.10 8.10 19.60 36.70 28.80 41.60 S.D. 39.38 25.14 29.86 25.17 14.29 11.15 8.48 9.05 20.01 43.02 39.89 58.17 N 10 10 10 10 10 10 10 10 10 10 10 10
Group III – 10.0 mg/kg Chlorpromazine Mean 14.80 8.10 8.80 12.30 7.40 5.70 8.20 25.70 12.80 12.00 8.60 17.70 S.D. 26.95 15.17 13.21 21.67 11.84 13.05 11.38 55.13 22.52 27.05 27.20 54.93 N 10 10 10 10 10 10 10 10 10 10 10 10

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Females Neurobehavioral Positive Control Data: Motor Activity Mean Motor Activity Values Postnatal Day 13 Appendix T

Interval 1 2 3 4 5 6 7 8 9 10 11 12
Group I – Control Mean 25.00 14.70 17.10 20.90 16.60 14.30 9.70 6.20 12.50 10.00 7.60 5.70 S.D. 32.00 17.81 24.47 19.77 19.29 14.19 11.61 8.98 13.82 10.72 11.35 4.27 N 10 10 10 10 10 10 10 10 10 10 10 10
Group II – 2.0 mg/kg Amphetamine Mean 36.30 19.40 29.10 24.00 30.40 26.40 41.30 33.20 38.90 52.00 49.90 31.90 S.D. 45.41 26.53 40.16 34.50 51.56 49.40 65.11 55.36 74.84 87.22 55.36 38.23 N 10 10 10 10 10 10 10 10 10 10 10 10
Group III – 10.0 mg/kg Chlorpromazine Mean 4.00 1.50 11.40 24.30 0.80 2.50 3.50 4.90 3.10 5.10 6.20 1.90 S.D. 8.07 3.75 19.41 50.71 1.62 4.06 5.76 9.34 5.51 10.79 10.25 2.88 N 10 10 10 10 10 10 10 10 10 10 10 10

### Huntingdon Life Sciences 00-6130 Page 956 211-DIPE-S Final Report

Males Neurobehavioral Positive Control Data: Motor Activity Mean Motor Activity Values Postnatal Day 17 Appendix T

Interval 1 2 3 4 5 6 7 8 9 0 11 12 Group I – Control Mean 75.00 55.80 49.40 50.90 36.30 36.00 39.10 35.20 32.00 31.00 33.20 14.50 S.D. 42.95 49.40 33.92 40.33 35.24 38.39 43.53 32.15 39.34 39.73 39.81 18.33 N 10 10 10 10 10 10 10 10 10 10 10 10
Group II – 2.0 mg/kg Amphetamine Mean 52.00 30.80 42.70 41.20 29.90 26.50 32.40 52.40 64.10 62.00 51.60 28.80 S.D. 45.11 34.37 45.49 30.49 26.91 30.95 34.10 46.85 69.09 59.57 55.35 29.13 N 10 10 10 10 10 10 10 10 10 10 10 10
Group III – 10.0 mg/kg Chlorpromazine Mean 13.10 10.80 12.30 8.20 13.40 9.20 10.30 18.30 10.70 17.30 10.60 11.60 S.D. 29.41 24.51 22.45 18.00 25.18 16.30 15.91 18.29 19.24 27.38 13.85 14.45 N 10 10 10 10 10 10 10 10 10 10 10 10

### Huntingdon Life Sciences 00-6130 Page 957 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Motor Activity Mean Motor Activity Values Postnatal Day 17 Appendix T

Interval 1 2 3 4 5 6 7 8 9 10 11 12
Group I – Control Mean 99.10 39.90 34.00 22.70 30.20 16.30 24.60 32.10 30.70 25.60 27.70 14.50 S.D. 37.91 43.36 30.81 43.34 36.66 25.23 29.95 42.49 36.06 39.64 39.21 22.14 N 10 10 10 10 10 10 10 10 10 10 10 10
Group II – 2.0 mg/kg Amphetamine Mean 85.20 47.40 30.20 47.40 34.00 15.80 17.50 18.40 26.50 33.60 19.10 34.40 S.D. 35.75 31.80 26.19 26.08 24.68 13.77 19.73 23.20 21.84 31.32 22.29 26.34 N 10 10 10 10 10 10 10 10 10 10 10 10
Group III – 10.0 mg/kg Chlorpromazine Mean 2.20 4.80 1.00 0.90 2.70 17.70 14.20 3.70 23.60 4.80 6.10 12.60 S.D. 3.16 6.09 2.31 2.51 6.96 52.49 37.67 5.85 46.08 9.17 18.94 28.88 N 10 10 10 10 10 10 10 10 10 10 10 10

### Huntingdon Life Sciences 00-6130 Page 958 211-DIPE-S Final Report

Males Neurobehavioral Positive Control Data: Motor Activity Mean Motor Activity Values Postnatal Day 21 Appendix T

Interval 1 2 3 4 5 6 7 8 9 10 11 12
Group I – Control Mean 73.60 50.10 25.30 17.10 16.50 28.50 25.70 14.60 27.00 16.10 7.90 8.80 S.D. 46.33 35.63 29.06 26.53 20.99 28.32 23.84 21.48 25.73 18.54 10.16 10.77 N 10 10 10 10 10 10 10 10 10 10 10 10
Group II – 2.0 mg/kg Amphetamine Mean 157.30 154.00 144.00 152.60 141.50 141.20 127.50 136.70 133.80 127.10 125.00 113.20 S.D. 30.36 22.58 9.13 36.00 25.08 28.27 27.20 26.42 28.39 23.50 31.85 25.57 N 10 10 10 10 10 10 10 10 10 10 10 10
Group III – 10.0 mg/kg Chlorpromazine Mean 14.20 6.90 0.70 2.60 5.10 4.50 4.20 11.50 8.60 10.50 5.20 5.20 S.D. 18.40 12.31 1.49 8.22 7.69 7.68 7.28 17.28 10.48 14.39 10.81 7.13 N 10 10 10 10 10 10 10 10 10 10 10 10

### Huntingdon Life Sciences 00-6130 Page 959 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Motor Activity Mean Motor Activity Values Postnatal Day 21 Appendix T

Interval 1 2 3 4 5 6 7 8 9 10 11 12
Group I – Control Mean 93.30 29.80 27.50 17.50 17.80 27.10 15.10 14.50 8.70 7.80 8.80 10.40 S.D. 51.26 23.93 20.88 25.10 29.56 30.45 23.90 20.60 14.43 12.41 17.87 21.70 N 10 10 10 10 10 10 10 10 10 10 10 10
Group II – 2.0 mg/kg Amphetamine Mean 149.10 133.50 114.30 118.80 118.40 116.60 108.60 104.90 113.20 111.30 108.00 95.00 S.D. 27.65 29.30 18.88 27.45 32.05 33.02 36.06 30.71 20.07 22.88 27.81 22.40 N 10 10 10 10 10 10 10 10 10 10 10 10
Group III – 10.0 mg/kg Chlorpromazine Mean 8.00 3.40 3.20 4.70 4.40 5.60 5.00 10.80 4.60 5.20 5.90 8.00 S.D. 11.19 6.92 4.78 5.72 7.79 7.73 12.27 12.46 7.44 12.89 12.75 9.43 N 10 10 10 10 10 10 10 10 10 10 10 10

### Huntingdon Life Sciences 00-6130 Page 960 211-DIPE-S Final Report

Males Neurobehavioral Positive Control Data: Motor Activity Mean Motor Activity Values Postnatal Day 62 Appendix T

Interval 1 2 3 4 5 6 7 8 9 10 11 12
Group I – Control Mean 233.1 211.1 182.2 156.5 157.3 120.3 82.4 80.1 65.4 51.3 44.2 18.3 S.D. 49.84 31.37 18.05 28.60 35.20 50.12 53.69 57.66 63.58 56.90 52.93 23.21 N 10 10 10 10 10 10 10 10 10 10 10 10
Group II – 2.0 mg/kg Amphetamine Mean 242.5 228.8 190.8 190.5 185.6 163.8 165 169.4 160.5 155.7 142.5 133.2 S.D. 61.10 44.69 47.22 55.66 32.24 41.13 48.15 53.80 49.25 35.92 38.54 42.49 N 10 10 10 10 10 10 10 10 10 10 10 10
Group III – 10.0 mg/kg Chlorpromazine Mean 28.8 31.3 26.4 20.3 16.8 19.7 10.6 9.1 5.9 7.4 6.3 4.2 S.D. 69.09 63.0 54.62 60.03 34.86 29.18 31.09 8.57 11.08 18.65 12.41 9.07 N 10 10 10 10 10 10 10 10 10 10 10 10

### Huntingdon Life Sciences 00-6130 Page 961 211-DIPE-S Final Report

Females Neurobehavioral Positive Control Data: Motor Activity Mean Motor Activity Values Postnatal Day 62 Appendix T

Interval 1 2 3 4 5 6 7 8 9 10 11 12
Group I – Control Mean 171.10 153.40 143.90 124.00 103.60 95.50 91.00 55.80 57.60 37.90 32.30 32.20 S.D. 24.85 23.08 37.22 35.57 29.28 37.63 28.77 30.97 42.84 39.45 42.60 30.80 N 10 10 10 10 10 10 10 10 10 10 10 10
Group II – 2.0 mg/kg Amphetamine Mean 150.10 144.90 133.60 132.00 137.80 150.50 120.30 130.40 124.00 134.10 143.30 105.30 S.D. 45.19 29.05 31.40 33.97 31.90 46.83 39.06 43.90 39.58 35.23 31.92 40.12 N 10 10 10 10 10 10 10 10 10 10 10 10
Group III – 10.0 mg/kg Chlorpromazine Mean 33.30 18.20 12.40 10.20 8.70 0.90 4.10 2.30 6.90 5.50 2.50 0.00 S.D. 43.27 30.08 33.27 13.49 23.79 2.51 8.57 2.63 9.69 8.45 5.04 0.00 N 10 10 10 10 10 10 10 10 10 10 10 10

### Huntingdon Life Sciences 00

- 6130 Page 962 211

- DIPE

- S Final Report
Neuropathology Historical Control Preface Appendix UHUNTINGDON LIFE SCIENCES PRINCETON RESEARCH CENTER

Supplier· Charles River

Termination Yea ICientltlcatJon Number

MICROSCOPIC FINDINGS BRAIN NUMBER EXAMINED Within Normal limits

CERVICAL SPINAL CORD NUMBER EXAMINED Within Normal limits

THORACIC SPINAL CORD NUMBER EXAMINED Within Normal limits

LUMBAR SPINAL CORD NUMBER EXAMINED Within Normal Limits

SCIATIC NERVE NUMBER EXAMINED Within Normal limits

SURAL NERVE NUMBER EXAMINED Within Normal limits

TIBIAL NERVE NUMBER EXAMINED Within Normal Limits

PERONEAL NERVES NUMBER EXAMINED Within Normal limits

GASSERION GANGLION NUMBER EXAMINED Within Normal Limits

DORSAL ROOT GANGLION NUMBER EXAMINED Within Normal Limits

DORSAL ROOT FIBER NUMBER EXAMINED Within Normal Limits

VENTRAL ROOT FIBER NUMBER EXAMINED Within Normal Limits

TRIGEMINAL GANGLION NUMBER EXAMINED Within Normal Limits NEUROPATHOLOGY HISTORICAL CONTROL DATA SPRAGUE DAWLEY RAT

Individual Study Data

- Male Animals 1993 1993 1995 1996 1997 1999 1999 TOTAL 92-4505 93-4507 94-4512 96-4084*** 98-4522* 98-4522- 98-4521-

37 5 5 5 6 6 5 5 37 5 5 5 6 6 5 5

37 5 5 5 6 6 5 5 37 5 5 5 6 6 5 5

31 5 5 5 0 6 5 5 31 5 5 5 0 6 5 5

37 5 5 5 6 6 5 5 37 5 5 5 6 6 5 5

37 5 5 5 6 6 5 5 37 5 5 5 6 6 5 5

35 5 5 5 6 6 3 5 35 5 5 5 6 6 3 5

36 5 5 5 6 6 4 5 36 5 5 5 6 6 4 5

6 0 0 0 6 0 0 0 6 0 0 0 6 0 0 0

12 0 0 0 6 6 0 0 12 0 0 0 6 6 0 0

6 0 0 0 0 6 0 0 6 0 0 0 0 6 0 0

6 0 0 0 0 6 0 

### o. 6 0 0 0 0 6 0 0

6 0 0 0 0 6 0 0 6 0 0 0 0 6 0 0

10 0 0 0 0 0 5 5 10 0 0 0 0 0 5 5

Printed: 6/26/2003 

### Page 963HUNTINGDON LIFE SCIENCES PRINCETON RESEARCH CENTER

Supplier: Charles River NEUROPATHOLOGY HISTORICAL CONTROL DATA SPRAGUE DAWLEY RAT

Individual Study Data

- Male Animals Termination Year 1993 1993 1995 ldentificatron Numoer TOTAL 92-4505 93-4507 94-4512

TRIGEMINAL NERVE NUMBER EXAMINED 5 0 0 0 Nerve fiber degeneration 1 0 0 0 Within Normal Limits 4 0 0 0

CERVICAL-DORSAL ROOT GANGLION NUMBER l;XAMINED 15 0 0 0 Within Normal Limits 15 0 0 0

CERVICAL-DORSAL ROOT FIBER NUMSER EXAMINED 16 0 0 0 Within Normal Limits 16 0 0 0

LUMBAR-DORSAL ROOT GANGLION NUMBER EXAMINED 13 0 0 0 Within Normal Limits 13 0 0 0

LUMBAR-DORSAL ROOT FIBER NUMBER EXAMINED 16 0 0 0 Within Normal Limits 16 0 0 0

EYES NUMBER EXAMINED 16 0 0 0 Within Normal Limits 16 0 0 0

OPTIC NERVE NUMBER EXAMINED 16 0 0 0 Within Normal Limits 16 0 0 0

*From positive control study data **Control animals received standard laboratory diet mixed with acetone ***Control animals received corn oil only 1996 1997 96-4084*** 98-4522*

0 0 0 0 0 0

6 0 6 0

6 0 6 0

6 0 6 0

6 0 6 0

6 0 6 0

6 0 6 0 1999 1999 98-4522** 98-4521**

5 0 1 0 4 0

5 4 5 4

5 5 5 5

3 4 3 4

5 5 5 5

5 5 5 5

5 5 5 5

Printed: 6/26/2003 

### Page 964HUNTINGDON LIFE SCIENCES PRINCETON RESEARCH CENTER

Supplier· Charles River NEUROPATHOLOGY HISTORICAL CONTROL DATA SPRAGUE DAWLEY RAT

Individual Study Data

- Female Animals Termination Date 1993 1993 1995 

### I 1997 1999 1999 study Number TOTAL 92-4505 93-4507 94-4512

. 198-4522* 98-4522** 98-4521**

MICROSCOPIC FINDINGS BRAIN NUMBER EXAMINED 31 5 5 5 6 5 5 Within Normal Limits 31 5 5 5 6 5 5

CERVICAL SPINAL CORD NUMBER EXAMINED 31 5 5 5 6 5 5 Within Normal Limits 31 5 5 5 6 5 5

THORACIC SPINAL CORD NUMBER EXAMINED 31 5 5 5 6 5 5 Within Normal Limits 31 5 5 5 6 5 5

LUMBAR SPINAL CORD NUMBER EXAMINED 31 5 5 5 6 5 5 Within Normal Limits 31 5 5 5 6 5 5

SCIATIC NERVE NUMBER EXAMINED 31 5 5 5 6 5 5 Nerve fiber degeneration 2 0 0 0 0 1 1 Within Normal Limits 29 5 5 5 6 4 4

SURAL NERVE NUMBER EXAMINED 30 5 5 5 6 4 5 Within Normal Limits 30 5 5 5 6 4 5

TIBIAL NERVE NUMBER EXAMINED 31 5 5 5 6 5 5 Nerve fiber degeneration 2 0 0 0 0 1 1 Within Normal Limits 29 5 5 5 6 4 4

GASSERION GANGLION NUMBER EXAMINED 6 0 0 0 6 0 0 Within Normal Limits 6 0 0 0 6 0 0

DORSAL ROOT GANGLION NUMBER EXAMINED 6 0 0 0 6 0 0 Within Normal Limits 6 0 0 0 6 0 0

DORSAL ROOT FIBER NUMBER EXAMINED 6 0 0 0 6 0 0 Within Normal Limits 6 0 0 0 6 0 0

VENTRAL ROOT FIBER NUMBER EXAMINED 6 0 0 0 6 0 0 Within Normal Limits 6 0 0 0 6 0 0

TRIGEMINAL GANGLION NUMBER EXAMINED 10 0 0 0 0 5 5 Within Normal limits 10 0 0 0 0 5 5

Printed: 6/26/2003 

### Page 965HUNTINGDON LIFE SCIENCES PRINCETON RESEARCH CENTER

Supplier: Charles River NEUROPATHOLOGY HISTORICAL CONTROL DATA SPRAGUE DAWLEY RAT

Individual Study Data

- Female Animals Termination Date 1993 1993 1995 1997 1999 1999 Study Number TOTAL 92-4505 93-4507 94-4512 98-4522* 98-4522** 98-4521**
TRIGEMINAL NERVE NUMBER EXAMINED 5 0 0 0 0 5 0 Nerve fiber degeneration 1 0 0 0 0 1 0 Within Normal Limits 4 0 0 0 0 4 0

CERVICAL-DORSAL ROOT GANGLION NUMBER EXAMINED 9 0 0 0 0 4 5 Within Normal Limits 9 0 0 0 0 4 5

CERVICAL-DORSAL ROOT FIBER NUMBER EXAMINED 10 0 0 0 0 5 5

· Within Normal Limits 10 0 0 0 0 5 5

LUMBAR-DORSAL ROOT GANGLION NUMBER EXAMINED 7 0 0 0 0 4 3 Within. Normal Limits 7 0 0 0 0 4 3

LUMBAR-DORSAL ROOT FIBER NUMBER EXAMINED 10 0 0 0 0 5 5 Within Normal Limits 10 0 0 0 0 5 5

EYES NUMBER EXAMINED 10 0 0 0 0 5 5 Within Normal Limits 10 0 0 0 0 5 5

OPTIC NERVE NUMBER EXAMINED 10 0 0 0 0 5 5 Within Normal Limits

. 10 0 0 0 0 5 5

*From positive control study data **Control animals received standard laboratory diet mixed with acetone

Printed: 6/26/2003 

### Page 966NEUROPATHOLOG,....t'UDYINFORMATIONStudyRouteofAgeatTerminationNumberLengthofStudyNumber/SexSupplierAdministrationInitiation InitiationDateDateSpeciesStrainCharlesRiver24, 25,28and92-45053Months5Kingston,NYdietary42days25-Mar-9329June1993ratSprague-Dawley11-15,1 8andIICharlesRiver19October93-45073Months5Kingston,NYdietary49days8-Jul-931993ratSprague-DawleyCharlesRiverStone30and3194-45123Months5Ridge,NYdietary43days1-Nov-94January1995ratSprague-DawleyCharlesRiver96-408428Days6*Portage,Mlgastricintubation45days6-Nov-964-Dec-96ratSprague-DawleyCharlesRiverintraperitoneal29and30May98-4522**10Days6Kingston,NYinjection7weeks19-May-971997ratSprague-DawleyCharlesRiver13-16,19-2398-452213Weeks5Kingston,NYdietary28days19-Jan-99Apri11999ratSprague-DawleyCharlesRiver19-22,25-2998-452113Weeks5Kingston,NYdietary___4._3days27-0ct-98Jan1999ratSprague-Dawley-·-----*malesonly**positivecontrolstudydataPage 967

### AppendixV

PROTOCOL

GASOLINE DIPE VAPOR CONDENSATE

A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS WITH NEUROTOXICITY ASSESSMENTS AND 

### 4-WEEKIN VIVO GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS



### HLS Study No.: 00-6130 Sponsor Study No.: 211-DIPE-S. Protocol No.: Final Date: 13 December 2001 

### Page 968

### lOO"d Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S**PROTOCOL~GNATURES/PREFACE** Page2 Final Protocol

( Information-to be distributed on a need-to-know 

### basis)

Study Title: Gasoline DIPE Vapor Condensate: A 13-: Week Whole-Body Inhalation Toxicity Study in 

### Rats with Neuroto:xici'ry Assessments and 4- Week 

### In V"zvo Genotoxicity and Irnmunotoxicity Assessments

HLS SrudyNo.: 00-6130

Sponsor Study No.: 211-DIPE-S



### This is 

### the Final Protocol. 

### It has been reviewed and approved by:

# ~

# Gory~~jll-< t7be-c.ot
Date Study Director Huntingdon Life Sciences (HLS)

Spons Representative Sponsor: American Petroleum Institute (API) Address: 1220 L Street, Northwest Washington, D.C. 20005-4070 Phone No.: 202-682-8480 Fax No.: 202-682-8270 

### Email: grayt@api o;cg

STJJDY SPECIFIC JNFORMATION

Ship unused Test Substance and Name: Michael C. Henley 

### empty Test Substance 

### Email: mche@chevron.com containers to: Address: Chevron Research and



### Oll3 l39 lOl Technology Company 100 Chevron Way Richmond, CA 94802-0627 Phone No.: I-510-242-3062 Fax No.: 1-510-242-5542



### 0 N019NIHSHM-OS3H-IdH go:ot CNOH)lOOl-ll-330 Page 969Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DJPE-S



### TABLE OF CONTENTS Page3 Final Protocol



### 1. INTRODUCTION

..................................................................................................... 5

2. STUDY PERSONNEL

.............................................................................................. 5

3. REGULATORY REFERENCES .•.......................................................................... 6

3.1. TEST GUIDELINE

................................................................................................. 6 3.2. GOOD LABORATORY PRACTICES

.................................................................. 7 3.3. FACILITIES MANAGEMENT/ANIMAL HUSBANDRY

................................... 7 3.4. ANIMAL WELFARE ACT COMPLIANCE

......................................................... 7 3.5. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE

........................... 8



### 4. QUALITY ASSURANCE MONITORING

............................................................ 8

5. ALTERATION OF DESIGN

................................................................................... 8

6. PROPOSED STUDY DATES

.................................................................................. 9



### 7. EXPERIMENTAL DESIGN

.................................................................................. tO

8. TEST SUBSTANCE

............................................................................................... 12

8.1. TEST SUBSTANCE

............................................................................................. 12 8.2. IDENTIFICATION OF TEST SUBSTANCE

...................................................... 12 8.3. ARCHIVAL SAMPLES

....................................................................................... 12 8.4. UNUSED TEST SUBSTANCE

........................................................................... 12


### 9. TEST ANIMALS

.................................................................................................... 12

9.1. SPECIES

............................................................................................................... 12 9 .2. SUPPLIER

............................................................................................................ 13 9.3. JUSTIFICATION FOR TEST SYSTEM SELECTION

....................................... 13 9.4. ANIMAL REQUIREMENTS/SPECIFICATIONS

.............................................. 13 9.5. ACCLIMATION PERIOD

................................................................................... 13 9.6. ANIMALHUSBANDRY/NON-EXPOSURE

..................................................... 14 9.7. ANIMALHUSBANDRYDURINGEXPOSURE

............................................... 15 9.8. SELECTION FOR STUDY

.................................................................................. 15 9.9. ANIMAL IDENTIFICATION

.............................................................................. 16



### 10. TEST SUBSTANCE ADMINISTRATION

.......................................................... 16

10.1. ROUTE OF ADMINISTRATION

.................................................................... 16 10.2. JUSTIFICATION FOR ROUTE OF ADMINISTRATION

............................. 16 10.3. FREQUENCY OF ADMINISTRATION

......................................................... 16 

### Page 970

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page4 Final Protocol

10.4. DURATION OF ADMINISTRATION

............................................................ 16 10.5. ADMINISTRATION OF TEST SUBSTANCE 16 10.6. EXPOSURE CONCENTRATION DETERMINATION

................................. 17 10.7. PARTICLE SIZE DISTRIBUTION ANALYSIS

............................................. 17 10.8. CHAMBER AND 

### EXPOSURE ROOM ENVIRONMENT

.............................. 18 10.9. SUMMARY OF CHAMBER ACTNITY

....................................................... 18

11. EXPERIMENTAL EV ALUATION ...............................................•...................... 19

11.1. OBSERVATIONS

............................................................................................ 19 11.2. BODY WEIGHTS

............................................................................................ 19 11.3. FEED CONSUMPTION

................................................................................... 19 11.4. OPHTHALMOSCOPIC EXAMINATION

...................................................... 20 11.5. NEUROBEHAVIORAL STUDIES

.................................................................. 20 11.6. CLINICAL LABORATORY STUDIES

........................................................... 22 11.7. GENOTOX EVALUATIONS

.......................................................................... 23 11.8. IMMUNOTOX EVALUATIONS

.................................................................... 24

12. POSTMORTEM

..................................................................................................... 25
12.1. MACROSCOPIC POSTMORTEM EXAMINATIONS

.................................. 25 12.2. TIME OF NECROPSY

..................................................................................... 26 12.3. METHOD OF EUTHANASIA

......................................................................... 26 12.4. ORGAN WEIGHTS

......................................................................................... 26 12.5. TISSUES PRESERVED

................................................................................... 26 12.6. MICROSCOPIC PATHOLOGY EVALUATIONS

......................................... 27 12.7. STAINS

............................................................................................................. 27 12.8. NEUROPATHOLOGY SACRIFICE

............................................................... 27 12.9. GFAPEVALUATION

...................................................................................... 29

13. 

### PRESERVATION OF RECORDS AND SPECIMENS

...................................... 29

14. STATISTICAL EVALUATIONS

......................................................................... 30

15. REPORTING

............................................................................................................................... 32

APPENDICES A {TISSUES PRESERVED/EXAMINED MICROSCOPICALLY)

..................... 33 B (MICRONUCLEUS EVALUATIONS) .........•.........•.................................. 36 C (SCE EVALUATIONS)

......................................................................... 46 D (IMMUNOTOXICITY EVALUATIONS)

................................................ 52 E (GFAP EV ALUATIONS)

..................................................................... 59 

### Page 971Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 5 Final Protocol



### 1. INTRODUCTION
HLS StudyNo.
Sponsor Study No.
Study Title

Testing Facility

Purpose



### 2. STUDY PERSONNEL Study Director:

Alternate Contact: 00-6130

211-DIPE-S

Gasoline DIPE Vapor Condensate: A 13- Week Whole-Body Inhalation Toxicity Study in Rats with Neurotoxicity Assessments and 4- Week 

### In Vivo Genotoxicity and Immunotoxicity Assessments

Huntingdon Life Sciences Princeton Research Center (PRC) POBox2360 Mettlers Road East Millstone, NJ 08875-2360

This study is designed to assess the potential inhalation toxicity of a test substance when administered via whole-body exposures to
rats for at least 13 weeks followed by a 4 week recovery period. The assessment will include routine toxicology parameters as well

as detailed evaluations of neurotoxicity, genotoxicity and immunotoxicity parameters.

GaryM. Hoffinan, B.A., D.A.B.T. Telephone: 732-873-2550 x2920 Fax: 732-873-3992 Email: hoffmang@princeton.huntingdon.com

CarolS. Auletta, M.B.A., D.A.B.T. x2690 Senior Director of Toxicology

Additional personnel will be documented in the project file and presented in the final report. 

### Page 972Huntingdon Life Sciences Study No. 00-6130 Sponsor StudyNo. 211-DIPE-S



### 3. REGULATORY REFERENCES

3.1. TEST GUIDELINE Page6 Final Protocol

This study is designed to meet or exceed the pertinent requirements of:

US EPA Vehicle Emissions fuhalation Exposure Guideline 79.61, CFR Vol. 59, No. 122, 27 June 1994.

US EPA Subchronic Toxicity Study with Specific Health Effect Assessments 79.62, CFR Vol. 59, No. 122,27 June 1994.

US EPA OPPTS Health Effects Test Guidelines 870.3465, 90-Day fuhalation Toxicity, August 1998.

US EPA OPPTS Health Effects Test Guidelines 870.1350, Acute fuhalation Toxicity with Histopathology (respiratory tract histopathology sections, only), Public Draft June 1996.

US EPA OPPTS Health Effects Test Guidelines 870.6200, Neurotoxicity Screening Battery, August 1998.

US EPA Neuropathology Assessments 79.66, CFR Vol. 59, No. 122, 27 June 1994.

US EPA GFAP Assay 79.67, CFR Vol. 59, No. 122,27 June 1994.

US EPA OPPTS Health Effects Test Guidelines 870.7800, Immunotoxicity, August 1998.

US EPA Micronucleus Assay 79.64, CFR Vol. 59, No. 122,27 June 1994.

US EPA OPPTS Health Effects Test Guidelines 870.5395, Mammalian Erythrocyte Micronucleus Test, August 1998.

US EPA SCE Assay 79.65, CFR Vol. 59, No. 122, 27 June 1994. 

### Page 973

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

3.2. GOOD LABORATORY PRACTICES Page 7 Final Protocol

This study will be conducted in accordance with 79.60, CFR Vol. 59, No. 122, 27 June 1994. This study will be performed according to protocol and Huntingdon Life Sciences' Standard Operating Procedures.

3.3. FACILITIES MANAGEMENT/ANIMAL HUSBANDRY Currently acceptable practices of good animal husbandry will be followed,
e.g., Guide for the Care and Use of Laboratory Animals; 

### National Academy Press, 1996. Huntingdon Life Sciences, Inc. is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

3.4. ANIMAL WELFARE ACT COMPLIANCE

This study will comply with all appropriate parts of the Animal Welfare Act regulations: 9 CFR Parts 1 and 2 Final Rules, Federal Register, Volume 54, No. 168, August 31, 1989, pp. 36112-36163 effective October 30, 1989 and 9CFR Part 3 Animal Welfare Standards; Final Rule, Federal Register, Volume 56, No. 32, February 15, 1991, pp. 6426-6505 effective March 18, 1991. The Sponsor should make particular note of the following:

1. The Sponsor's signature on this protocol documents for the study described, there are no generally accepted non-animal alternatives and the study does not unnecessarily duplicate previous experiments.

2. All procedures used in this study have been designed to avoid discomfort, distress and pain to the animals. All methods are described in this study protocol or in written laboratory standard operating procedures.

3. 

### Any procedures outlined in this study protocol which are expected to

cause more than momentary or slight pain or distress to the animals will be performed with appropriate sedatives, analgesics or anesthetics unless the withholding of these agents is justified for scientific reasons, in writing, by the Sponsor and the Study Director and approved by the IACUC; in which case the procedure will continue for the minimum time necessary. Documentation of the justification for withholding
treatment for pain or distress and IACUC approval of the procedures will be made prior to study initiation on the IACUC Protocol Review form. 

### Page 974

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 8 Final Protocol

4. Animals experiencing more than momentary or slight pain or distress due
to emergency situations such as injury or illness will be treated by the Testing Facility's veterinarian staff with approved analgesics or agents to relieve pain. If possible, the Study Director will be consulted prior to treatment; however, the veterinary staff is authorized to admiriister
emergency treatment as necessary. Any subsequent treatment or euthanasia will be administered after consultation with the Study Director. The Sponsor will be advised by the Study Director of all
emergency situations in as timely a manner as possible.

5. Methods of euthanasia used during this study are in conformance with the above referenced regulations.

6. The number of animals in the protocol is considered the minimum
necessary to satisfy the regulatory requirement for studies of this type including assessments of routine toxicology parameters as well as detailed evaluations of neurotoxicity, genotoxicity and immunotoxicity parameters.

3.5. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE

The IACUC Protocol Review Subcommittee has reviewed this protocol and found it to be in compliance with all appropriate regulations.



### 4. QUALITY ASSURANCE MONITORING

The Huntingdon Life Sciences Quality Assurance Unit will monitor the facilities, equipment, personnel, methods, practices, records and controls used in this study to

assure that they are in conformance with this protocol, company SOP's, and the appropriate Good Laboratory Practice regulations.



### 5. ALTERATION OF DESIGN
Alterations of this protocol may be made as the study progresses. No changes in the protocol will be made without the consent of the Sponsor. In the event that the Sponsor authorizes a protocol change verbally, such changes will be honored by the Testing Facility and will be followed by a written verification. All protocol modifications will be signed by the Study Director and a Sponsor representative. Any modifications potentially affecting animal welfare will also be signed by two members of the Institutional Animal Care and Use Committee prior to the modification's implementation. 

### Page 975Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S



### 6. PROPOSED STUDY DATES*

Study Initiation Date:

Receipt of test animals:

Initiation ofExposures: (Experimental Start Date)

Termination ofExposures:

Necropsy:

Submission of draft final report:

Experimental Termination:

Study Completion Date: Page 9 Final Protocol

Date Study Director signs protocol

24 January 2002

12 February 2002

16May2002

13-17 May 2002 (Terminal/Neuropathology) 14 June 2002 (Recovery)

25 October 2002

Date of last data collection (Date final report is signed by Pathologist)

Date fmal report is signed by Study Director

*See Appendices B, C, D and E for schedules for the Genotoxicity, Immunotoxicity and GF AP Satellite Studies 

### Page 976HuntingdonLifeSciencesStudyNo.00-6130SponsorStudyNo.211-DIPE-S7.EXPERIMENTALDESIGNGroupExposureLevel3Initialb(mg/m3)MFI(control)0(aironly)2020II(low)2,0001010Ill(mid)10,0001010Page10FinalProtocolNumberofAnimals(MAINandRECOVERYSTUDIES)ClinicalLaboratoryStudiesNecropsydMicroscopicPathology4Weeks&RecoverycTerminalRecoverycTerminalRecoveryTerminalMFMFMFMFMFMF1010A.N.A.N.101010101010A.R.A.R.1010--1010--A.R.A.R.--1010--1010--A.R.A.R.--•IV(high)20,00020201010A.N. A.N.101010101010A.R.A.R.I•Exposureswillbe6hours/day,generally5days/weekfor13weeksforatleast65exposuresforMainandRecoveryStudies."Thisnumberofanimalsincludes10/sex/groupdesignatedfortheTerminalSacrifice(MainStudyanimals)and10/sex/group(I&IVonly)designatedforRecoveryaftertheTerminalSacrifice(RecoveryStudyanimals)."Therecoveryperiodwillbeatleast4weeks.dCompletepostmortemevaluationswillalsobeperformedonanimalswhicharefounddeadoreuthanatizedinamoribundconditionduringthecourseofthestudy.A.N.=AsNeeded(additionalcost)basedonresultsofTerminalSacrificeevaluations.A.R.=AsRequired:1)Targetorgans/tissuesidentifiedbyGroupIVevaluationsatTerminalSacrifice(additionalcost);2)LungswillbeexaminedmicroscopicallyforallanimalsinallgroupsattheTerminalsacrificeinterval;3)Alltissuesfromanimalsfounddeadoreuthanatizedinamoribundconditionduringthecourseofthestudy(additionalcost);4)Grosslesions(additionalcost).M=Male;F=Female;ThefirstdayofexposurewillbeDay0.Page 977HuntingdonLifeSciencesStudyNo.00-6130SponsorStudyNo.211-DlPE-SEXPERIMENTALDESIGN(CONTINUED)GroupExposureLevetNumberofAnimals(SATELLITESTUDIES)InitialbNeuropathologyGFAPImmunotoxicity(mg/m3)MFMFMFMFI(control)0(aironly)152555 5 5-10ll(low)2,0001525555 5-10ill(mid)10,00015255555-10N(high)20,0001525555 5-10Page11FinalProtocolGenotoxicity(MicronucleusandSCE)MF55555555•Exposureswillbe6hours/day,generally5days/weekfor13weeksforatleast65exposuresforNeuropathologyandGFAPStudies.Exposureswillbe6hours/day,generally5days/weekfor4weeksforatleast20exposuresforGenotoxicityandIrnrnunotoxicityStudies.hrhisnumberofanimalsincludes5/sex/groupdesignatedforneuropathologyaftertheTenninalSacrifice,5/sex/groupdesignatedforGFAPevaluationaftertheTenninalSacrifice,10females/groupdesignatedforIrnrnunotoxicity(PFC)evaluationsfollowinga 4weekexposureperiodand5/sex/groupdesignatedforGenotoxicity(MicronucleusandSisterChromatidExchange)evaluationsfollowinga 4weekexposureperiod.ForthelrnrnunotoxicityandGenotoxicityevaluations,separategroupsofanimals(10femalesand5/sex/evaluation,respectively)willbeusedaspositivecontrolanimalsfortheseevaluations.ForIrnrnunotoxicityevaluations,positivecontrolratswillbegivenadoseof50mglkgCyclophosphamide(CP)intraperitoneallyonceperdayfor4dayspriortosacrifice.ForGenotoxicityevaluations,positivecontrolratswillbegivenadoseof5mglkg(SCE)and40mglkg(Micronucleus)CPintraperitoneallywithin24hourspriortosacrifice.Thesepositivecontrolanimalswillnotbechamberexposedpriortodosingandsacrifice.TheCPsolutionspreparedfordosingwillnotbeassayedforpurity,unifonnityandstabilityasperGLP's.M=Male;F=Female;ThefirstdayofexposurewillbeDay0.GFAP=GlialFibrillaryAcidicProtein;SCE=SisterChromatidExchange;PFC=PlaqueFormingCellPage 978Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S



## 8. TESTSUBSTANCE Page 12 Final Protocol



### 8.1. TEST SUBSTANCE Gasoline DIPE Vapor Condensate Description, lot number, storage, expiration date (if available) and handling procedures, as well as other pertinent information will be documented in the study data.



### 8.2. IDENTIFICATION OF TEST SUBSTANCE

Unless otherwise noted, the identity, strength, composition, stability and method of synthesis, fabrication and/or derivation of each batch of the test substance will be documented by the Sponsor before its use in the study. This documentation will be maintained by the Sponsor at the address indicated on page 2 of this protocol. The Sponsor will conduct a purity analysis of the test substance by GC prior to the
start of this study.



### 8.3. ARCHIVAL SAMPLES

An archival sample from each lot of test substance will be taken and stored in the Archives of the Testing Facility. If multiple studies are conducted with the same substance, a common archival sample may be taken and appropriately labeled.



### 8.4. UNUSED TEST SUBSTANCE

The unused portion of the test substance as well as any empty test substance containers will be returned to the Sponsor's designee (see page 2) following completion of the study. Empty test substance containers will be returned to the Sponsor's designee on an as needed basis. The Sponsor will be responsible for tracking their disposition.

In the event the Sponsor wishes the Testing FaCility to arrange for disposal, a cost for this service will be provided.



## 9. TEST ANIMALS

Albino Rats (Outbred) V AF/Plus®



### 9.1. SPECIES Sprague Dawley- derived

### (CD~ [Crl: CD® IGS BR] 

### Page 979Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

9.2. SUPPLIER

Charles River Laboratories Kingston, NY

9.3. JUSTIFICATION FOR TEST SYSTEM SELECTION Page 13 Final Protocol

The rat is a rodent animal model commonly utilized in toxicity studies as recommended in the referenced guidelines. ill addition, a historical database is available for comparative evaluation.

9.4. ANIMAL REQUIREMENTS/SPECIFICATIONS

9.4.1. NUMBER ON TEST

Total Males Females 310 130 180

9.4.2. AGE

Age at Receipt Age at Exposures fuitiation Study Phase (weeks) (weeks) Main**~6 ~8**  Neuropathology**~6 ~8**  GFAP**~6 ~8**  Genotoxicity**~4 ~6**  Immunotoxicity**~4 ~6** 

Note: Females will be nulliparous and non-pregnant.

9.4.3. WEIGHT Approximately 175 to 350 grams at first exposure for animals that are**~8** weeks of age. Approximately 150 to 250 grams at first exposure for animals that are**~6** weeks of age. Animals outside of this range may be used at the discretion of the Study Director.

9.5. ACCLIMATION PERIOD Approximately two weeks; all animals will be checked for viability twice daily. Prior to assignment to study all animals will be examined by a staff member to ascertain suitability for study. 

### Page 980Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S



### 9.6. ANIMAL HUSBANDRY/NON-EXPOSURE

9.6.1. · HOUSING Page 14 Final Protocol

Animals younger than 5 weeks at receipt will be housed 2/cage during the initial week of acclimation. Animals 5 weeks or older will be housed individually in suspended stainless steel wire mesh cages.



### 9.6.2. FEED & FEED ANALYSIS

Certified Rodent Diet, No. 5002; (Meal) (PMI Feeds, Inc., St. Louis, MO) ad libitum.

Analytical certification of batches of feed will be maintained on file by the manufacturer. There are no known contaminants in the feed that are expected to interfere with the results of this study.



### 9.6.3. WATER& WATER ANALYSIS
Elizabethtown Water Company, Westfield, NJ; ad libitum, via automated watering system.

Monthly water analyses are conducted by Elizabethtown Water Company to

assure that water meets standards specified under the EPA Federal Safe Drinking Water Act Regulations (40 CPR Part 141). Water analysis, provided by the supplier, will be maintained on file at the Testing Facility. In addition, internal biannual chemical and microbiological water samples collected from representative rooms in this facility are assayed. Results are maintained on file. There are no known contaminants in the water that are expected to interfere with the results of this study.



### 9.6.4. VETERINARY CARE

Animals are monitored by the technical staff for any conditions requiring possible veterinary care. If any such conditions are identified, a staff veterinarian will be notified for an examination and evaluation. Animals will be treated as outlined in the Animal Welfare Act Compliance section of this protocol. 

### Page 981

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

9.6.5. ENVIRONMENTAL CONDITIONS



### Light/Dark Cycle Page 15 Final Protocol

Twelve hour light/dark cycle provided via automatic timer.



### Temperature

Temperature will be monitored in accordance with Testing Facility SOPs
to ensure that the desired range of 18-26°C is maintained to the maximum
extent possible.

Humidity

Humidity will be monitored in accordance with Testing Facility SOPs to

ensure that the desired range of 30 to 70% is maintained to the maximum
extent possible.

9.7. ANIMAL HUSBANDRY DURING EXPOSURE

9.7.1. HOUSING Individually in cages.

9.7.2. FEED None.

9.7.3. WATER None.

9.8. SELECTION FOR STUDY

More animals than required for the study will be purchased and acclimated. Animals considered suitable for study on the basis of pretest physical examinations, body weight data and any other pretest evaluations will be randomly assigned, by sex, to control or treated groups in an attempt to equalize mean group body weights. Individual weights of animals placed on test shall be within+/- 20% of the mean weight for each sex for each study. The Main/Recovery study and each of the Neuropathology and GF AP and Genotoxicity and hnmunotoxicity studies will be randomized separately. Disposition of all animals not utilized in the study will be maintained in the study file. 

### Page 982

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

9.9. ANIMAL IDENTIFICATION 

### Page 16 Final Protocol

Each animal will be assigned a temporary identification number upon receipt. After selection for study, each animal will be ear-tagged by the Testing Facility with a number assigned by the Testing Facility. The number assigned by the Testing Facility plus the study number will comprise the unique animal number for each animal. If 

### the tag is lost, it will be replaced or the animal will be tattooed for identification. Each cage will be provided with a cage card that will be color-coded for exposure level identification and will contain the study number and animal number.



### 10. TEST SUBSTANCE ADMINISTRATION

10.1. ROUTE OF ADMINISTRATION


### Inhalation via whole-body exposures.

10.2. JUSTIFICATION FOR ROUTE OF ADMINISTRATION


### The inhalation route is one of the potential routes of human exposure to this test substance and is the route specified in the referenced guidelines.

10.3. FREQUENCY OF ADMINISTRATION



### Once daily, 6 hours/day.

10.4. -nuRATION OF ADMINISTRATION


### The test substance will be administered for 6 hours/ day, generally 5 days per week for 13 weeks for at least 65 exposures for Main!Recovery/Neuropathology/GF AP Studies. Animals for the Genotoxicity and hnmunotoxicity evaluations will be exposed for 6 hours/day, generally 5 days per week for 4 weeks for at least 20
exposures. Test substance administration will continue through the day prior to necropsy for all animals except the animals in Groups I 

### and IV 

### designated for
recovery will be held for at least 4 

### weeks prior to necropsy.

10.5. ADMINISTRATION OF TEST SUBSTANCE



### The test substance will be administered as a vapor in the breathing air of the animals. The test atmosphere will be generated by an appropriate procedure determined during pre-study trials. The trials will be performed to evaluate the optimal set of conditions and equipment to generate a stable atmosphere at the target exposure levels and maintain uniform conditions throughout the exposure chambers. The method will be described in the raw data of the study and in the report. 

### Page 983

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 17 Final Protocol

The whole-body exposure chambers will each have a volume of approximately 1000 liters. Two chambers will be used for each exposure level, in general one for male animals and one for female animals. Each chamber will be operated at a minimum flow rate of 200 liters per minute. The fmal airflow will be set to provide at least

one air change in 5.0 minutes (12 air changes/hour) and a T99 equilibrium time of at
most 23 minutes. This chamber size and airflow rate is considered adequate to maintain the oxygen level at least 19% and the animal loading factor below 5%. At the end of each exposure, all animals will remain in the chamber for a minimum of 30 minutes. During this time, the chamber will be operated at approximately the

same flow rate using clean air only.



### 10.6. EXPOSURE CONCENTRATION DETERMINATION

A nominal exposure concentration will be calculated. The flow of air through the chamber will be monitored using appropriate calibrated equipment. The test substance consumed during the exposure will be divided by the total volume of air passing through the chamber (volumetric flow rate times total exposure time) to give the nominal concentration.

During each exposure, measurements of airborne concentrations will be performed in the animals' breathing zone at least 4 times using an appropriate sampling procedure and IR analytical procedure. Also, one sample per chamber per week will be analyzed by gas chromatography (GC) to characterize at least 10 major components (comprising at least 80% by weight of the test substance) to show test substance stability and comparison between the neat liquid test substance and the vaporized test atmospheres.

If more than the normal amount of trials is required because of test substance generation or monitoring problems (80 technician hours), the Sponsor will be consulted prior to additional trials (additional cost).



### 10.7. PARTICLE SIZE DISTRIBUTION ANALYSIS

During each week of exposure, particle size determinations will be performed using

a TSI Aerodynamic Particle Sizer to characterize the aerodynamic particle size distribution of any aerosol present. 

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### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 18 Final Protocol

10.8. CHAMBER AND EXPOSURE ROOM ENVIRONMENT

Chamber temperature, humidity, airflow rate and static pressure will be monitored continuously and recorded every 30 minutes during exposure. Chamber temperature and relative humidity will be maintained, to the maximum extent possible, between 20 to 24°C and 40 to 60%, respectively. Chamber oxygen levels (maintained at least 19%) will be measured pretest and at the beginning, middle and ehd of the study.

Air samples will be taken in the vapor generation area pretest and at the beginning, middle and end of the study. Light (maintained approximately 30-40 foot-candles at 1.0 meter above the floor) and noise levels (maintained below 85 decibels) in the exposure room will be measured pretest and at the beginning, middle and end of the study.

10.9. SUMMARY OF CHAMBER ACTIVITY

The minimum frequency of chamber activity is summarized below:

Activity
Measured Test Substance Concentration

Measured Test Substance Characterization

Particle Size
Temperature

Relative Humidity

Airflow Rate

Static Pressure

Nominal Test Substance Concentration (excluding the air control chamber)

Rotation Pattern of Exposure Cages Loading/Unloading Verification Frequency/chamber 4X/day

1X/week

1X/week 13X/day 13X/day 13X/day 13X/day 1X/day

1X/week 1X/day 

### Page 985Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S



### 11. EXPERIMENTAL EVALUATION

11.1. OBSERVATIONS

11.1.1. VIABILITY CHECKS (IN-CAGE) Page 19 Final Protocol

Observations for mortality, general appearance and signs of severe toxic or pharmacological effects will be made at least twice daily. Animals in extremely poor health or in a possible moribund condition will be identified for further monitoring and possible euthanasia.



### 11.1.2. PHYSICAL EXAMINATIONS

All animals will be observed as a group at least once during each exposure.

Each animal (except Neuropathology, GFAP, Genotoxicity and Immunotoxicity animals) will be removed from its cage and examined twice pretest and once weekly during the study period. Examinations will include observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluations of respiration, palpation for tissue masses, circulatory effects, autonomic effects, central nervous system effects, changes in motor activity, and reactivity to handling or sensory stimuli. Neuropathology, GFAP, Genotoxicity and Immunotoxicity animals will be removed from their cage and examined once pretest before selection onto study.


### 11.2. BODY WEIGHTS Body weights will be recorded at least twice pretest, weekly during the study and at termination.



### 11.3. FEED CONSUMPTION

Feed consumption will be measured (weighed) during the week prior to treatment initiation and weekly throughout the study. Feed will be available without restriction 7 days/week except during inhalation exposures and when fasting prior
to blood collection. Animals will be presented with weighed feeders at the scheduled intervals. After 7 days, the feeders will be reweighed and the resulting weight subtracted from the initial feeder weight to obtain the grams of feed consumed per animal per week. The grams consumed per kilogram of body weight per day will then be calculated for each animal. 

### Page 986

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

11.4. OPHTHALMOSCOPIC EXAMINATION 

### Page 20 Final Protocol

All animals (except Genotoxicity and Immunotoxicity animals) will be examined pretest and at study termination (optional at recovery, additional cost). Eyelids, lacrimal··· apparatus and conjunctiva will be examined grossly; cornea, anterior chamber, lens, vitreous humor, retina and optic disc will be examined by indirect ophthalmoscopy. The eyes will be examined after instillation of a mydriatic such as tropicamide.

11.5. NEUROBEHAVIORAL STUDIES 

### Testing will be staggered over several sessions and will be conducted on nonexposure days at least 16 hr 

### post-exposure. Each session will consist of 10 animals/sex/exposure group. Testing will be performed on the Neuropathology and the GFAP animals. Noise level will be maintained within a level of 55 to 65 decibels by a white noise generator. Temperature, humidity and illumination will be measured and recorded to ensure that variations in environmental conditions

are minimal during all evaluations. The functional observational battery will be performed for all animals before evaluation of motor activity.

11.5.1. MOTOR ACTIVITY 

### Activity will be monitored using an automated Photobeam Activity System (San Diego Instruments, Inc.). Sessions will be 60 minutes in length; each session will be divided into 12, 5-minute intervals. The time of testing will be balanced across treatment groups. Positive historical control data demonstrating the sensitivity of the procedure will be documented in the final report.

Number of Animals/Intervals 

### 10/sex/group pretest and during the 4th, 8th and 13th weeks of exposure (optional
at recovery, additional cost)

11.5.2. FUNCTIONAL OBSERVATIONAL BATTERY



### The functional observational battery will include assessments in the home
cage, open field, and reflex testing. The time of testing will be balanced

across treatment groups. All observations will be performed by trained observers who will be unaware of the animals' treatment. Positive historical control data demonstrating the sensitivity of the procedure will be documented in the final report.

Number of Animals/Intervals 

### 10/sex/group pretest and during the 4th, 8th and 13th weeks of exposure (optional 

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### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 21 Final Protocol

at recovery, additional cost)

Parameters Evaluated


### Evaluations will be performed according to the Testing Facility's Standard Operating Procedures which include defined scales for each function listed below. In 

### addition, descriptive comments can be made during the testing period about behaviors that are not adequately described by the defined scales. Testing will proceed from least to the most interactive with the subject.

1. Assessment of signs of autonomic function:

• Ranking and degree of lacrimation and salivation

• Presence or absence of piloerection and exophthalmos

• Count of urination and defecation

• Pupillary function and degree of palpebral closure

2. Description, incidence and severity of any convulsions, tremors or abnormal movements both in home cage and the open field

3. Ranking of reactivity to general stimuli, such as removal from cage

or handling, with a range of severity scores from no reaction to hyperactivity.

4. Ranking of the subjects general level of activity during observations of the unperturbed subject in the open field, with a range of severity scores from unresponsive to hyperactive.

5. Description and incidence of posture and gait evaluations in the home cage and open field.

6. Ranking of gait abnormalities

7. Forelimb and hindlimb grip strength measurements

8. Quantitative measure oflanding foot splay

9. Sensorimotor responses to visual and auditory stimuli and the
response to a tail pinch

10. Body weight

11. Description and incidence of any abnormal behaviors, excessive or repetitive actions, altered fur appearance, or red/crusty deposits around the eyes, nose, or mouth, and any other observations that
may facilitate interpretation of the data.

12. Ranking of righting ability 

### Page 988Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

11.6. CLINICAL LABORATORY STUDIES Page 22 Final Protocol

Clinical pathology procedures and parameters are based on those recommended in guidelines published by the Joint Scientific Committee for International Harmonization of Clinical Pathology Testing in "Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies", Fund. Appl. Tox.: 29, 198-201 (1996).

Blood will be obtained from lightly anesthetized (carbon dioxide/oxygen; 60%/40%) animals via puncture of the orbital sinus (retrobulbar). Rats will be fasted overnight prior to blood collection. The Main Study animals will be bled at the Interim (**~4** weeks) and Terminal intervals. The Recovery Study animals will be bled (optional)
at the Recovery interval. Blood will be collected and studies performed as follows:·
11.6.1. HEMATOLOGY AND COAGULATION STUDIES

Blood**(~0.25** mL) for hematology studies will be collected into tubes containing EDTA anticoagulant. Blood**(~1.0** mL) for coagulation studies will be collected into tubes containing sodium citrate anticoagulant.

Number of Animals 1 0/sex/group

Collection Intervals**~4** weeks, termination & recovery (optional, additional cost)

Parameters Evaluated (If inadequate volume of sample available, these parameters are in priority order) erythrocyte count hematocrit hemoglobin concentration

mean corpuscular volume

mean corpuscular hemoglobin

mean corpuscular hemoglobin concentration leukocyte count (total and differential) platelet count prothrombin time activated partial thromboplastin time reticulocyte count erythrocyte morphology (from peripheral blood smears)

11.6.2. CLINICAL CHEMISTRY

Blood (**~** 1.5 mL for all animals**except~** 1.0 mL for the female animals at the 4th week interval) for clinical chemistry studies will be collected into tubes with no anticoagulant, allowed to clot, and centrifuged to obtain serum. 

### Page 989Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

Number of Animals 1 0/sex/group Page 23 Final Protocol

Collection Intervals**~4** weeks, Termination & Recovery (optional, additional cost)

Parameters Evaluated (If inadequate volume of sample available, these parameters are in priority order)

alanine aminotransferase alkaline phosphatase aspartate aminotransferase creatine kinase gamma-glutamyl transferase lactate dehydrogenase sorbitol dehydrogenase

urea nitrogen bilirubin (total) bilirubin conjugated (direct) bilirubin unconjugated (indirect) glucose total protein albumin globulin (calculated as total protein

- albumin = globulin) albumin/globulin ratio (calculated) creatinine sodium potassium chloride calcium phosphorus, inorganic bile acids cholesterol (total)

11.6.3. RETENTION/STORAGE OF SPECIMENS Any remaining (frozen) serum, which may have limited storage stability, will be stored for up to six months after completion of assays and will then be discarded. Peripheral blood smears will be retained and archived with the study.

11.7. GENOTOXICITY EVALUATIONS

Note: The same animals will be used for both of the following evaluations except a 

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### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 24 Final Protocol

separate positive control group will be used for each evaluation. The cyclophosphamide will be received from BioReliance as acquired from Sigma Chemical Company. The identity, strength, purity and composition or other characteristics to define the positive control article will not be determined by the Testing Facility. The positive control article will be characterized as per the Certificate of Analysis on file with the Testing Facility. The stability of the positive control article will not be determined by the Testing Facility. Analyses
to determine the uniformity (as applicable) or concentration of the positive control mixture will not be performed by the Testing Facility. The stability of the positive control article mixture will not be determined by the Testing Facility.

SCE Evaluations Sprague Dawley-derived (CD®) rats, 5/sex/group, will be exposed to Gasoline DIPE Vapor Condensate via inhalation for a 4 week (5 days per week) exposure period by Huntingdon Life Sciences (PRC). PRC personnel will also perform the
treatment of non-exposed (5/sex) positive control animals (5 mg/kg cyclophosphamide, IP with a 0.5 mg/mL solution @ 10 mL/kg, within 24 hours prior to sacrifice). BioReliance personnel will sacrifice (isoflurane inhalation) the animals on the day after the final exposure, collect blood (2-4 mL into sodium heparin tubes) from the abdominal aorta and transport the samples on wet ice to BioReliance, Rockville, MD (attn: Ramadevi Gudi, Ph.D.). Upon receipt, samples will be processed and evaluated for determination of SCE response as detailed in the referenced guidelines and procedures (see Appendix C). The Testing Facility will be responsible for the GLP compliance of this subcontractor.

Micronucleus Evaluations Sprague Dawley-derived (CD®) rats, 5/sex/group, will be exposed to Gasoline DIPE Vapor Condensate via inhalation for a 4 week (5 days per week) exposure period by Huntingdon Life Sciences (PRC). PRC personnel will also perform the
treatment o( 

### non-exposed (5/sex) positive control animals (40 mglkg cyclophosphamide, IP with a 4.0 mg/mL solution @ 10 mL/kg, within 24 hours prior to sacrifice). The positive control animals will be sacrificed under C02 anesthesia. BioReliance personnel will remove the femurs immediately after the blood sample collection for the SCE evaluation and fixed, unstained slides will be prepared and HLS personnel will ship via overnight delivery to Eye Research Centre, Eye, Suffolk, UK (attn: Christine Mason, B.Sc.). Upon receipt, slides will be stained and evaluated for determination of micronucleus response as detailed in the referenced guidelines and procedures (see Appendix B). The Testing Facility will be responsible for the GLP compliance of this subcontractor.



### 11.8. IMMUNOTOXICITY EVALUATIONS

Female Sprague Dawley-derived (CD®) rats, 10/group, will be exposed to Gasoline DIPE Vapor Condensate via inhalation for a 4 week (5 days per week) 

### Page 991

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 25 Final Protocol

exposure period by Huntingdon Life Sciences (PRC). PRC personnel will also perform the treatment of non-exposed (10 females) positive control animals (50 mg/kg cyclophosphamide, IP with a 5 mg/mL solution @ 10 mL/kg, once per day for four days prior to sacrifice). Four days prior to sacrifice, animals will be sensitised with sheep red blood cells (2 x 1 0 8 ) 

### via tail vein injection by hnmunoTox, Inc. personnel. PRC personnel will, on the day after the final
exposure, collect and freeze (::S-20.C) blood (serum) samples (orbital collection anesthetized via carbon dioxide/oxygen inhalation), and then sacrifice (carbon dioxide inhalation) the animals, thymus will be removed, weighed, and preserved (1 0% neutral buffered formalin) for possible histopathology, spleens will be aseptically removed, weighed and shipped intact in HEPES/EBSS/Gentamicin solution (prepared at PRC) in individual shipping containers on wet ice (2-8.C) via overnight delivery to hnmunoTox., Inc., Richmond, VA (attn: Vanessa Peachee, M.S.). Upon receipt, spleen samples will be further processed for determination of PFC response as detailed in the referenced guidelines and procedures (see Appendix D). The serum samples will be retained by the Testing Facility until the spleen samples are evaluated for all subchronic inhalation studies in this test program and then discarded if not needed by the subcontractor. The Testing Facility will be responsible for the GLP compliance of this subcontractor.

The cyclophosphamide will be received from Sigma Chemical Company. The identity, strength, purity and composition or other characteristics to define the positive control article will not be determined by the Testing Facility. The

. positive control article will be characterized as per the Certificate of Analysis on file with the Testing Facility. The stability of the positive control article will not be determined by the Testing Facility. Analyses to determine the uniformity (as applicable) or concentration of the positive control mixture will not be performed by the Testing Facility. The stability of the positive control article mixture will
not be determined by the Testing Facility.



### 12. POSTMORTEM

12.1. MACROSCOPIC POSTMORTEM EXAMINATIONS

Complete macroscopic postmortem examinations will be performed on all Main/Recovery/Neuropathology animals including animals euthanatized in a moribund condition or found dead; all abnormal observations will be recorded. Complete macroscopic postmortem examinations will only be performed on all GF AP/Genotoxicity/hnmunotoxicity animals euthanatized in a moribund condition

or found dead; all abnormal observations will be recorded. The necropsy will include examination of the external surface and all orifices; the external surfaces of the brain and spinal cord; the organs and tissues of the cranial, thoracic, abdominal and pelvic cavities and neck; and the remainder ofthe carcass. 

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### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S



### 12.2. TIME OF NECROPSY

12.2.1. MORIBUND ANIMALS Page 26 Final Protocol

Animals showirig signs of severe debility, particularly if death appears imminent, will be euthanatized to prevent loss of tissues through autolysis.



### 12.2.2. TERMINAL & 

### RECOVERY NECROPSY Necropsy of all animals surviving to study termination will be performed after animals have been treated and/or held for the appropriate duration. Necropsy schedules will be established in order to assure that approximately equal numbers of males and females from each group will be examined on each day of necropsy and that examinations of animals of both sexes from each group will be performed at similar times of the day throughout the necropsy period.



### 12.3. METHOD OF EUTHANASIA Exsanguination following carbon dioxide inhalation for Main Study and Recovery Study animals. See sections 11.7, 11.8, 12.8 and 12.9 for methods for all other animals.



### 12.4. ORGAN WEIGHTS Organs indicated below will be taken from all Main Study and Recovery Study animals at the scheduled necropsy, weighed, recorded and organ/body and organ/brain weight ratios calculated. Organs will not be weighed for animals found dead or euthanatized in a moribund condition during the course of the study. Prior
to weighing, all organs will be carefully dissected and properly trimmed to remove fat and other contiguous tissue in a uniform manner. Organs will be weighed as

soon as possible after dissection to avoid drying. Paired organs will be weighed together: adrenals brain epididymides heart kidneys liver lungs

ovanes



### 12.5. TISSUES PRESERVED prostate seminal vesicles spleen testes thymus
uterus

Tissues listed in Appendix A will be obtained at necropsy and preserved for all Main Study and Recovery Study animals.



### 12.5.1. PRESERVATIVES Eyes

- glutaraldehyde/paraformaldeyhde Testes/epididymides- modified Davidson's solution 

### Page 993

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

All other tissues

- 10% neutral buffered formalin. Page 27 Final Protocol

Note: Lungs and urinary bladder will be infused with formalin to insure fixation.



### 12.6. MICROSCOPIC PATHOLOGY EVALUATIONS

Slides of tissues listed in Appendix A (under Microscopic Examination) will be prepared and examined microscopically for all Main Study animals from the Terminal Sacrifice as indicated. Target organs will be designated by the Study Director, pathologist and/or Sponsor based on experimental findings; authorization will be obtained from the Sponsor prior to performing examinations. Note: Any abnormalities not noted during macroscopic postmortem examinations which are

seen during histological processing will be recorded. Gross lesions will be examined at additional cost. Fibers from the sciatic nerve sample may be teased apart for further analysis under the microscope (optional, additional cost).

Screening for alpha 2u-globulin Nephropathy: A preliminary diagnosis will be made on hematoxylin and eosin stained slides of the kidney and/or based on elevated kidney weights at the Terminal Sacrifice. The sections will then be stained (additional cost) with Mallory Heidenhain stain, if the following lesions are seen with H&E: angular hyalin inclusions in the proximal convoluted tubule, tubular degeneration and regeneration, and/or globular casts at the corticomedullary junction.


### 12.7. STAINS

Standard stains used: hematoxylin and eosin on paraffin sections, toluidine blue on epoxy resin sections. Special stains may be employed on selected tissues to aid in making a diagnosis at the discretion of the Study Pathologist. Special stains may be employed at the request of the Sponsor (additional cost).



### 12.8. NEUROPATHOLOGY SACRIFICE

Animals (5/sex/group) will be anesthetized with an intramuscular injection**(~1.5** 

### mL/kg) of ketamine/xylazine/acepromazine and transcardially perfused with phosphate buffered saline followed by 1% glutaraldehyde and 4% paraformaldehyde in the same buffer. After perfusion, the required tissues will be dissected out. Measurement of the size (length and width) and weight of the whole brain (cerebrum, cerebellum and pons-medulla) shall be made. All tissues will then be placed into a fresh solution of the same fixative prior to processing.

Tissues listed below will be preserved for all designated neuropathology animals (5/sex/group) after 13 weeks oftreatment: 

### Page 994Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

Tissue

brain (forebrain, central cerebrum, hippocampus, basal ganglia, midbrain, cerebellum and pons, medulla)
eye with optic nerve spinal cord (cervical, thoracic, lumbar, cross and longitudinal sections) sciatic nerve (cross and longitudinal sections) tibial nerve (cross and longitudinal sections) sural nerve (cross and longitudinal sections) trigeminal ganglia

dorsal root ganglia (from C3- C6 and L4-L6) dorsal root fibers (from C3-C6 and L4-L6) ventral root fibers (from C3-C6 and L4-L6) lungs and trachea tissues with macroscopic findings Preserved (Groups) All

X

X

X

X

X

X X

X

X

X X

X Page 28 Final Protocol

Microscopic Examination

(Groups) land IV II and ill

X

X

X

X

X

X X

X

X

X X X

X X

Peripheral nerves will be post-fixed in 1% osmium tetroxide, processed and embedded in epoxy resin and microtomed at approximately 2 microns. All other tissues, including the brain, eye with optic nerve, spinal cord, trigeminal ganglia, dorsal root ganglia, dorsal and ventral root fibers, lungs and trachea will be processed by standard techniques, embedded in paraffin and microtomed at approximately 6 microns.

The tissues listed above will be examined microscopically for all animals as indicated. Tissues with macroscopic lesions will be examined in all animals (additional cost). Any abnormalities not noted during macroscopic postmortem examinations which are seen during histological processing will be recorded. If microscopic findings indicative of an effect of test substance administration are

seen in high-exposure animals, then examinations will be made of these tissues/organs for low- and mid-exposure animals (additional cost). Additional 

### Page 995

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 29 Final Protocol

examinations will be made only after consultation with the Sponsor and receipt of authorization from the Sponsor.

If any evidence of neuropathology is found, then a subjective diagnosis will be performed to evaluate exposure-response relationships (additional cost). All regions of the nervous system exhibiting any evidence of neuropathological changes will be included in the analysis. Sections from all exposure groups from each region will be coded and examined in a randomized order without knowledge of the code. The frequency and severity of each lesion will be recorded. After all samples have been rated, the code will be broken and statistical analysis performed to evaluate exposure-response relationships.

12.9. GFAP EVALUATION Sprague Dawley-derived (CD®) rats, 5/sex/group, will be exposed to Gasoline DIPE Vapor Condensate via inhalation for a 13 week (5 days per week) exposure period by Huntingdon Life Sciences (PRC). US Center for Disease Control (CDC) Health Effects Laboratory personnel will sacrifice (carbon dioxide inhalation) the animals after the 13 week exposure period, and remove, weigh and process the brains. PRC personnel will ship the samples on dry 

### ice to the US Center for Disease Control Health Effects Laboratory, Morgantown, WV (attn: James P. O'Callaghan, Ph.D.). Upon receipt, samples will be further processed for determination of GF AP response as detailed in the referenced guidelines and procedures (see Appendix E). The Sponsor will be responsible for the spirit of GLP compliance ofthis subcontractor.



### 13. PRESERVATION OF RECORDS AND SPECIMENS

All data documenting experimental details and study procedures and observations will be recorded and maintained as raw data.

At the completion of the study, all reports, raw data, preserved specimens and retained samples will be maintained in the Testing Facility's Archives for a period of one year after submission of the signed final report.

The Sponsor will be contacted in 

### order to determine the final disposition of these materials. The Sponsor is responsible for all costs associated with the storage of these materials beyond one year from the issuance of the final report and for any costs associated with the shipment of these materials to the Sponsor or to any other facility designated by the Sponsor. 

### Page 996Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S



### 14. STATISTICAL EVALUATIONS
The following items will be analyzed statistically in the final report: Page 30 Final Protocol

mean body weight values and body weight changes (from pretest)

mean food consumption values (presented as grams of food/kg of body weight/day)

mean clinical laboratory values

mean terminal organ weights, organ/body weight ratios and organ/brain weight ratios

mean motor activity counts

mean forelimb and hindlimb grip strength measurements

mean landing foot splay measurements

Mean values of all exposure groups will be compared to the mean value for the control
group at each time interval.

Evaluation of equality of group means will be made by the appropriate statistical method, followed by a multiple comparison test if needed. Bartlett's test (Bartlett, 1937; Sokal and Rohlf, 1995) will be performed to determine if groups have equal variances. For all parameters except organ weights, if the variances are equal, parametric procedures will be used; if not, nonparametric procedures will be used. Organ weight data will be analyzed only by parametric methods. The parametric method will be the standard one-way analysis of variance (ANOV A) using the F ratio to assess significance (Armitage, 1971; Dunlap and Duffy, 1975). If significant differences among the means are indicated, additional tests will be used to determine which means are significantly different from the control: Dunnett's (Dunlap et al., 1981; Dunnett, 1955, 1964), Williams (Williams, 1971, 1972), or Cochran and Cox's modified t-test (Cochran and Cox, 1959). The nonparametric method will be the Kruskal-Wallis test (Kruskal and Wallis, 1952, 1953) and if differences are indicated, Shirley's test (Shirley, 1977), Steel's test (Steel, 1959) or Pairwise Comparison with Bonferroni Correction (Games and Howell, 1976) will be used
to determine which means differ from control. Bartlett's test for equality of variance will be conducted at the 1% significance level; all other statistical tests will be conducted at the 5% and 1% significance levels.

References for these procedures are: Armitage, P. 1971. 

### Statistical Methods in Medical Research. Oxford, UK: Blackwell Scientific Publications; Bartlett, M.S. 1937. Properties of sufficiency and statistical tests. 

### Proceedings of the Royal Society, Series A, 160: 268-282; Cochran, W.G. and Cox, G.M. 1959. 

### Experimental Designs, New York: John Wiley, pp. 100-102; Dunlap, W.P. and Duffy, J.A. 1975. Fortran 

### N Functions for Calculating Exact Probabilities Associated with Z, Chi-Square, T and F Values. 

### Behav. Res. Methods and Instrumentations 7:59-60; Dunlap, W.P., Marx, M.S. and Agamy, G.G. 1981. Fortran N 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 

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### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DJPE-S Page 31 Final Protocol

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 

### 20-3: 482-491; Games, P.A. and Howell, J.F. 1976. Pairwise multiple comparison procedures with unequal n's and/or variances: a monte-carlo study. Journal of Educational Statistics 1: 113-125; Kruskal, W.H. and Wallis, W.A. 1952. Use of Ranks in one-criterion 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; Shirley, E.A.C. 1977. A nonparametric equivalent of Williams' test for contrasting increasing dose levels of a treatment. Biometrics 33: 386-389; Sokal, R.R. and Rohlf, F.J. 1995. Biometry. 3rd Edition. San Francisco: W.H. Freeman pp. 369-371; Steel, R.G.D. 1959. A multiple comparison rank sum test: treatment versus control. Biometrics 

### 15: 560-572.; Williams, D.A. 1971. A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27: 103-117; Williams, D.A. 1972. The comparison of several dose levels with a zero dose control. Biometrics 28: 519-531.

Motor Activity Data



### The data will be analyzed using split-plot repeated measures ANOV A with model terms for group, animal within group, interval and group by interval interaction. If the group x interval interaction is statistically significant (p=0.05), indicating non-parallelism in the behavioral profile between groups, a separate one-way ANOV A for group effects will be performed at each interval. If the response data passes on the parallel hypothesis, an ANOVA (using summed responses over intervals) will be used to test for the overall
treatment effect which constitutes the level hypothesis. If any significant overall
treatment group effect is found by any of the above ANOVAs, Dunnett's t-test will be used to find groups that differed from control. Analyses will be performed for sexes separately and combined. Treatment group effects will be deemed significant at the p=0.05 level. Plots, tables, listings, and analyses will be generated using SAS® version 6.12 for WINDOWS. Analyses will be conducted by CATO Research, 200 Westpark Corporate Center, 4364 South Alston Avenue, Durham, NC 27713-2280. The Testing Facility will be responsible for the GLP compliance of this subcontractor. 

### Page 998Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S



### 15. REPORTING

15.1. STATUS REPORTS Page 32 Final Protocol

Periodic verbal and written updates on study progress will be provided by the Study Director. In general, written status reports will be submitted weekly and at termination of the Main/Neuropathology Study and Recovery Study.



### 15.2 FINAL REPORT One unbound hard copy and one electronic copy of a draft report will be submitted following termination of the study. The Main/Recovery/Neuropathology Study and each other satellite study will be separately reported. After receipt and review of the Sponsor's comments, appropriate changes will be made and two hard copies and one electronic copy of a signed, final report will be issued. (Additional copies will be provided at additional cost). The Main/Recovery/Neuropathology Study report will minimally include:

Compliance Statement (including Sponsor signature line) Quality Assurance Statement Abstract Introduction Experimental Design Materials and Methods Discussion of study results Conclusion and No Observed Adverse Effect Level (NOAEL) statement Mortality data Individual animal termination history Summary of daily animal observations (as appropriate) Summary of weekly physical examination data Tables of exposure data Tables of mean and individual body weights and body weight changes Tables of mean and individual feed consumption Graphs of body weights and feed consumption Tables of mean and individual hematology and clinical chemistry values Tables of individual differential leukocyte counts Tables of mean and individual organ weights, organ/body and organ/brain weight ratios Tables of mean and individual motor activity counts Tables of mean and individual functional observational battery results Summary of Positive Control Neurobehavioral Studies Individual and summary tables of macroscopic pathology findings Individual and summary tables of microscopic pathology findings Individual and summary tables of neuropathology findings References for experimental methodology Senior personnel participating in the study Protocol and any protocol amendments 

### Page 999Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S



### APPENDIX A Page 33 Final Protocol



### Tissues Preserved/Examined Microscopically

Tissue a Preserved Microscopic Examination

I (Groups)

I, IV 

### II, III

adrenal gland X X

aorta (thoracic) X X

bone (sternum/femur with articular surface) X X

bone marrow (femur)b X 

### xc

brain (medulla/pons, cerebrum and cerebellum) X X
epididymides X X
esophagus X X

eye (with optic nerve) X XC

heart X X

kidneys X X

large intestine (cecum, colon, and rectum) X X

lacrimal gland X 

## xc

larynx d X X

liver X X

lungs (with rnainstem bronchi) X X X

lymph node (mediastinal and mesenteric) X X

mammary gland X XC

muscle (biceps femoris) X XC

nasopharyngeal tissue e X X

nerve (sciatic) X X

ovaries X X

pancreas X X

pituitary X XC

prostate X X
salivary gland with submandibular lymph node X X

seminal vesicles X X 

### Page 1000Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

Tissue a

skin

small intestine (duodenum, jejumnn, ilewn)
spinal cord (cervical, thoracic, lwnbar)
spleen

stomach

testes
thymic region
thyroid (with parathyroids)

trachea
uninary bladder

uterus (body/horns with cervix)
Zymbal 's gland

gross lesions and tissue masses f

target organsf,g Preserved

X

X

X

X

X

X

X

X

X

X

X

X

X

X Page 34 Final Protocol

Microscopic Examination

(Groups)
I, IV 

### II, III

XC

X

X

X

X

X

X

X

X

X

X

XC

X X

X X

a These tissues will also be examined for animals in Groups IT and ill which die prior to study termination (at
additional cost).

b Qualitative examination (no differential count). Bone marrow smears will be prepared and archived. They
will only be evaluated (Sponsor approval, additional cost) if needed.

c Tissues need only be examined microscopically if indicated by signs of toxicity or target organ involvement (additional cost).

d The laryngeal mucosa shall be examined. Sections of the larynx to be examined include the epitheliwn covering the base of the epiglottis, the ventral pouch and the medial surfaces of the vocal processes of the arytenoid cartilages.

e Four sections of the nasopharyngeal tissue shall be examined. This shall include sections through the nasal cavity and examinations of the squamous, transitional, respiratory and olfactory epithelia.

r Examinations performed at additional cost.**~arget** organs will be designated by the Study Director, Pathologist and/or Sponsor based on experimental fmdings. 

### Page 1001Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S



### APPENDIXB Micronucleus Evaluations Page 35 Final Protocol 

### Page 1002Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 36 Final Protocol

SATELLITE PROCEDURE

GASOLINE DIPE VAPOR CONDENSATE

RAT MICRONUCLEUS TEST

Subcontractor's Sponsor Huntingdon Life Sciences Princeton Research Center (PRC) Mettlers Road East Millstone, NJ 08875-2360 Subcontractor Huntingdon Life Sciences Ltd Eye Research Centre (ERC) Eye Suffolk IP23 7PX England 

### Page 1003

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

STUDY DETAILS PAGE 

### Study number: 00-6130 Study title: Rat micronucleus test Page 37 Final Protocol

Test Substance: Gasoline DIPE Vapor Condensate

Route of administration: Inhalation Stability of test substance: Assessed by the Study Sponsor Analysis of achieved concentration: Principal illvestigator:

Head ofDepartment: Study Director: Assessed at PRC

Christine Mason, B.Sc. at ERC

Gillian Clare, Ph.D. at ERC Gary M. Hoffman, BA, DABT at PRC

ill the temporary absence of the Principal illvestigator, The Head of Department may make decisions affecting the conduct of the study, in keeping with their training and experience or will appoint another scientist with appropriate training and experience to do so. The Principal illvestigator on her return will ratify all decisions taken in her absence.

Location of study:

Proposed study dates: Start: 15 March 2002 Completion: 11 

### April 2002 Draft report: 25 October 2002 ill-Life: PRC

Genetic Toxicology: ERC except statistical analysis at Huntingdon Research Centre (HRC) 

### Page 1004

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

1. 

### INTRODUCTION Page 38 Final Protocol

The object of this study is to assess the mutagenic potential of the test substance in an in vivo 

### cytogenetic test system which complies with the following guidelines:
US EPA Micronucleus Assay 79.64, CFR Vol. 59, No. 122, 27 June 1994.
US EPA (1998) Health Effects Test Guidelines; OPPTS 870.5395 Mammalian Erythrocyte Micronucleus Test.



### 2. BACKGROUND

The bone marrow micronucleus test, originally developed by Matter and Schmid (1971), is a widely employed and internationally accepted short-term assay for identification of genotoxic effects (chromosome damage and aneuploidy) associated with mutagens and carcinogens (Mavoumin et al 

### 1990). This in vivo 

### system allows consideration of various factors including pharmacokinetics, metabolism and DNA repair which cannot be accurately modelled in an in vitro 

### system. Young adult rats are chosen for use because of the high rate of cell division in the bone marrow, because of the wealth of background data on this species, and because of their general suitability for toxicological investigations.

In 

### mitotic cells in which chromosomal breakage has been caused by the test substance or its metabolites, acentric fragments of the chromosomes do not separate at the anaphase stage of cell division. After telophase these fragments may not be included in the nuclei of the daughter cells and hence will form single or multiple micronuclei (Howell-Jolly bodies) in the cytoplasm of these cells. Micronuclei are seen in a wide variety of cells, but erythrocytes are chosen for examination since micronuclei are not obscured by the main nucleus and are therefore easily detected in this cell type (Boller and Schmid 1970).

Micronucleated immature erythrocytes appear in the bone marrow approximately 24 hours after induction of chromosome damage. These immature erythrocytes can be differentiated by a variety of staining techniques which rely on their relatively high content of residual RNA. Using the Feulgen!H&E method, they stain slate-blue while mature erythrocytes (which contain little RNA)

are counterstained orange. An 

### increased incidence of micronucleated immature erythrocytes is indicative of recent exposure to a chromosome-damaging agent. A simultaneous marked increase in the incidence of micronucleated mature erythrocytes is not expected and may be indicative of micronucleus-like artifacts (Schmid 1976).

Substances which interfere with the mitotic spindle apparatus will cause non-disjunction (unequal separation of the chromosomes at anaphase resulting in aneuploidy) or lagging 

### Page 1005

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 39 Final Protocol

chromosomes at anaphase which may not be incorporated into the daughter nuclei. These lagging chromosomes are not excluded from the erythroblast with the main nucleus and hence also give rise to micronuclei.

Any toxic effects of the test substance on the nucleated cells may lead either to a reduction in cell division or to cell death. These effects in tum lead to a reduction in the number of nucleated cells and immature erythrocytes; to compensate for this, peripheral blood is shunted into the bone marrow (von Ledebur and Schmid 1973). If the proportion of immature erythrocytes is found to be significantly less than the control value, this is taken as being indicative of toxicity. A very large decrease in the proportion would be indicative of a cytostatic

or cytotoxic effect.

3. EXPERIMENTAL PROCEDURE

Animal management



### Species
Strain
Source Age Weight range Acclimatization

Housing

Diet

Positive control 

### Rat Sprague Dawley- derived**(CD~** [Crl: CD® IGS BR]
Charles River Laboratories, Kingston, NY**~6** 

### weeks old (at exposures initiation)**~** 

### 150-250 g (at exposures initiation); within ±20% of mean per sex Approximately two weeks; all animals will be checked for viability twice daily. Prior to assignment to study all animals will be examined by a senior staff member to ascertain suitability for study.

Animals will be housed individually in suspended stainless steel wire mesh cages. The animal room will provide a controlled environment with the thermostat set at 18-26°C and relative humidity set at 30- 70%. The room will be illuminated by artificial light for 12 hours per day. An auxiliary power supply is available in the event of main supply failure.

Certified Rodent Diet, No. 5002; (Meal) (PMI Feeds, Inc., St. Louis, MO) ad libitum. 

### Elizabethtown Water Company, Westfield, NJ; ad libitum, 

### via automated watering system. There are no known contaminants in the feed or water which are expected to interfere with the results of this study. 

### Page 1006Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

Identity Cyclophosphamide Page 40 Final Protocol

Supplier Aldrich Chemical Co Ltd or other suitable supplier Appearance White crystalline powder
Vehicle Purified water Dosage preparation Solution prepared just prior to use

The study design is shown below: Animals in air control and test substance groups will be exposed for 4 weeks (5 days per week) by the route specified in the study details section. Animals in the positive control group will receive 40 mg/kg cyclophosphamide intraperitoneally within 24 hours prior to sacrifice. Animals will be regularly examined for adverse clinical signs until the time of sacrifice.

Material Exposure Sampling No. of animals Level Time After Last Exposure or Dose (hours) M F

Air Control Omg/m 3**~24** 5 5

Test substance 2,000mg/m 3**~24** 5 5

Test substance 10,000 mg/m 3**~24** 5 5

Test substance 20,000 mg/m 3**~24** 5 5

Cyclophosphamide 40mg/kgiP**~24** 5 5

PREPARATION OF BONE MARROW SMEARS

At approximately 24 hours after administration of the final exposure, five male and five female animals from each group 

### will be killed by humane methods. The femurs will be dissected from each animal and the distal heads removed. The proximal heads will be kept intact and as much tissue as possible will be removed from the bones. The contents of each femur will be eluted in 

### Page 1007

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 41 Final Protocol

fetal bovine serum by aspiration through an 18g needle fitted to a plastic syringe. The resulting cell suspensions will be spun at 1000 rpm (150 x g) for 5 minutes using a centrifuge. The final cell pellet will be resuspended in a small volume of fetal bovine serum to facilitate smearing in

. the conventional manner on glass microscope slides (Schmid 1976). Four smears will be

prepared from each animal. Two smears from each animal will be shipped unstained to ERC where they will be stained and examined. The remaining 2 smears and the cell pellet (refrigerated) will be held in reserve at PRC in case of technical problems with the first 2

smears.
Due to the presence of mast cell granules in rat bone smears, which appear identical to micronuclei when stained using the Romanowsky methods, a modified Feulgen staining method is employed for the rat micronucleus test in this laboratory. This method specifically stains DNA-containing bodies deep purple while leaving mast cell granules unstained. The method also allows reasonable differentiation of mature and immature erythrocytes and produces permanent preparations.

Staining of slides

1. 

### Fixed for 3 to 10 minutes in absolute methanol.
2. Hydrolysed in Bouin's fluid at room temperature for 30 hours.
3. Washed three times in purified water (5 minutes per wash).
4. Stained in Schiffs reagent for one hour at room temperature
5. Washed three times in purified water (5 minutes per wash).
6. Counter-stained for 10 minutes in very dilute (approximately 0.06 g/1) aqueous Eosin yellowish. The dilution and staining time in Eosin may be adjusted to give optimum differentiation between mature and immature erythrocytes.
7. Washed for five minutes in purified water.
8. Stained for 30 minutes in Mayer's Haemalum diluted 9 volumes: 1 volume with aqueous acridine orange solution (1 mg/ml).
9. Rinsed in purified water briefly.
10. Briefly rinsed in running tap water.
11. Washed for 5 minutes in purified water.
12. Air-dried.
13. Slides will be mounted with coverslips using DPX mountant.
14. The mountant will be allowed to harden at approximately 37°C.

NB All stains and Bouin's fluid will be filtered immediately prior to use to remove particulate material. 

### Page 1008Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

Microscopic examination Page 42 Final Protocol

Coded slides will be examined by light microscopy and 2000 immature erythrocytes per animal will be examined for the presence of micronuclei. Usually only one smear is examined per animal,
any remaining smear being held temporarily in reserve in case of technical problems with the first

smear. Micronuclei are identified by the following criteria: Large enough to discern morphological characteristics
Should possess a generally rounded shape with a clearly defined outline
Should be deeply stained and similar in color to the nuclei of other cells

- not black
Should lie in the same focal plane as the cell
Lack internal structure, ie they are pyknotic
There should be no micronucleus-like debris in the area surrounding the cell

The proportion of immature erythrocytes will be assessed by examination of a total of at least 1 000 erythrocytes per animal.



### 4. ASSESSMENT OF RESULTS

The results obtained for each treatment group will be compared with the results obtained for the concurrent air control group using non-parametric statistical methods. Non-parametric statistical methods are chosen for analysis of results because: They are suited to analysis of data consisting of discrete/integer values with ties such as the incidence of micronucleated immature erythrocytes.
The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of non-parametric analysis and classical analysis of variance are very similar).
"Outliers" are frequently found in the proportion of immature erythrocytes for both control and treated animals; non-parametric analysis based on rank does not give these values an undue weighting.

Unless there is a substantial difference in response between sexes (which occurs only rarely) results for the two sexes are combined to facilitate interpretation and maximize the power of statistical analysis.

For incidences ofmicronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachussetts). Comparison of several dose levels is made with the concurrent control using the Linear by 

### Page 1009

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 43 Final Protocol

Linear Association test for trend in a step-down fashion if significance is detected (Agresti et a!. 

### 1990); for individual inter-group comparisons (eg 

### the positive control group) this procedure simplifies to a straightforward permutation test (Gibbons 1985). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie 

### exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

A positive response is normally indicated by a statistically significant exposure-level-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (P<0.01); individual and/or group mean values should exceed the laboratory historical control range (Morrison and Ashby 1995). A negative result is indicated where individual and group mean incidences of micronucleated immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (P>0.01) and where these values fall within the historical control range. An 

### equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.

Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant exposure-level-related decrease in the proportion of immature erythrocytes (P<0.01). Additional slide reading or animal testing may very occasionally be required to clarify unclear or equivocal results.

5. REPORTING



### The report will contain details of the test substance, methodology, results and the interpretation of the data. Tabulated results will show individual animal results, and group mean results. Good Laboratory Practice and Quality Assurance statements will be included.

6. MAINTENANCE OF RECORDS



### All raw data, samples and specimens (if appropriate) arising from the performance of this study will remain the property of the Sponsor. Types of sample and specimen which are unsuitable, by reason of instability, for long term retention and archiving may be disposed of after the periods stated in Huntingdon Life Sciences Standard Operating Procedures. All other samples and specimens and all raw data will be retained by Huntingdon Life Sciences PRC in its archive for a period of one year from the date on which the Study Director signs the final report. After such time, the Sponsor will be contacted and his advice sought on the return, disposal or further retention of the materials. If requested, Huntingdon Life Sciences will continue to retain the materials subject to a reasonable fee being agreed with the Sponsor. Huntingdon Life Sciences will retain the Quality Assurance records relevant to this in its archive indefinitely. 

### Page 1010Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page44 Final Protocol



### 7. GOOD LABORATORY PRACTICE

The study will be conducted in compliance with the principles of Good Laboratory Practice Standards as set forth in:
US EPA 79.60, CPR Vol. 59, No. 122,27 June 1994



### 8. QUALITY ASSURANCE

The following will be inspected or audited in relation to this study.

Protocol Audit

Process based inspections

Report Audit Study specific protocol and study specific pages.

Routine and repetitive procedures will be inspected on representative studies, not necessarily on this study.

The draft report and study data will be audited before issue of the draft report to the Sponsor.

QA findings will be reported to the Study Director and Company Management promptly on completion of each action, except for process based inspections which will be reported to appropriate Company Management only.



### 9. REFERENCES

AGRESTI, A., MEHTA, C.R. and PATEL, N.R. (1990) Exact inference for contingency tables with ordered categories. Journal of the American Statistical Association, 85,453.

BOLLER, K. and SCHMID, W. (1970) Chemical mutagenesis in mammals. The bone marrow of the Chinese hamster as an in vivo test system. Haematological findings after treatment with Trenimon (translation). Humangenetik, 11, 34.

CYTEL (1995) StatXact 3 for Windows: Statistical Software for Exact Nonparametric Inference. Cytel Software Corporation, NC, USA.

GIBBONS, J.D. (1985) Nonparametric Statistical Inference, 2nd edition, Marcel Dekker, New York.

JONCKHEERE, A.R. (1954) A distribution-free k-sample test against ordered alternatives. Biometrics, 41, 133-145.

KRUSKAL, W.H. and WALLIS, W.A. (1952) Use of Ranks in One-Criterion Variance Analysis. Journal of the American Statistical Association, 47, 583-621. 

### Page 1011

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 45 Final Protocol

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

### 47, 583-621.

MacGREGOR, J.T., HEDDLE, J.A., HITE, M., MARGOLIN, B.H., RAMEL, C., SALAMONE, M.F., TICE, R.R. and WILD, D. (1987) Guidelines for the conduct of micronucleus assays in mammalian bone marrow erythrocytes. Mutation Research, 189, 

### 103.

MATTER, B. and SCHMID, W. (1971) Trenimon-induced chromosomal damage in bone

marrow cells of six mammalian species, evaluated by the micronucleus test. Mutation Research, 12, 

### 417.

MA VOURNIN, K.H., BLAKEY, D.H., CIMINO, M.C., SALAMONE, M.F. and HEDDLE, J.A. (1990) The in vivo 

### micronucleus assay in mammalian bone marrow and peripheral blood. A report of the US Environmental Protection Agency Gene- Tox Program. Mutation Research, 

### 239,29.

MORRISON, V. and ASHBY, J. (1995) High resolution rodent bone marrow micronucleus
assays of 1 ,2-dimethylhydrazine : implication of systemic toxicity and individual responders. Mutagenesis, 10, 

### 129.

SAS INSTITUTE (1989) SASISTAT User's Guide, Version 6, Fourth Edition, Vo/.2. 

### SAS Institute Inc., Cary, NC, USA.

SAS INSTITUTE (1996) SAS/STAT Software: Changes and Enhancements through Release 

### 6.11. SAS Institute, Cary, NC, USA.

SAS INSTITUTE (1996) SASISTAT Software: 

### Changes and Enhancements for Release 6.12. SAS Institute, Cary, NC, USA.

SCHMID, W. (1976) The micronucleus test for cytogenetic analysis. In: HOLLANDER, A. ( ed.) Chemical Mutagens, Principles and Methods for their Detection, 4, 

### 31. Published by Plenum Press, New York.

von LEDEBUR, M. and SCHMID, W. (1973) The micronucleus test. Methodological aspects. Mutation Research, 19, 

### 109.

WILCOXON, F. (1945). Individual comparisons by ranking methods. Biometrics Bulletin, 1, 

### 80- 83. 

### Page 1012Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S



### APPENDIXC SCE Evaluations Page 46 Final Protocol 

### Page 1013Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 47 Final Protocol

Satellite Procedure In Vivo-In Vitro Rat Peripheral Lymphocyte Sister Chromatid Exchange Assay

1.0 PURPOSE

2.0

3.0 The purpose of this study is to evaluate the potential of the test substance to induce sister chromatid exchanges (SCEs) in peripheral lymphocytes following inhalation exposure of rats.

FACILITIES and STAFF

2.1 Subcontractor's Sponsor: Huntingdon Life Sciences Princeton Research Center (PRC) Mettlers Road East Millstone, New Jersey 08875-2360

2.2 Study Director: Gary M. Hoffman, B.A., DABT Huntingdon Life Sciences Phone: (732) 873-2550 Ext. 2920 Fax: (732) 873-3992 E-mail: hoffmang@princeton.huntingdon.com

2.3 Principal Investigator: Ramadevi Gudi, Ph.D. BioReliance Phone: (301) 610-2169 Fax: (301) 738-2362 E-mail: rgudi@bioreliance.com

2.4 Testing Facility Project: 00-6130

IDENTIFICATION OF TEST AND CONTROL SUBSTANCES

3.1 Test Substance: Gasoline DIPE Vapor Condensate

3.2 Controls: Negative: Positive: Air control Cyclophosphamide (CP)

3.3 Test Substance Retention Sample

The retention of a reserve sample of the test substance will be the responsibility of Huntingdon Life Sciences. 

### Page 1014

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

4.0 SUBCONTRACTOR AND KEY PERSONNEL

4.1 Name:

4.2 Address:

4.3 Principal Investigator: BioReliance

9630 Medical Center Drive Rockville, MD 20850

Ramadevi Gudi, Ph.D.

4.4 Regulatory Requirements/Good Laboratory Practice This study will be conducted in accordance with: US EPA SCE Assay 79.65, CFR Vol. 59, No. 122,27 June 1994. US EPA GLP 79.60, CFR Vol. 59, No. 122,27 June 1994.

5.0 TEST SCHEDULE

5.1 Proposed Experimental Initiation Date: 15 March 2002 Page48 Final Protocol

5.2 Proposed Experimental Completion Date: 11 April2002 (in-life) May 2002 (evaluation)

5.3 Proposed Draft Report Date: 25 October 2002

6.0 TEST SYSTEM

Closed-colony, random-bred rodents are acceptable models for mutagenicity studies. SpragueDawley rats were selected because of the availability of historical control data. Information on the
test system is provided by Huntingdon Life Sciences and is contained in their files.

' 

### 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY

Information on the experimental design and methodology has been provided by Huntingdon Life Sciences and is contained in their files. All in vivo 

### portions of the study, including inhalation
exposures, will be performed by Huntingdon Life Sciences. Animals will be anesthetized and blood samples will be collected by BioReliance personnel on site at Huntingdon Life Sciences. The blood samples will be transported to BioReliance on the day of collection. Within 24 hours after blood collection, the blood samples will be cultured in appropriate growth medium. Lymphocytes will be grown in the presence of 5-bromo-2'-deoxy-uridine (BrdU) for two cell cycles and dividing cells will be arrested in metaphase and harvested for microscopic evaluation of 

### Page 1015Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page49 Final Protocol

SCEs. The potential of the test substance to increase SCEs in a dose-responsive manner will be measured relative to a control group.

The study design will be as follows:
Number per Sex to be Sacrificed After Test Substance Group Administration

Negative Air Control 5

Test Substance (mg/m 3 ) 2,000 5 10,000 5 20,000 5

Positive Control Cyclophosphamide 5

7.1 Controls

7.1.1 Negative control

The negative control group of animals will be exposed to air only.

7 .1.2 Positive control

Cyclophosphamide monohydrate (CP, CAS number 6055-19-2) will be administered as the positive control at a dose of 5 mg/kg. CP will be administered by intraperitoneal injection within 24 hours prior to harvest.

7.2 Blood Collection and Cell Culture

Approximately 24 hours after the end of a 4 week (5 days per week) exposure period, 2-4 ml of blood will be collected from the abdominal aorta from each anesthetized (isoflurane inhalation) rat including rats treated with positive control article. Whole blood cultures in duplicate per animal will be set up in RPMI 1640 culture medium supplemented with 10% fetal bovine serum, antibiotics (penicillin G, 100 u/ml and streptomycin sulfate, 0.1 mg/ml), 25 mM HEPES buffer, 20 ug/ml phytohemagglutinin and an additional 2 mM L-glutamine. Cultures will be initiated by inoculating 

### Page 1016

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211- D IPE-S Page 50 Final Protocol

approximately 0.5 ml of whole blood per 5.0 ml of complete culture medium in T-25 sterile disposable tissue culture flasks and incubated at 37°C. Approximately 21 hours after initiation of the cultures, the cells will be exposed to 5.0 J..lg/mL of BrdU (Sigma Co., St. Louis, MO).

7.3 Cells Harvest and Staining

At approximately 68 hours, colcemid at 0.2 J..lg/ml will be added to each flask and incubated for approximately 4 hours. The cells will be swollen by hypotonic treatment (0.075 M KCl), fixed and washed with methanol:acetic acid (3:1), capped and stored overnight or longer at 2-6°C. To prepare slides, the cells will be collected by centrifugation and resuspended in fresh fixative. One to two drops of fixed cells will be dropped onto a glass microscope slide and air-dried. The slide will be identified by the study number, animal number and date. One to two slides will be prepared from each flask. The slides will be stained using a modified fluorescence-plus-Giemsa technique (Perry and Wolff, 1974). Slides will be stained in Hoechst 33258, 5 J..lg/ml for 10 minutes, mounted in phosphate buffer, pH 6.8, exposed to a U.V. lamp at approximately 60°C for 4-6 minutes, and then stained in 5% Giemsa for 6-10 minutes (BioReliance SOP# OPGT0442). Slides will be air dried and permanently mounted.

7.4 Scoring

Slides will be coded using random numbers by an individual not involved with the scoring process. Metaphase cells will be examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 25 second division metaphases per animal will be scored for SCEs. At least 100 consecutive metaphase cells will be scored for the number of cells in first-, second- or third-division metaphase for each animal as an indicator of toxicity (cell cycle delay) (BioReliance SOP# OPGT0442 and OPGT0336). At least 1000 cells will scored for mitotic index per animal.

8.0 Criteria for Determination of a Valid Test

The mean SCEs/cell/animal for the positive control animals must be statistically increased relative to the negative control.

9.0 Evaluation of Test Results

The number of SCEs per second-division metaphase cell will be presented for each group along with the range of SCEs per animal within a group. SCE frequencies as the grand mean 

### ± 

### Page 1017

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 51 Final Protocol

standard deviation will be presented for each group. The Average Generation Time (AGT) is estimated as: 

### = Number of hours in BrdU X 100/ (number M1 cells x 1 )+(number 

### Mz cells x 2)+(number M3 cells x 3)]. A Dunnett's t test for multiple comparisons will be performed to compare the SCE frequency of test exposure levels to the negative control frequency, and if p:::0.05, that exposure level will be considered significant. The conclusion of the study will be based on the Principal Investigator's evaluation of all the data, including the biological as well as statistical significance. The following criteria will be used as guidelines in evaluation of this study.

The test substance will be considered positive if an exposure-level responsive and statistically significant increase is observed over a minimum of two exposure levels. A statistically significant increase at the high exposure level with an exposure-level responsive although not statistically significant increase at lower exposure levels will be assessed as suspect. A statistically significant increase at one or more exposure levels with no evidence of a exposure level response will be assessed as equivocal or as negative according to the magnitude of the
response and the number of exposure levels affected.

10.0 Report

A report of the results of this portion of the study conducted at BioReliance will be prepared by BioReliance and will accurately describe all methods used for generation and analysis of the data. Results presented will include, but not be limited to: cells used.
test conditions: composition of medium, incubation time, concentration of BrdU, Colcemid7, positive and negative controls. number of cell cultures. mitotic index and number of metaphases analyzed for sister chromatid exchanges (SCE). cell cyCle kinetics relative to the negative control.

11.0 Records and Archives

All raw data, the protocol and all reports generated at BioReliance will be maintained according
to Standard Operating Procedure OPQP3040 by the BioReliance RAQA unit headquartered at: BioReliance, 14920 Broschart Road, Rockville, MD 20850. After submission of the final report, all ofthe above will be shipped to Huntingdon Life Sciences to be archived.

12.0 Specimens All specimens, such as microscope slides, will be held in storage as long as the quality of the preparation affords evaluation or in accordance with the relevant Good Laboratory Practice Regulations. 

### Page 1018Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S



### APPENDIXD lmmunotoxicity Evaluations Page 52 Final Protocol 

### Page 1019Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 53 Final Protocol

IMMUNOTOX, INC. In Vitro & In Vivo Immunotoxicological Assessments

Satellite Procedure Title:

Immunotoxicological Evaluation of Gasoline DIPE Vapor Condensate Using the Plaque Forming Cell Assay

Subcontractor's Sponsor:

Huntingdon Life Sciences (HLS)
Mettlers Road
East Millstone, New Jersey 08875

Principal Investigator:

Kimber L. White, Jr., Ph.D.

Subcontractor Facility:

ImmunoTox, Inc. Virginia BioTechnology Research Park
800 E. Leigh Street, Suite 209 Riclnnond, VA 23219-1534

Study Identification:

HLS Study Number: 00-6130 ImmunoTox, Inc. Project Number: ITI 1001 

### Page 1020Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 54 Final Protocol

PURPOSE: The purpose of this study is to provide evaluation of immunological parameters for Huntingdon Life
Sciences Study No. 00-6130. In this study, the ability of the test substance, Gasoline DIPE Vapor Condensate, will be evaluated for its ability to affect the humoral immune component of the immune system, when evaluated in the antibody- forming cell response to the T -dependent antigen sheep ef)'to/ocytes.

lillNTINGDON STUDY NUMBER: 00-6130

IMMUNOTOX, INC. PROCEDURE NUMBER: 00-6130

IMMUNOTOX, INC. PROJECT NUMBER: ITI 1001

SECURITY: Industrial

PROPOSED STUDY INITIATION DATE: (Date Protocol Signed By Study Director) To Be Supplied by Huntingdon Life Sciences

PROPOSED EXPERIMENTAL START DATE: (First Day of Treatment) 14 February 2002

PROPOSED EXPERIMENTAL TERMINATION 14 March 2002
DATE: (Date ofFinal Sacrifice)

PROPOSED DRAFT REPORT DATE: 25 October 2002

SUBCONTRACTOR'S SPONSOR: Huntingdon Life Sciences
Princeton Research Center (PRC)
Mettlers Road

STUDY DIRECTOR:

SUB CONTRACTOR: East Millstone, NJ 08875-2360

Gary M. Hoffman, B.A., DABT

ImmunoTox, Inc. Virginia BioTechnology Research Park
800 East Leigh Street, Suite 209 Richmond, VA 23219-1534 Telephone: (804) 828-6880
Fax: (804) 828-6881 

### Page 1021Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 55 Final Protocol

SUB CONTRACTOR FACILITY: ImmunoTox, Inc. Virginia BioTechnology Research Park
800 East Leigh Street, Suite 209 Richmond, VA 23219-1534

IMMUNOTOX. INC. PERSONNEL RESPONSIBILITIES: Principal Investigator:
Assistant Principal Investigator: Quality Assurance: Kimber L. White, Jr., Ph.D.
Vanessa L. Peachee, M.S.
Kuei-Lan Chuang, M.P.H.

STUDY GUIDELINES: The study will be conducted in accordance US EPA OPPTS Health Effects Test Guidelines 870.7800, Immunotoxicity, August 1998, in compliance with the 79.60, CFR Vol. 59, No. 122, 27 June 1994 and in
accordance with agreements between Huntingdon Life Sciences and ImmunoTox, Inc.

BACKGROUND:

The humoral immune component of the acquired immune response is associated with the production of antibody to specific antigens. The antigen used in this assay is sheep eythrocytes (sRBC). A plaque, occurring from the lysis of sRBC, is elicited as a result of the interaction of complement and antibodies (produced in response to the i.v. sensitization) directed against sRBC. Each plaque is generated from a single IgM antibody-producing B cell, permitting
the number of antibody-forming cells (AFC) present in the whole spleen to be calculated. The sRBC is aT-dependent antigen requiring T cells, B cells and macrophages to function properly in order to obtain an antibody-forming cell (AFC) response. If a test agent affects any of these cell-types to a significant degree, an altered response will be
observed. As a result, the T-dependent IgM response to sRBC is one of the most sensitive immunotoxicological assays currently in use. A significant modulation in the IgM AFC response, when compared to the appropriate controls,
indicates that the test agent is capable of modifYing the humoral immune response in the whole animal and, thus, has
the potential for immunotoxicity. This assay is one of the Tier I assays used by the NTP 1
•

OVERALL STUDY DESIGN:

Female Sprague Dawley-derived**(CD~** rats, 10/group, will be exposed to Gasoline DIPE Vapor Condensate via
inhalation for 4 weeks (5 days per week) by Huntingdon Life Sciences (PRC). Four days prior to sacrifice, animals will
be immunized via tail vein injection by ImmunoTox, Inc. personnel. Animals will be sacrificed by PRC personnel on the day after the final exposure, spleens will be aseptically removed, weighed, and shipped intact in individual shipping
containers at 2-8'c via overnight delivery to ImmunoTox., Inc. Upon receipt, spleens will be further processed for
determination of IgM response as detailed in procedures below. 

### Page 1022Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

Exposure Level (mg!m)
0 2,000 10,000 20,000
Positive Control* Page 56 Final Protocol

Number of Female Rats
10
10
10
10
10 *50mg/kg cyclophosphamide, IP, 4 days prior to sacrifice. These animals

will not be chamber exposed.

PROCEDURE (lmrnunoTox. Inc.SOP/ADM/030, SOP/CST/046 SOP/PFC/008).

The primary IgM response to sRBC will be enumerated using a modified hemolytic plaque assay of Jeme 8 which
indicates the number of antibody-forming cells (AFC) present.

1. Rats will be sensitized with 2x10 8 sRBC i.v. four days prior to sacrifice by ImmunoTox, Inc. personnel as described in the main protocol.

2. Positive control animals will be treated with 50 mg/kg cyclophosphamide (IP) with a 5 mg/mL solution @ 10 mL/kg once each day for four days prior to sacrifice. Huntingdon Life Sciences personnel will perform the treatment of positive control animals.

3. Huntingdon Life Sciences (PRC) personnel will, on the day after the fmal exposure, collect into tubes without anticoagulant (centrifuged at -3000 rpm at 2-8·C) and freeze**(~-20·C)** blood (-3.0 mL of
whole blood; -1.5 mL of serum) samples (orbital collection anesthetized via carbon dioxide/oxygen inhalation), and then sacrifice (carbon dioxide inhalation) the animals. Spleens will be removed, weighed and shipped in HEPES/EBSS/Gentamicin solution (prepared at PRC) at the time of sacrifice by PRC personnel to ImrnunoTox, Inc. for immunotoxicological evaluation. Thymus will be removed and weighed and preserved (10% neutral buffered formalin) for possible histopathology. Animals will

not be allowed to recover between blood collection and spleen collection.

4. Upon arrival at the subcontractor facility, spleens will be accessioned in accordance with the SOP for receipt of biological samples. Single cell suspensions will be prepared from each spleen using a Stomacher® 80 Lab Blender in accordance with the SOP for rat spleens. Cell suspensions will then
be centrifuged and resuspended in Earle's Balanced Salt Solution with HEPES. Viability of splenocytes will be determined using propidium iodide (PI) and the Coulter EPICS XL-MCL Flow Cytometer.

5. The cells will be resuspended in a 6 ml volume, and 1:50 and 1:150 dilutions are prepared. 

### Page 1023Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 57 Final Protocol

6. An 0.1 ml aliquot of spleen cells from each suspension will be added to separate test tubes each containing 25 111 guinea pig complement, 25 111 sRBC, and 0.5 ml of warm agar (0.5%). After thoroughly mixing, each test tube mixture will be plated onto a separate petri dish, covered with a microscope cover slip, and incubated at approximately 36-38°C for approximately 3 hours.
7. One replicate per animal will be evaluated.
8. Spleen cell counts will be performed on samples using a Model Z1 Coulter Counter. The plaques
which develop will be counted using a Bellco plaque viewer. The data are expressed as number of cells/spleen, specific activity (AFC/10 6 spleen cells) and total spleen activity (AFC/spleen).
9. Blood samples will be shipped on dry ice to ImmunoTox, Inc. and evaluated if the spleen sample results are unsatisfactory

.

. STATISTICAL ANALYSES OF IMMUNOTOXICOLOGICAL DATA(SOP/CSA/006): The data obtained in the

immunotoxicological studies will be analyzed in accordance with standard operating procedures. Data will be first
tested for homogeneity of variances using the Bartlett's Chi Square Test 2 • Homogeneous data will be evaluated by a parametric one-way analysis of variance 3 • When significant differences occur, the treatment groups will be compared to the vehicle control group using the Dunnett's t Test 4 • Non-homogeneous data will be evaluated using a non-parametric analysis ofvariance 3

. When significant differences occur, the treatment groups will be compared to vehicle control using the Gehan- Wilcoxon Test 5 when appropriate. The Jonckheere's Test 6 will be used to test for dose-related trends across the vehicle and treatment groups. The positive control data will be evaluated using the
Student's t Tese and compared to the vehicle control group. The criteria for accepting the results of the positive
control in the assay will be a statistically significant (P:S0.05) decrease in the response as compared to the vehicle
control group.

IMMUNOTOXICOLOGICAL REPORT: The report on the immunotoxicological results will contain all procedures and/or references to published procedures used during the study. Methods used for statistical analysis
will also be included.

IMMUNOTOXICOLOGICAL DATA AND RECORD RETENTION: All data and records pertaining to this immunotoxicological study will be submitted to Huntingdon Life Sciences (PRC) upon acceptance of the fmal report. Upon completion of this study, the report and raw data for this study will be maintained in the archives of Huntingdon Life Sciences.

PERSONNEL: Curriculum vitae for the key personnel involved in this study are maintained in the individual's training record maintained at ImmunoTox, Inc.'s office.

SAFETY: The appropriate safety precautions for the assay are described in SOP/SAF/004. 

### Page 1024Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

REFERENCES: Page 58 Final Protocol

1. Luster, M.I., Munson, A.E., Thomas, P., Holsapple, M.P., Fenters, J., White, K.L., Jr., Lauer, L.D, and Dean, J.D. (1988). Development of a testing battery to assess chemical-induced immunotoxicity. Fund. Appl. Toxicol. 10:2-19.

2. Bartlett, M.S. (1937). Sub-sampling for attributes. J. Roy. Stat. Soc. Suppl. 4:131-135.

3. Kruskal, W.H., and Wallis, W.A. (1952). Use of ranks in one-criterion variance analysis. J. Amer. Stat. Assoc. 47:583-621.

4. Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer.
Stat. Assoc. 50:1096-1121.

5. Gross, A.J., and Clark, V.A. (1975). Gehan-Wilcoxon Test. In Survival Distribution: Reliability Applications in
Biomedical Sciences. A.J. Gross and V.A. Clark, eds. John Wiley and Sons, New York, p.120-123.

6. Hollander, M. and Wolfe, D.A. (1975). Jonckheere's Test:Non-Parametric Statistics Methods, eds. M. Hollander
and D.A. Wolfe, John Wiley and Sons, New York, p.124-129.

7. Sokal, R.R. and Rohlf, F.J. (1981 ). Biometry. Freeman, San Francisco, p.226-231.

8. Jeme NK, Henry C, Nordin AA, Fun H, Koros MC, and Lefkovits I (1974). Plaque-forming cells: Methodology and theory. Tmspl. Rev. 18:130-191. 

### Page 1025Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S



### APPENDIXE GF AP Evaluations Page 59 Final Protocol 

### Page 1026

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 60 Final Protocol

GFAP SATELLITE PROCEDURE



### 1.0 PURPOSE

The purpose of this study is to evaluate the potential of the test substance to induce GF AP following inhalation exposure of rats.

2.0 FACILITIES and STAFF

2.1 Subcontractor's Sponsor: Huntingdon Life Sciences

2.2 Study Director: Princeton Research Center (PRC) Mettlers Road East Millstone, New Jersey 08875-2360

Gary M. Hoffman, B.A., DABT Huntingdon Life Sciences Phone: (732) 873-2550 Ext. 2920

. Fax: (732) 873-3992

E-mail: hoffmang@princeton.huntingdon.com

2.3 Principal Investigator: James P. O'Callaghan, Ph.D. US Centers for Disease Control (CDC) and Prevention Health Effects Laboratory Division

- NIOSH 1095 Willowdale Road Morgantown, WV 26505-2888 Phone No.: 304-285-6079 or 6233 Fax No.: 304-285-6220 Email: jdo5@cdc.gov

2.4 Testing Facility Project: 00-6130

3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES

3.1 Test Substance: Gasoline DIPE Vapor Condensate

3.2 Control: Negative: Air control 

### Page 1027

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

3.3 Test Substance Retention Sample Page 61 Final Protocol

The retention of a reserve sample of the Test substance will be the responsibility of Huntingdon Life Sciences.

4.0 SUBCONTRACTOR AND KEY PERSONNEL

4.1 Name:

4.2 Address: US Centers for Disease Control and Prevention Health Effects Laboratory Division

- NIOSH

1095 Willowdale Road Morgantown, WV 26505-2888

4.3 Principal fuvestigator: James P. O'Callaghan, Ph.D.

5.0 TEST SCHEDULE

5.1 Proposed Experimental fuitiation Date: 12 February 2002

5.2 Proposed Experimental Completion Date: 15 May 2002

5.3 Proposed Draft Report Date: 25 October 2002

6.0 TEST SYSTEM

Closed-colony, random-bred rodents are acceptable models for GF AP studies. Sprague- Dawley
rats were selected because of the availability of historical control data. fuformation on the test system is provided by Huntingdon Life Sciences and is contained in their files.

7.0 EXPERIMENTAL DESIGN AND METHODOLOGY

Information on the experimental design and methodology has been provided by Huntingdon Life Sciences and is contained in their files. All in vivo 

### portions of the study, including inhalation
exposures for 13 weeks (5 days per week), will be performed by Huntingdon Life Sciences. Animals will be sacrificed and brain samples will be collected by CDC personnel on site at Huntingdon Life Sciences. The brain samples will be shipped to CDC and processed and evaluated

as indicated in the following procedures. The potential of the test substance to increase GF AP in a dose-responsive manner will be measured relative to a control group. 

### Page 1028Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

The study design will be as follows:

Group

Negative Air Control

Test Substance (mglm 3 ) 2,000 10,000 20,000

7.1 Exposure Levels Selection Page 62 Final Protocol

Number per Sex

5

5 5 5

The exposure levels for the GF AP assay will be as shown in the above table.

7.2 Controls: The negative control group of animals will be exposed to air only.

7.3 Brain Collection and Processing and Evaluations

See the following procedures.

8.0 Report

A report of the results of this portion of the study conducted at CDC will be prepared and will accurately describe all methods used for generation, results and analysis of the data.

9.0 Records and Archives

All raw data, the protocol and all reports generated at CDC will be maintained. After submission of the final report, all of the above will be shipped to Huntingdon Life Sciences to be archived.

10.0 Specimens

All specimens will be held in storage as long as the quality of the preparation affords evaluation

or in accordance with the relevant Good Laboratory Practice Regulations. 

### Page 1029Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S

11.0 Regulatory Requirements/Good Laboratory Practice

This study will be conducted in accordance with the spirit of: US EPA GFAP Assay79.67, CFR Vol. 59, No. 122,27 June 1994. US EPA GLP 79.60, CFR Vol. 59, No. 122,27 June 1994.



### GFAP ELISA ASSAY Standard Operating Procedure

1 SCOPE OF APPLICATION Page 63 Final Protocol

The following is an SOP to be used in the quantification of the levels of glial fibrillary acidic protein (GF AP) in rat or mouse brain tissue. This procedure will be conducted by: Principal Investigator: Dr. James P. O'Callaghan US Centers for Disease Control and Prevention Health Effects Laboratory Division

- NIOSH



### 2 PREREQUISITES



## 2.1 EQUIPMENT 

## and SUPPLIES



## 2.1.1 EQIDPMENT

Ultrasonic Cell Disruptor Pipettes Hot/Stir Plate Plate Reader



## 2.1.2 CHEMICAL REAGENTS aGlial Fibrillary Acidic Protein (DAKO Z0334)

- Lot# 096 (302) Carnation Non-Fat Dry Milk aGlial Fibrillary Acidic Protein (Chemicon MAB 3402-Lot 14119825-07) Phosphate Buffered Saline (Pierce #28374) Alkaline Phosphatase conjugated amouse IgG(Jackson ImmunoResearch #315-055-003) Alkaline Phosphatase Substrate Kit (Bio-Rad #172-1 063) Triton X-100 (Bio-Rad #161-0407) Sodium Hydroxide (Fisher #S320) 

### Page 1030

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S



## 2.1.3 SUPPLIES

Microtiter Plates (hnmulon 2, Dynatech) Pipet tips


## 2.2 TRAINING REQUIREMENTS Page 64 Final Protocol

Able to use a plate reader and to accurately dilute tissue samples.



### 3 CAUTIONARY NOTES OR SPECIAL CONSIDERATIONS

1. Powdered milk should be purchased from a grocery store and used within six months. Old powdered milk gives a yellow color in all samples and masks any GF AP results.

2. When preparing the alkaline phosphatase substrate (p-nitrophenylphosphate), observe the color. The color should be a faint yellow, if it is darker a new kit should be used.



### 4 PROCEDURE

4.1 STANDARD CURVE PREPARATION



### 1. Remove a tube of the GFAP standard (see O'Callaghan et al., 1990 for method of preparation) from the freezer, thaw at room temperature and vortex prior to aliquoting.

2. Preparation of the standard curve requires dilution of the GF AP standard. The standard has

a known total protein concentration (11.5 mg/ml) and a known concentration of GF AP (2.4flg GFAP/ mg total protein). The total protein value is used to prepare a standard curve in PBS with0.5% Triton X-100. The protein values for the standard curve should be between 0.25 to 10 flg/100fll (i.e. 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, 10flg/100fll). 

### Page 1031Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211- DIPE-S

7.5!-lg (.018!-lg) 13.0!11 5.0!lg(.Ol2!lg)

2.5!lg(.006!lg)

l.O!lg(.0024!lg)

0.5!lg(.0012!lg)

0.25!lg(.0006!lg)



## 4.2 SAMPLEPREPARATION l.Omlfrom tube #1 (lOll g)

500!11 from tube #3(5!-lg)

500!11 from tube #3(5!-lg)

lml from tube #5(1!-lg)

500!11 from tube #6(0.5!-lg) Page 65 Final Protocol

1987!11

lml

500!11

2ml

lml

500!11

1. Control and experimental animals are sacrificed, brain regions are dissected, weighed, and placed in the appropriately labeled tube.

2. At this point, the samples are sonified in 10 volumes (i.e. a volume that is 1 OX the weight of the tissue e. g. tissue weight = .045 grams lOX volume= 450!11 1% SDS) of hot 1% SDS (85-95°C) is added and then sonified. In both cases, the samples are stored within 24 hours at -70°C.

3. Before the frozen tissues can be used in the GF AP ELISA, they are removed from the freezer, placed on dry ice, then sonified in hot 1% SDS (85- 950C) lOX volume (step2). Continue with step 4.

4. Experimental samples already sonified in 1% SDS are removed from the freezer, allowed to thaw at room temperature, and diluted in PBS with 0.5% 

### Page 1032Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 66 Final Protocol

Triton X-1 00 to a concentration of 1 OJlg total protein/1 00111 of PBS with 0.5% Triton X-100. Samples high in GFAP (e. g. cerebellum) may need to be diluted to a concentration of 5Jlg total protein/1 00111 of PBS with 0.5% Triton X-100. Samples low in GFAP (e.g. striatum) may need to be diluted to 20J1g total protein/lOOJll ofPBS with 0.5% Triton X-100. The dilution of the samples is critical because the optical density readings for each samples needs
to fall on the linear portion of the standard curve.

5. Samples are diluted according to the total protein value of the sample (e.g. Sample 

### = 11.5mg/ml total protein; the dilution for this sample will be 8.70 111 991.3 111 ofPBS with 0.5% Triton X-100.)

6. If samples have to be diluted to a lower concentration, a 

### 1: 1 dilution of the 10J1g/100J1l dilution results in the 5J1g/100J1l (200J1l of the 10J1g/100J1l dilution plus 200111 of the PBS with 0.5% Triton X-100).


## 4.3 REAGENT PREPARATION

1. Volumes Required- The volumes of reagents are 1 OOJ11/well (1 Oml per plate and the volume of wash is 200J1llwell (20ml per plate).

2. Phosphate Buffered Saline (PBS)- One packet of PBS is mixed thoroughly with 500ml of deionized water.

3. PBS+O.S% Triton X-100- 2.5ml of Triton X-100 is added to 500ml ofPBS.

4. Blotto- PBS+ 5% powdered milk. Step 4 uses BLOTTO without Triton X100, in steps 7 and 9, BLOTTO contains 0.5% TritonX-100. Before the assay is started, the total volume of BLOTTO needed for the entire assay should be determined (total volume should be made fresh on the day of the assay). The larger volume allows a good mix for the addition of the Triton X-1 00 detergent. PBS may be warmed slightly; this will facilitate getting the powdered milk into solution.

5. Polyclonal anti-GFAP (Dako)- 25J11/10ml ofPBS

6. Monoclonal anti-GFAP (Chemicon)- 1:500 20J11!10ml Blotto+0.5% Triton X-100

7. Alkaline Phosphatase- 1:3000 3.3J1ll10ml Blotto+0.5% Triton X-100 

### Page 1033Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 67 Final Protocol

8. P-nitrophenylphosphate substrate- 2 ml of diethanolamine buffer, 2 pnitrophenylphosphate tablets in 8ml of deionized water.



## 4.4 ASSAY PROCEDURE

1. Coat Immulon-2 flat bottom plates with aGF AP, 1.0jlg total immunoglobulin protein 

### I 100111 PBS 

### I well. (25!11 of aGFAP, Dako Z0334, in 10ml of PBS is the quantity needed per plate).

2. Incubate the coated plate at 37°C for 1 hour. This step may be done at the begining of the assay or it may be done the night before and refrigerated overnight at 4°C.

3. Empty plate, tap on absorbent paper to remove excess (this procedure is important to eliminate the possibility of any reagent carry-over between steps). Wash plates 4X with PBS (200jll per well), tapping and blotting between each wash.

4. Block 1 hour with BLOTTO (5% wlv non-fat powdered milk in PBS) 100111
per well.

5. Empty plate, tap on absorbent paper to remove excess, load diluted standard

curve and samples in a volume of 100111 per well. Incubate for 1 hour at room temperature. The template below is an example of a typical plate for this labs GF AP Assay.

Std 1 2 10 10 18 18 26 34 34 Std2 3 3 11 11 19 19 27 27 35 35 Std 3 4 4 12 12 20 20 28 28 36 36 Std4 5 5 13 13 21 21 29 29 37 37 Std 5 6 6 14 14 22 22 30 30 38 38 Std6 7 7 15 15 23 23 31 31 39 39 Std 7 8 8 16 16 24 24 32 32 40 40
6. Wash 4X with PBS+ 0.5% Triton X-100, 200jll/well.

7. Incubate for 1 hour in monoclonal aGFAP (Chemicon) at a dilution of 1:500 plus alkaline phosphatase conjugated anti-mouse IgG 

### Page 1034

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 68 Final Protocol

(Jackson hnmunoResearch) at a dilution of 1:3000 made up in BLOTTO plus 0.5% Triton X-100, 100).11/well.made up in BLOTTO plus 0.5% Triton X-100, 100 )11/well.

8. Wash 4X with PBS+ 0.5% Triton X-100.

9. Wash 4X with PBS+ 0.5% Triton X-100, 200)11/well.

10. Add P-nitrophenylphosphate substrate(Bio-Rad) in a volume of 100)11/well and incubate for 20 minutes.

11. Stop reaction with 0.4N NaOH 1 

### 00)11/well.

12. Check wells for any bubbles, read plate at 405nm.


## 4.5 CALCULATIONS



### 1. Plates are read on a Molecular Devices Spectra Max Plus microplate reader. The plate reader is coupled to a PC running the Soft:Max Pro(Molecular Devices) program.

2. A template is set up for each plate in the assay. The template includes the position of the standard curve and value for each point, and the name, number, and dilution factor of the unknown samples. The dilution factor for the samples is determined by the amount of total protein loaded into the wells (e.g. 10).tg of total protein=100x dilution factor, 5).tg of total protein=200x dilution factor, etc. The dilution factor is the multiplier needed to bring the total protein value up to 1mg of total protein in the final calculations.) 

### Page 1035Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S



## GFAP STANDARD CURVE

Curve Fit: 4-Parameter


### y = (A-D)/(1 + (x/C)"B) + D

A= -0.00391 B = 1.29 

### c = 0.00618 D = 0.658
0.6 -1-

-rOD



### _,_

0 0.0001 Log scale of ug Page 69 Final Protocol

Corr. Coeff: -1 .00

1**~-~~w~~~~~-y:::,t;?::;>Y~t.~~~:i&;~;%i~~ti~;~~sw:.:gn%;Jf~'::ri~g~t~f{~;~:iiJ)s!~:J?e_\f,** 

## ·· ·•· · ··cv·

. :**~:fflQ:J** 

### .. • · ·. 6.00e-4ug B1 0.033 0.03 0.004 14.14 B2 0.027 0.0012ug C1 0.065 0.065 ***** ***** C2 0.065 0.0024ug D1 0.146 0.145 7.07E-04 0.486 D2 0.145 0.006ug El 0.326 0.327 0.001 0.432 E2 0.328 0.012ug Fl 0.463 0.454 0.013 2.963 F2 0.444 0.018ug G1 0.53 0.526 0.006 1.075 G2 0.522 STD07 0.024ug Hl 0.546 0.561 0.022 3.904 H2 0.577 

### Page 1036

### Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 70 Final Protocol

3. The above standard curve is a typical curve generated from the rat hippocampus standard. The OD values for unknown samples need to fall on the straight portion of the curve, between STD 3 and STD 5.

4. The SoftMax Pro program automatically subtracts the blank from each OD reading (plate blank information is given in the table above), means the two readings for each sample, and multiplies the value obtained from the curve for this OD by the dilution factor for the unknown sample.

5. The report generated by the So:ft:Max Pro program may be imported into an Excel spreadsheet or data may be entered manually. The chart below is an example of the report generated in Softmax Pro for 10 unknown hippocampus samples that has been imported into

an Excel spreadsheet. These samples were diluted to 2.5J.!g of total protein/lOOJ.!l, therefore the dilution factor for all ten samples is 400(this factor may be included as another column

on the spreadsheet).

'·; 

### UN:KN'OWNS' 

## , • :/**M:e~~'~'; ~,sicJ:p~Y:,;~\**  /.:**·~** Wen· 

### OD Value 

### >)J.?Y. 

# · , .**mi'tii~'** 

# ·. 2.633 0.114 4.323 A3 0.334 2.552 A4 0.347 2.713


## ··.· iflp();i 2.482 0.099 3.984 B3 0.334 2.552

. . . o·:·**~:** B4 0.322 2.412 HIP03 3.099 ***** ***** C3 0.375 3.099 C4 0.375 3.099 

### ltJPQ4 2.792 0.093 3.34 D3 0.358 2.858 D4 0.348 2.726 

### Iiii>6s 2.206 0.074 3.338 E3 0.298 2.154 E4 0.308 2.259 

### HIP0.6 2.681 0.027 0.999 F3 0.343 2.662 F4 0.346 2.7 IDPQ7.··. 3.077 0.094 3.048 G3 0.369 3.011 G4 0.378 3.144 

### ID:Poik 2.631 0.044 1.663 H3 0.338 2.601 H4 0.343 2.662

:;•" ':::::· H:IP09:.**.~** -- 2.934 0.108 3.697 A5 0.369 3.011**~~__:-~** ;:**~--_\\:~;_-~:;~:·:::~-:\** A6 0.358 2.858 2.626 0.087 3.325 B5 0.345 2.688 B6 0.335 2.564



### 6. If sample dilutions were prepared by pipetting the exact amount of total protein, the value for GF AP has been calculated and no further calculations need to be done. 

### Page 1037Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 71 Final Protocol

7. If sample dilutions were prepared by pipetting 10111 volumes for all samples, the exact
amount of total protein needs to be included in the final calculations. This may be done on the Excel spreadsheet using the formula below: GF AP value+ 1110 Total protein value=11g GF AP/mg Total Protein

Unadjusted GFAP Value for Hippo #3 = 3.099

Protein Value for Hippo #3 = 9.883

3.099 + 9.883 x 10 = 3.136 jlg GFAP/mg Total Protein



### 5 REFERENCES

Characterization of the origins of the astrocyte response to injury using the dopaminergic neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. O'Callaghan, J.P.
, Miller, D.B., and Reinhard, J.F., Jr. Brain Res. 521: 73-80, 1990.

Quantification of Glial Fibrillary Acidic Protein: Comparison of Slot-Immunobinding Assay with a Novel Sandwich ELISA, O'Callaghan, J.P., Neurotoxicology and Teratology, Vol. 13, pp. 275-281, Pergamon Press plc. 1991

Soft:Max User's Manaual, Molecular Devices Corporation, Sunnyvale, CA



### Brain Tissue Preparation Standard Operating Procedure



### 1 SCOPE OF APPLICATION

The following is an SOP to be used for preparing rat or mouse brain tissue to be assayed for total protein concentration and the concentration of glial fibrillary acidic protein (GF AP). 

### Page 1038Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S 2 PREREQUISITES 2.1 EQUIPMENT and SUPPLIES 2.1.1 EQUIPMENT Ultrasonic Cell Disruptor Pipettes Hot/Stir Plate Plate Reader 2.1.2 CHEMICAL REAGENTS Sodium Dodecyl Sulfate, Bio-Rad #161-0302 2.1.3 SUPPLIES Microfuge tubes Pipet tips 2.2 TRAINING REQUIREMENTS Ability to dissect discrete regions of the rat brain.

Page 72 Final Protocol 3 CAUTIONARY NOTES OR SPECIAL CONSIDERATIONS SDS needs to be heated to a temperature of (85-95°C). 4 PROCEDURE 4.1 REAGENT PREPARATION 1% SDS A stock solution of 10% SDS is prepared w/v, 100 gms of SDS in 1 litre of deionized water. The 1% SDS is a dilution of the stock- 1 Oml of 1 OX stock in 90ml of deionized water.

Page 1039Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 73 Final Protocol 4.2 TISSUE PREPARATION 1. Control and Test substance exposed animals are sacrificed, brains then are rapidly removed from the skull case with the aid of pointed scissors (to open the brain case) and curved blunt forceps (to remove the brain). 2. The brain is placed on a cold (approximately 4°C) dissecting platform (Thermoelectrics cold plate, Aldrich Chemical Co., St. Louis, MO) and individual areas are dissected along neuroanatomical landmarks using fine curved forceps and a #ll scalpel blade. The areas dissected include: cerebellum, cortex, hippocampus, hypothalamus, striatum, thalamus, rest of brain. fu addition, olfactory bulbs and pituitary are removed from their position in the skull case after brain removal. 3. The dissected brain parts then are weighed in a previously tared and appropriately labelled tube. At this point, each brain region is immersed in 10 volumes of hot 1% SDS (85-95°C)

(i.e. 1 OX volume the weight of the tissue; e.g. tissue weight = .045 grams, 1 OX volume=**450~1** 1 %SDS) and then homogenized by sanification with an ultrasonification microprobe (Heat Systems, Farmingdale, NY). The samples then are stored within 24 hours at -70°C prior to assay. Sample Storage/Handling and Shipping Standard Operating Procedure 1 SCOPE OF APPLICATION 2 2.1

The following is an SOP to be used for storing/handling and shipping of brain tissue homogenates that serve as standards and unknowns for determining total protein concentration and the concentration of glial fibrillary acidic protein (GF AP) in each homogenate. PREREQUISITES EQUIPMENT and SUPPLIES 2.1.1 EQUIPMENT -70 °C Freezer 2.1.2 CHEMICAL REAGENTS 2.1.3 SUPPLIES

2.2 TRAINING REQUIREMENTS

Page 1040Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Ability to handle samples frozen at -70°C

Page 74 Final Protocol 3 CAUTIONARY NOTES OR SPECIAL CONSIDERATIONS Wear protective gloves when removing samples from -70°C freezers. 4 PROCEDURE 4.1 STORAGE/HANDLING Homogenates will be stored frozen at -70°C prior to shipping to CDC-NIOSH and shipped frozen on dry ice. 4.2 SIDPPING Homogenates will be shipped on dry ice in protective Styrofoam containers to CDC-NIOSH. BCA PROTEIN ASSAY Standard Operating Procedure 1 SCOPE OF APPLICATION The following is an SOP to be used in the determination of total protein in brain tissue

by the Pierce BCA method. BSA (Bovine Serum Albumin) is used as the standard and samples are prepared by sonification in 1% (w/v) SDS. 2 PREREQUISITES 2.1 EQUIPMENT and SUPPLIES 2.1.1 EQUIPMENT Plate Reader Pipettes Incubator Vortex-Mixer 2.1.2 CHEMICAL REAGENTS BCA Protein Kit, Pierce #23223 Bovine Serum Albumin (BSA), Sigma A 7888

Page 1041Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Sodium Dodecyl Sulfate, Bio-Rad #161-0302 2.1.3 SUPPLIES Microtubes 1.2ml 96 well microtiter plates Pipette Tips 2.2 TRAINING REQUIREMENTS Able to use a plate reader and to accurately dilute tissue samples.

Page 75 Final Protocol 3 CAUTIONARY NOTES OR SPECIAL CONSIDERATIONS BCA reagent is stable for up to orie week; standard practice dictates the use of 50 ml quantities, enough to do two full microtiter plates. SDS serves as a wetting agent and pipetting SDS-containing samples can lead to pipetting errors. Therefore, use only a single pipette tip per sample and withdraw the sample only a single

time per tip. This procedure holds for SDS and all SDS-containing samples. 4 PROCEDURE 4.1 REAGENT PREPARATION 1.1% SDS A stock solution of 10% SDS is prepared w/v, 100 gms of SDS in 1 litre of deionized water. The 1% SDS is a dilution of the stock- lOml of lOX stock in 90 ml deionized water. 2. BCA Reagent This reagent is made according to the directions provided by Pierce, 50ml of solution A and lml

of solution B. Thoroughly mix while adding solution B. 4.2 STANDARD PREPARATION Protein Standards: Bovine Serum Albumin (BSA) in 1% (w/v) SDS (sodium dodecyl sulfate). 1. Prepare lmg/ml BSA in 1% SDS. Weigh out 0.100 gms of dry BSA on an analytical balance. Add the BSA to a 100 ml volumetric flask then gradually add 1% SDS to the flask. Mix gently

Page 1042Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DJPE-S Page 76 Final Protocol after each addition of SDS to dissolve the BSA without a lot of bubbling. Continue adding the SDS until the total 100 ml has been added. 2. Aliquot a volume into microfuge tubes (600 )ll is enough volume to do one standard curve). Store the tubes at -70°C . Thaw as needed, but do not re-freeze. 3. Blank: Remove 200).11 of 1% SDS, place it in a dilution tube. 4. l.Of.lg/lOf.ll: Remove 20).11 of the prepared BSA standard(1mg/ml), place it in a dilution tube and add 180).11 of 1% SDS. 5. 2.5f.1g/l0f.1l: Remove 50).11 of the prepared BSA standard, place it in a dilution tube and add 150).11 of 1% SDS. 6. S.Of.lg/1 Of.ll: Remove 1 OO)ll of the prepared BSA standard, place it in a dilution tube and add 1 OO)ll of 1% SDS. 7. 7.5f.lg/l0f.1l: Remove 150).11 of the prepared BSA standard, place it in a dilution tube and add 50!ll of 1% SDS. 8. lO.Of.lg/lOf.ll: Remove 200).11 of the prepared BSA standard and place it in a dilution tube. BSA Standard Curve l.O)lg 20).11 180).11 2.5)lg 50).11 150).11 5.0)lg 100111 100).11 7.5)lg 150).11 50).11 10.0)lg 200).11 0 9. Repeat steps 3 to 8. This generates a second standard curve, independent ofthe first.

Page 1043Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 77 Final Protocol 10. Vortex each tube of the diluted BSA standard, remove 10!-ll ofthe diluted standard and place it in the designated well on a microtiter plate. 4.3 SAMPLE PREPARATION A : ;, B**!""~** \...__. l"**~"** LJ .i:.**r_~** c 1-1

The unknown experimental samples are diluted two times, independently. 1. Remove samples from the freezer and allow to thaw at room temperature. 2. Vortex and then remove a 1 OJ.ll aliquot from the sample and place it into a dilution tube. Add 190J.1l of 1% SDS to each sample tube. 4. Repeat step 2. This becomes the second dilution for this sample. 4. Vortex the dilution tubes and remove a 1 OJ.ll aliquot and place it in the appropriate well on the microtiter plate. 5. Repeat steps 1 through 4 for each experimental sample. 6. The following chart is an example of a typical microtiter plate layout, standard curve and 42 unknown samples. Microtiter Plate Template "' 4 .) 6

-- 8 9 : {; j: I·J i "-' t.. '-./ ..t' j.;_ Blk Blk 3 3 11 11 19 19 27 27 35 35

Std 1 Std 1 4 4 12 12 20 20 28 28 36 36 Std2 Std2 5 5 13 13 21 21 29 29 37 37

Std 3 Std 3 6 6 14 14 22 22 30 30 38 38 Std4 Std4 7 7 15 15 23 23 31 31 39 39

Std 5 Std 5 8 8 16 16 24 24 32 32 40 40 1 1 9 9 17 17 25 25 33 33 41 41 2 2 1_0 __ 10 18 18 26 26 34 34 42 42**~**

-**-~** 4.4 PROTEIN ASSAY 1. Add 200J.1l of reagent to each of the wells containing either the standard curve or sample. 2. Place the plate in the plate reader and mix the plate using the automatic mix button on the plate reader or vortex gently prior to placing the plate into the reader.

Page 1044Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S 3. Incubate the plate at 37°C for 30 minutes.

Page 78 Final Protocol 4. At the completion of the incubation period, the plate is checked for bubbles and then read in the plate reader at a wavelength of 562nm. "Pop" any bubbles with a needle or a pipette tip prior to reading the plate. 4.5 CALCULATIONS 1. Software programs coupled to specific plate readers are used to calculate protein values based on comparing OD readings of the unknowns with those obtained from the standard curve. OD values and standard protein values are plotted in a linear fashion on both axes. The following is a typical standard curve for the BCA Protein Assay when bovine serum albumin is u,sed for the standard. The table gives the actual values for each point on the standard curve and their corresponding OD readings. BCA Total Protein Standard Curve Curve Fit: Linear y=A+B*x A = 0.0240 8 = 0.0395 0 0

0.4

Carr. Coeff: 0.999 0 10 1 Linear scale of 1-'9

Page 1045Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Page 79 Final Protocol STD01 STD02 STD03 STD04 STD05

1.000 )Jg 2.500 )Jg 5.000 )Jg 7.500 )Jg 10.00 )Jg A12

/''**"'~;'lEO,~~~~,.~;;;:;~' /~~C:"1.?~~:;~~~"** **-:~::{~;t(~--~=f~~~.~~-~"~** 5 J**f#~~:~~y~-~& ~~;0TT'~"'t?~**  lm}-1ti?"·'T&:"'·**,>~!ihw,;z;l,;:p** ..**:,\Vq·";\)l1ii~~xu··** ·.· .....**~""~'!t"~I>!~":J"** •. ··**ft~** · ·

-- • ···' H:1>;:"'1 .... :;**!:N~~~** .... -1:'**~"'?~<"):_" ',.~\~'<"-** **~ 'r-"'"'~**  .. t"**-;:;:~,~-"~~~b<">f"'~;;,-:.~<"'** ;.<::**).,~** A3 0.06 0.058 0.002 3.626 A4 0.057 AS 0.131 0.131 7.07E-04 0.542 A6 0.13 A7 0.22 0.218 0.003 1.297 A8 0.216 A9 0.311 0.321 0.014 4.406 A10 0.331 A11 0.419 0.418 0.001 0.338 0.417 2. The blank can be automatically subtracted from all other OD readings or it must be done manually if this feature is not available. 3. The dilution factors can be entered into the programs, otherwise they must be considered in the final calculations.

4. The final printout will include a plot of the standard curve and the protein concentration of each unknown expressed as mg/ml or Jlg/Jll. The table below gives the calculated protein values

for unknown hippocampus samples in mg/ml or Jlg/Jll. These values include the subtraction of the blank value and a dilution factor of 2.

Page 1046Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S 10.44   ** *** * 9.883 0.072 0.725 11.18 1.254 11.23 11.45 0.717 6.258

82 0.205 83 0.23 84 0.23 85 0.218 86 0.22 87 0.227 88 0.262 89 0.24 810 0.26 5. Example of calculations, if samples are prepared as listed above:

Page 80 Final Protocol 9.173 10.44 10.44 9.832 9.934 10.29 12.06 10.95 11.96 a. Hippo #3 is prepared as a 1 :20 dilution (1 OJ.!l of standard or

2 2 2 2 unknown sample are placed in a dilution tube and brought up to a final volume of 200J.!l) b. OD reading for this sample is .219 c. OD of .219 corresponds**to~** 4.942J.!g/10J.!l or .4942J.!g/J.!l total protein d. This sample has been diluted 1:20 so the value is actually .4942J.!g/J.!l x 20= 9.883J.!g/J.!l (since the protein sample was loaded in 10 J.!l volumes, the value can be determined by using a dilution factor of 2, 4.942J.!g/10J.!l x2=9.883J.!g/J.!l). ' 5 REFERENCES BCA Protein Assay Reagent (instruction manual), Pierce, Rockford, IL. So:ft:Max Pro User's Manual, Molecular Devices Corporation. Sunnyvale, CA.

Page 1047Huntingdon Life Sciences Study No. 0045130 Sponsor Study No. 211-DJPE-S Page 1 of 1 Final Protocol Protocol Amendment No. 1 Study Title: Gasoline DlPE Vapor Condensate: A 13- Week Whole-Body Inhalation Toxicity Study in Rats with Neurotoxicity Assessments and 4- Week In Vivo Genotoxicity and Immunotoxicity Assessments Changes Page 10, Experimental Design:

Revise: One male animal designated for the Gl'Oup ll Main Study will be redesignated for the Group ll GF AP evaluation. Reasons for Changes A Group IT male animal (#2070) was euthanized on 29Apri102 as a result of an accidental injury. Since this animal was 1 of 5 male animals designated for GFAP evaluation after 13 weeks of exposure, it was decided to replace this animal with 1 of 10 male animals designated for Main Study evaluations. Amendment approved by:**L~** Gary M. Hoffman,**~.A.,** DABT Study Director**6~0L** Date**~,.?'&>??....!**

Page 1048Huntingdon Life Sciences Study No. ()()..6130 §ponsor Study No. 211-Dn'E·S PrQJ:ocol Amendment No. 2

Page 1 of2 Final Protocol Srudy Tide: Gasoline DIPE Vapor Condensate: A 13-Week Whole-Body Inhalation Toxicity Study in Rats with Neurotoxicity Assessments and 4- Week In Vrvo Oenotoxicity and Irmnunotoxicity Assessments Changes Statistical Evaluations, page 31: Replace for the section on alJ Motor Activity Data and add for all Functional Observational Data including grip strength and foot splay: The data Cor analyses are the**lDO~r** activity data and the functional observational battery (FOB) data. All analyses will lnchide sex as an independent variable. In those instances where there are significant etJects of sex separate analyses by sex may be done to**~lain** the nature ofthe·effed:.. The analyses will be performed by Mark J. Nicolich, PhD, Statistician, ExxonMobil Biomedical Sciences, Inc, Annandale, NJ. The Sponsor will be responsible for the SubcOntractor's GLP**c:omplian~e.** Arter submisSion of the final report, an· of the records of the Subcontractor will be shipped to Huntingdon Life Sciences**to~** archived. Motor Activity Data: The analysis of the motor adi.vity will use a prafUe repeated**measur~** analysis, with post-hoc tests to test the polynomial response pattern between the control and each treated grotJp [O'Brien, et al, 1985]. The profile will be the 12 repeated 5 minute readings of beam breaks taken on each anilnal over the 60 minutes of testing. The residuals from. the model will be tested for normality by the Sfulpiro· Wdk test for normality at the p<O.Ollevel of significance [Shapiro- Wilk, 1965]. If the residuals al"e not normally distributed, the dependent variable (number of beam

breaks) wil1 be converted to a function of its percentile rank then transformed by the inverse normal distribution (the transformation is known as Blom's transformation

where the rank order is repla£ed by [rank-0. 37S]J[n+0.25]) and reanalyzed [Blom, 1958]. Functional Observational Data: Some of the sensorimotor and observational battery variables are measured on a continuous scale and some are nominal o-r eonnt scale. AU**~** will be classified as either continuous or nominal. The a.naJysis of the continuous variables will be oondlJde(l by a repeated measures analysis of covariance

(ANCOV A) with the initial (pretest) measures as the covariate [Snedeco.- and Cochnm, 1989] •. The reslduaJs from the model will be tested for normality by the Shapiro- Wilk test [Shapiro• Wilkt 1.9()5]. Those variables that do not exhibit normally distributed residuals at the O.Ollevel wiJI be transformed by Blom's normalized rank transformation and reanaly%Cd [Blom, 1958]. The nominal data af the functional

Page 1049Huntingdon Life Sciences Study No. ()()..6130 SponSQt Srudy No. 211-DIPE..S Protocol Amendment No.2

Page2of2 Fmal Protocol observational battery will be analyzed by a aunulative lo.;t repeated measures**anal~** with contrast terms to test for differences between control and dosed groups [Agresti, 1989]. For the measures that are count data (such as number of rears or number of urinations), the count responses may be grouped into several data ranges to allow sufficient data in the cells. For example, the count of urinations might be separated into three groups of 0, 1 to 4, S or more, and analyzed. References: Agresti, Alan, "A survey of models for repeated ordered categorlcaJ response data", Statistics jn Medicine, vol 8, 1989, pg 1209-1224. Blom, G., Statistical Estimates and Transformed Beta Variables, J. Wiley and Sons,

NY, 1958. O'BrieJi, R.G. and Kaiser, M..K., "MANOVA method for analyzing repeated measures designs: an extensive primer", PSychological Bulletin, vol97 -2, 1985, pg 316- 333. Shapiro, S.S. and Wdk, M.B., "An analysis of variance test for normality (complete samples}'', Bio-roetrika, 52, 1965, pg 591-611. (Shapiro- Wilk test). Snedecor, G.W. and Cochran, W.(7., Statistical Methods, 8th Edition, Iowa State University Press, Ames, Iowa, 1989. Reasons**fo~** Changes As per request of the BP A. an evaluation of all motor activity and FOB data with**consid~tion** to differences by $eX and time is :required. 17.c-t4xf0 ,. Gary M. Hoffman; B.A.. DABT Date s 144'1 Date

Page 1050Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Protocol Amendment No. 3

Study Title: Gasoline DIPE Vapor Condensate: A 13-Week Whole-Body Inhalation Toxicity Study in Rats with Neurotoxicity Assessments and 4- Week In Vivo Genotoxicity and Jmmunotoxicity Assessments Changes to supercede Protocol Amendment No. 2 Statistical Evaluations, page 31:

Page 1 of2 Final Protocol Replace for the section on all Motor Activity Data and add for all Functional Observational Data including grip strength and foot splay: The data for analyses are the motor activity data and the functional observational battery (FOB) data. All analyses will include sex as an independent variable. In those instances where there are significant effects of sex separate analyses by sex may be done to explain the nature of the effect. The analyses will be performed by Mark J. Nicolich, PhD, Statistician, ExxonMobil Biomedical Sciences, Inc, Annandale, NJ. The Sponsor will be responsible for the Subcontractor's GLP compliance. After submission of the fmal report, all of the records of the Subcontractor will be shipped to Huntingdon Life Sciences to be archived.

Motor Activity Data: The analysis of the motor activity will use a mixed model analysis of covariance with an unstructured error relationship among the 5 minute periods, and a first order autoregressive error structure on weeks. The pretest response will be used as

the covariate. The residuals from the model will be tested for normality at the p<O.Ol level of significance by the Kolmogorov-Smirnov test for normality. H the residuals are

not normally distributed the dependent variable (number of beam breaks) will be converted to a function of its percentile rank, then transformed by the inverse normal distribution and reanalyzed as above. The transformation is known as BJorn's transformation where the rank order is replaced by [rank-0. 375]/[n+0.25]), and reanalyzed [Blom, 1958]. Functional Observational Data: Some of the sensorimotor and observational battery

variables are measured on a continuous scale and some are nominal or count scale. All measures will be classified as either continuous or nominal. The analysis of the continuous variables will be conducted by a mixed model analysis of covariance with a first order autoregressive error structure on the time points. The pretest response was used as the covariate. The residuals from the model will be tested for normality at the p<0.01 level of significance. Those variables that do not exhibit normally distributed residuals at the 0.01level will be transformed by Blom's normalized rank transformation and reanalyzed [Blom, 1958). The nominal data of the functional observational battery will be analyzed by a cumulative logit repeated measures analysis with contrast terms to test for differences between control and dosed groups [Agresti, 1989]. For the measures that are count data (such as number of rears or number of urinations), the count

Page 1051Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Protocol Amendment No. 3

Page 2 of2 Final Protocol responses may be grouped into several data ranges to allow sufficient data in the cells. For example, the count of urinations might be separated into three groups of 0, 1 to 4, 5

or more, and analyzed. References: Agresti, Alan, 11 A survey of models for repeated ordered categorical response data 11 , Statistics in Medicine, vol8, 1989, pg 1209-1224. Blom, G., Statistical Estimates and Transformed Beta Variables, J, Wiley and Sons, NY, 1958. Galecki, AT, "General class of covariance structures for two or more repeated factors in longitudinal data analysis," Communications in Statistics- Theory and Methods, 23(11), 1994 pg 3105 -3119. Pinheiro, JC and Bates, DM, Mixed-Effects Models inS and S-PLUS, Springer-Verlag, NY,2002. Shapiro, S.S. and Wilk, M.B., "An analysis of variance test for normality (complete samples)", Biometrika, 52,1965, pg 591-611. (Shapiro-Wilk test). Stephens, M.A., 'EDF statistics for goodness of fit and some comparisons', Journal of the American Statistical Association, vol69, 1974, pg 730-737 (Kolmogorov-Smirnov test).

Reasons for Changes As per request of the EPA, an evaluation of all motor activity and FOB data with consideration to differences by sex and time is required. Also, the Kolmogorov-Smirnov test replaces Shapiro- Wilk test for the motor activity residual analysis due to computer program limitations. Amendment approved by:**¥~** Gary M. Hoffnlan, ts.A., lJA.tS 1 Study, Direator

lo&fh?t Date /it. 7'**2t~oy** I Date

Page 1052·\ ..

Huntingdon Life Sciences Study No. 00-6130 Sponsor Study No. 211-DIPE-S Protocol Amendment No.4

Page 1 ofl Final Protocol Study Title: Gasoline DIPE Vapor Condensate: A 13.:Week Whole-Body Inhalation Toxicity Study in Rats with Neurotoxicity Assessments and 4-Week In Vivo Genotoxicity and Immunotoxicity Assessments Changes Page 10, Experimental Design and Page 22, Clinical Labomtory Studies: Add: The hematology and clinical chemistry evaluations were performed on all animals designated for the Recovery sacrifice.

Reasons for Changes Possibly exposure-related effects were seen in these evaluations at the Terminal interval. Amendment approved by:**GmyMHo~** Study Director**/"2-~** a o4- Date 1,(**~,lt:1() ~**  Date

Page 1053Page 1054Page 1055

### Huntingdon Life Sciences 00

- 6130 Page 1056 211

- DIPE

- S Final Report

Testing Facility Personnel Appendix W

TITLE/DEPARTMENT NAME/DEGREE SENIOR VICE PRESIDENT,

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

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

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

DIRECTOR, TOXICOLOGY

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

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

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

STUDY MONITOR Georgia Burnett, A.A.S.

VETERINARIAN Theresa Kusznir, D.V.M.

MANAGERS /SUPERVISORS
Inhalation/Pharmacy Clinical Pathology Analytical Services Necropsy Ellen H. Whiting, AALAS LAT Lisa A. Spall, B.S., M.T. (ASCP) Kay Saladdin, B.S. Thomas M. Niebojewski, B.S.

SUBCONTRACTOR

O phthalmology Lionel F. Rubin, V.M.P., A.C.V.O.