### STUDY NO. 00-6131 SPONSOR NO. 211-TBA-S

GASOLINE TBA VAPOR CONDENSATE:

A 13-WEEK WHOLE-BODY INHALATION TOXICITY STUDY IN RATS

WITH NEUROTOXICITY ASSESSMENTS AND 4-WEEK IN VIVO

GENOTOXICITY AND IMMUNOTOXICITY ASSESSMENTS

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., DABT

Date: 5 March 2013

Page 841 of 1470

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TABLE OF CONTENTS

Volume III

COVER PAGE .................................................................................................................841 TABLE OF CONTENTS .................................................................................................842

APPENDICES N. Equipment List ...........................................................................................................843 O. Miran Calib ration .......................................................................................................846 P. Chamber Distribu tion Records ..................................................................................849 Q. Testing Room and Chambers Environmental Monitoring .........................................852 R. Animal Termina tion History ......................................................................................854 S. Analytical Report .......................................................................................................871 T. Neurobehavioral Positiv e Control Data .....................................................................898 U. Protocol and Protocol Amendments ..........................................................................991 V. Protocol and Protocol Amendments ..........................................................................997 W. Testing Facility Personnel .......................................................................................1084

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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 insert with nichrome wire (Crown Glass Co., Inc.) Flowmeter with built-in valve, size 0-65mm, tube #’s 6G02R 3, 6G03R3, 6G04R3 (Key Instruments) Variable Autotransformer, Type 3PN 1010 (Statco Energy Products Company) Minitrol Autotransformer, Type PL-312 (Glas-col ® 

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

### Microfibre TM Disposable Filter Units, No. L9933-05 (Grade DQ) Quick-disconnect fitting with toggle valve (Rego ® 

### )

Compound Reservoir 5 gallon cylinder (Manchester Tank)

Balance Mettler PM30000K (Mettler Instrument Corporation)

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

### ) Top Trak TM Mass Flow Meter, size 0 - 1 Lpm, Model 821-1, (Sierra Instruments), calibrated prestudy with a Gilibrator ®



### Bubble Generator, PN D-00286, SN 569- S and Gilibrator ®



### base unit, PN D-800268 (Gilian Instruments)

### Huntingdon Life Sciences 00-6131 Page 844 211-TBA-S Final Report

Equipment List Appendix N

Pressure/Vacuum Gauges Norgreen backpressure gauge, PN 9892K23 and PN R07-219RGKA Ashcroft backpressure gauge, PN 733-47 Matheson backpressure gauge, PN 63-3161 USG backpressure gauge, PN 12672-1 GAST ® 

### vacuum gauge, Model 169-01 Dwyer ®



### Magnehelic ® 

### gauge (Dwyer ®



### Instruments Inc.) Union Carbide vacuum gauge, Model SG6310

Regulators Norgreen, PN 9892K23 and PN R07-219RGKA

Valves Metering Valve, Model SS-4L Series (Nupro ® 

### Co.)

Tubing Plastic, size 1⁄4”, 1⁄2”, 3/16” (Norton) Teflon ® 

### /Tygon ® 

### , size 1/8”, 1⁄4”, 1⁄2” Stainless steel, size 1⁄2” Stainless steel “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 Benchtop Digital Multimeter No. 22-195

Particle Sizer/Analyzer TSI Aerodynamic Particle Sizer, Model 311001 and a DELL computer, Model 486P/25, equipped with an Epson LQ-570+ Dot matrix printer, Model P6308B

Absorbent Tube Sampling Charcoal Tubes, Lot # 13843/15350 (Supelco) Charcoal Tubes, ORBO-32, Lot # 2000 (Supelco)

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Equipment List Appendix N

Vacuum Pumps Thomas Industries Inc., Model 707CM50 Thomas Industries Inc., Model 107CA183 Becton-Dickinson, Model 1182

Timers Gralab Universal Timer, Model 171

Environmental Monitoring VWR Big-Digit Hygrometer/T hermometer, Model No. 61161-378 Oxygen/Gas Analyzer, Model 1214S (Gastech) Digital Sound Level Meter, Model 840029 (Sper Scientific) Photometer 1 Light Mete r (Quantum Instruments)

Miscellaneous Ball and Socket joint Gas-tight syringe, size 0 - 25 mL, Model 1025 (Hamilton Company) Gas Bags (Calibrated Instruments)

### Huntingdon Life Sciences 00-6131 Page 846 211-TBA-S Final Report

Miran Calibration Appendix O

Methodology for Gasoline TBA Vapor Condensate

Settings: The instrument settings for the unit are summarized below:

wavelength, microns 10.4 pathlength, dial setting 5.68 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 uni t was zeroed and the calibration series was performed as shown below. The resu ltant data were plotted to obtain a calibration curve. Each observer used a separate syringe for calibration.

Injection Calculated Absorbance Volume Concentration 1 

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

9 1053 0.0383 0.0435 0.0409 17 1989 0.0725 0.0852 0.0789 86 10064 0.341 0.331 0.336 170 19894 0.645 0.688 0.667 210 24575 0.789 0.788 0.789

1 

### Calculated Conc. (mg/m) = Injection vo lume (μL) x Density (mg/μL) x 1000 L/m 5.64 L where density = 0.66 mg/μL 5.64 L = volume of Miran cell

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Miran Calibration Appendix O

Calibration 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 Reading Range (μL) (mg/m 3 

### ) (volts) (volts)

17 1989 0.0789 0.0671 – 0.0907 86 10064 0.336 0.2856 – 0.386 170 19894 0.667 0.567 – 0.767

The absorbance was recorded after eac h injection. The absorbance was considered satisfactory if it was within 15% of the original calibration series. If any of the absorbance values fell out side the 15% range, the injection was rechecked as follows. The volume fo r 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 curv e. If the average of the pair was within the 15% range, the original was accept ed. If the value of the average was outside the 15% range, the Study Direct or decided if a new graph was to be prepared.

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Miran Calibration Appendix O

Calibration Curve for Gasoline TBA Vapor Condensate

y = 79230x 4 - 118555x 3 + 56322x 2 + 21544x

R 2 = 0.9987

0 5000 10000 15000 20000 25000 30000

0.0000 0.2000 0.4000 0.6000 0.8000 1.0000 Absorba nce (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 

### ) 5-Jun-02 H-1 2060 1.00 H-2 1990 0.97 H-7 1800 0.87 H-8 1510 0.73 H-1 2080 1.00 H-14 2080 1.00 H-12 1990 0.96 H-11 1960 0.94 H-13 2190 1.05 11-Jun-02 H-1 2010 1.00 H-7 1710 0.85 12-Jun-02 H-1 1990 1.00 H-7 1860 0.93 H-8 1530 0.77 14-Jun-02 H-1 2000 1.00 H-7 1830 0.92 H-8 1610 0.81 H-1 2030 1.00 H-7 1940 0.96 H-8 1680 0.83 H-1 2080 1.00 H-14 2310 1.11

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Chamber Distribution Records Appendix P

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

### ) Ratio to H-1

IIB (2000 mg/m 3 

### ) 5-Jun-02 H-1 2100 1.00 H-2 2030 0.97 H-7 1970 0.94 H-8 2000 0.95 H-1 2010 1.00 H-14 2340 1.16 H-13 2010 1.00 H-12 2100 1.04 H-11 2210 1.10

IIIA (10000 mg/m 3 

### ) 6-Jun-02 H-1 10700 1.00 H-2 10800 1.01 H-7 10800 1.01 H-8 10700 1.00 H-1 9350 1.00 H-14 9510 1.02 H-12 9920 1.06 H-11 9890 1.06 H-1 9420 1.00 H-13 9160 0.97

IIIB (10000 mg/m 3 

### ) 6-Jun-02 H-1 9730 1.00 H-2 9510 0.98 H-7 10500 1.08 H-8 10700 1.10 H-1 9610 1.00 H-14 9920 1.03 H-13 10400 1.08 H-12 11000 1.14 H-11 11500 1.20

### Huntingdon Life Sciences 00-6131 Page 851 211-TBA-S Final Report

Chamber Distribution Records Appendix P

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

### ) Ratio to H-1

IVA (20000 mg/m 3 

### ) 10-Jun-02 H-1 20400 1.00 H-2 20300 1.00 H-7 19200 0.94 H-8 19600 0.96 H-1 19300 1.00 H-14 18000 0.93 H-13 19800 1.03 H-12 20100 1.04 H-1 20100 1.00 H-11 19900 0.99

IVB (20000 mg/m 3 

### ) 10-Jun-02 H-1 20500 1.00 H-2 20700 1.01 H-7 20700 1.01 H-8 20100 0.98 H-1 19500 1.00 H-14 20200 1.04 H-13 19800 1.02 H-12 20700 1.06 12-Jun-02 H-1 19400 1.00 H-11 19900 1.03

### Huntingdon Life Sciences 00-6131 Page 852 211-TBA-S Final Report

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 36.5 60.3 - Back Room 0 37.2 58.0 - Group IA Chamber - - - 20 4.50 x 10 - 3 Group IB Chamber - - - 20 4.27 x10 - 3 Group IIA Chamber - - - 20 6.23 x10 - 3 Group IIB Chamber - - - 20 5.23 x 10 - 3 Group IIIA Chamber - - - 20 6.17 x10 - 3 Group IIIB Chamber - - - 20 7.62 x10 - 3 Group IVA Chamber - - - 20 7.30 x10 - 3 Group IVB Chamber - - - 20 6.49 x10 - 3

Week 0 Front Room 0 37.1 61.0 - Back Room 0 38.2 58.7 - 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

Week 6 Front Room 0 37.8 58.5 - Back Room 0 37.9 61.5 - 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

### Huntingdon Life Sciences 00-6131 Page 853 211-TBA-S Final Report

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 12 Front Room 0 38.7 61.5 - Back Room 0 38.2 58.9 - 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

### Huntingdon Life Sciences 00-6131 Page 854 211-TBA-S Final Report

Animal Termination History Preface Appendix R

The following animals were replaced pretest due to FOB observations for 1580 and 4580, and because 4079 was missing for 24 hours from the room:

Group Males Females Animal Replaced Replacement Animal Replaced Replacement

I - - 1580 1596 IV 4079 4086 4580 4596Page 855Page 856Page 857Page 858Page 859Page 860Page 861Page 862Page 863Page 864Page 865Page 866Page 867Page 868Page 869Page 870Page 871Page 872Page 873Page 874Page 875Page 876

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Neurobehavioral Positive Control Data Preface Appendix T

Table of Contents

Functional Observational Battery ...................................................................... 899 Motor Acti vity .................................................................................................... 964

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Neurobehavioral Positive Control Data: Functional Observational Battery Preface Appendix T

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

Table of Contents

Introducti on ...................................................................................................... 900 Materials and Methods .................................................................................... 900 Results and Di scussion ................................................................................... 919 Conclusi on ....................................................................................................... 924 Mean Body Weights, Grip Strength and Landing Foot Splay Values ............... 925 Incidence Summary of Functional Obse rvational Battery Evaluations ............. 936

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Neurobehavioral Positive Control Data: Functional Observational Battery Appendix T

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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Neurobehavioral Positive Control Data: Functional Observational Battery Appendix T

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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Neurobehavioral Positive Control Data: Functional Observational Battery Appendix T

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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Neurobehavioral Positive Control Data: Functional Observational Battery Appendix T

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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Neurobehavioral Positive Control Data: Functional Observational Battery Appendix T

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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Neurobehavioral Positive Control Data: Functional Observational Battery Appendix T

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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Neurobehavioral Positive Control Data: Functional Observational Battery Appendix T

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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Neurobehavioral Positive Control Data: Functional Observational Battery Appendix T

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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Neurobehavioral Positive Control Data: Functional Observational Battery Appendix T

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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Neurobehavioral Positive Control Data: Functional Observational Battery Appendix T

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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Neurobehavioral Positive Control Data: Functional Observational Battery Appendix T

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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Neurobehavioral Positive Control Data: Functional Observational Battery Appendix T

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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Neurobehavioral Positive Control Data: Functional Observational Battery Appendix T

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 signific ant 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 signific ant 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 signific ant 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 signific ant 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 signific ant 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 signific ant 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 signific ant 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 signific ant 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

### Huntingdon Life Sciences 00-6131 Page 933 211-TBA-S Final Report

Females Neurobehavioral Positive Control Data: Functional Observational Battery Mean Body Weights, Grip Strength

and Landing Foot Splay Values Appendix T

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

### Huntingdon Life Sciences 00-6131 Page 934 211-TBA-S Final Report

Repeats Neurobehavioral Positive Control Data: Functional Observational Battery Mean Body Weights, Grip Strength

and Landing Foot Splay Values Appendix T

%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

### Huntingdon Life Sciences 00-6131 Page 935 211-TBA-S Final Report

Repeats Neurobehavioral Positive Control Data: Functional Observational Battery Mean Body Weights, Grip Strength

and Landing Foot Splay Values Appendix T

%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

### Huntingdon Life Sciences 00-6131 Page 936 211-TBA-S Final Report

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

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.

### Huntingdon Life Sciences 00-6131 Page 937 211-TBA-S Final Report

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

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

### Huntingdon Life Sciences 00-6131 Page 938 211-TBA-S Final Report

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

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

### Huntingdon Life Sciences 00-6131 Page 939 211-TBA-S Final Report

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

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

### Huntingdon Life Sciences 00-6131 Page 940 211-TBA-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

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

### Huntingdon Life Sciences 00-6131 Page 941 211-TBA-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

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 00-6131 Page 942 211-TBA-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 00-6131 Page 943 211-TBA-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 00-6131 Page 944 211-TBA-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 00-6131 Page 945 211-TBA-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 00-6131 Page 946 211-TBA-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 00-6131 Page 947 211-TBA-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 00-6131 Page 948 211-TBA-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 00-6131 Page 949 211-TBA-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 00-6131 Page 950 211-TBA-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 00-6131 Page 951 211-TBA-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 00-6131 Page 952 211-TBA-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 00-6131 Page 953 211-TBA-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 00-6131 Page 954 211-TBA-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 00-6131 Page 955 211-TBA-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 00-6131 Page 956 211-TBA-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

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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

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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

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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

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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

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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

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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

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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 ...................................................................................................... 965 Materials and Methods .................................................................................... 965 Result s ............................................................................................................ 980 Discussi on ....................................................................................................... 981 Conclusi on ....................................................................................................... 981 Mean Motor Activi ty Val ues ............................................................................. 982

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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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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.

### Huntingdon Life Sciences 00-6131 Page 981 211-TBA-S Final Report

Neurobehavioral Positive Control Data: Motor Activity Appendix T

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.

### Huntingdon Life Sciences 00-6131 Page 982 211-TBA-S Final Report

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 Hypothesi

s (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

### Huntingdon Life Sciences 00-6131 Page 983 211-TBA-S Final Report

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

### Huntingdon Life Sciences 00-6131 Page 984 211-TBA-S Final Report

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 00-6131 Page 985 211-TBA-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 00-6131 Page 986 211-TBA-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 00-6131 Page 987 211-TBA-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 00-6131 Page 988 211-TBA-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 00-6131 Page 989 211-TBA-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

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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

- 613 1 Page 991 211

- TBA

- S Final Report

Neuropathology Historical Control Preface Appendix UPage 992Page 993Page 994Page 995Page 996

### PROTOCOL

GASOLINE TBA VAPOR CONDENSATE

A 13-WEEK WHOLE-BODY INHALATI ON TOXICITY STUDY IN RATS WITH NEUROTOXICITY A SSESSMENTS AND 4-WEEK IN VIVO GENOTOXICITY AND IMMUNO TOXICITY ASSESSMENTS

HLS Study No.: 00-6131 Sponsor Study No.: 211-TBA-S Protocol No.: Final Date: 29 April 2002 Page 997Page 998

### Huntingdon Life Sciences Study No. 00-6131 Page 3 Sponsor Study No. 211-TBA-S Fina l Protocol

TABLE OF CONTENTS

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

6. PROPOSED STUDY DATES .................................................................................. 9

7. EXPERIMENTAL DESIGN .................................................................................. 10

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. ANIMAL HUSBANDRY/NON-EXPOSURE ..................................................... 14 9.7. ANIMAL HUSBANDRY DURING EXPOSURE ............................................... 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 999

### Huntingdon Life Sciences Study No. 00-6131 Page 4 Sponsor Study No. 211-TBA-S Fina l 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 ACTIVITY ........................................................ 18

11. EXPERIMENTAL EVALUATION ...................................................................... 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. GENOTOXICITY EVALUATIONS ................................................................ 23 11.8. IMMUNOTOXICITY 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. GFAP EVALUATION ...................................................................................... 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)......................................................35 C (SCE EVALUATIONS).........................................................................46 D (IMMUNOTOXICITY EVALUATIONS)................................................52 E (GFAP EVALUATIONS).....................................................................59 Page 1000

### Huntingdon Life Sciences Study No. 00-6131 Page 5 Sponsor Study No . 211-TBA-S Fi nal Protocol



### 1. INTRODUCTION HLS Study No. 00-6131
Sponsor Study No. 211-TBA-S

Study Title Gasoline TBA Vapor C ondensate: A 13- Week Whole-Body Inhalation Toxicity Study in Rats with Neurotoxicity Assessments and 4-Week In Vivo Genotoxicity and Immunotoxicity Assessments

Testing Facility Huntingdon Life Sciences Princeton Research Center (PRC) PO Box 2360 Mettlers Road East Millstone, NJ 08875-2360

Purpose 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.



### 2. STUDY PERSONNEL Study Director: Gary M. Hoffman, B.A., D.A.B.T. Telephone: 732-873-2550 x2920 Fax: 732-873-3992 Email: hoffmang@princeton.huntingdon.com



### Alternate Contact: Carol S. 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 1001

### Huntingdon Life Sciences Study No. 00-6131 Page 6 Sponsor Study No . 211-TBA-S Fi nal Protocol



### 3. REGULATORY REFERENCES

3.1. TEST GUIDELINE This study is designed to meet or exceed the pertinent requirements of:

US EPA Vehicle Emissions Inhalation 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 Inhalation Toxicity, August 1998.

US EPA OPPTS Health Effects Test Guidelines 870.1350, Acute Inhalation Toxicity with Histopathology (respir atory 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 1002

### Huntingdon Life Sciences Study No. 00-6131 Page 7 Sponsor Study No . 211-TBA-S Fi nal Protocol

3.2. GOOD LABORATORY PRACTICES 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 Accred itation of Laboratory Animal Care International (AAALAC).

3.4. ANIMAL WELFARE ACT COMPLIANCE This study will comply with all appropria te 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 Standa rds; 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 initiati on on the IACUC Protocol Review form. Page 1003

### Huntingdon Life Sciences Study No. 00-6131 Page 8 Sponsor Study No . 211-TBA-S Fi nal Protocol

4. Animals experiencing more than mome ntary or slight pain or distress due to test substance or emergency situati ons 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 administer 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 situ ations 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 (see page 6) for studies of this type including assessments of routine toxicology parameters (10/sex/group) as well as detailed evaluations of neurotoxicity (10/sex/group), genotoxicity (5/sex/group) and immunotoxicity (10/group) parameters.

3.5. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE

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



### 4. QUALITY ASSURANCE MONITORING

The Huntingdon Life Sciences Quality A ssurance 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. Page 1004

### Huntingdon Life Sciences Study No. 00-6131 Page 9 Sponsor Study No . 211-TBA-S Fi nal Protocol



### 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 ve rbally, 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.



### 6. PROPOSED STUDY DATES*

Study Initiation Date: Date Study Director signs protocol

Receipt of test animals: 6 June 2002

Initiation of Exposures: 25 June 2002 (Experimental Start Date)

Termination of Exposures: 26 September 2002

Necropsy: 23-27 Sept. 2002 ( Terminal/Neuropathology 

### ) 23 October 2002 (Recovery)

Submission of draft final report: 11 February 2003

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

Study Completion Date: Date final report is signed by Study Director

*See Appendices B, C, D and E for schedules for the Genotoxicity, Immunotoxicity and GFAP Satellite Studies Page 1005Huntingdon Life Sciences Study No. 00-6131Page 10Sponsor Study No. 211-TBA-SFinal Protocol7.EXPERIMENTAL DESIGNGroup ExposureLevelaNumber of Animals (MAIN and RECOVERY STUDIES)(mg/m3)InitialbClinical Laboratory StudiesNecropsydMicroscopicPathology4 Weeks &TerminalRecoverycTerminal RecoverycTerminal RecoveryM F M F M F M F M F M F M FI (control) 0 (air only) 20 20 10 10 A.N. A.N. 10 10 10 10 10 10 A.R. A.R.II(low) 2,000 10 10 10 10 - - 10 10 - - A.R. A.R. - -III(mid) 10,000 10 10 10 10 - - 10 10 - - A.R. A.R. - -IV(high) 20,000 20 20 10 10 A.N. A.N. 10 10 10 10 10 10 A.R. A.R.aExposures will be 6 hours/day, generally 5 days/week for 13 weeks for at least 65 exposures for Main and Recovery Studies.bThis number of animals includes 10/sex/groupdesignated for the Terminal Sacrifice (Main Study animals) and 10/sex/group (I & IV only) designated for Recovery after the TerminalSacrifice (Recovery Study animals).cThe recovery period will be at least 4 weeks.dComplete postmortem evaluations will alsobe performed on animals which are found dead or euthanatized in a moribund conditionduring the course of the study.A.N. = As Needed (additional cost) based on results of Terminal Sacrifice evaluations.A.R. = As Required: 1) Targetorgans/tissues identified by Group IV evaluations atTerminal Sacrifice (additional cost); 2) Lungs will be examined microscopically for all animals in allgroups at the Terminal sacrifice interval; 3) All tissues from animals found dead or euthanatized in a moribund condition during the course of the study (additional cost); 4) Grosslesions (additional cost).M = Male; F = Female; The first day of exposure will be Day 0.Page 1006Huntingdon Life Sciences Study No. 00-6131Page 11Sponsor Study No. 211-TBA-SFinal ProtocolEXPERIMENTAL DESIGN (CONTINUED)Group ExposureLevelaNumber of Animals (SATELLITE STUDIES)(mg/m3)InitialbNeuropathology GFAP Immunotoxicity Genotoxicity(Micronucleus and SCE)M F M F M F M F M FI (control) 0 (air only) 15 25 5 5 5 5 - 10 55II(low) 2,000 15 25 5 5 5 5 - 10 5 5III(mid) 10,000 15 25 5 5 5 5 - 10 5 5IV(high) 20,000 15 25 5 5 5 5 - 10 5 5aExposures will be 6 hours/day, generally 5 days/week for 13 weeks for at least 65 exposures forNeuropathology and GFAP Studies. Exposures will be 6 hours/day, generally 5days/week for 4 weeks for at least 20 exposuresfor Genotoxicity and Immunotoxicity Studies.bThis number of animals includes 5/sex/groupdesignated for neuropathology after the Terminal Sacrifice, 5/sex/group designatedfor GFAP evaluation after the Terminal Sacrifice, 10females/group designated for Immunotoxicity(PFC) evaluations following a 4 week exposure period and 5/sex/group designated forGenotoxicity (Micronucleus and Sister ChromatidExchange) evaluations following a 4 week exposure period. For the Immunotoxicity and Genotoxicityevaluations, separate groupsof animals (10 femalesand 5/sex/evaluation,respectively) will be used as positive control animals for these evaluations. For Immunotoxicity evaluations, positive control rats will be given a dose of 50 mg/kg Cyclophosphamide(CP) intraperitoneally once per day for 4 days prior to sacrifice. For Genotoxicity evaluations, positive control rats will begiven a dose of 5 mg/kg (SCE) and 40 mg/kg (Micronucleus)CP intraperitoneally within 24 hours prior tosacrifice. These positivecontrol animals will not be chamber exposed prior to dosing and sacrifice. The CPsolutions prepared for dosingwill not be assayed for purity, uniformity and stability as per GLP’s.M = Male; F = Female; The first day of exposure will be Day 0.GFAP = Glial Fibrillary Acidic Protein; SCE = Sister Chromatid Exchange; PFC = Plaque Forming CellPage 1007Huntingdon Life Sciences Study No. 00-6131 Page 12 Sponsor Study No. 211-TBA-S Final Protocol



### 8. TEST SUBSTANCE

8.1. TEST SUBSTANCE Gasoline TBA Vapor Condensate Description, lot number, storage, expira tion date (if available) and handling procedures, as well as other pertinent in formation 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 in dicated 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 multip le studies are conducted with the same substance, a common archival sample ma y be taken and appropriately labeled.

8.4. UNUSED TEST SUBSTANCE The unused portion of the test substanc e as well as any empty test substance containers will be returned to the Spon sor’s designee (see page 2) following completion of the subsequent reproducti on study 00-4206. Empty test substance containers will be returned to the Sponso r’s designee on an as needed basis. The Sponsor will be responsible fo r tracking their disposition.

In the event the Sponsor wishes the Testin g Facility to arrange for disposal, a cost for this service will be provided.



### 9. TEST ANIMALS

Albino Rats (Out bred) VAF/Plus ®

9.1. SPECIES Sprague Dawley - derived (CD ® ) [Crl: CD ® IGS BR] Page 1008Huntingdon Life Sciences Study No. 00-6131 Page 13 Sponsor Study No. 211-TBA-S Final Protocol

9.2. SUPPLIER

Charles River Laboratories Kingston, NY

9.3. JUSTIFICATION FOR TEST SYSTEM SELECTION

The rat is a rodent animal model comm only utilized in toxicity studies as recommended in the referenced guidelines. In 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

Study Phase Age at Receipt (weeks) Age at Exposures Initiation (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. An imals 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 1009Huntingdon Life Sciences Study No. 00-6131 Page 14 Sponsor Study No. 211-TBA-S Final Protocol

9.6. ANIMAL HUSBANDRY/NON-EXPOSURE

9.6.1. HOUSING Animals younger than 5 weeks at recei pt will be housed 2/cage during the initial week of acclimat ion. Animals 5 weeks or older will be housed individually in suspended stainl ess 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 conducte d by Elizabethtown Water Company to assure that water meets standards specified under the EPA Federal Safe Drinking Water Act Regulations (40 CFR Part 141). Water analysis, provided by the supplier, will be maintained on file at the Testing Facility. In addition, internal biannual chemi cal and microbiological water samples collected from representative rooms in th is 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 1010Huntingdon Life Sciences Study No. 00-6131 Page 15 Sponsor Study No. 211-TBA-S Final Protocol

9.6.5. ENVIRONMENTAL CONDITIONS

Light/Dark Cycle

Twelve hour light/dark cycle provided via automatic timer.

Temperature

Temperature will be monitored in accord ance 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 Individual in suspended, stai nless steel wire mesh 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 th e basis of pretest physical examinations, body weight data and any other pretest eval uations 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 te st shall be within +/- 20% of the mean weight for each sex for each study. Th e Main/Recovery study and each of the Neuropathology and GFAP and Genotoxicity and Immunotoxicity studies will be randomized separately. Disposition of all animals not utilized in the study will be maintained in the study file. Page 1011Huntingdon Life Sciences Study No. 00-6131 Page 16 Sponsor Study No. 211-TBA-S Final Protocol

9.9. ANIMAL IDENTIFICATION Each animal will be assigned a temporar y 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 comp rise the unique animal number for each animal. If the tag is lost, it will be re placed 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 wi ll 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. DURATION 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/Neu ropathology/GFAP Studies. Animals for the Genotoxicity and Immunotoxicity evaluations will be exposed for 6 hours/day, generally 5 days per week for 4 weeks for at least 20 exposures. Test substance administrati on 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 tr ials will be performed to evaluate the optimal set of conditions and equipment to generate a stable atmosphere at the target exposure levels and maintain un iform conditions throughout the exposure chambers. The method will be described in the raw data of the study and in the report. Page 1012Huntingdon Life Sciences Study No. 00-6131 Page 17 Sponsor Study No. 211-TBA-S Final Protocol

The whole-body exposure chambers will each have a volume of approximately 1000 liters. Two chambers will be used fo r 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 final airflow will be set to provide at least one air change in 5.0 mi nutes (12 air changes/hour) and a T 99 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, a ll 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 cal culated. 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 fl ow rate times total exposure time) to give the nominal concentration.

During each exposure, measurem ents 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, on e sample per chamber per week will be analyzed by gas chromatography (GC) to ch aracterize 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 li quid 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 hour s), the Sponsor will be consulted prior to additional trials (additional cost).

10.7. PARTICLE SIZE DISTRIBUTION ANALYSIS

During each week of exposure, particle si ze determinations will be performed using a TSI Aerodynamic Particle Sizer to characterize the aerodynamic particle size distribution of any aerosol present. Page 1013Huntingdon Life Sciences Study No. 00-6131 Page 18 Sponsor Study No. 211-TBA-S 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 measur ed pretest and at the beginning, middle and end of the study.

Air samples will be taken in the va por 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 floo r) 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 Frequency/chamber
Measured Test Substance Concentration 4X/day

Measured Test Substance Characterization 1X/week

Particle Size 1X/week
Temperature 13X/day
Relative Humidity 13X/day

Airflow Rate 13X/day

Static Pressure 13X/day

Nominal Test Substance Concentration (excluding the air control chamber) 1X/day

Rotation Pattern of Exposure Cages 1X/week
Loading/Unloading Verification 1X/day Page 1014Huntingdon Life Sciences Study No. 00-6131 Page 19 Sponsor Study No. 211-TBA-S Final Protocol



### 11. EXPERIMENTAL EVALUATION

11.1. OBSERVATIONS

11.1.1. VIABILITY CHECKS (IN-CAGE)

Observations for mortality, general ap pearance 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 monitori ng 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 Neuropat hology, 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 conditi on, skin and fur, eyes, nose, oral cavity, abdomen and external genita lia as well as evaluations of respiration, palpation for tissue mass es, circulatory effects, autonomic effects, central nervous system eff ects, changes in motor activity, and reactivity to handling or sensory stimuli. Neur opathology, GFAP, Genotoxicity and Immunotoxicity anim als 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 twi ce pretest, weekly during the study and at termination (fasted).

11.3. FEED CONSUMPTION Feed consumption will be measured (weighe d) during the week prior to treatment initiation and weekly throughout the st udy. Feed will be available wit hout restriction 7 days/week except during inha lation exposures and when fasting prior to blood collection. Animals will be presented with weighed feeders at the scheduled intervals. After 7 days, the fe eders will be reweighed and the resulting weight subtracted from the initial feed er 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 1015Huntingdon Life Sciences Study No. 00-6131 Page 20 Sponsor Study No. 211-TBA-S Final Protocol

11.4. OPHTHALMOSCOPIC EXAMINATION All animals (except Genotoxicity and Immuno toxicity 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. Te mperature, humidity and illumination will be measured and recorded to ensure that variations in environmental conditions are minimal during all evaluations. The f unctional 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 in to 12, 5-minute intervals. The time of testing will be balanced across trea tment groups. Positive historical control data demonstrating the sens itivity 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 observationa l battery will include assessments in the home cage, open field, and reflex testing. The time of testing will be balanced across treatment groups. All observat ions will be performed by trained observers who will be unaware of th e 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 Page 1016Huntingdon Life Sciences Study No. 00-6131 Page 21 Sponsor Study No. 211-TBA-S Final Protocol

at recovery, additional cost)

Parameters Evaluated Evaluations will be perf ormed according to the Testing Facility’s Standard Operating Procedures which include defined scales for each function listed below. In addition, descript ive 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 l acrimation and salivation

• Presence or absence of pi loerection 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 hom e cage and the open field

3. Ranking of reactivity to general st imuli, such as removal from cage or handling, with a range of severity scores from no reaction to hyperactivity.

4. Ranking of the subjects genera l level of activity during observations of the unperturbed subj ect in the open field, with a range of severity scores from unresponsive to hyperactive.

5. Description and incidence of postu re 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 of landing 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 a ppearance, 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 1017Huntingdon Life Sciences Study No. 00-6131 Page 22 Sponsor Study No. 211-TBA-S Final Protocol

11.6. CLINICAL LABORATORY STUDIES Clinical pathology procedures and parame ters are based on those recommended in guidelines published by the Joint Scie ntific Committee for International Harmonization of Clinical Pathology Testing in “Harmonization of Animal Clinical Pathology Testing in T oxicity 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 orb ital sinus (retrobulbar). Rats will be fasted overnight prior to bl ood collection. The Main St udy animals will be bled at the Interim (~4 weeks) and Terminal interv als. The Recovery Study animals will be bled (optional) at the Recovery interv al. Blood will be collected and studies performed as follows:

11.6.1. HEMATOLOGY AND COAGULATION STUDIES

Blood (~0.25 mL) for hematology studi es 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 10/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 4 th week interval) for clinical chemistr y studies will be collected into tubes with no anticoagulant, allowed to cl ot, and centrifuged to obtain serum. Page 1018Huntingdon Life Sciences Study No. 00-6131 Page 23 Sponsor Study No. 211-TBA-S Final Protocol

Number of Animals 10/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)

alanine aminotransferase alkaline phosphatase aspartate aminotransferase creatine kinase gamma-glutamyl transferase lactate dehydrogenase sorbitol dehydrogenase urea nitrogen bilirubin (total) bilirubin conjugated (direct) bilirubin unconju gated (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 af ter completion of assays and will then be discarded. Peripheral blood smears w ill 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 Page 1019Huntingdon Life Sciences Study No. 00-6131 Page 24 Sponsor Study No. 211-TBA-S 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, stre ngth, purity and composition or other characteristics to define the positive c ontrol article will not be determined by the Testing Facility. The positive control ar ticle 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 determ ined by the Testing Facility. Analyses to determine the uniformity (as applicab le) or concentration of the positive control mixture will not be performed by th e Testing Facility. The stability of the positive control article mixture wi ll not be determined by the Testing Facility.

SCE Evaluations Sprague Dawley-derived (CD  ) rats, 5/sex/group, will be exposed to Gasoline TBA Vapor Condensate via i nhalation 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). BioRe liance personnel will sa crifice (isoflurane inhalation) the animals on the day after the final exposur e, 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: Ramade vi Gudi, Ph.D.). Upon receipt, samples will be processed and evaluated for determ ination of SCE res ponse as detailed in the referenced guidelines and procedures (see Appendix C). The Testing Facility will be responsible for the GLP co mpliance of this subcontractor.

Micronucleus Evaluations Sprague Dawley-derived (CD  ) rats, 5/sex/group, will be exposed to Gasoline TBA Vapor Condensate via i nhalation 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 (40 mg/kg cyclophosphamide, IP with a 4.0 mg/mL solution @ 10 mL/kg, within 24 hours prior to sacrifice). The positive cont rol animals will be sacrificed under CO 2 anesthesia. BioReliance personnel will remove the femu rs immediately after the blood sample collection for the SCE evaluati on and fixed, unstained slides will be prepared and HLS personnel will ship vi a overnight delivery to Eye Research Centre, Eye, Suffolk, UK (attn: Christine Mason, B.Sc.). Upon receipt, slides will be stained and evaluated for determinati on 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 TBA Vapor Condensate via inhalation for a 4 week (5 days per week) Page 1020Huntingdon Life Sciences Study No. 00-6131 Page 25 Sponsor Study No. 211-TBA-S Final Protocol

exposure period by Huntingdon Life Sciences (PRC). PRC pers onnel 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 da ys prior to sacrifice, animals will be sensitised with sheep red blood cells (2 x 10 8 ) via tail vein injection by ImmunoTox, Inc. personnel. PRC pers onnel will, on the day after the final exposure, collect and freeze (<-20 o C) blood (serum) samples (orbital collection anesthetized via carbon dioxide/oxygen i nhalation), and then sacrifice (carbon dioxide inhalation) the animals, the thymus will be removed, weighed, and preserved (10% neutral buffered form alin) for possible histopathology, the spleens will be aseptically remove d, weighed and shipped intact in HEPES/EBSS/Gentamicin solution (prepa red at PRC) in individual shipping containers on wet ice (2-8 o C) via overnight delivery to ImmunoTox., 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 (s ee Appendix D). The serum samples will be retained by the Testing Facility until th e 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 dete rmined by the Testing Facility. The positive control article will be characterized as per the Certificate of Analysis on file with the Testing Facility. The stabil ity 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 mixt ure 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 in cluding animals euthanatized in a moribund condition or found dead; all abnormal observations will be recorded. Complete macroscopic postmortem examinat ions will only be performed on all GFAP/Genotoxicity/Immunotoxicity 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 ti ssues of the cranial, thoracic, abdominal and pelvic cavities and neck; and the remainder of the carcass. Page 1021Huntingdon Life Sciences Study No. 00-6131 Page 26 Sponsor Study No. 211-TBA-S Final Protocol

12.2. TIME OF NECROPSY

12.2.1. MORIBUND ANIMALS Animals showing signs of severe debility, particularly if death appears imminent, will be euthanatized to prev ent 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 establis hed in order to assure that approximately equal numbers of ma les and females from each group will be examined on each day of necropsy a nd 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 carbo n 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, we ighed, recorded and organ/body and organ/brain weight ratios calculated. Orga ns will not be weighed for animals found dead or euthanatized in a moribund conditio n 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 dissect ion to avoid drying. Pair ed organs will be weighed together: adrenals kidneys p rostate testes brain liver seminal vesicles thymus epididymides heart lungs ovaries spleen uterus

12.5. TISSUES PRESERVED

Tissues listed in Appendix A will be obt ained at necropsy and preserved for all Main Study and Recovery Study animals.

12.5.1. PRESERVATIVES Eyes - glutaraldehy de/paraformaldeyhde Testes/epididymides – modified Davidson’s solution Page 1022Huntingdon Life Sciences Study No. 00-6131 Page 27 Sponsor Study No. 211-TBA-S Final Protocol

All other tissues - 10% ne utral buffered formalin. 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 experiment al findings; authorization will be obtained from the Sponsor prior to performing examinations. Note: Any abnormalities not noted during macroscopi c 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 micros cope (optional, additional cost).

Screening for alpha 2u-globulin Nephropat hy: A preliminary diagnosis will be made on hematoxylin and eosin stained sl ides 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 followi ng lesions are seen with H&E: angular hyalin inclusions in the proximal convoluted tubule, tubular degeneration and regenerati on, 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 require d tissues will be dissected out. Measurement of the size (l ength 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 of treatment: Page 1023Huntingdon Life Sciences Study No. 00-6131 Page 28 Sponsor Study No. 211-TBA-S Final Protocol

Tissue Preserved (Groups) Microscopic Examination

(Groups) All I and IV II and III brain (forebrain, central cerebrum, hippocampus, basal ganglia, midbrain, cerebellum and pons, medulla) X X

eye with optic nerve X X spinal cord (cervical, thoracic, lumbar, cross and longitudinal sections) X X

sciatic nerve (cross and longitudinal sections) X X tibial nerve (cross and longitudinal sections) X X sural nerve (cross and longitudinal sections) X X trigeminal ganglia X X dorsal root ganglia (from C 3 - C 6 and L 4 -L 6 ) X X dorsal root fibers (from C 3 -C 6 and L 4 -L 6 ) X X ventral root fibers (from C 3 -C 6 and L 4 -L 6 ) X X lungs and trachea X X X tissues with macroscopic findings 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 r oot fibers, lungs and trachea will be processed by standard techniques, embe dded in paraffin and microtomed at approximately 6 microns.

The tissues listed above will be examin ed 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 histologi cal processing will be recorded. If microscopic findings indicative of an effect of test s ubstance administration are seen in high-exposure animals, then ex aminations will be made of these tissues/organs for low- and mid-exposure animals (additional cost). Additional Page 1024Huntingdon Life Sciences Study No. 00-6131 Page 29 Sponsor Study No. 211-TBA-S Final Protocol

examinations will be made only after consultation with the Sponsor and receipt of authorization from the Sponsor.

If any evidence of neuropa thology is found, then a subj ective diagnosis will be performed to evaluate exposure-response relationships (additional cost). All regions of the nervous system exhibi ting any evidence of neuropathological changes will be included in the analysis. Sections from all exposure groups from each region will be coded and examin ed in a randomized order without knowledge of the code. The frequency a nd severity of each lesion will be recorded. After all samples have been rated, the code will be broken and statistical analysis performed to ev aluate exposure-res ponse relationships.

12.9. GFAP EVALUATION Sprague Dawley-derived (CD  ) rats, 5/sex/group, will be exposed to Gasoline TBA Vapor Condensate via inha lation 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 peri od, 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 Laborat ory, Morgantown, WV (attn: James P. O’Callaghan, Ph.D.). Upon receipt, samp les will be furt her processed for determination of GFAP response as deta iled in the referenced guidelines and procedures (see Appendix E). The Sponsor w ill be responsible for the spirit of GLP compliance of this subcontractor.



### 13. PRESERVATION OF RECORDS AND SPECIMENS

All data documenting experimental details an d 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 dete rmine the final disposition of these materials. The Sponsor is responsible for all costs asso ciated with the storage of these materials beyond one year from the issuance of the final re port and for any costs associated with the shipment of these materials to the Sponsor or to any other facility designated by the Sponsor. Page 1025Huntingdon Life Sciences Study No. 00-6131 Page 30 Sponsor Study No. 211-TBA-S Final Protocol



### 14. STATISTICAL EVALUATIONS The following items will be analyzed statistically in the final report:

mean body weight values and body weight changes (from pretest) mean food consumption values (prese nted 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 need ed. Bartlett’s test (Bartlett, 1937; Sokal and Rohlf, 1995) will be performed to determin e if groups have equal variances. For all parameters except organ weights, if the vari ances are equal, parametric procedures will be used; if not, nonparametric procedures w ill be used. Organ weight data will be analyzed only by parametric methods. The pa rametric method will be the standard oneway analysis of variance (ANOVA) using the F ratio to assess significance (Armitage, 1971; Dunlap and Duffy, 1975). If signif icant 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 indi cated, Shirley’s test (Shirley, 1977), Steel’s test (Steel, 1959) or Pairwi se Comparison with Bonferr oni 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 conducte d 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 st atistical tests. Proceedings of the Roya l 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 IV Functions for Ca lculating Exact Probabi lities 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 IV functions for calculating probabilities associated with Dunnett’s test. Behav. Res. Methods and Instrumentation 13: 363-366; Dunnett, C.W. 1955. A multipl e comparison procedure for comparing Page 1026Huntingdon Life Sciences Study No. 00-6131 Page 31 Sponsor Study No. 211-TBA-S Final Protocol

several treatments with a control. Journal of the American Statistical Association 50: 1096-1121; Dunnett, C.W. 1964. New tables for mu ltiple comparisons with a control. Biometrics 20-3: 482-491; Games, P.A. and Ho well, 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; Krus kal, W.H. and Wallis, W.A. 1952. Use of Ranks in one-criterion variance analysis. Journa l 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 Associat ion 48: 907-911; Shirley, E.A.C. 1977. A non-parametric 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 tr eatment means when several dose levels are compared with a zero dose control. Biomet rics 27: 103-117; Williams, D.A. 1972. The comparison of several dose levels with a ze ro dose control. Biometrics 28: 519-531.

Motor Activity Data

The data will be analyzed using split-plot repeated measures ANOVA with model terms for group, animal within group, interval a nd group by interval interaction. If the group x interval interaction is statis tically significant (p=0.05), i ndicating non-parallelism in the behavioral profile between groups, a separate one-way ANOVA for gr oup effects will be performed at each interval. If the response data passes on the pa rallel hypothesis, an ANOVA (using summed responses over intervals) will be used to test for the overall treatment effect which constitutes the le vel 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 cont rol. Analyses will be performed for sexes separately and combined. Treatment group ef fects will be deemed significant at the p=0.05 level. Plots, tables, listings, and an alyses will be generate d using SAS® version 6.12 for WINDOWS. Analyses will be co nducted 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 1027Huntingdon Life Sciences Study No. 00-6131 Page 32 Sponsor Study No. 211-TBA-S Final Protocol



### 15. REPORTING

15.1. STATUS REPORTS Periodic verbal and written updates on st udy progress will be provided by the Study Director. In general, written status reports will be subm itted weekly and at termination of the Main/Neuropa thology Study and R ecovery Study.

15.2 FINAL REPORT One unbound hard copy and one electronic co py of a draft report will be submitted following termination of the study. Th e 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 re port will be issued. (Additional copies will be provided at additional cost). The Main/Recovery/Neuropathology Study report will minimally include:

Compliance Statement (includi ng Sponsor signature line) Quality Assurance Statement Abstract Introduction Experimental Design Materials and Methods Discussion of study results Conclusion and No Observed Advers e Effect Level (NOAEL) statement Mortality data Individual animal termination history Summary of daily animal observations (as appropriate) Summary of weekly physical examination data Ophthalmology 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 hema tology and clinical chemistry values Tables of individual differ ential leukocyte counts Tables of mean and individual organ we ights, organ/body and organ/brain weight ratios Tables of mean and individual motor activity counts Tables of mean and individual func tional observational battery results Summary of Positive Control Neurobehavioral Studies Individual and summary tables of macroscopic pa thology findings Individual and summary tables of microscopic pathology findings Individual and summary tables of neuropathology findings References for expe rimental methodology Senior pe rsonnel participating in the study Protocol and any protocol amendments Page 1028Huntingdon Life Sciences Study No. 00-6131 Page 33 Sponsor Study No. 211-TBA-S Final Protocol



### APPENDIX A Tissues Preserved/Examined Microscopically

Tissue a Preserved Microscopic Examination

(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 X c

brain (medulla/pons, cerebrum and cerebellum) X X

epididymides X X

esophagus X X

eye (with optic nerve) X X c

heart X X

kidneys X X

large intestine (cecum, colon, and rectum) X X

lacrimal gland X X c

larynx d X X

liver X X

lungs (with mainstem bronchi) X X X

lymph node (mediastinal and mesenteric) X X

mammary gland X X c

muscle (biceps femoris) X X c

nasopharyngeal tissue e X X

nerve (sciatic) X X

ovaries X X

pancreas X X

pituitary X X c

prostate X X
salivary gland with submandibular lymph node X X

seminal vesicles X X Page 1029Huntingdon Life Sciences Study No. 00-6131 Page 34 Sponsor Study No. 211-TBA-S Final Protocol

Tissue a Preserved Microscopic Examination

(Groups)

I, IV II, III

skin X X c

small intestine (duodenum, jejunum, ileum) X X

spinal cord (cervical, thoracic, lumbar) X X

spleen X X

stomach X X

testes X X

thymic region X X

thyroid (with parathyroids) X X

trachea X X

uninary bladder X X

uterus (body/horns with cervix) X X

Zymbal’s gland X X c

gross lesions and tissue masses f X X X

target organs f,g X X X

a These tissues will also be examined for animals in Groups II and III 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 epithelium 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.

f Examinations performed at additional cost.

g Target organs will be designated by the Study Director, Pathologist and/or Sponsor based on experimental findings. Page 1030Huntingdon Life Sciences Study No. 00-6131 Page 35 Sponsor Study No. 211-TBA-S Final Protocol



### APPENDIX B Micronucleus Evaluations Page 1031Huntingdon Life Sciences Study No. 00-6131 Page 36 Sponsor Study No. 211-TBA-S Final Protocol

SATELLITE PROCEDURE

GASOLINE TBA 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 1032

### Huntingdon Life Sciences Study No. 00-6131 Page 37 Sponsor Study No . 211-TBA-S Fi nal Protocol

STUDY DETAILS PAGE Study number: 00-6131 Study title: Rat micronucleus test

Test Substance: Gasoline TBA Vapor Condensate

Route of administration: Inhalation Stability of test substance: Assessed by the Study Sponsor Analysis of achieved concentration: Assessed at PRC Principal Investigator: Christine Mason, B.Sc. at ERC Head of Department: Gillian Clare, Ph.D. at ERC Study Director: Gary M. Hoffman, BA, DABT at PRC

In the temporary absence of the Principal Investigator, 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 Investigator on her return will ratify all decisions taken in her absence. Location of study: In-Life: PRC Genetic Toxicology: ERC except statistical analysis at Huntingdon Research Centre (HRC) Proposed study dates: Start: 26 July 2002 Completion: 22 August 2002 Draft report: 11 February 2003 Page 1033

### Huntingdon Life Sciences Study No. 00-6131 Page 38 Sponsor Study No . 211-TBA-S Fi nal Protocol

1. 

### INTRODUCTION The object of this study is to assess the muta genic 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 Gu idelines; 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 (Mavournin 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 w ealth 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 chromosome s do not separate at the anaphase stage of cell division. After telophase these fragments ma y not be included in the nuclei of the daughter cells and hence will form single or multiple mi cronuclei (Howell-Jolly bodies) in the cytoplasm of these cells. Micronuclei are seen in a wide va riety 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 relativ ely 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 inciden ce of micronucleated immature erythrocytes is indicative of recent exposure to a chromosome-damaging agent. A simultaneous marked increase in the incidence of micronucleated mature erythroc ytes 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 1034

### Huntingdon Life Sciences Study No. 00-6131 Page 39 Sponsor Study No . 211-TBA-S Fi nal Protocol

chromosomes at anaphase which may not be in corporated into the daughter nuclei. These lagging chromosomes are not excluded from the er ythroblast with the main nucleus and hence also give rise to micronuclei.

Any toxic effects of the test s ubstance on the nucleated cells may lead either to a reduction in cell division or to cell death. These effects in turn 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 Schm id 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 decreas e in the proportion would be indicative of a cytostatic or cytotoxic effect.

3. EXPERIMENTAL PROCEDURE

Animal management

Species Rat Strain Sprague Dawley - derived (CD ® 

### ) [Crl: CD ® 

### IGS BR] Source Charles River Laboratories, Kingston, NY Age

~ 6 weeks old (at exposures initiation) Weight range ~150-250 g (at exposures initiation); within ±20% of mean per sex Acclimatization 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.

Housing 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 18-26oC 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.

Diet Certified Rodent Diet, No. 5002; (M eal) (PMI Feeds, Inc., St. Louis, MO) ad libitum. Elizabethtown Water Company, Westfield, NJ; a d libitum , via automated watering system. There are no known contaminants in the feed or water wh ich are expected to interfere with the results of this study.

Positive control Page 1035

### Huntingdon Life Sciences Study No. 00-6131 Page 40 Sponsor Study No . 211-TBA-S Fi nal Protocol

Identity Cyclophosphamide 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 sec tion. 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 Level Sampling Time After Last Exposure or Dose (hours) No. of animals

M F

Air Control 0 m g / m 3 

### ~24 5 5

Test substance 2,000 m g / m 3 

### ~24 5 5

Test substance 10,000 m g / m 3 

### ~24 5 5

Test substance 20,000 m g / m 3 

### ~24 5 5

C y clo p hos p hamide 40 m g /k g IP ~24 5 5

PREPARATION OF BONE MARROW SMEARS At approximately 24 hours after administration of th e final exposure, five male and five female animals from each group will be euthanized 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 Page 1036

### Huntingdon Life Sciences Study No. 00-6131 Page 41 Sponsor Study No . 211-TBA-S Fi nal Protocol

eluted in 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 sm all volume of fetal bovine serum to facilitate smearing in the conventional manner on glass mi croscope 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 at ~4oC) 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 Schiff's 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 dilu te (approximately 0.06 g/l) 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 37oC.

Note: All stains and Bouin's fluid will be filtered i mmediately prior to use to remove particulate material. Page 1037

### Huntingdon Life Sciences Study No. 00-6131 Page 42 Sponsor Study No . 211-TBA-S Fi nal Protocol

Microscopic examination Coded slides will be examined by light micros copy 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 1000 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 und erlying 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 fac ilitate interpretation and maximize the power of statistical analysis.

For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CY TEL Software Corporation, Cambridge, Massachussetts). Comparison of several dose levels is made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected Page 1038

### Huntingdon Life Sciences Study No. 00-6131 Page 43 Sponsor Study No . 211-TBA-S Fi nal Protocol

(Agresti et al. 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 st atistically 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 norma lly 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 indi vidual 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 Sta ndard 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, Hun tingdon Life Sciences will continue to retain the materials subject to a reasonable fee being agr eed with the Sponsor. Huntingdon Life Sciences will retain the Quality Assurance records relevant to this in its archive indefinitely. Page 1039

### Huntingdon Life Sciences Study No. 00-6131 Page 44 Sponsor Study No . 211-TBA-S Fi nal 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, CFR Vol. 59, No. 122, 27 June 1994

8. QUALITY ASSURANCE

The following will be inspected or audited in relation to this study.

Protocol Audit : Study specific protocol and study specific pages.

Process based inspections : Routine and repetitive procedures will be inspected on representative studies, not necessarily on this study.

Report Audit : 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 Direct or 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 So ftware 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 dist ribution-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 1040

### Huntingdon Life Sciences Study No. 00-6131 Page 45 Sponsor Study No . 211-TBA-S Fi nal 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) Trenim on-induced chromosomal damage in bone marrow cells of six mammalian species, evaluated by the micronucleus test. Mutation Research

, 12 , 417.

MAVOURNIN, 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 Pr otection 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) SAS/STAT User's Guide, Version 6, Fourth Edition, Vol.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) SAS/STAT 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 co mparisons by ranking methods. Biometrics Bulletin, 1 , 80- 83. Page 1041

### Huntingdon Life Sciences Study No. 00-6131 Page 46 Sponsor Study No . 211-TBA-S Fi nal Protocol



### APPENDIX C SCE Evaluations Page 1042Page 1043

### Huntingdon Life Sciences Study No. 00-6131 Page 48 Sponsor Study No . 211-TBA-S Fi nal Protocol

4.0 SUBCONTRACTOR AND KEY PERSONNEL

4.1 Name: BioReliance

4.2 Address: 9630 Medical Center Drive Rockville, MD 20850

4.3 Principal Investigator: 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: 26 July 2002

5.2 Proposed Experimental Completion Date: 22 August (in-life) September 2002 (evaluation)

5.3 Proposed Draft Report Date: 11 February 2003

6.0 TEST SYSTEM

Closed-colony, random-bred rodents are acceptable models for mutagenicity studies. SpragueDawley rats were selected because of the availab ility 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 pe rsonnel 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 culture d 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 harves ted for microscopic evaluation of SCEs. The Page 1044

### Huntingdon Life Sciences Study No. 00-6131 Page 49 Sponsor Study No . 211-TBA-S Fi nal Protocol

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:

Group Number per Sex to be Sacrificed After Test Substance Administration

Negative Air Control 5
Test Substance (mg/m 3 

### ) 2,000 10,000 20,000 5 5 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 dos e 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) e xposure period, 2-4 ml of blood will be collected from the abdominal aorta from each anesthetized (isoflurane inhalation) rat including rats tr eated with positive control article. Whole blood cultures in duplicate per animal w ill 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 1045

### Huntingdon Life Sciences Study No. 00-6131 Page 50 Sponsor Study No . 211-TBA-S Fi nal Protocol

approximately 0.5 ml of whole blood per 5.0 ml of complete culture medium in T-25 sterile disposable tissue culture flasks a nd incubated at 37oC. Approximately 21 hours after initiation of the cultures, the cells will be exposed to 5.0 μg/mL of BrdU (Sigma Co., St. Louis, MO).

7.3 Cells Harvest and Staining

At approximately 68 hours, colcemid at 0.2 μg/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 2-6oC. To prep are 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-d ried. 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 w ill be stained in Hoechst 33258, 5 μg/ml for 10 minutes, mounted in phosphate buffer, pH 6.8, exposed to a U.V. lamp at approximately 60 o 

### 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 po ssible, 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 be scored for mitotic index per animal.

8.0 Criteria for Determination of a Valid Test

The mean SCEs/cell/animal for the positive contro l 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 1046

### Huntingdon Life Sciences Study No. 00-6131 Page 51 Sponsor Study No . 211-TBA-S Fi nal 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 M 1 

### cells x 1)+(number M 2 

### cells x 2)+(number M 3 

### cells x 3)]. A Dunnett's t test for multiple comparisons will be performed to compare the SCE frequency of test exposure leve ls to the negative control frequency, and if p<0.05, that exposure level will be considered significant. The conclusion of the st udy 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 expos ure 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, Colcemid 7

, 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 genera ted 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 of the 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 accordan ce with the relevant Good Laboratory Practice Regulations. Page 1047

### Huntingdon Life Sciences Study No. 00-6131 Page 52 Sponsor Study No . 211-TBA-S Fi nal Protocol



### APPENDIX D Immunotoxicity Evaluations Page 1048

### Huntingdon Life Sciences Study No. 00-6131 Page 53 Sponsor Study No . 211-TBA-S Fi nal Protocol

IMMUNOTOX, INC. In Vitro & In Vivo Immunotoxicological Assessments

Satellite Procedure Title:

Immunotoxicological Evaluation of Gasoline TBA 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 Richmond, VA 23219-1534

Study Identification :

HLS Study Number: 00-6131 ImmunoTox, Inc. Project Number: ITI 1101 Page 1049

### Huntingdon Life Sciences Study No. 00-6131 Page 54 Sponsor Study No . 211-TBA-S Fi nal Protocol

PURPOSE: The purpose of this study is to provide evaluation of immunological parameters for Huntingdon Life Sciences Study No. 00-6131. In this study, the ab ility of the test substance, Gasoline TBA Vapor Condensate, will be evaluated for its ability to aff ect the humoral immune co mponent of the immune system, when evaluated in the antibody-forming cell response to the T-dependent antigen sheep erythrocytes.

HUNTINGDON STUDY NUMBER: 00-6131

IMMUNOTOX, INC. PROCEDURE NUMBER: 00-6131

IMMUNOTOX, INC. PROJECT NUMBER: ITI 1101

SECURITY: Industrial Confidential

PROPOSED STUDY INITIATION DATE: To Be Supplied by Huntingdon Life Sciences (Date Protocol Signed By Study Director)

PROPOSED EXPERIMENTAL START DATE: 25 June 2002 (First Day of Treatment)

PROPOSED EXPERIMENTAL TERMINATION 23 July 2002 DATE: (Date of Final Sacrifice)

PROPOSED DRAFT REPORT DATE: 11 February 2003

SUBCONTRACTOR’S SPONSOR: Huntingdon Life Sciences Princeton Research Center (PRC)
Mettlers Road East Millstone, NJ 08875-2360

STUDY DIRECTOR: Gary M. Hoffman, B.A., DABT

SUB CONTRACTOR: 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 1050

### Huntingdon Life Sciences Study No. 00-6131 Page 55 Sponsor Study No . 211-TBA-S Fi nal 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: Kimber L. White, Jr., Ph.D. Assistant Principal Investigator: Vanessa L. Peachee, M.S. Quality Assurance: 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 accordan ce with agreements between Hunti ngdon 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 comple ment 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 a Tdependent antigen requiring T cells, B cells and macrophages to function properly in order to obtain an antibodyforming cell (AFC) response. If a test agent affects any of th ese 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 significan t 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 TBA 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 exposu re, spleens will be aseptically remove d, weighed, and shipped intact in individual shipping containers at 2-8 o C via overnight delivery to ImmunoTox., Inc. Upon receipt, spleens will be further processed for determination of IgM re sponse as detailed in procedures below. Page 1051

### Huntingdon Life Sciences Study No. 00-6131 Page 56 Sponsor Study No . 211-TBA-S Fi nal Protocol

Exposure Level (mg/m 3 ) Number of Female Rats

0 10 2,000 10 10,000 10 20,000 10

Positive Control* 10 *50mg/kg cyclophosphamide, IP, 4 days prior to sacrifice. These animals will not be chamber exposed.

PROCEDURE (ImmunoTox, 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 Jerne 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 final exposure, collect into tubes without anticoagulant (centrifuged at ~3000 rpm at 2-8 o C) and freeze (<-20 o 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 ImmunoTox, Inc. for imm unotoxicological evaluation. Thymus will be removed and weighed and preserve d (10% neutral buffered formalin) for possible histopathology.
Animals will not be allowed to recover betw een blood collection and spleen collection.

4. Upon arrival at the subcontractor facility, spleens will be accessione d in accordance with the SOP for receipt of biological samples. Si ngle cell suspensions will be pr epared from each spleen using a
Stomacher® 80 Lab Blender in accord ance with the SOP for rat spleen s. Cell suspen sions 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 volum e, and 1:50 and 1:150 dilutions are prepared. Page 1052

### Huntingdon Life Sciences Study No. 00-6131 Page 57 Sponsor Study No . 211-TBA-S Fi nal Protocol

6. An 0.1 ml aliquot of spleen cells from each susp ension will be added to separate test tubes each containing 25 μl guinea pi g complement, 25 μl sRBC, and 0.5 ml of warm agar (0.5%). After thoroughly mixing, each test tube mi xture will be plated onto a separate petri dish, covered with a microscope cover slip, and incubated at appr oximately 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 Coulte r 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 st udies will be analyzed in accord ance with standard operating pr ocedures. 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 of variance 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 vehi cle and treatment groups. The positive c ontrol data will be evaluated using
the Student's t Test 7 and compared to the vehicle control group. Th e criteria for accepting the results of the positive control in the assay will be a statistically significant (P<0 .05) decrease in the response as compared to the vehicle control group.

IMMUNOTOXICOLOGICAL REPORT: The report on the immunotoxicological resu lts will contain all procedures and/or references to published procedures us ed 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 st udy will be submitted to Huntingdon Life Sc iences (PRC) upon acceptance of the final report. Upon completion of this study, the report and raw data for th is study will be maintain ed 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 1053

### Huntingdon Life Sciences Study No. 00-6131 Page 58 Sponsor Study No . 211-TBA-S Fi nal Protocol

REFERENCES:

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 co mparing 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. Jerne NK, Henry C, Nordin AA, Fun H, Koros MC, and Lefkovits I (1974). Plaque-forming cells: Methodology and theory. Trnspl. Rev. 18:130-191. Page 1054

### Huntingdon Life Sciences Study No. 00-6131 Page 59 Sponsor Study No . 211-TBA-S Fi nal Protocol



### APPENDIX E GFAP Evaluations Page 1055Page 1056

### Huntingdon Life Sciences Study No. 00-6131 Page 61 Sponsor Study No . 211-TBA-S Fi nal Protocol

3.3 Test Substance Retention Sample

The retention of a reserve sample of th e Test substance will be the responsibility of Huntingdon Life Sciences.

4.0 SUBCONTRACTOR AND KEY PERSONNEL

4.1 Name: US Centers for Disease Control and Prevention Health Effects Laboratory Division – NIOSH

4.2 Address: 1095 Willowdale Road Morgantown, WV 26505-2888

4.3 Principal Investigator: James P. O’Callaghan, Ph.D.

5.0 TEST SCHEDULE

5.1 Proposed Experimental Initiation Date: 25 June 2002

5.2 Proposed Experimental Completion Date: 25 September 2002

5.3 Proposed Draft Report Date: 11 February 2003

6.0 TEST SYSTEM

Closed-colony, random-bred rodents are acceptabl e models for GFAP studies. Sprague-Dawley 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 for 13 weeks (5 days per week), will be performed by Huntingdon Life Sciences. Animals will be sacrificed and brain sample s 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 GFAP in a dose-responsive manner will be measured relative to a control group. Page 1057

### Huntingdon Life Sciences Study No. 00-6131 Page 62 Sponsor Study No . 211-TBA-S Fi nal Protocol

The study design will be as follows:

Group Number per Sex

Negative Air Control 5

Test Substance (mg/m 3 

### ) 2,000 10,000 20,000 5 5 5

7.1 Exposure Levels Selection

The exposure levels for the GFAP 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 ge nerated 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 1058

### Huntingdon Life Sciences Study No. 00-6131 Page 63 Sponsor Study No . 211-TBA-S Fi nal Protocol

11.0 Regulatory Requirements/Good Laboratory Practice

This study will be conducted in accordance with the spirit of: US EPA GFAP Assay 79.67, CFR Vol. 59, No. 122, 27 June 1994. US EPA GLP 79.60, CFR Vol. 59, No. 122, 27 June 1994.



### GFAP ELISA ASSAY Standa rd Operating Procedure

1 SCOPE OF APPLICATION

The following is an SOP to be used in the quantif ication of the levels of glial fibrillary acidic protein (GFAP) 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 E QUIPMENT 

## and S UPPLIES



## 2.1.1 E QUIPMENT

Ultrasonic Cell Disruptor Pipettes Hot/Stir Plate Plate Reader



## 2.1.2 C HEMICAL 

## R EAGENTS

α Glial Fibrillary Acidic Protein (DAKO Z0334) - Lot # 096 (302) Carnation Non-Fat Dry Milk

α Glial Fibrillary Acidic Protein (Chemicon MAB 3402-Lot 14119825-07) Phosphate Buffered Saline (Pierce #28374) Alkaline Phosphatase conjugated α mouse IgG(Jackson ImmunoResearch #315-055-003) Alkaline Phosphatase Substrate Kit (Bio-Rad #172-1063) Triton X-100 (Bio-Rad #161-0407) Sodium Hydroxide (Fisher #S320) Page 1059

### Huntingdon Life Sciences Study No. 00-6131 Page 64 Sponsor Study No . 211-TBA-S Fi nal Protocol



## 2.1.3 S UPPLIES

Microtiter Plates (Immulon 2, Dynatech) Pipet tips



## 2.2 T RAINING 

## R EQUIREMENTS

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 gr ocery store and used with in six months. Old powdered milk gives a yellow color in all samples and masks any GFAP 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 S TANDARD 

### C URVE 

### P REPARATION



### 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 dilu tion of the GFAP standard. The standard has a known total protein concentration (11.5 mg /ml) and a known concentration of GFAP (2.4μg GFAP/ mg total protein). The total prot ein 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 μg/100μl (i.e. 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, 10μg/100μl). Page 1060

### Huntingdon Life Sciences Study No. 00-6131 Page 65 Sponsor Study No . 211-TBA-S Fi nal Protocol

GFAP Standard Curve Preparation



### Tube # μg of Protein/100μl/well (μg of GFAP) μl of Hippo Standard Serial Dilution μl of PBS+0.5% Triton X100
1 10μg (.024μg) 17.8 μl 1982μl

2 7.5μg (.018μg) 13.0μl 1987μl 3 5.0μg(.012μg) 1.0ml from tube #1(10μg) 1ml

4 2.5μg(.006μg) 500μl from tube #3(5μg) 500μl

5 1.0μg(.0024μg) 500μl from tube #3(5μg) 2 ml

6 0.5μg(.0012μg) 1ml from tube #5(1μg) 1ml

7 0.25μg(.0006μg) 500μl from tube #6(0.5μg) 500μl



## 4.2 S AMPLE 

### P REPARATION



### 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 10X the weight of the tissue e. g. tissue weight = .045 grams 10X volume= 450μl 1% SDS) of hot 1% SDS (85-95 o 

### C) is added and then sonified. In both cases, the samples are stored within 24 hours at -70 o 

### C.

3. Before the frozen tissues can be used in the GFAP ELISA, they are removed from the freezer, placed on dry ice, then sonified in hot 1% SDS (85- 95 o 

### C) 10X 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 1061

### Huntingdon Life Sciences Study No. 00-6131 Page 66 Sponsor Study No . 211-TBA-S Fi nal Protocol

Triton X-100 to a concentration of 10μg total protein/ 100μl of PBS with 0.5% Triton X-100. Samples high in GFAP (e. g. cerebellum) may need to be diluted to a concentration of 5μg total protein/ 100μl of PBS with 0.5% Triton X-100. Samples low in GFAP (e.g. striatum) may need to be diluted to 20μg total protein/ 100μl of PBS 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 μl 991.3 μl of PBS with 0.5% Triton X-100.)

6. If samples have to be diluted to a lower concentration, a 1:1 dilution of the 10μg/100μl dilution results in the 5μg/100μl (200μl of the 10μg/100μl dilution plus 200μl of the PBS with 0.5% Triton X-100).



## 4.3 R EAGENT PREPARATION



### 1. Volumes Required- The volumes of reagents are 100μ l/well (10ml per plate and the volume of wash is 200μl/well (20ml per plate).

2. Phosphate Buffered Saline (PBS) - One packet of PBS is mixed thoroughly with 500ml of deionized water.

3. PBS+0.5% Triton X-100 - 2.5ml of Triton X-100 is added to 500ml of PBS.

4. B lotto - 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 th e assay). The larger volume allows a good mix for the addition of the Triton X-100 deterg ent. PBS may be warmed slightly; this will facilitate getting the powdered milk into solution.

5. Polyclonal anti-GFAP (Dako)- 25μl/10ml of PBS

6. Monoclonal anti-GFAP (Chemicon)- 1:500 20μl/10ml Blotto+0.5% Triton X-100

7. Alkaline Phosphatase - 1:3000 3.3μl/10ml Blotto+0.5% Triton X-100 Page 1062

### Huntingdon Life Sciences Study No. 00-6131 Page 67 Sponsor Study No . 211-TBA-S Fi nal Protocol

8 . P-nitrophenylphosphate substrate - 2 ml of diethanolamine buffer, 2 pnitrophenylphosphate tablets in 8ml of deionized water.



## 4.4 A SSAY PROCEDURE



### 1. Coat Immulon-2 flat bottom plates with α GFAP, 1.0μg total immunoglobulin protein / 100μl PBS / well. (25μl of α GFAP, Dako Z0334, in 10ml of PBS is the quantity needed per plate).

2. Incubate the coated plate at 37 o 

### 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 o 

### C.

3. Empty plate, tap on absorbent paper to remove excess (this procedure is important to eliminate the possibility of any reagent carry-ove r between steps). Wash plates 4X with PBS (200μl per well), tapping and blotting between each wash.

4. Block 1 hour with BLOTTO (5% w/v non-fat powdered milk in PBS) 100μl per well.

5. Empty plate, tap on absorbent paper to remove excess, load diluted standard curve and samples in a volume of 100μl per well. Incubate for 1 hour at room temperature. The template below is an example of a typical plate for this labs GFAP Assay.

Microtiter Plate Template
1 2 3 4 5 6 7 8 9 10 11 12 A Blk Blk 1 1 9 9 17 17 25 25 33 33 B Std 1 Std 1 2 2 10 10 18 18 26 26 34 34 C Std 2 Std 2 3 3 11 11 19 19 27 27 35 35 D Std 3 Std 3 4 4 12 12 20 20 28 28 36 36 E Std 4 Std 4 5 5 13 13 21 21 29 29 37 37 F Std 5 Std 5 6 6 14 14 22 22 30 30 38 38 G Std 6 Std 6 7 7 15 15 23 23 31 31 39 39 H Std 7 Std 7 8 8 16 16 24 24 32 32 40 40 6. Wash 4X with PBS + 0.5% Triton X-100, 200μl/well.

7. Incubate for 1 hour in monoclonal α GFAP (Chemicon) at a dilution of 1:500 plus alkaline phosphatase conjugated anti-mouse IgG Page 1063

### Huntingdon Life Sciences Study No. 00-6131 Page 68 Sponsor Study No . 211-TBA-S Fi nal Protocol

(Jackson ImmunoResearch) at a dilution of 1:3000 made up in BLOTTO plus 0.5% Triton X-100, 100μl/well.made up in BLOTTO plus 0.5% Triton X-100, 100μl /well.

8. Wash 4X with PBS + 0.5% Triton X-100.

9. Wash 4X with PBS + 0.5% Triton X-100, 200μl/well.

10. Add P-nitrophenylphosphate substrate(Bio-Rad) in a volume of 100μl/well and incubate for 20 minutes.

11. Stop reaction with 0.4N NaOH 100μl/well.

12. Check wells for any bubbles, read plate at 405nm.



## 4.5 C ALCULATIONS



### 1. Plates are read on a Molecular Devices Spect ra Max Plus microplate reader. The plate reader is coupled to a PC running the SoftMax Pro(Molecular Devices) program.

2. A template is set up for each pl ate in the assay. The template includes the position of the standard curve and value for each point, and th e 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. 10μg of to tal protein=100x dilution factor , 5μg 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 1064

### Huntingdon Life Sciences Study No. 00-6131 Page 69 Sponsor Study No . 211-TBA-S Fi nal Protocol



## GFAP S TANDARD 

## C URVE

PLATE

BLANK Well OD Mean Std Dev CV

BL A1 0.096 0.095 0.001 1.489

A2 0.094

STANDARDS Value Well OD Mean Std Dev CV STD01 6.00e-4ug B1 0.033 0.03 0.004 14.14 B2 0.027 STD02 0.0012ug C1 0.065 0.065 ***** ***** C2 0.065 STD03 0.0024ug D1 0.146 0.145 7.07E-04 0.486 D2 0.145 STD04 0.006ug E1 0.326 0.327 0.001 0.432 E2 0.328 STD05 0.012ug F1 0.463 0.454 0.013 2.963 F2 0.444 STD06 0.018ug G1 0.53 0.526 0.006 1.075 G2 0.522 STD07 0.024ug H1 0.546 0.561 0.022 3.904 H2 0.577 C urve Fit: C orr. C oeff: 4-Param eter

A = -0.00391 B = 1.29 C = 0.00618 D = 0.658 y = (A-D)/(1 + (x/C)^B ) + D -1.00

0.0001 1 Log scale of ug 0 0.6

O D Page 1065

### Huntingdon Life Sciences Study No. 00-6131 Page 70 Sponsor Study No . 211-TBA-S Fi nal Protocol

3. The above standard curve is a typical curve generated from the rat hippocampus standard. The OD values for unknown samples need to fa ll 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 SoftMax 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 hippocam pus samples that has been imported into an Excel spreadsheet. These samples were dilu ted to 2.5μg of total protein/100μl, therefore the dilution factor for all ten samples is 400(th is factor may be included as another column on the spreadsheet).

UNKNOWNS Mean Std Dev CV Well OD Value HIP01 2.633 0.114 4.323 A3 0.334 2.552 A4 0.347 2.713 HIP02 2.482 0.099 3.984 B3 0.334 2.552 B4 0.322 2.412 HIP03 3.099 ***** ***** C3 0.375 3.099 C4 0.375 3.099 HIP04 2.792 0.093 3.34 D3 0.358 2.858 D4 0.348 2.726 HIP05 2.206 0.074 3.338 E3 0.298 2.154 E4 0.308 2.259 HIP06 2.681 0.027 0.999 F3 0.343 2.662 F4 0.346 2.7 HIP07 3.077 0.094 3.048 G3 0.369 3.011 G4 0.378 3.144 HIP08 2.631 0.044 1.663 H3 0.338 2.601 H4 0.343 2.662 HIP09 2.934 0.108 3.697 A5 0.369 3.011 A6 0.358 2.858 HIP10 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 GFAP has been calculated and no further calculations need to be done. Page 1066

### Huntingdon Life Sciences Study No. 00-6131 Page 71 Sponsor Study No . 211-TBA-S Fi nal Protocol

7. If sample dilutions were prepared by pipetting 10μl 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: GFAP value ÷ 1/10 Total protein value=μg GFAP/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 μg 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 Prot ein: 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

SoftMax User’s Manaual, Molecular Devices Corporation, Sunnyvale, CA



### Brain Tissue Preparation Sta ndard 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 (GFAP). Page 1067

### Huntingdon Life Sciences Study No. 00-6131 Page 72 Sponsor Study No . 211-TBA-S Fi nal Protocol



## 2 PREREQUISITES



## 2.1 E QUIPMENT 

### and S UPPLIES



## 2.1.1 E QUIPMENT



### Ultrasonic Cell Disruptor Pipettes Hot/Stir Plate Plate Reader



## 2.1.2 C HEMICAL 

### R EAGENTS



### Sodium Dodecyl Sulfate, Bio-Rad #161-0302



## 2.1.3 S UPPLIES



### Microfuge tubes Pipet tips



## 2.2 T RAINING 

### R EQUIREMENTS



### Ability to dissect discrete regions of the rat brain.



## 3 CAUTIONARY NOTES OR SPECIAL CONSIDERATIONS

SDS needs to be heated to a temperature of (85-95oC).



## 4 PROCEDURE



## 4.1 R EAGENT 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- 10ml of 10X stock in 90ml of deionized water. Page 1068

### Huntingdon Life Sciences Study No. 00-6131 Page 73 Sponsor Study No . 211-TBA-S Fi nal Protocol



## 4.2 TISSUE P REPARATION



### 1. Control and Test substance exposed animals are sacrificed, brains then are rapidly removed from the skull case with the aid of pointed sci ssors (to open the brain case) and curved blunt forceps (to remove the brain).

2. The brain is placed on a cold (approximately 4oC) 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, hippocam pus, hypothalamus, striatum, thalamus, rest of brain. In 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 i mmersed in 10 volumes of hot 1% SDS (85-95 o 

### C) (i.e. 10X volume the weight of the tissue; e.g. tissue weight = .045 grams, 10X volume= 450μl 1%SDS) and then homogenized by sonifica tion with an ultrasonification microprobe (Heat Systems, Farmingdale, NY). The sample s then are stored within 24 hours at -70 o 

### C prior to assay.



### Sample Storage/Handling and Shipping Standard Operating Procedure

1 SCOPE OF APPLICATION The following is an SOP to be used for st oring/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 (GFAP) in each homogenate.



## 2 PREREQUISITES

2.1 E QUIPMENT 

### and S UPPLIES



### 2.1.1 E QUIPMENT



### -70 o 

### C Freezer



### 2.1.2 C HEMICAL 

### R EAGENTS



### 2.1.3 S UPPLIES



### 2.2 T RAINING 

### R EQUIREMENTS Page 1069

### Huntingdon Life Sciences Study No. 00-6131 Page 74 Sponsor Study No . 211-TBA-S Fi nal Protocol

Ability to handle samples frozen at -70oC



## 3 CAUTIONARY NOTES OR SPECIAL CONSIDERATIONS

Wear protective gloves when removing samples from -70oC freezers.



## 4 PROCEDURE

4.1 S TORAGE 

### /H ANDLING



### Homogenates will be stored frozen at -70oC prior to shipping to CDC-NIOSH and shipped frozen on dry ice.



## 4.2 S HIPPING



### Homogenates will be shipped on dry ice in protective Styrofoam containers to CDC-NIOSH.



### BCA PROTEIN ASSAY Standard Operating Pr ocedure

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 E QUIPMENT 

### and S UPPLIES



## 2.1.1 E QUIPMENT



### Plate Reader Pipettes Incubator Vortex-Mixer



## 2.1.2 C HEMICAL 

### R EAGENTS



### BCA Protein Kit, Pierce #23223 Bovine Serum Albumin (BSA), Sigma A7888 Page 1070

### Huntingdon Life Sciences Study No. 00-6131 Page 75 Sponsor Study No . 211-TBA-S Fi nal Protocol

Sodium Dodecyl Sulfate, Bio-Rad #161-0302



## 2.1.3 S UPPLIES



### Microtubes 1.2ml 96 well microtiter plates Pipette Tips



## 2.2 T RAINING 

### R EQUIREMENTS



### Able to use a plate reader and to accurately dilute tissue samples.



## 3 CAUTIONARY NOTES OR SPECIAL CONSIDERATIONS

BCA reagent is stable for up to one 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 R EAGENT 

### P REPARATION



### 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- 10ml of 10X stock in 90 ml deionized water.

2. BCA Reagent This reagent is made according to the directi ons provided by Pierce, 50ml of solution A and 1ml of solution B. Thoroughly mix while adding solution B.



## 4.2 S TANDARD 

### P REPARATION



### Protein Standards: Bovine Serum Albumin (BSA) in 1% (w/v) SDS (sodium dodecyl sulfate).

1. Prepare 1mg/ml BSA in 1% SDS. Wei gh out 0.100 gms of dry BSA on an analytical balance. Add the BSA to a 100 ml volumetric flas k then gradually add 1% SDS to the flask. Page 1071

### Huntingdon Life Sciences Study No. 00-6131 Page 76 Sponsor Study No . 211-TBA-S Fi nal Protocol

Mix gently 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 μl is enough volume to do one standard curve). Store the tubes at -70oC . Thaw as needed, but do not re-freeze.

3. Blank: Remove 200μl of 1% SDS, place it in a dilution tube.

4. 1.0μg/10μl: Remove 20μl of the prepared BSA standa rd(1mg/ml), place it in a dilution tube and add 180μl of 1% SDS.

5. 2.5μg/10μl: Remove 50μl of the prepared BSA standard, place it in a dilution tube and add 150μl of 1% SDS.

6 . 5.0μg/10μl: Remove 100μl of the prepared BSA standard, place it in a dilution tube and add 100μl of 1% SDS.

7. 7.5μg/10μl: Remove 150μl of the prepared BSA standard, place it in a dilution tube and add 50μl of 1% SDS.

8. 10.0μg/10μl: Remove 200μl of the prepared BSA standard and place it in a dilution tube.
BSA Standard Curve Tube

# μg Protein / μl of 1mg/ml

BSA μl of 1%SDS

10μl/well Standard 1 Blank 0 200μl

2 1.0μg 20μl 180μl

3 2.5μg 50μl 150μl

4 5.0μg 100μl 100μl

5 7.5μg 150μl 50μl

6 10.0μg 200μl 0

9. Repeat steps 3 to 8. This generates a second standard curve, independent of the first. Page 1072

### Huntingdon Life Sciences Study No. 00-6131 Page 77 Sponsor Study No . 211-TBA-S Fi nal Protocol

10. Vortex each tube of the diluted BSA standard, remove 10μl of the diluted standard and place it in the designated well on a microtiter plate.



## 4.3 S AMPLE 

### P REPARATION



### 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 10μl aliquot from the sample and place it into a dilution tube. Add 190μl 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 10μ l 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

1 2 3 4 5 6 7 8 9 10 11 12

A Blk Blk 3 3 11 11 19 19 27 27 35 35
B Std 1 Std 1 4 4 12 12 20 20 28 28 36 36 C Std 2 Std 2 5 5 13 13 21 21 29 29 37 37
D Std 3 Std 3 6 6 14 14 22 22 30 30 38 38
E Std 4 Std 4 7 7 15 15 23 23 31 31 39 39
F Std 5 Std 5 8 8 16 16 24 24 32 32 40 40 G 1 1 9 9 17 17 25 25 33 33 41 41
H 2 2 10 10 18 18 26 26 34 34 42 42



## 4.4 P ROTEIN 

### A SSAY



### 1. Add 200μl of reagent to each of the wells containing either the standard curve or sample.

2. Place the plate in the plate reader and mi x the plate using the automatic mix button on the plate reader or vortex gently prior to placing the plate into the reader. Page 1073

### Huntingdon Life Sciences Study No. 00-6131 Page 78 Sponsor Study No . 211-TBA-S Fi nal Protocol

3. Incubate the plate at 37oC for 30 minutes.

4. At the completion of the incubation period, the plate is checked for bubbles and then read in the plate reader at a wavelengt h of 562nm. “Pop” any bubbles with a needle or a pipette tip prior to reading the plate.



## 4.5 C ALCULATIONS



### 1. Software programs coupled to specific plate r eaders 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 used for the standard. The table gives the actua l values for each point on the standard curve and their corresponding OD readings.



## BCA Total Protein Standard Curve

Curve Fit: Corr. Coeff: Linear

A = 0.0240 B = 0.0395 

### y = A + B * x 0.999

1 10 Linear scale of μg 0 0.4OD Page 1074

### Huntingdon Life Sciences Study No. 00-6131 Page 79 Sponsor Study No . 211-TBA-S Fi nal Protocol

Plate

Blank Well OD Mean Std Dev CV

BL A1 0.083 0.082 0.001 1.725

A2 0.081

STANDARD Value Well OD Mean Std Dev CV

STD01 1.000 μg A3 0.06 0.058 0.002 3.626

A4 0.057

STD02 2.500 μg A5 0.131 0.131 7.07E-04 0.542

A6 0.13

STD03 5.000 μg A7 0.22 0.218 0.003 1.297

A8 0.216

STD04 7.500 μg A9 0.311 0.321 0.014 4.406

A10 0.331

STD05 10.00 μg A11 0.419 0.418 0.001 0.338

A12 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 th e standard curve and the protein concentration of each unknown expressed as mg/ml or μg/μl. Th e table below gives the calculated protein values for unknown hippocampus samples in mg/m l or μg/μl. These values include the subtraction of the blank value and a dilution factor of 2. Page 1075

### Huntingdon Life Sciences Study No. 00-6131 Page 80 Sponsor Study No . 211-TBA-S Fi nal Protocol

UNKNOW Mean Std Dev CV Well OD Value Dil.

Factor

UNK01 9.325 0.215 2.306 B1 0.211 9.477 2

B2 0.205 9.173

UNK02 10.44 ****

* ***** B3 0.23 10.44 2

B4 0.23 10.44

UNK03 9.883 0.072 0.725 B5 0.218 9.832 2

B6 0.22 9.934

UNK04 11.18 1.254 11.23 B7 0.227 10.29 2

B8 0.262 12.06

UNK05 11.45 0.717 6.258 B9 0.24 10.95 2

B10 0.26 11.96



### 5. Example of calculations, if samples are prepared as listed above:

a. Hippo #3 is prepared as a 1:20 dilution (10μl of standard or unknown sample are placed in a dilution t ube and brought up to a final volume of 200μl) b. OD reading for this sample is .219 c. OD of .219 corresponds to ≈ 4.942μg/10μl or .4942μg/μl total protein d. This sample has been diluted 1:20 so the value is actually .4942μg/μl x 20= 9.883μg/μl (since the protein sample was loaded in 10 μl volumes, the value can be determined by using a dilution factor of 2, 4.942μg/10μl x2=9.883μg/μl).



## 5 R EFERENCES



### BCA Protein Assay Reagent (instruction manual), Pierce, Rockford, IL.

SoftMax Pro User’s Manual, Molecular Devices Corporation. Sunnyvale, CA. Page 1076Page 1077Page 1078Page 1079Page 1080Page 1081Page 1082Page 1083

### Huntingdon Life Sciences 00

- 6131 Page 1084 211

- TBA

- 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, SAFETY

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

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

DIRECTOR, TOXICOLOGY

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

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

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

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

STUDY MONITOR Georgia Burnett, A.A.S.

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

MANAGER/SUPERVISOR

Rodent/Inhalation Toxicology Analytical Services Clinical Pathology Histology Necropsy Robert Faust, B.S. Kay S aladdin, B.S. Lisa A. Spall, B.S., M.T. (ASCP) Janet Kusisto, B.S., H.T. (ASCP) G. Elizabeth Baxter, B.S.

SUBCONTRACTORS

OPHTHALMOLOGY Lionel F. Rubin, V.M.D., A.C.V.O.