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 Calibration .......................................................................................................846 

P. Chamber Distribution Records ..................................................................................849 

Q. Testing Room and Chambers Environmental Monitoring .........................................852 

R. Animal Termination History ......................................................................................854 

S. Analytical Report .......................................................................................................871 

T. Neurobehavioral Positive 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 6G02R3, 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® MicrofibreTM 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 TrakTM 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) 
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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/Thermometer, Model No. 61161-378 

Oxygen/Gas Analyzer, Model 1214S (Gastech) 

Digital Sound Level Meter, Model 840029 (Sper Scientific) 

Photometer 1 Light Meter (Quantum Instruments) 

Miscellaneous 

Ball and Socket joint 

Gas-tight syringe, size 0 - 25 mL, Model 1025 (Hamilton Company) 

Gas Bags (Calibrated Instruments) 
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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 unit was zeroed and the calibration series 

was performed as shown below. The resultant data were plotted to obtain a 

calibration curve. Each observer used a separate syringe for calibration. 

Injection Calculated Absorbance 

Volume Concentration1 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 volume (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/m3) (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 each injection. The absorbance was 

considered satisfactory if it was within 15% of the original calibration series. If 

any of the absorbance values fell outside the 15% range, the injection was 

rechecked as follows. The volume for the value that was out of range was 

reinjected twice. The closer pair of the three injections were averaged and the 

results were compared to the original curve. If the average of the pair was within 

the 15% range, the original was accepted. If the value of the average was 

outside the 15% range, the Study Director decided if a new graph was to be 

prepared. 
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Miran Calibration Appendix O 

Calibration Curve for Gasoline TBA Vapor Condensate 

y = 79230x4 - 118555x3 + 56322x2 + 21544x

R2

 = 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/m3

) 

Ratio to H-1

IIA (2000 mg/m3) 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/m3

) 

Ratio to H-1

IIB (2000 mg/m3) 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/m3) 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/m3) 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 

 
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Chamber Distribution Records Appendix P 

Group 

(target) 

Date Port IR Conc 

(mg/m3

) 

Ratio to H-1

IVA (20000 mg/m3) 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/m3) 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 

 
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 Testing Room and Chambers 

Environmental Monitoring Appendix Q 

Interval Location Test 

Substance

(mg/m3

) 

Light 

(Ft Candles) 

Noise 

(dB) 

Oxygen 

(%) 

Particle 

Sizing 

(mg/m3

)

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 
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 Testing Room and Chambers 

Environmental Monitoring Appendix Q 

Interval Location Test 

Substance

(mg/m3

) 

Light 

(Ft Candles) 

Noise 

(dB) 

Oxygen 

(%) 

Particle 

Sizing 

(mg/m3

)

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 
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 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 4596 
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 Neurobehavioral Positive Control Data 

Preface Appendix T 

Table of Contents 

Functional Observational Battery ...................................................................... 899 

Motor Activity .................................................................................................... 964 
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Functional Observational Battery 

Preface Appendix T 

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

Table of Contents 

Introduction ...................................................................................................... 900 

Materials and Methods .................................................................................... 900 

Results and Discussion ................................................................................... 919 

Conclusion ....................................................................................................... 924 

Mean Body Weights, Grip Strength and Landing Foot Splay Values ............... 925 

Incidence Summary of Functional Observational Battery Evaluations ............. 936 
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Functional Observational Battery Appendix T 

1. INTRODUCTION 

This study was designed to train technicians 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 Research 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 provide 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 compliance with Part 160 of 40 CFR 

(EPA/FIFRA Good Laboratory Practice 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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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 training 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 Dosesa Initial 

Functional 

Observational 

Battery 

Dose 

(mg/kg)

Volume 

(mL/kg)

Conc. 

(mg/mL) M F M F 

1b 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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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 observational 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 Dosesa Initial 

Functional 

Observational 

Battery 

Dose 

(mg/kg)

Volume 

(mL/kg)

Conc. 

(mg/mL) M F M F 

1b 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; subcutaneous 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 article 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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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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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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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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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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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 article 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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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 females/chemical) were used. This 

was considered the minimum number necessary to demonstrate the 

range of effects of the chemical 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 group 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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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 twenty 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 technical staff for any conditions 

requiring possible veterinary care. 
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2.11. HUSBANDRY 

2.11.1. HOUSING 

Animals were doubly housed in elevated, stainless steel, wire mesh 

cages during the first week of the acclimation period and 

individually housed thereafter. 

2.11.2. FEED 

Certified Rodent Diet, No. 5002; (Meal) (PMI Nutrition 

International, St. Louis, Missouri) was available without 

restriction. Fresh feed was presented weekly. 

2.11.3. FEED ANALYSIS 

Analysis of each feed lot used during this study was performed by 

the manufacturer. Results are maintained on file. 

2.11.4. WATER 

Water was available without restriction via an automated watering 

system (Elizabethtown Water Company, Westfield, New Jersey). 

2.11.5. WATER ANALYSIS 

Water analyses are conducted by Elizabethtown Water Company, 

Westfield, New Jersey (Raritan-East Millstone Plant) to ensure 

that water meets standards specified under the EPA Federal Safe 

Drinking Water Act Regulations (40 CFR Part 141). In addition, 

water samples are collected biannually from representative rooms 

in the Testing Facility; chemical and microbiological water 

analyses are conducted on these samples by a subcontract 

laboratory. Results of all water analyses are maintained on file at 

the Testing Facility. 

2.11.6. CONTAMINANTS 

There were no known contaminants in the feed or water which 

were expected to interfere with the results of this study. 
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2.11.7. ENVIRONMENTAL CONDITIONS 

Light/Dark Cycle 

A twelve hour light/dark cycle was 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 articles were mixed with 0.9% saline to 

achieve the desired concentrations. Dosing solutions were prepared either 

the day prior to or the day of dose administration and were stored 

refrigerated when not in use. 

2.13. ANALYSIS OF DOSE SOLUTIONS 

Analysis of the dosing solutions was not performed for this study. 
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2.14. TEST ARTICLE ADMINISTRATION 

2.14.1. ROUTE OF ADMINISTRATION 

Chlorpromazine, D-Amphetamine Sulfate and saline were 

administered by intraperitoneal injection into the abdominal cavity 

near the inguinal region using a sterile needle and syringe of 

appropriate size. Doses were calculated using the most recent 

body weights available. A volume of 5 mL/kg of body weight was 

administered. 

Physostigmine was administered by intraperitoneal injection into 

the abdominal cavity near the inguinal region or by subcutaneous 

injection into an area on the back, using a sterile needle and 

syringe of appropriate size. Intraperitoneal 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 administered 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/NEUROBEHAVIORAL 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 training 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 based 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 landing foot splay. This was done 

by determining the mean and standard deviation for the control 

animals during the performance evaluation for each technician and 

calculating the coefficient of variance [(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 level 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 performed 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 were 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 different 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 test (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 occurred 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 exceeded 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 retrained in the performance of the FOB 

evaluations and subsequently tested for their ability to identify the 

behavioral pattern produced by three 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 presented 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 after 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 animal lying on its side or flattened; 

effects on gait and posture in the open 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 also noted effects on palpebral closure 

in the home cage, with eyelids slightly drooping or half-closed and 

moderate or extreme lacrimation. Six of the eight observers also noted no 

response to an auditory stimulus and 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 predominant 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 effects 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 stimulation 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 noted 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 incidence scores for Observer No. 8 were 

lower than those recorded by the other observers. Since the scores were 

low for all measures of cholinergic activity, 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 16.0b 8.7 8.8 21.1b 

Hindlimb grip strength 15.3 25.3 32.8a

27.2a

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 9.8c 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 repeated with ten untreated 

animals. 

4. CONCLUSION 

Treatment of Sprague-Dawley rats with Chlorpromazine, D-Amphetamine 

Sulfate, or Physostigmine produced patterns of behavior consistent with their 

expected neurological effects. These data provide evidence that the procedures 

used for the Functional Observational Battery evaluations in the Testing Facility 

are capable of detecting major neurotoxic endpoints. In addition, the 

neurobehavioral profiles for each compound were correctly identified by 

Observers 1-2 and 4-9, who were blinded 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 the performance of forelimb and hindlimb 

grip strength measurements and landing foot splay measurements were achieved 

by all observers. In conclusion, Observers 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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Males 

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 significant differences from control mean. 

%CV = (Control Group S.D./Control 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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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 significant differences from control mean. 

%CV = (Control Group S.D./Control 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 

 
Huntingdon Life Sciences 00-6131 Page 927 

 211-TBA-S Final Report 

Males 

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 significant differences from control mean. 

%CV = (Control Group S.D./Control 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 

 
Huntingdon Life Sciences 00-6131 Page 928 

 211-TBA-S Final Report 

Males 

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 significant differences from control mean. 

%CV = (Control Group S.D./Control 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 

 
Huntingdon Life Sciences 00-6131 Page 929 

 211-TBA-S Final Report 

Males 

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 significant differences from control mean. 

%CV = (Control Group S.D./Control 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 

 
Huntingdon Life Sciences 00-6131 Page 930 

 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 significant differences from control mean. 

%CV = (Control Group S.D./Control 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 

 
Huntingdon Life Sciences 00-6131 Page 931 

 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 significant differences from control mean. 

%CV = (Control Group S.D./Control 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 

 
Huntingdon Life Sciences 00-6131 Page 932 

 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 significant differences from control mean. 

%CV = (Control Group S.D./Control 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 significant differences from control mean. 

%CV = (Control Group S.D./Control 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./Control 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./Control 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 describing 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 number 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 Approaches, 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 Avoid, 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 Approaches, 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 Avoid, 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 Approaches, 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 Avoid, 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 Approaches, 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 Avoid, 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 determined 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 Posturea

 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 Approaches, 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 Avoid, 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 Approaches, 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 Avoid, 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 
Huntingdon Life Sciences 00-6131 Page 957 

 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 

Reflex Assessments 

Approach Response Normal: Slowly Approaches, 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 Avoid, or attacks and/or bites 0 0 0 0 

 

Audition Normal: Flinches or Flicks Ears 5 3 5 5 

 No Reaction 0 2 0 0 

 Exaggerated; Jumps, Flips, Bites 0 0 0 0 

 

Pain Normal: Turns/Bites or Walks Forward/Away 5 5 5 4 

 No Reaction 0 0 0 1 

 Apparent Increased Pain 0 0 0 0 

 

Pupil Response Pupil Constricts 5 4 5 0 

 No Constriction 0 1 0 0 

 Miosis 0 0 0 5 

 Mydriasis 0 0 0 0 

 

Pinna Ear Flattens Against Head 3 0 4 0 

 Animal Shakes Head 2 5 1 5 

 No Response 0 0 0 0 

 

Proprioception Normal: Returns Leg to Original Position 5 4 5 5 

 Returns Leg Only Partially to Original Position 0 1 0 0 

 No Response 0 0 0 0 

 

Air Righting Reflex Normal: Lands on All Four Feet 4 5 4 1 

 Slightly Uncoordinated 1 0 1 2 

 Lands on Side 0 0 0 2 

 Lands on Back 0 0 0 0 
Huntingdon Life Sciences 00-6131 Page 958 

 211-TBA-S Final Report 

Females 

Neurobehavioral Positive Control Data: 

Functional Observational Battery 

Incidence Summary of Functional 

Observational Battery Evaluations Appendix T 

Observer 8 Group: 1 2 3 4 

 

Number of Animals Examined 5 5 5 5 

Home Cage Evaluations 

Posture Sitting or Standing Normally 0 4 0 3 

 Alert, Oriented Toward Observer 4 0 4 1 

 Asleep 1 0 0 0 

 Lying on Side 0 0 0 0 

 Flattened 0 1 0 1 

 Crouched 0 0 0 0 

 

Vocalizations Not Present 5 5 5 5 

 Present 0 0 0 0 

Palpebral Closure Eyelids Open 4 1 5 5 

 Eyelids Completely Closed 1 0 0 0 

 Eyelids Slightly Drooping 0 4 0 0 

 Eyelids Half Closed 0 0 0 0 

 

Handling Evaluations 

Ease of Removal Very Easy 3 4 4 3 

 Easy 2 1 1 2 

 Moderately Difficult 0 0 0 0 

 Freezes 0 0 0 0 

 Difficult 0 0 0 0 

 

Ease of Handling Normal (easy, alert) 4 5 5 5 

 Moderately Difficult 1 0 0 0 

 Freezes 0 0 0 0 

 Difficult 0 0 0 0 

 Totally limp 0 0 0 0 

 

Chromodacryorrhea Not Present 5 4 5 5 

 Present 0 1 0 0 

Lacrimation Not Present 5 2 5 5 

 Moderate 0 2 0 0 

 Extreme 0 1 0 0 

 

Coat Normal 5 5 5 5 

 Slightly Soiled 0 0 0 0 

 Moderately Soiled 0 0 0 0 

 Extremely Soiled 0 0 0 0 

 

Salivation Not Present 5 5 5 4 

 Slight 0 0 0 1 

 Moderate 0 0 0 0 

 Extreme 0 0 0 0 

 
Huntingdon Life Sciences 00-6131 Page 959 

 211-TBA-S Final Report 

Females 

Neurobehavioral Positive Control Data: 

Functional Observational Battery 

Incidence Summary of Functional 

Observational Battery Evaluations Appendix T 

Observer 8 Group: 1 2 3 4 

 

Number of Animals Examined 5 5 5 5 

Open Field Evaluations 

Gait and Posture Normal 5 1 5 4 

 Ataxia 0 0 0 0 

 Body Drags 0 3 0 1 

 Limbs Splayed or Dragging 0 2 0 0 

 Hunched Posture 0 0 0 0 

 

Locomotion Normal 5 0 1 4 

 Increased Movement 0 0 4 0 

 Decreased Movement 0 4 0 1 

 None 0 1 0 0 

 

Arousal Normal 5 0 1 4 

 Low, Slight Stupor 0 1 0 1 

 Very Low, Stupor 0 4 0 0 

 High, Slight Excitement, Tense 0 0 4 0 

 Very High, Hyper-Alert 0 0 0 0 

 

Piloerection Not Present 5 5 5 5 

 Present 0 0 0 0 

Exophthalmia Not Present 5 5 5 5 

 Present 0 0 0 0 

Feces Number of Boluses 0 4 0 0 

 Unformed Stool 0 0 0 0 

Urine Number of Pools 0 0 0 1 

 Polyuria 0 0 0 0 

Stereotypic Behavior 

Stereotypic Behavior Not Present 5 5 1 4 

 Present 0 0 4 1 

 

Motor Movements 

Fasciculations Not Present 5 5 5 4 

 Slight 0 0 0 1 

 Severe 0 0 0 0 

 

Convulsions Not Present 5 5 5 5 

 Slight 0 0 0 0 

 Severe 0 0 0 0 

 

Tremors Not Present 5 5 5 5 

 Slight 0 0 0 0 

 Severe 0 0 0 0 
Huntingdon Life Sciences 00-6131 Page 960 

 211-TBA-S Final Report 

Females 

Neurobehavioral Positive Control Data: 

Functional Observational Battery 

Incidence Summary of Functional 

Observational Battery Evaluations Appendix T 

Observer 8 Group: 1 2 3 4 

 

Number of Animals Examined 5 5 5 5 

Reflex Assessments 

Approach Response Normal: Slowly Approaches, 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 Avoid, or attacks and/or bites 0 0 0 0 

 

Audition Normal: Flinches or Flicks Ears 5 3 5 5 

 No Reaction 0 2 0 0 

 Exaggerated; Jumps, Flips, Bites 0 0 0 0 

 

Pain Normal: Turns/Bites or Walks Forward/Away 5 4 5 5 

 No Reaction 0 1 0 0 

 Apparent Increased Pain 0 0 0 0 

 

Pupil Response Pupil Constricts 5 3 4 2 

 No Constriction 0 2 0 1 

 Miosis 0 0 0 2 

 Mydriasis 0 0 1 0 

 

Pinna Ear Flattens Against Head 0 1 1 0 

 Animal Shakes Head 5 4 4 5 

 No Response 0 0 0 0 

 

Proprioception Normal: Returns Leg to Original Position 5 5 5 5 

 Returns Leg Only Partially to Original Position 0 0 0 0 

 No Response 0 0 0 0 

 

Air Righting Reflex Normal: Lands on All Four Feet 5 5 5 4 

 Slightly Uncoordinated 0 0 0 0 

 Lands on Side 0 0 0 1 

 Lands on Back 0 0 0 0 
Huntingdon Life Sciences 00-6131 Page 961 

 211-TBA-S Final Report 

Females 

Neurobehavioral Positive Control Data: 

Functional Observational Battery 

Incidence Summary of Functional 

Observational Battery Evaluations Appendix T 

Observer 9 Group: 1 2 3 4 

 

Number of Animals Examined 5 5 5 5 

Home Cage Evaluations 

Posture Sitting or Standing Normally 4 1 4 0 

 Alert, Oriented Toward Observer 1 0 1 0 

 Asleep 0 0 0 0 

 Lying on Side 0 0 0 0 

 Flattened 0 4 0 5 

 Crouched 0 0 0 0 

 

Vocalizations Not Present 5 5 5 5 

 Present 0 0 0 0 

Palpebral Closure Eyelids Open 5 1 5 5 

 Eyelids Completely Closed 0 0 0 0 

 Eyelids Slightly Drooping 0 4 0 0 

 Eyelids Half Closed 0 0 0 0 

 

Handling Evaluations 

Ease of Removal Very Easy 3 4 4 4 

 Easy 2 1 1 1 

 Moderately Difficult 0 0 0 0 

 Freezes 0 0 0 0 

 Difficult 0 0 0 0 

 

Ease of Handling Normal (easy, alert) 5 5 5 5 

 Moderately Difficult 0 0 0 0 

 Freezes 0 0 0 0 

 Difficult 0 0 0 0 

 Totally limp 0 0 0 0 

 

Chromodacryorrhea Not Present 5 4 5 5 

 Present 0 1 0 0 

Lacrimation Not Present 5 1 5 3 

 Moderate 0 2 0 2 

 Extreme 0 2 0 0 

 

Coat Normal 5 5 4 2 

 Slightly Soiled 0 0 1 3 

 Moderately Soiled 0 0 0 0 

 Extremely Soiled 0 0 0 0 

 

Salivation Not Present 5 5 5 3 

 Slight 0 0 0 2 

 Moderate 0 0 0 0 

 Extreme 0 0 0 0 

 
Huntingdon Life Sciences 00-6131 Page 962 

 211-TBA-S Final Report 

Females 

Neurobehavioral Positive Control Data: 

Functional Observational Battery 

Incidence Summary of Functional 

Observational Battery Evaluations Appendix T 

Observer 9 Group: 1 2 3 4 

 

Number of Animals Examined 5 5 5 5 

Open Field Evaluations 

Gait and Posture Normal 5 1 5 0 

 Ataxia 0 0 0 2 

 Body Drags 0 2 0 3 

 Limbs Splayed or Dragging 0 3 0 1 

 Hunched Posture 0 0 0 0 

 

Locomotion Normal 3 0 1 0 

 Increased Movement 0 0 4 0 

 Decreased Movement 2 1 0 3 

 None 0 4 0 2 

 

Arousal Normal 3 0 0 0 

 Low, Slight Stupor 2 1 0 0 

 Very Low, Stupor 0 4 0 5 

 High, Slight Excitement, Tense 0 0 5 0 

 Very High, Hyper-Alert 0 0 0 0 

 

Piloerection Not Present 5 5 5 5 

 Present 0 0 0 0 

Exophthalmia Not Present 5 5 5 5 

 Present 0 0 0 0 

Feces Number of Boluses 0 3 0 0 

 Unformed Stool 0 0 0 0 

Urine Number of Pools 0 2 0 0 

 Polyuria 0 0 0 0 

Stereotypic Behavior 

Stereotypic Behavior Not Present 5 5 0 0 

 Present 0 0 5 5 

 

Motor Movements 

Fasciculations Not Present 5 5 5 0 

 Slight 0 0 0 3 

 Severe 0 0 0 2 

 

Convulsions Not Present 5 5 5 5 

 Slight 0 0 0 0 

 Severe 0 0 0 0 

 

Tremors Not Present 5 5 5 5 

 Slight 0 0 0 0 

 Severe 0 0 0 0 
Huntingdon Life Sciences 00-6131 Page 963 

 211-TBA-S Final Report 

Females 

Neurobehavioral Positive Control Data: 

Functional Observational Battery 

Incidence Summary of Functional 

Observational Battery Evaluations Appendix T 

Observer 9 Group: 1 2 3 4 

 

Number of Animals Examined 5 5 5 5 

Reflex Assessments 

Approach Response Normal: Slowly Approaches, 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 Avoid, 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 
Huntingdon Life Sciences 00-6131 Page 964 

 211-TBA-S Final Report 

 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 

Introduction ...................................................................................................... 965 

Materials and Methods .................................................................................... 965 

Results ............................................................................................................ 980 

Discussion ....................................................................................................... 981 

Conclusion ....................................................................................................... 981 

Mean Motor Activity Values ............................................................................. 982 
Huntingdon Life Sciences 00-6131 Page 965 

 211-TBA-S Final Report 

 Neurobehavioral Positive Control Data: 

Motor Activity Appendix T 

1. INTRODUCTION 

This study was designed to assess the effect 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 Positive 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 provide 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 compliance 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 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. 
Huntingdon Life Sciences 00-6131 Page 966 

 211-TBA-S Final Report 

 Neurobehavioral Positive Control Data: 

Motor Activity Appendix T 

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 DIRECTOR1

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 Director. George Brown, 

M.S. assumed all Study Director responsibilities following Dr. Patyna’s departure from Huntingdon Life 

Sciences, effective 22 December 2000. Effective 10 Nov 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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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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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 for the duration of the study. Each 

cage was also provided with a cage card which contained the study 

number and animal number. 
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2.13. VETERINARY CARE 

Animals were monitored by the technical staff for any conditions 

requiring possible veterinary care. 

2.14. ANIMAL HUSBANDRY 

2.14.1. HOUSING 

Each female rat and litter, from 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 (Raritan-East Millstone Plant) to 

ensure that water meets standards specified under the EPA Federal 

Safe Drinking Water Act Regulations (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 water analyses were conducted on 

these samples by a subcontract laboratory. Results were 

maintained on file at the Testing 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. Contaminant 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 more 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 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. 

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 counted on Postnatal Days 10,13,17, 

and 21. 

2.17.2. WEANING 

Pups were weaned on Postnatal Day 21 and housed two or three of 

the same sex per cage. 

2.17.3. LOCOMOTOR ACTIVITY BEHAVIORAL 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 female rats were euthanized via an 

overdose of carbon dioxide and discarded. After the locomotor activity 

behavioral assessment on 62, the rats were 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, all 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 occurred 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. However, 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 the 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 washout 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 Pathologist Associates International 
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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 overall 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 investigation 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 locomotor activity for male and female rats 

combined, on Postnatal Days 17, 21, and 62, when compared to the control group. 

Statistically significant decreases in locomotor activity were observed for Group 

III male and female rats individually, on Postnatal Days 17, 21, and 62 only. 
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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 were observed equally in both male and 

female rats in the treatment groups. 

Rats treated with Amphetamine exhibited 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 exhibited 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 Postnatal 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 activity in this study suggests the importance 

of evaluating behavioral endpoints at several 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 intraperitoneal 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 locomotor activity alterations in young Sprague 

Dawley rats. 
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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 significant differences from control 

x: a>b indicates mean for group a significantly greater than mean for group b in interval x. 

I = control, II = amphetamine, III = chlorpromazine 
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Males 

Neurobehavioral Positive Control Data: 

Motor Activity 

Mean Motor Activity Values 

Postnatal Day 13 Appendix T 

 Interval 

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

 

Group I – Control 

Mean 27.70 12.30 18.40 23.60 10.60 14.10 12.90 7.40 7.10 9.40 8.80 9.90 

S.D. 21.88 15.32 30.96 40.81 12.43 20.80 12.81 10.24 11.32 10.15 7.18 11.90

N 10 10 10 10 10 10 10 10 10 10 10 10 

 

Group II – 2.0 mg/kg Amphetamine 

Mean 37.70 21.10 18.40 17.20 10.20 9.20 6.10 8.10 19.60 36.70 28.80 41.60

S.D. 39.38 25.14 29.86 25.17 14.29 11.15 8.48 9.05 20.01 43.02 39.89 58.17

N 10 10 10 10 10 10 10 10 10 10 10 10 

 

Group III – 10.0 mg/kg Chlorpromazine 

Mean 14.80 8.10 8.80 12.30 7.40 5.70 8.20 25.70 12.80 12.00 8.60 17.70

S.D. 26.95 15.17 13.21 21.67 11.84 13.05 11.38 55.13 22.52 27.05 27.20 54.93

N 10 10 10 10 10 10 10 10 10 10 10 10 
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Females 

Neurobehavioral Positive Control Data: 

Motor Activity 

Mean Motor Activity Values 

Postnatal Day 13 Appendix T 

Interval 

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

 

Group I – Control 

Mean 25.00 14.70 17.10 20.90 16.60 14.30 9.70 6.20 12.50 10.00 7.60 5.70 

S.D. 32.00 17.81 24.47 19.77 19.29 14.19 11.61 8.98 13.82 10.72 11.35 4.27 

N 10 10 10 10 10 10 10 10 10 10 10 10 

 

Group II – 2.0 mg/kg Amphetamine 

Mean 36.30 19.40 29.10 24.00 30.40 26.40 41.30 33.20 38.90 52.00 49.90 31.90

S.D. 45.41 26.53 40.16 34.50 51.56 49.40 65.11 55.36 74.84 87.22 55.36 38.23

N 10 10 10 10 10 10 10 10 10 10 10 10 

 

Group III – 10.0 mg/kg Chlorpromazine 

Mean 4.00 1.50 11.40 24.30 0.80 2.50 3.50 4.90 3.10 5.10 6.20 1.90 

S.D. 8.07 3.75 19.41 50.71 1.62 4.06 5.76 9.34 5.51 10.79 10.25 2.88 

N 10 10 10 10 10 10 10 10 10 10 10 10 

 
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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 
Huntingdon Life Sciences 00-6131 Page 990 

 211-TBA-S Final Report 

Females 

Neurobehavioral Positive Control Data: 

Motor Activity 

Mean Motor Activity Values 

Postnatal Day 62 Appendix T 

 Interval 

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

 

Group I – Control 

Mean 171.10 153.40 143.90 124.00 103.60 95.50 91.00 55.80 57.60 37.90 32.30 32.20

S.D. 24.85 23.08 37.22 35.57 29.28 37.63 28.77 30.97 42.84 39.45 42.60 30.80

N 10 10 10 10 10 10 10 10 10 10 10 10 

 

Group II – 2.0 mg/kg Amphetamine 

Mean 150.10 144.90 133.60 132.00 137.80 150.50 120.30 130.40 124.00 134.10 143.30 105.30

S.D. 45.19 29.05 31.40 33.97 31.90 46.83 39.06 43.90 39.58 35.23 31.92 40.12

N 10 10 10 10 10 10 10 10 10 10 10 10 

 

Group III – 10.0 mg/kg Chlorpromazine 

Mean 33.30 18.20 12.40 10.20 8.70 0.90 4.10 2.30 6.90 5.50 2.50 0.00 

S.D. 43.27 30.08 33.27 13.49 23.79 2.51 8.57 2.63 9.69 8.45 5.04 0.00 

N 10 10 10 10 10 10 10 10 10 10 10 10 
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211-TBA-S Final Report

Neuropathology Historical Control

Preface Appendix U




Page 996
PROTOCOL

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 

 

HLS Study No.: 00-6131 

Sponsor Study No.: 211-TBA-S 

Protocol No.: Final 

Date: 29 April 2002 

Page 997

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Sponsor Study No. 211-TBA-S Final 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 

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Sponsor Study No. 211-TBA-S Final Protocol 

10.4. DURATION OF ADMINISTRATION ............................................................ 16 

10.5. ADMINISTRATION OF TEST SUBSTANCE ................................................ 16 

10.6. EXPOSURE CONCENTRATION DETERMINATION ................................. 17 

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

10.8. CHAMBER AND EXPOSURE ROOM ENVIRONMENT ............................. 18 

10.9. SUMMARY OF CHAMBER 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

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Sponsor Study No. 211-TBA-S Final Protocol 

1. INTRODUCTION

HLS Study No. 00-6131 

Sponsor Study No. 211-TBA-S 

Study Title 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 

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. 

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Sponsor Study No. 211-TBA-S Final 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 (respiratory tract histopathology sections, 

only), Public Draft June 1996. 

US EPA OPPTS Health Effects Test Guidelines 870.6200, Neurotoxicity 

Screening Battery, August 1998. 

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

June 1994. 

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

US EPA OPPTS Health Effects Test Guidelines 870.7800, Immunotoxicity, 

August 1998. 

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

US EPA OPPTS Health Effects Test Guidelines 870.5395, Mammalian 

Erythrocyte Micronucleus Test, August 1998. 

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

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Sponsor Study No. 211-TBA-S Final 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 Accreditation of Laboratory Animal Care 

International (AAALAC). 

3.4. ANIMAL WELFARE ACT COMPLIANCE 

This study will comply with all appropriate parts of the Animal Welfare Act 

regulations: 9 CFR Parts 1 and 2 Final Rules, Federal Register, Volume 54, 

No. 168, August 31, 1989, pp. 36112-36163 effective October 30, 1989 and 

9CFR Part 3 Animal Welfare Standards; Final Rule, Federal Register, 

Volume 56, No. 32, February 15, 1991, pp. 6426-6505 effective March 18, 

1991. The Sponsor should make particular note of the following: 

1. The Sponsor's signature on this protocol documents for the study 

described, there are no generally accepted non-animal alternatives and 

the study does not unnecessarily duplicate previous experiments. 

2. All procedures used in this study have been designed to avoid 

discomfort, distress and pain to the animals. All methods are described 

in this study protocol or in written laboratory standard operating 

procedures. 

3. Any procedures outlined in this study protocol which are expected to 

cause more than momentary or slight pain or distress to the animals will 

be performed with appropriate sedatives, analgesics or anesthetics 

unless the withholding of these agents is justified for scientific reasons, 

in writing, by the Sponsor and the Study Director and approved by the 

IACUC; in which case the procedure will continue for the minimum 

time necessary. Documentation of the justification for withholding 

treatment for pain or distress and IACUC approval of the procedures 

will be made prior to study initiation on the IACUC Protocol Review 

form. 

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Sponsor Study No. 211-TBA-S Final Protocol 

4. Animals experiencing more than momentary or slight pain or distress due 

to test substance or emergency situations such as injury or illness will be 

treated by the Testing Facility's veterinarian staff with approved 

analgesics or agents to relieve pain. If possible, the Study Director will 

be consulted prior to treatment; however, the veterinary staff is 

authorized to 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 situations in as timely a manner as 

possible. 

5. Methods of euthanasia used during this study are in conformance with 

the above referenced regulations. 

6. The number of animals in the protocol is considered the minimum 

necessary to satisfy the regulatory requirement (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 Subcommittee has reviewed this protocol and 

found it to be in compliance with all appropriate regulations. 

4. QUALITY ASSURANCE MONITORING 

The Huntingdon Life Sciences Quality Assurance Unit will monitor the facilities, 

equipment, personnel, methods, practices, records and controls used in this study to 

assure that they are in conformance with this protocol, company SOP's, and the 

appropriate Good Laboratory Practice regulations. 

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Sponsor Study No. 211-TBA-S Final 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 verbally, such changes will be honored by the 

Testing Facility and will be followed by a written verification. All protocol 

modifications will be signed by the Study Director and a Sponsor representative. 

Any modifications potentially affecting animal welfare will also be signed by two 

members of the Institutional Animal Care and Use Committee prior to the 

modification's implementation. 

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

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Huntingdon Life Sciences Study No. 00-6131 

Sponsor Study No. 211-TBA-S 

7. EXPERIMENTAL DESIGN 

Group Exposure 

Levela

Number of Animals (MAIN and RECOVERY STUDIES) 

(mg/m3) 

Initialb Clinical Laboratory Studies Necropsyd Mic

4 Weeks & 

Terminal 

Recoveryc Terminal Recoveryc Term

M F M F M F M F M F M 

I (control) 0 (air only) 20 20 10 10 A.N. A.N. 10 10 10 10 10 II(low) 2,000 10 10 10 10 - - 10 10 - - A.R. III(mid) 10,000 10 10 10 10 - - 10 10 - - A.R. IV(high) 20,000 20 20 10 10 A.N. A.N. 10 10 10 10 10 

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/group designated for the Terminal Sacrifice (Main Study animals) and 10/sex/group (I & IV only) designated fo

Sacrifice (Recovery Study animals). 

cThe recovery period will be at least 4 weeks. dComplete postmortem evaluations will also be performed on animals which are found dead or euthanatized in a moribund condition during the course of t

A.N. = As Needed (additional cost) based on results of Terminal Sacrifice evaluations. A.R. = As Required: 1) Target organs/tissues identified by Group IV evaluations at Terminal Sacrifice (additional cost); 2) Lungs will be examined micro

groups at the Terminal sacrifice interval; 3) All tissues from animals found dead or euthanatized in a moribund condition during the course of the stu

lesions (additional cost). M = Male; F = Female; The first day of exposure will be Day 0. 
Huntingdon Life Sciences Study No. 00-6131 

Sponsor Study No. 211-TBA-S EXPERIMENTAL DESIGN (CONTINUED) 

Group Exposure 

Levela

Number of Animals (SATELLITE STUDIES) 

(mg/m3) 

Initialb Neuropathology GFAP Immunotoxicity 

(M

M F M F M F M F M 

I (control) 0 (air only) 15 25 5 5 5 5 - 10 II(low) 2,000 15 25 5 5 5 5 - 10 III(mid) 10,000 15 25 5 5 5 5 - 10 IV(high) 20,000 15 25 5 5 5 5 - 10 

 aExposures will be 6 hours/day, generally 5 days/week for 13 weeks for at least 65 exposures for Neuropathology and GFAP Studies. Exposures wi

days/week for 4 weeks for at least 20 exposures for Genotoxicity and Immunotoxicity Studies.

bThis number of animals includes 5/sex/group designated for neuropathology after the Terminal Sacrifice, 5/sex/group designated for GFAP evaluation a

females/group designated for Immunotoxicity (PFC) evaluations following a 4 week exposure period and 5/sex/group designated for Genotoxicity (Micro

Exchange) evaluations following a 4 week exposure period. For the Immunotoxicity and Genotoxicity evaluations, separate groups of animals (10 

respectively) will be used as positive control animals for these evaluations. For Immunotoxicity evaluations, positive control rats will be given a dose of 

(CP) intraperitoneally once per day for 4 days prior to sacrifice. For Genotoxicity evaluations, positive control rats will be given a dose of 5 mg/kg (SCE) CP intraperitoneally within 24 hours prior to sacrifice. These positive control animals will not be chamber exposed prior to dosing and sacrifice. The CP 

will 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 Cell 
Huntingdon 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, expiration date (if available) and handling 

procedures, as well as other pertinent information will be documented in the study 

data. 

8.2. IDENTIFICATION OF TEST SUBSTANCE 

Unless otherwise noted, the identity, strength, composition, stability and method of 

synthesis, fabrication and/or derivation of each batch of the test substance will be 

documented by the Sponsor before its use in the study. This documentation will be 

maintained by the Sponsor at the address indicated on page 2 of this protocol. The 

Sponsor will conduct a purity analysis of the test substance by GC prior to the 

start of this study. 

8.3. ARCHIVAL SAMPLES 

An archival sample from each lot of test substance will be taken and stored in the 

Archives of the Testing Facility. If multiple studies are conducted with the same 

substance, a common archival sample may be taken and appropriately labeled. 

8.4. UNUSED TEST SUBSTANCE 

The unused portion of the test substance as well as any empty test substance 

containers will be returned to the Sponsor’s designee (see page 2) following 

completion of the subsequent reproduction study 00-4206. Empty test substance 

containers will be returned to the Sponsor’s designee on an as needed basis. The 

Sponsor will be responsible for tracking their disposition. 

In the event the Sponsor wishes the Testing Facility to arrange for disposal, a cost 

for this service will be provided. 

9. TEST ANIMALS 

Albino Rats (Outbred) VAF/Plus® 

9.1. SPECIES 

Sprague Dawley - derived (CD®) 

[Crl: CD® IGS BR] 

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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 commonly 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. Animals outside of this range may be 

used at the discretion of the Study Director. 

9.5. ACCLIMATION PERIOD 

Approximately two weeks; all animals will be checked for viability twice daily. 

Prior to assignment to study all animals will be examined by a staff member to 

ascertain suitability for study. 

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Sponsor Study No. 211-TBA-S Final Protocol 

9.6. ANIMAL HUSBANDRY/NON-EXPOSURE 

9.6.1. HOUSING 

Animals younger than 5 weeks at receipt will be housed 2/cage during the 

initial week of acclimation. Animals 5 weeks or older will be housed 

individually in suspended stainless steel wire mesh cages. 

9.6.2. FEED & FEED ANALYSIS 

Certified Rodent Diet, No. 5002; (Meal) (PMI Feeds, Inc., St. Louis, MO) 

ad libitum. 

Analytical certification of batches of feed will be maintained on file by the 

manufacturer. There are no known contaminants in the feed that are 

expected to interfere with the results of this study. 

9.6.3. WATER & WATER ANALYSIS 

Elizabethtown Water Company, Westfield, NJ; ad libitum, via automated 

watering system. 

Monthly water analyses are conducted by Elizabethtown Water Company to 

assure that water meets standards specified under the EPA Federal Safe 

Drinking Water Act Regulations (40 CFR Part 141). Water analysis, 

provided by the supplier, will be maintained on file at the Testing Facility. 

In addition, internal biannual chemical and microbiological water samples 

collected from representative rooms in this facility are assayed. Results are 

maintained on file. There are no known contaminants in the water that are 

expected to interfere with the results of this study. 

9.6.4. VETERINARY CARE 

Animals are monitored by the technical staff for any conditions requiring 

possible veterinary care. If any such conditions are identified, a staff 

veterinarian will be notified for an examination and evaluation. Animals 

will be treated as outlined in the Animal Welfare Act Compliance section 

of this protocol. 

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9.6.5. ENVIRONMENTAL CONDITIONS 

Light/Dark Cycle

Twelve hour light/dark cycle provided via automatic timer. 

Temperature

Temperature will be monitored in accordance with Testing Facility SOPs 

to ensure that the desired range of 18-26°C is maintained to the maximum 

extent possible. 

Humidity

Humidity will be monitored in accordance with Testing Facility SOPs to 

ensure that the desired range of 30 to 70% is maintained to the maximum 

extent possible. 

9.7. ANIMAL HUSBANDRY DURING EXPOSURE 

9.7.1. HOUSING Individual in suspended, stainless 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 the basis of pretest physical examinations, 

body weight data and any other pretest evaluations will be randomly assigned, by 

sex, to control or treated groups in an attempt to equalize mean group body weights. 

 Individual weights of animals placed on test shall be within +/- 20% of the mean 

weight for each sex for each study. The Main/Recovery study and each of the 

Neuropathology and 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. 

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9.9. ANIMAL IDENTIFICATION 

Each animal will be assigned a temporary identification number upon receipt. After 

selection for study, each animal will be ear-tagged by the Testing Facility with a 

number assigned by the Testing Facility. The number assigned by the Testing 

Facility plus the study number will comprise the unique animal number for each 

animal. If the tag is lost, it will be replaced or the animal will be tattooed for 

identification. Each cage will be provided with a cage card that will be color-coded 

for exposure level identification and will contain the study number and animal 

number. 

10. TEST SUBSTANCE ADMINISTRATION 

10.1. ROUTE OF ADMINISTRATION 

Inhalation via whole-body exposures. 

10.2. JUSTIFICATION FOR ROUTE OF ADMINISTRATION 

The inhalation route is one of the potential routes of human exposure to this test 

substance and is the route specified in the referenced guidelines. 

10.3. FREQUENCY OF ADMINISTRATION 

Once daily, 6 hours/day. 

10.4. 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/Neuropathology/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 administration will continue through the day prior to 

necropsy for all animals except the animals in Groups I and IV designated for 

recovery will be held for at least 4 weeks prior to necropsy. 

10.5. ADMINISTRATION OF TEST SUBSTANCE 

The test substance will be administered as a vapor in the breathing air of the 

animals. The test atmosphere will be generated by an appropriate procedure 

determined during pre-study trials. The trials will be performed to evaluate the 

optimal set of conditions and equipment to generate a stable atmosphere at the 

target exposure levels and maintain uniform conditions throughout the exposure 

chambers. The method will be described in the raw data of the study and in the 

report. 

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The whole-body exposure chambers will each have a volume of approximately 

1000 liters. Two chambers will be used for each exposure level, in general one for 

male animals and one for female animals. Each chamber will be operated at a 

minimum flow rate of 200 liters per minute. The final airflow will be set to provide 

at least one air change in 5.0 minutes (12 air changes/hour) and a T99 equilibrium 

time of at most 23 minutes. This chamber size and airflow rate is considered 

adequate to maintain the oxygen level at least 19% and the animal loading factor 

below 5%. At the end of each exposure, all animals will remain in the chamber for 

a minimum of 30 minutes. During this time, the chamber will be operated at 

approximately the same flow rate using clean air only. 

10.6. EXPOSURE CONCENTRATION DETERMINATION 

A nominal exposure concentration will be calculated. The flow of air through the 

chamber will be monitored using appropriate calibrated equipment. The test 

substance consumed during the exposure will be divided by the total volume of air 

passing through the chamber (volumetric flow rate times total exposure time) to 

give the nominal concentration. 

During each exposure, measurements of airborne concentrations will be performed in 

the animals’ breathing zone at least 4 times using an appropriate sampling procedure 

and IR analytical procedure. Also, one sample per chamber per week will be 

analyzed by gas chromatography (GC) to characterize at least 10 major components 

(comprising at least 80% by weight of the test substance) to show test substance 

stability and comparison between the neat liquid test substance and the vaporized test 

atmospheres. 

If more than the normal amount of trials is required because of test substance 

generation or monitoring problems (80 technician hours), the Sponsor will be 

consulted prior to additional trials (additional cost). 

10.7. PARTICLE SIZE DISTRIBUTION ANALYSIS 

During each week of exposure, particle size determinations will be performed using 

a TSI Aerodynamic Particle Sizer to characterize the aerodynamic particle size 

distribution of any aerosol present. 

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10.8. CHAMBER AND EXPOSURE ROOM ENVIRONMENT 

Chamber temperature, humidity, airflow rate and static pressure will be monitored 

continuously and recorded every 30 minutes during exposure. Chamber 

temperature and relative humidity will be maintained, to the maximum extent 

possible, between 20 to 24°C and 40 to 60%, respectively. Chamber oxygen levels 

(maintained at least 19%) will be measured pretest and at the beginning, middle 

and end of the study. 

Air samples will be taken in the vapor generation area pretest and at the 

beginning, middle and end of the study. Light (maintained approximately 30-40 

foot-candles at 1.0 meter above the floor) and noise levels (maintained below 85 

decibels) in the exposure room will be measured pretest and at the beginning, 

middle and end of the study. 

10.9. SUMMARY OF CHAMBER ACTIVITY 

The minimum frequency of chamber activity is summarized below: 

Activity 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 

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11. EXPERIMENTAL EVALUATION 

11.1. OBSERVATIONS 

11.1.1. VIABILITY CHECKS (IN-CAGE) 

Observations for mortality, general appearance and signs of severe toxic 

or pharmacological effects will be made at least twice daily. Animals in 

extremely poor health or in a possible moribund condition will be 

identified for further monitoring and possible euthanasia. 

11.1.2. PHYSICAL EXAMINATIONS 

All animals will be observed as a group at least once during each exposure. 

Each animal (except Neuropathology, GFAP, Genotoxicity and 

Immunotoxicity animals) will be removed from its cage and examined 

twice pretest and once weekly during the study period. Examinations will 

include observations of general condition, skin and fur, eyes, nose, oral 

cavity, abdomen and external genitalia as well as evaluations of 

respiration, palpation for tissue masses, circulatory effects, autonomic 

effects, central nervous system effects, changes in motor activity, and 

reactivity to handling or sensory stimuli. Neuropathology, GFAP, 

Genotoxicity and Immunotoxicity animals will be removed from their 

cage and examined once pretest before selection onto study. 

11.2. BODY WEIGHTS 

Body weights will be recorded at least twice pretest, weekly during the study and at 

termination (fasted). 

11.3. FEED CONSUMPTION 

Feed consumption will be measured (weighed) during the week prior to treatment 

initiation and weekly throughout the study. Feed will be available without 

restriction 7 days/week except during inhalation exposures and when fasting prior 

to blood collection. Animals will be presented with weighed feeders at the 

scheduled intervals. After 7 days, the feeders will be reweighed and the resulting 

weight subtracted from the initial feeder weight to obtain the grams of feed 

consumed per animal per week. The grams consumed per kilogram of body 

weight per day will then be calculated for each animal. 

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11.4. OPHTHALMOSCOPIC EXAMINATION 

All animals (except Genotoxicity and Immunotoxicity animals) will be examined 

pretest and at study termination (optional at recovery, additional cost). Eyelids, 

lacrimal apparatus and conjunctiva will be examined grossly; cornea, anterior 

chamber, lens, vitreous humor, retina and optic disc will be examined by indirect 

ophthalmoscopy. The eyes will be examined after instillation of a mydriatic such as 

tropicamide. 

11.5. NEUROBEHAVIORAL STUDIES 

Testing will be staggered over several sessions and will be conducted on nonexposure days at least 16 hr post-exposure. Each session will consist of 10 

animals/sex/exposure group. Testing will be performed on the Neuropathology 

and the GFAP animals. Noise level will be maintained within a level of 55 to 65 

decibels by a white noise generator. Temperature, humidity and illumination will 

be measured and recorded to ensure that variations in environmental conditions 

are minimal during all evaluations. The functional observational battery will be 

performed for all animals before evaluation of motor activity. 

11.5.1. MOTOR ACTIVITY 

Activity will be monitored using an automated Photobeam Activity 

System (San Diego Instruments, Inc.). Sessions will be 60 minutes in 

length; each session will be divided into 12, 5-minute intervals. The time 

of testing will be balanced across treatment groups. Positive historical 

control data demonstrating the sensitivity of the procedure will be 

documented in the final report. 

Number of Animals/Intervals 10/sex/group pretest and 

during the 4th, 8th and 13th 

weeks of exposure (optional 

at recovery, additional cost) 

11.5.2. FUNCTIONAL OBSERVATIONAL BATTERY 

The functional observational battery will include assessments in the home 

cage, open field, and reflex testing. The time of testing will be balanced 

across treatment groups. All observations will be performed by trained 

observers who will be unaware of the animals’ treatment. Positive 

historical control data demonstrating the sensitivity of the procedure will 

be documented in the final report. 

Number of Animals/Intervals 10/sex/group pretest and 

during the 4th, 8th and 13th 

weeks of exposure (optional 

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at recovery, additional cost) 

Parameters Evaluated 

Evaluations will be performed according to the Testing Facility’s Standard 

Operating Procedures which include defined scales for each function 

listed below. In addition, descriptive comments can be made during the 

testing period about behaviors that are not adequately described by the 

defined scales. Testing will proceed from least to the most interactive with 

the subject. 

1. Assessment of signs of autonomic function: 

• Ranking and degree of lacrimation and salivation 

• Presence or absence of piloerection and exophthalmos 

• Count of urination and defecation 

• Pupillary function and degree of palpebral closure 

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

abnormal movements both in home cage and the open field 

3. Ranking of reactivity to general stimuli, such as removal from 

cage or handling, with a range of severity scores from no reaction 

to hyperactivity. 

4. Ranking of the subjects general level of activity during 

observations of the unperturbed subject in the open field, with a 

range of severity scores from unresponsive to hyperactive. 

5. Description and incidence of posture and gait evaluations in the 

home cage and open field. 

6. Ranking of gait abnormalities 

7. Forelimb and hindlimb grip strength measurements 

8. Quantitative measure 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 appearance, or red/crusty deposits 

around the eyes, nose, or mouth, and any other observations that 

may facilitate interpretation of the data. 

12. Ranking of righting ability 

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11.6. CLINICAL LABORATORY STUDIES 

Clinical pathology procedures and parameters are based on those recommended in 

guidelines published by the Joint Scientific Committee for International 

Harmonization of Clinical Pathology Testing in “Harmonization of Animal 

Clinical Pathology Testing in Toxicity and Safety Studies”, Fund. Appl. Tox.: 29, 

198-201 (1996). 

Blood will be obtained from lightly anesthetized (carbon dioxide/oxygen; 

60%/40%) animals via puncture of the orbital sinus (retrobulbar). Rats will be 

fasted overnight prior to blood collection. The Main Study animals will be bled at 

the Interim (~4 weeks) and Terminal intervals. The Recovery Study animals will be 

bled (optional) at the Recovery interval. Blood will be collected and studies 

performed as follows: 

11.6.1. HEMATOLOGY AND COAGULATION STUDIES 

Blood (~0.25 mL) for hematology studies will be collected into tubes 

containing EDTA anticoagulant. Blood (~1.0 mL) for coagulation studies 

will be collected into tubes containing sodium citrate anticoagulant. 

Number of Animals 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 

4th week interval) for clinical chemistry studies will be collected into tubes 

with no anticoagulant, allowed to clot, and centrifuged to obtain serum. 

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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 unconjugated (indirect) 

glucose 

total protein 

albumin 

globulin (calculated as total protein - albumin = globulin) 

albumin/globulin ratio (calculated) 

creatinine 

sodium 

potassium 

chloride 

calcium 

phosphorus, inorganic 

bile acids 

cholesterol (total) 

11.6.3. RETENTION/STORAGE OF SPECIMENS 

Any remaining (frozen) serum, which may have limited storage stability, 

will be stored for up to six months after completion of assays and will then 

be discarded. Peripheral blood smears will be retained and archived with 

the study. 

11.7. GENOTOXICITY EVALUATIONS 

 Note: The same animals will be used for both of the following evaluations except a 

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 separate positive control group will be used for each evaluation. The 

cyclophosphamide will be received from BioReliance as acquired from Sigma 

Chemical Company. The identity, strength, purity and composition or other 

characteristics to define the positive control article will not be determined by 

the Testing Facility. The positive control article will be characterized as per the 

Certificate of Analysis on file with the Testing Facility. The stability of the 

positive control article will not be determined by the Testing Facility. Analyses 

to determine the uniformity (as applicable) or concentration of the positive 

control mixture will not be performed by the Testing Facility. The stability of 

the positive control article mixture will not be determined by the Testing 

Facility. 

SCE Evaluations 

Sprague Dawley-derived (CD) rats, 5/sex/group, will be exposed to Gasoline 

TBA Vapor Condensate via inhalation for a 4 week (5 days per week) exposure 

period by Huntingdon Life Sciences (PRC). PRC personnel will also perform the 

treatment of non-exposed (5/sex) positive control animals (5 mg/kg 

cyclophosphamide, IP with a 0.5 mg/mL solution @ 10 mL/kg, within 24 hours 

prior to sacrifice). BioReliance personnel will sacrifice (isoflurane inhalation) the 

animals on the day after the final exposure, collect blood (2-4 mL into sodium 

heparin tubes) from the abdominal aorta and transport the samples on wet ice to 

BioReliance, Rockville, MD (attn: Ramadevi Gudi, Ph.D.). Upon receipt, samples 

will be processed and evaluated for determination of SCE response as detailed in 

the referenced guidelines and procedures (see Appendix C). The Testing Facility 

will be responsible for the GLP compliance of this subcontractor. 

Micronucleus Evaluations 

Sprague Dawley-derived (CD) rats, 5/sex/group, will be exposed to Gasoline 

TBA Vapor Condensate via inhalation for a 4 week (5 days per week) exposure 

period by Huntingdon Life Sciences (PRC). PRC personnel will also perform the 

treatment of non-exposed (5/sex) positive control animals (40 mg/kg 

cyclophosphamide, IP with a 4.0 mg/mL solution @ 10 mL/kg, within 24 hours 

prior to sacrifice). The positive control animals will be sacrificed under CO2

anesthesia. BioReliance personnel will remove the femurs immediately after the 

blood sample collection for the SCE evaluation and fixed, unstained slides will be 

prepared and HLS personnel will ship via overnight delivery to Eye Research 

Centre, Eye, Suffolk, UK (attn: Christine Mason, B.Sc.). Upon receipt, slides will 

be stained and evaluated for determination of micronucleus response as detailed 

in the referenced guidelines and procedures (see Appendix B). The Testing Facility 

will be responsible for the GLP compliance of this subcontractor. 

11.8. IMMUNOTOXICITY EVALUATIONS 

Female Sprague Dawley-derived (CD) rats, 10/group, will be exposed to 

Gasoline TBA Vapor Condensate via inhalation for a 4 week (5 days per week) 

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exposure period by Huntingdon Life Sciences (PRC). PRC personnel will also 

perform the treatment of non-exposed (10 females) positive control animals (50 

mg/kg cyclophosphamide, IP with a 5 mg/mL solution @ 10 mL/kg, once per day 

for four days prior to sacrifice). Four days prior to sacrifice, animals will be 

sensitised with sheep red blood cells (2 x 108

) via tail vein injection by 

ImmunoTox, Inc. personnel. PRC personnel will, on the day after the final 

exposure, collect and freeze (<-20o

C) blood (serum) samples (orbital collection 

anesthetized via carbon dioxide/oxygen inhalation), and then sacrifice (carbon 

dioxide inhalation) the animals, the thymus will be removed, weighed, and 

preserved (10% neutral buffered formalin) for possible histopathology, the 

spleens will be aseptically removed, weighed and shipped intact in 

HEPES/EBSS/Gentamicin solution (prepared at PRC) in individual shipping 

containers on wet ice (2-8o

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 (see Appendix D). The serum samples will be 

retained by the Testing Facility until the spleen samples are evaluated for all 

subchronic inhalation studies in this test program and then discarded if not needed 

by the subcontractor. The Testing Facility will be responsible for the GLP 

compliance of this subcontractor. 

The cyclophosphamide will be received from Sigma Chemical Company. The 

identity, strength, purity and composition or other characteristics to define the 

positive control article will not be determined by the Testing Facility. The 

positive control article will be characterized as per the Certificate of Analysis on 

file with the Testing Facility. The stability of the positive control article will not 

be determined by the Testing Facility. Analyses to determine the uniformity (as 

applicable) or concentration of the positive control mixture will not be performed 

by the Testing Facility. The stability of the positive control article mixture will 

not be determined by the Testing Facility. 

12. POSTMORTEM 

12.1. MACROSCOPIC POSTMORTEM EXAMINATIONS 

Complete macroscopic postmortem examinations will be performed on all 

Main/Recovery/Neuropathology animals including animals euthanatized in a 

moribund condition or found dead; all abnormal observations will be recorded. 

Complete macroscopic postmortem examinations will only be performed on all 

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 tissues of the cranial, thoracic, abdominal 

and pelvic cavities and neck; and the remainder of the carcass. 

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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 prevent loss of tissues through autolysis. 

12.2.2. TERMINAL & RECOVERY NECROPSY 

Necropsy of all animals surviving to study termination will be performed 

after animals have been treated and/or held for the appropriate duration. 

Necropsy schedules will be established in order to assure that 

approximately equal numbers of males and females from each group will 

be examined on each day of necropsy and that examinations of animals of 

both sexes from each group will be performed at similar times of the day 

throughout the necropsy period. 

12.3. METHOD OF EUTHANASIA 

Exsanguination following carbon dioxide inhalation for Main Study and Recovery 

Study animals. See sections 11.7, 11.8, 12.8 and 12.9 for methods for all other 

animals. 

12.4. ORGAN WEIGHTS 

Organs indicated below will be taken from all Main Study and Recovery Study 

animals at the scheduled necropsy, weighed, recorded and organ/body and 

organ/brain weight ratios calculated. Organs will not be weighed for animals found 

dead or euthanatized in a moribund condition during the course of the study. Prior 

to weighing, all organs will be carefully dissected and properly trimmed to remove 

fat and other contiguous tissue in a uniform manner. Organs will be weighed as 

soon as possible after dissection to avoid drying. Paired organs will be weighed 

together: 

adrenals kidneys prostate testes 

brain liver seminal vesicles thymus 

epididymides 

heart 

lungs 

ovaries 

spleen uterus 

12.5. TISSUES PRESERVED 

Tissues listed in Appendix A will be obtained at necropsy and preserved for all 

Main Study and Recovery Study animals. 

12.5.1. PRESERVATIVES 

Eyes - glutaraldehyde/paraformaldeyhde 

Testes/epididymides – modified Davidson’s solution 

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All other tissues - 10% neutral 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 experimental findings; authorization 

will be obtained from the Sponsor prior to performing examinations. Note: Any 

abnormalities not noted during macroscopic postmortem examinations which are 

seen during histological processing will be recorded. Gross lesions will be 

examined at additional cost. Fibers from the sciatic nerve sample may be teased 

apart for further analysis under the microscope (optional, additional cost). 

Screening for alpha 2u-globulin Nephropathy: A preliminary diagnosis will be 

made on hematoxylin and eosin stained slides of the kidney and/or based on 

elevated kidney weights at the Terminal Sacrifice. The sections will then be stained 

(additional cost) with Mallory Heidenhain stain, if the following lesions are seen 

with H&E: angular hyalin inclusions in the proximal convoluted tubule, tubular 

degeneration and regeneration, and/or globular casts at the corticomedullary 

junction. 

12.7. STAINS 

Standard stains used: hematoxylin and eosin on paraffin sections, toluidine blue on 

epoxy resin sections. Special stains may be employed on selected tissues to aid in 

making a diagnosis at the discretion of the Study Pathologist. Special stains may be 

employed at the request of the Sponsor (additional cost). 

12.8. NEUROPATHOLOGY SACRIFICE 

Animals (5/sex/group) will be anesthetized with an intramuscular injection (~1.5 

mL/kg) of ketamine/xylazine/acepromazine and transcardially perfused with 

phosphate buffered saline followed by 1% glutaraldehyde and 4% 

paraformaldehyde in the same buffer. After perfusion, the required tissues will be 

dissected out. Measurement of the size (length and width) and weight of the 

whole brain (cerebrum, cerebellum and pons-medulla) shall be made. All tissues 

will then be placed into a fresh solution of the same fixative prior to processing. 

Tissues listed below will be preserved for all designated neuropathology animals 

(5/sex/group) after 13 weeks of treatment: 

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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 C3-

C6 and L4-L6) X X 

dorsal root fibers (from C3-C6

and L4-L6) X X 

ventral root fibers (from C3-C6

and L4-L6) 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 root fibers, lungs and trachea will be 

processed by standard techniques, embedded in paraffin and microtomed at 

approximately 6 microns. 

The tissues listed above will be examined microscopically for all animals as 

indicated. Tissues with macroscopic lesions will be examined in all animals 

(additional cost). Any abnormalities not noted during macroscopic postmortem 

examinations which are seen during histological processing will be recorded. If 

microscopic findings indicative of an effect of test substance administration are 

seen in high-exposure animals, then examinations will be made of these 

tissues/organs for low- and mid-exposure animals (additional cost). Additional 

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

authorization from the Sponsor. 

If any evidence of neuropathology is found, then a subjective diagnosis will be 

performed to evaluate exposure-response relationships (additional cost). All 

regions of the nervous system exhibiting any evidence of neuropathological 

changes will be included in the analysis. Sections from all exposure groups from 

each region will be coded and examined in a randomized order without 

knowledge of the code. The frequency and severity of each lesion will be 

recorded. After all samples have been rated, the code will be broken and 

statistical analysis performed to evaluate exposure-response relationships. 

12.9. GFAP EVALUATION 

Sprague Dawley-derived (CD) rats, 5/sex/group, will be exposed to Gasoline 

TBA Vapor Condensate via inhalation for a 13 week (5 days per week) exposure 

period by Huntingdon Life Sciences (PRC). US Center for Disease Control (CDC) 

Health Effects Laboratory personnel will sacrifice (carbon dioxide inhalation) the 

animals after the 13 week exposure period, and remove, weigh and process the 

brains. PRC personnel will ship the samples on dry ice to the US Center for 

Disease Control Health Effects Laboratory, Morgantown, WV (attn: James P. 

O’Callaghan, Ph.D.). Upon receipt, samples will be further processed for 

determination of GFAP response as detailed in the referenced guidelines and 

procedures (see Appendix E). The Sponsor will be responsible for the spirit of GLP 

compliance of this subcontractor. 

13. PRESERVATION OF RECORDS AND SPECIMENS 

All data documenting experimental details and study procedures and observations will be 

recorded and maintained as raw data. 

At the completion of the study, all reports, raw data, preserved specimens and retained 

samples will be maintained in the Testing Facility's Archives for a period of one year after 

submission of the signed final report. 

The Sponsor will be contacted in order to determine the final disposition of these materials. 

The Sponsor is responsible for all costs associated with the storage of these materials 

beyond one year from the issuance of the final report and for any costs associated with the 

shipment of these materials to the Sponsor or to any other facility designated by the 

Sponsor. 

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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 (presented as grams of food/kg of body 

weight/day) 

mean clinical laboratory values 

mean terminal organ weights, organ/body weight ratios and 

organ/brain weight ratios 

mean motor activity counts 

mean forelimb and hindlimb grip strength measurements 

mean landing foot splay measurements 

Mean values of all exposure groups will be compared to the mean value for the control 

group at each time interval. 

Evaluation of equality of group means will be made by the appropriate statistical method, 

followed by a multiple comparison test if needed. Bartlett’s test (Bartlett, 1937; Sokal 

and Rohlf, 1995) will be performed to determine if groups have equal variances. For all 

parameters except organ weights, if the variances are equal, parametric procedures will 

be used; if not, nonparametric procedures will be used. Organ weight data will be 

analyzed only by parametric methods. The parametric method will be the standard oneway analysis of variance (ANOVA) using the F ratio to assess significance (Armitage, 

1971; Dunlap and Duffy, 1975). If significant differences among the means are 

indicated, additional tests will be used to determine which means are significantly 

different from the control: Dunnett’s (Dunlap et al., 1981; Dunnett, 1955, 1964), 

Williams (Williams, 1971, 1972), or Cochran and Cox’s modified t-test (Cochran and 

Cox, 1959). The nonparametric method will be the Kruskal-Wallis test (Kruskal and 

Wallis, 1952, 1953) and if differences are indicated, Shirley’s test (Shirley, 1977), Steel’s 

test (Steel, 1959) or Pairwise Comparison with Bonferroni Correction (Games and 

Howell, 1976) will be used to determine which means differ from control. Bartlett’s test 

for equality of variance will be conducted at the 1% significance level; all other statistical 

tests will be conducted at the 5% and 1% significance levels. 

References for these procedures are: 

Armitage, P. 1971. Statistical Methods in Medical Research. Oxford, UK: Blackwell 

Scientific Publications; Bartlett, M.S. 1937. Properties of sufficiency and statistical tests. 

Proceedings of the Royal Society, Series A, 160: 268-282; Cochran, W.G. and Cox, G.M. 

1959. Experimental Designs, New York: John Wiley, pp. 100-102; Dunlap, W.P. and 

Duffy, J.A. 1975. Fortran IV Functions for Calculating Exact Probabilities Associated 

with Z, Chi-Square, T and F Values. Behav. Res. Methods and Instrumentations 7:59-60; 

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

probabilities associated with Dunnett’s test. Behav. Res. Methods and Instrumentation

13: 363-366; Dunnett, C.W. 1955. A multiple comparison procedure for comparing 

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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 multiple comparisons with a control. 

Biometrics 20-3: 482-491; Games, P.A. and Howell, J.F. 1976. Pairwise multiple 

comparison procedures with unequal n’s and/or variances: a monte-carlo study. Journal 

of Educational Statistics 1: 113-125; Kruskal, W.H. and Wallis, W.A. 1952. Use of 

Ranks in one-criterion variance analysis. Journal of the American Statistical Association 

47: 583-621; Kruskal, W.H. and Wallis, W.A. 1953. Errata for Kruskal-Wallis (1952) 

Journal of the American Statistical Association 48: 907-911; Shirley, E.A.C. 1977. A 

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 treatment means when several dose levels are 

compared with a zero dose control. Biometrics 27: 103-117; Williams, D.A. 1972. The 

comparison of several dose levels with a zero dose control. Biometrics 28: 519-531. 

Motor Activity Data 

The data will be analyzed using split-plot repeated measures ANOVA with model terms 

for group, animal within group, interval and group by interval interaction. If the group x 

interval interaction is statistically significant (p=0.05), indicating non-parallelism in the 

behavioral profile between groups, a separate one-way ANOVA for group effects will be 

performed at each interval. If the response data passes on the parallel hypothesis, an 

ANOVA (using summed responses over intervals) will be used to test for the overall 

treatment effect which constitutes the level hypothesis. If any significant overall 

treatment group effect is found by any of the above ANOVAs, Dunnett’s t-test will be 

used to find groups that differed from control. Analyses will be performed for sexes 

separately and combined. Treatment group effects will be deemed significant at the 

p=0.05 level. Plots, tables, listings, and analyses will be generated using SAS® version 

6.12 for WINDOWS. Analyses will be conducted by CATO Research, 200 Westpark 

Corporate Center, 4364 South Alston Avenue, Durham, NC 27713-2280. The Testing 

Facility will be responsible for the GLP compliance of this subcontractor. 

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15. REPORTING 

15.1. STATUS REPORTS 

Periodic verbal and written updates on study progress will be provided by the 

Study Director. In general, written status reports will be submitted weekly and at 

termination of the Main/Neuropathology Study and Recovery Study. 

15.2 FINAL REPORT 

One unbound hard copy and one electronic copy of a draft report will be submitted 

following termination of the study. The Main/Recovery/Neuropathology Study and 

each other satellite study will be separately reported. After receipt and review of the 

Sponsor's comments, appropriate changes will be made and two hard copies and 

one electronic copy of a signed, final report will be issued. (Additional copies will 

be provided at additional cost). The Main/Recovery/Neuropathology Study report 

will minimally include: 

Compliance Statement (including Sponsor signature line) 

Quality Assurance Statement 

Abstract 

Introduction 

Experimental Design 

Materials and Methods 

Discussion of study results 

Conclusion and No Observed Adverse Effect Level (NOAEL) statement 

Mortality data 

Individual animal termination history 

Summary of daily animal observations (as appropriate) 

Summary of weekly physical examination data 

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 hematology and clinical chemistry values 

Tables of individual differential leukocyte counts 

Tables of mean and individual organ weights, organ/body and organ/brain weight 

ratios 

Tables of mean and individual motor activity counts 

Tables of mean and individual functional observational battery results 

Summary of Positive Control Neurobehavioral Studies 

Individual and summary tables of macroscopic pathology findings 

Individual and summary tables of microscopic pathology findings 

Individual and summary tables of neuropathology findings 

References for experimental methodology 

 Senior personnel participating in the study 

 Protocol and any protocol amendments 

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APPENDIX A 

Tissues Preserved/Examined Microscopically 

Tissuea 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 Xc

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

epididymides X X 

esophagus X X 

eye (with optic nerve) X Xc 

heart X X 

kidneys X X 

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

lacrimal gland X Xc 

larynx d X X 

liver X X 

lungs (with mainstem bronchi) X X X 

lymph node (mediastinal and mesenteric) X X 

mammary gland X Xc 

muscle (biceps femoris) X Xc 

nasopharyngeal tissue e X X 

nerve (sciatic) X X 

ovaries X X 

pancreas X X 

 pituitary X Xc

 prostate X X

 salivary gland with submandibular lymph node X X 

 seminal vesicles X X 

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Tissuea Preserved Microscopic 

Examination 

(Groups)

 I, IV II, III 

skin X Xc 

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 Xc 

gross lesions and tissue massesf X X X 

target organsf,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. 

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APPENDIX B 

Micronucleus Evaluations 

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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 

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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 

 

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1. INTRODUCTION 

The object of this study is to assess the mutagenic potential of the test substance in an in vivo

cytogenetic test system which complies with the following guidelines: 

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

US EPA (1998) Health Effects Test Guidelines; OPPTS 870.5395 Mammalian Erythrocyte 

Micronucleus Test. 

2. BACKGROUND 

The bone marrow micronucleus test, originally developed by Matter and Schmid (1971), is a 

widely employed and internationally accepted short-term assay for identification of genotoxic 

effects (chromosome damage and aneuploidy) associated with mutagens and carcinogens 

(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 wealth of background data on this species, and because of their 

general suitability for toxicological investigations. 

In mitotic cells in which chromosomal breakage has been caused by the test substance or its 

metabolites, acentric fragments of the chromosomes do not separate at the anaphase stage of 

cell division. After telophase these fragments may not be included in the nuclei of the daughter 

cells and hence will form single or multiple micronuclei (Howell-Jolly bodies) in the cytoplasm 

of these cells. Micronuclei are seen in a wide variety of cells, but erythrocytes are chosen for 

examination since micronuclei are not obscured by the main nucleus and are therefore easily 

detected in this cell type (Boller and Schmid 1970). 

Micronucleated immature erythrocytes appear in the bone marrow approximately 24 hours after 

induction of chromosome damage. These immature erythrocytes can be differentiated by a variety 

of staining techniques which rely on their relatively high content of residual RNA. Using the 

Feulgen/H&E method, they stain slate-blue while mature erythrocytes (which contain little RNA) 

are counterstained orange. An increased incidence of micronucleated immature erythrocytes is 

indicative of recent exposure to a chromosome-damaging agent. A simultaneous marked increase 

in the incidence of micronucleated mature erythrocytes is not expected and may be indicative of 

micronucleus-like artifacts (Schmid 1976). 

Substances which interfere with the mitotic spindle apparatus will cause non-disjunction 

(unequal separation of the chromosomes at anaphase resulting in aneuploidy) or lagging 

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chromosomes at anaphase which may not be incorporated into the daughter nuclei. These 

lagging chromosomes are not excluded from the erythroblast with the main nucleus and hence 

also give rise to micronuclei. 

Any toxic effects of the test substance on the nucleated cells may lead either to a reduction in 

cell division or to cell death. These effects in 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 Schmid 1973). If the proportion of immature 

erythrocytes is found to be significantly less than the control value, this is taken as being 

indicative of toxicity. A very large decrease in the proportion would be indicative of a 

cytostatic or cytotoxic effect. 

3. EXPERIMENTAL PROCEDURE 

Animal management 

 Species 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; (Meal) (PMI Feeds, Inc., St. Louis,

MO) ad libitum. Elizabethtown Water Company, Westfield, NJ; ad

libitum, via automated watering system. There are no known 

contaminants in the feed or water which are expected to interfere with 

the results of this study. 

Positive control 

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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 section. Animals in the positive control group will 

receive 40 mg/kg cyclophosphamide intraperitoneally within 24 hours prior to sacrifice. 

Animals will be regularly examined for adverse clinical signs until the time of sacrifice. 

Material Exposure 

Level 

Sampling 

Time After 

Last 

Exposure or 

Dose (hours) 

No. of animals 

 M F 

Air Control 0 mg/m3 ~24 5 5 

Test substance 2,000 mg/m3 ~24 5 5 

Test substance 10,000 mg/m3 ~24 5 5 

Test substance 20,000 mg/m3 ~24 5 5 

Cyclophosphamide 40 mg/kg IP ~24 5 5 

 PREPARATION OF BONE MARROW SMEARS 

At approximately 24 hours after administration of the final exposure, five male and five female 

animals from each group will be 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 

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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 small volume of fetal bovine serum to facilitate 

smearing in the conventional manner on glass microscope slides (Schmid 1976). Four smears 

will be prepared from each animal. Two smears from each animal will be shipped unstained to 

ERC where they will be stained and examined. The remaining 2 smears and the cell pellet 

(refrigerated 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 dilute (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 immediately prior to use to remove particulate 

material. 

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Microscopic examination 

Coded slides will be examined by light microscopy and 2000 immature erythrocytes per animal 

will be examined for the presence of micronuclei. Usually only one smear is examined per 

animal, any remaining smear being held temporarily in reserve in case of technical problems with 

the first smear. 

Micronuclei are identified by the following criteria: 

Large enough to discern morphological characteristics 

Should possess a generally rounded shape with a clearly defined outline 

Should be deeply stained and similar in color to the nuclei of other cells - not black 

Should lie in the same focal plane as the cell 

Lack internal structure, ie they are pyknotic 

There should be no micronucleus-like debris in the area surrounding the cell 

The proportion of immature erythrocytes will be assessed by examination of a total of at least 

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 underlying distribution of data and therefore 

the values do not require transformation to fit a theoretical distribution (where data can be 

approximately fitted to a normal distribution, the results of non-parametric analysis and 

classical analysis of variance are very similar). 

“Outliers” are frequently found in the proportion of immature erythrocytes for both control 

and treated animals; non-parametric analysis based on rank does not give these values an 

undue weighting. 

Unless there is a substantial difference in response between sexes (which occurs only rarely) 

results for the two sexes are combined to facilitate interpretation and maximize the power of 

statistical analysis. 

For incidences of micronucleated immature erythrocytes, exact one-sided p-values are 

calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, 

Massachussetts). Comparison of several dose levels is made with the concurrent control using 

the Linear by Linear Association test for trend in a step-down fashion if significance is detected 

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Sponsor Study No. 211-TBA-S Final 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 statistically significant exposure-level-related 

increase in the incidence of micronucleated immature erythrocytes for the treatment group 

compared with the concurrent control group (P<0.01); individual and/or group mean values 

should exceed the laboratory historical control range (Morrison and Ashby 1995). A negative 

result is indicated where individual and group mean incidences of micronucleated immature 

erythrocytes for the group treated with the test substance are not significantly greater than 

incidences for the concurrent control group (P>0.01) and where these values fall within the 

historical control range. An equivocal response is obtained when the results do not meet the 

criteria specified for a positive or negative response. 

Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically 

significant exposure-level-related decrease in the proportion of immature erythrocytes (P<0.01). 

Additional slide reading or animal testing may very occasionally be required to clarify unclear 

or equivocal results. 

5. REPORTING 

The report will contain details of the test substance, methodology, results and the interpretation 

of the data. Tabulated results will show individual animal results, and group mean results. 

Good Laboratory Practice and Quality Assurance statements will be included. 

6. MAINTENANCE OF RECORDS 

All raw data, samples and specimens (if appropriate) arising from the performance of this study 

will remain the property of the Sponsor. Types of sample and specimen which are unsuitable, 

by reason of instability, for long term retention and archiving may be disposed of after the 

periods stated in Huntingdon Life Sciences Standard Operating Procedures. All other samples 

and specimens and all raw data will be retained by Huntingdon Life Sciences PRC in its archive 

for a period of one year from the date on which the Study Director signs the final report. After 

such time, the Sponsor will be contacted and his advice sought on the return, disposal or further 

retention of the materials. If requested, Huntingdon Life Sciences will continue to retain the 

materials subject to a reasonable fee being agreed with the Sponsor. Huntingdon Life Sciences 

will retain the Quality Assurance records relevant to this in its archive indefinitely. 

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Sponsor Study No. 211-TBA-S Final Protocol 

7. GOOD LABORATORY PRACTICE 

The study will be conducted in compliance with the principles of Good Laboratory Practice 

Standards as set forth in: 

US EPA 79.60, 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 Director and Company Management promptly on 

completion of each action, except for process based inspections which will be reported to 

appropriate Company Management only. 

9. REFERENCES 

AGRESTI, A., MEHTA, C.R. and PATEL, N.R. (1990) Exact inference for contingency tables 

with ordered categories. Journal of the American Statistical Association, 85, 453. 

BOLLER, K. and SCHMID, W. (1970) Chemical mutagenesis in mammals. The bone marrow 

of the Chinese hamster as an in vivo test system. Haematological findings after treatment with 

Trenimon (translation). Humangenetik, 11, 34. 

CYTEL (1995) StatXact 3 for Windows: Statistical Software for Exact Nonparametric Inference. 

Cytel Software Corporation, NC, USA. 

GIBBONS, J.D. (1985) Nonparametric Statistical Inference, 2nd edition, Marcel Dekker, New 

York. 

JONCKHEERE, A.R. (1954) A distribution-free k-sample test against ordered alternatives. 

Biometrics, 41, 133-145. 

KRUSKAL, W.H. and WALLIS, W.A. (1952) Use of Ranks in One-Criterion Variance Analysis. 

Journal of the American Statistical Association, 47, 583-621. 

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Sponsor Study No. 211-TBA-S Final Protocol 

KRUSKAL, W.H. and WALLIS, W.A. (1953) Errata for Kruskal-Wallis (1952). Journal of the 

American Statistical Association, 47, 583-621. 

MacGREGOR, J.T., HEDDLE, J.A., HITE, M., MARGOLIN, B.H., RAMEL, C., 

SALAMONE, M.F., TICE, R.R. and WILD, D. (1987) Guidelines for the conduct of 

micronucleus assays in mammalian bone marrow erythrocytes. Mutation Research, 189, 103. 

MATTER, B. and SCHMID, W. (1971) Trenimon-induced chromosomal damage in bone 

marrow cells of six mammalian species, evaluated by the micronucleus test. Mutation 

Research, 12, 417. 

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 Protection Agency Gene-Tox Program. Mutation Research, 

239, 29. 

MORRISON, V. and ASHBY, J. (1995) High resolution rodent bone marrow micronucleus 

assays of 1,2-dimethylhydrazine : implication of systemic toxicity and individual responders. 

Mutagenesis, 10, 129. 

SAS INSTITUTE (1989) 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 comparisons by ranking methods. Biometrics Bulletin, 1, 80-

83. 

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Sponsor Study No. 211-TBA-S Final Protocol 

APPENDIX C 

SCE Evaluations 

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Sponsor Study No. 211-TBA-S Final 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 availability of historical control data. Information on the 

test system is provided by Huntingdon Life Sciences and is contained in their files. 

7.0 EXPERIMENTAL DESIGN AND METHODOLOGY 

 Information on the experimental design and methodology has been provided by Huntingdon Life 

Sciences and is contained in their files. All in vivo portions of the study, including inhalation 

exposures, will be performed by Huntingdon Life Sciences. Animals will be anesthetized and 

blood samples will be collected by BioReliance personnel on site at Huntingdon Life Sciences. The 

blood samples will be transported to BioReliance on the day of collection. Within 24 hours after 

blood collection, the blood samples will be cultured in appropriate growth medium. Lymphocytes 

will be grown in the presence of 5-bromo-2'-deoxy-uridine (BrdU) for two cell cycles and dividing 

cells will be arrested in metaphase and harvested for microscopic evaluation of SCEs. The 

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Sponsor Study No. 211-TBA-S Final 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/m3) 

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 dose of 5 mg/kg. CP will be administered 

by intraperitoneal injection within 24 hours prior to harvest. 

 

 7.2 Blood Collection and Cell Culture 

Approximately 24 hours after the end of a 4 week (5 days per week) exposure period, 2-4 

ml of blood will be collected from the abdominal aorta from each anesthetized 

(isoflurane inhalation) rat including rats treated with positive control article. Whole 

blood cultures in duplicate per animal will be set up in RPMI 1640 culture medium 

supplemented with 10% fetal bovine serum, antibiotics (penicillin G, 100 u/ml and 

streptomycin sulfate, 0.1 mg/ml), 25 mM HEPES buffer, 20 ug/ml phytohemagglutinin 

and an additional 2 mM L-glutamine. Cultures will be initiated by inoculating 

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Sponsor Study No. 211-TBA-S Final Protocol 

approximately 0.5 ml of whole blood per 5.0 ml of complete culture medium in T-25 

sterile disposable tissue culture flasks and incubated at 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 prepare slides, the cells will be collected by 

centrifugation and resuspended in fresh fixative. One to two drops of fixed cells will be 

dropped onto a glass microscope slide and air-dried. The slide will be identified by the 

study number, animal number and date. One to two slides will be prepared from each 

flask. The slides will be stained using a modified fluorescence-plus-Giemsa technique 

(Perry and Wolff, 1974). Slides will be stained in Hoechst 33258, 5 μg/ml for 10 

minutes, mounted in phosphate buffer, pH 6.8, exposed to a U.V. lamp at approximately 

60o

C for 4-6 minutes, and then stained in 5% Giemsa for 6-10 minutes (BioReliance 

SOP# OPGT0442). Slides will be air dried and permanently mounted. 

7.4 Scoring 

Slides will be coded using random numbers by an individual not involved with the 

scoring process. Metaphase cells will be examined under oil immersion without prior 

knowledge of treatment groups. Whenever possible, a minimum of 25 second division 

metaphases per animal will be scored for SCEs. At least 100 consecutive metaphase 

cells will be scored for the number of cells in first-, second- or third-division metaphase 

for each animal as an indicator of toxicity (cell cycle delay) (BioReliance SOP# 

OPGT0442 and OPGT0336). At least 1000 cells will be scored for mitotic index per 

animal. 

8.0 Criteria for Determination of a Valid Test 

The mean SCEs/cell/animal for the positive control animals must be statistically increased 

relative to the negative control. 

9.0 Evaluation of Test Results 

The number of SCEs per second-division metaphase cell will be presented for each group along 

with the range of SCEs per animal within a group. SCE frequencies as the grand mean ±

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Sponsor Study No. 211-TBA-S Final Protocol 

standard deviation will be presented for each group. The Average Generation Time (AGT) is 

estimated as: = Number of hours in BrdU X 100/ (number M1 cells x 1)+(number M2 cells x 

2)+(number M3 cells x 3)]. A Dunnett's t test for multiple comparisons will be performed to 

compare the SCE frequency of test exposure levels to the negative control frequency, and if 

p<0.05, that exposure level will be considered significant. The conclusion of the study will be 

based on the Principal Investigator’s evaluation of all the data, including the biological as well as 

statistical significance. The following criteria will be used as guidelines in evaluation of this study. 

 

The test substance will be considered positive if an exposure-level responsive and statistically 

significant increase is observed over a minimum of two exposure levels. A statistically 

significant increase at the high exposure level with an exposure-level responsive although not 

statistically significant increase at lower exposure levels will be assessed as suspect. A 

statistically significant increase at one or more exposure levels with no evidence of a exposure 

level response will be assessed as equivocal or as negative according to the magnitude of the 

response and the number of exposure levels affected. 

10.0 Report 

A report of the results of this portion of the study conducted at BioReliance will be prepared by 

BioReliance and will accurately describe all methods used for generation and analysis of the 

data. Results presented will include, but not be limited to: 

cells used. 

test conditions: composition of medium, incubation time, concentration of BrdU, Colcemid7, 

positive and negative controls. 

number of cell cultures. 

mitotic index and number of metaphases analyzed for sister chromatid exchanges (SCE). 

cell cycle kinetics relative to the negative control. 

11.0 Records and Archives 

All raw data, the protocol and all reports generated at BioReliance will be maintained according 

to Standard Operating Procedure OPQP3040 by the BioReliance RAQA unit headquartered at: 

BioReliance, 14920 Broschart Road, Rockville, MD 20850. After submission of the final 

report, all 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 accordance with the relevant Good Laboratory Practice 

Regulations. 

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Sponsor Study No. 211-TBA-S Final Protocol 

APPENDIX D 

Immunotoxicity Evaluations 

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Huntingdon Life Sciences Study No. 00-6131 Page 53 

Sponsor Study No. 211-TBA-S Final 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 

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Sponsor Study No. 211-TBA-S Final 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 ability of the test substance, Gasoline TBA Vapor 

Condensate, will be evaluated for its ability to affect the humoral immune component of the immune 

system, when evaluated in the antibody-forming cell response to the T-dependent antigen sheep 

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 

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Huntingdon Life Sciences Study No. 00-6131 Page 55 

Sponsor Study No. 211-TBA-S Final Protocol 

SUB CONTRACTOR FACILITY: ImmunoTox, Inc. 

 Virginia BioTechnology Research Park 

 800 East Leigh Street, Suite 209 

 Richmond, VA 23219-1534 

IMMUNOTOX, INC. PERSONNEL RESPONSIBILITIES: 

 Principal Investigator: 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 accordance with agreements between Huntingdon Life Sciences and ImmunoTox, Inc. 

BACKGROUND: 

 

The humoral immune component of the acquired immune response is associated with the production of antibody to 

specific antigens. The antigen used in this assay is sheep eythrocytes (sRBC). A plaque, occurring from the lysis of 

sRBC, is elicited as a result of the interaction of complement and antibodies (produced in response to the i.v. 

sensitization) directed against sRBC. Each plaque is generated from a single IgM antibody-producing B cell, 

permitting the number of antibody-forming cells (AFC) present in the whole spleen to be calculated. The sRBC is 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 these cell-types to a significant degree, an altered response 

will be observed. As a result, the T-dependent IgM response to sRBC is one of the most sensitive 

immunotoxicological assays currently in use. A significant modulation in the IgM AFC response, when compared to 

the appropriate controls, indicates that the test agent is capable of modifying the humoral immune response in the 

whole animal and, thus, has the potential for immunotoxicity. This assay is one of the Tier I assays used by the NTP1

. 

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 exposure, spleens will be aseptically removed, weighed, and shipped intact in 

individual shipping containers at 2-8o

C via overnight delivery to ImmunoTox., Inc. Upon receipt, spleens will be 

further processed for determination of IgM response as detailed in procedures below. 

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Sponsor Study No. 211-TBA-S Final Protocol 

Exposure Level (mg/m3) 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 Jerne8 which 

indicates the number of antibody-forming cells (AFC) present. 

1. Rats will be sensitized with 2x108

 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 2-8o

C) and freeze (<-20oC) 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 immunotoxicological evaluation. Thymus will be 

removed and weighed and preserved (10% neutral buffered formalin) for possible histopathology. 

Animals will not be allowed to recover between blood collection and spleen collection. 

4. Upon arrival at the subcontractor facility, spleens will be accessioned in accordance with the SOP for 

receipt of biological samples. Single cell suspensions will be prepared from each spleen using a 

Stomacher® 80 Lab Blender in accordance with the SOP for rat spleens. Cell suspensions will then 

be centrifuged and resuspended in Earle’s Balanced Salt Solution with HEPES. Viability of 

splenocytes will be determined using propidium iodide (PI) and the Coulter EPICS XL-MCL Flow 

Cytometer. 

5. The cells will be resuspended in a 6 ml volume, and 1:50 and 1:150 dilutions are prepared. 

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Sponsor Study No. 211-TBA-S Final Protocol 

6. An 0.1 ml aliquot of spleen cells from each suspension will be added to separate test tubes each 

containing 25 μl guinea pig complement, 25 μl sRBC, and 0.5 ml of warm agar (0.5%). After 

thoroughly mixing, each test tube mixture will be plated onto a separate petri dish, covered with a 

microscope cover slip, and incubated at approximately 36-38°C for approximately 3 hours. 

7. One replicate per animal will be evaluated. 

8. Spleen cell counts will be performed on samples using a Model Z1 Coulter Counter. The plaques 

which develop will be counted using a Bellco plaque viewer. The data are expressed as number of 

cells/spleen, specific activity (AFC/106

 spleen cells) and total spleen activity (AFC/spleen). 

9. Blood samples will be shipped on dry ice to ImmunoTox, Inc. and evaluated if the spleen sample 

results are unsatisfactory. 

STATISTICAL ANALYSES OF IMMUNOTOXICOLOGICAL DATA(SOP/CSA/006): The data obtained in the 

immunotoxicological studies will be analyzed in accordance with standard operating procedures. Data will be first 

tested for homogeneity of variances using the Bartlett's Chi Square Test2

. Homogeneous data will be evaluated by 

a parametric one-way analysis of variance3

. When significant differences occur, the treatment groups will be 

compared to the vehicle control group using the Dunnett's t Test4

. Non-homogeneous data will be evaluated using 

a non-parametric analysis of variance3

. When significant differences occur, the treatment groups will be compared 

to vehicle control using the Gehan-Wilcoxon Test5

 when appropriate. The Jonckheere's Test6 will be used to test 

for dose-related trends across the vehicle and treatment groups. The positive control data will be evaluated using 

the Student's t Test7

 and compared to the vehicle control group. The criteria for accepting the results of the positive 

control in the assay will be a statistically significant (P<0.05) decrease in the response as compared to the vehicle 

control group. 

IMMUNOTOXICOLOGICAL REPORT: The report on the immunotoxicological results will contain all 

procedures and/or references to published procedures used during the study. Methods used for statistical analysis 

will also be included. 

IMMUNOTOXICOLOGICAL DATA AND RECORD RETENTION: All data and records pertaining to this 

immunotoxicological study will be submitted to Huntingdon Life Sciences (PRC) upon acceptance of the final 

report. Upon completion of this study, the report and raw data for this study will be maintained in the archives of 

Huntingdon Life Sciences. 

PERSONNEL: Curriculum vitae for the key personnel involved in this study are maintained in the individual’s 

training record maintained at ImmunoTox, Inc.’s office. 

SAFETY: The appropriate safety precautions for the assay are described in SOP/SAF/004. 

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Sponsor Study No. 211-TBA-S Final 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 comparing several treatments with a control. J. Amer. 

Stat. Assoc. 50:1096-1121. 

5. Gross, A.J., and Clark, V.A. (1975). Gehan-Wilcoxon Test. In Survival Distribution: Reliability Applications in 

Biomedical Sciences. A.J. Gross and V.A. Clark, eds. John Wiley and Sons, New York, p.120-123. 

6. Hollander, M. and Wolfe, D.A. (1975). Jonckheere's Test:Non-Parametric Statistics Methods, eds. M. Hollander 

and D.A. Wolfe, John Wiley and Sons, New York, p.124-129. 

7. Sokal, R.R. and Rohlf, F.J. (1981). Biometry. Freeman, San Francisco, p.226-231. 

8. 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. 

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Sponsor Study No. 211-TBA-S Final Protocol 

APPENDIX E 

GFAP Evaluations 

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Sponsor Study No. 211-TBA-S Final Protocol 

 3.3 Test Substance Retention Sample 

 The retention of a reserve sample of the Test substance will be the responsibility of 

Huntingdon Life Sciences. 

 4.0 SUBCONTRACTOR AND KEY PERSONNEL 

 4.1 Name: 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 acceptable 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 samples will be collected by CDC personnel on site at 

Huntingdon Life Sciences. The brain samples will be shipped to CDC and processed and evaluated 

as indicated in the following procedures. The potential of the test substance to increase GFAP in a 

dose-responsive manner will be measured relative to a control group. 

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Sponsor Study No. 211-TBA-S Final Protocol 

 The study design will be as follows: 

 Group Number per Sex 

Negative Air Control 5 

Test Substance (mg/m3) 

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 generated at CDC will be maintained. After 

submission of the final report, all of the above will be shipped to Huntingdon Life Sciences to 

be archived. 

10.0 Specimens 

All specimens will be held in storage as long as the quality of the preparation affords evaluation 

or in accordance with the relevant Good Laboratory Practice Regulations. 

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Sponsor Study No. 211-TBA-S Final 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 Standard Operating Procedure 

1 SCOPE OF APPLICATION 

The following is an SOP to be used in the quantification 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 EQUIPMENT and SUPPLIES

 

2.1.1 EQUIPMENT

 Ultrasonic Cell Disruptor 

 Pipettes 

 Hot/Stir Plate 

 Plate Reader 

 

2.1.2 CHEMICAL REAGENTS

 αGlial Fibrillary Acidic Protein (DAKO Z0334) - Lot # 096 (302) 

 Carnation Non-Fat Dry Milk 

 αGlial Fibrillary Acidic Protein (Chemicon MAB 3402-Lot 14119825-07) 

 Phosphate Buffered Saline (Pierce #28374) 

 Alkaline Phosphatase conjugated αmouse 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) 

 

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Sponsor Study No. 211-TBA-S Final Protocol 

2.1.3 SUPPLIES

 Microtiter Plates (Immulon 2, Dynatech) 

 Pipet tips 

2.2 TRAINING REQUIREMENTS

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

3 CAUTIONARY NOTES OR SPECIAL CONSIDERATIONS

 1. Powdered milk should be purchased from a grocery store and used within six months. Old 

powdered milk gives a yellow color in all samples and masks any 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 STANDARD CURVE PREPARATION

1. Remove a tube of the GFAP standard (see O’Callaghan et al., 1990 for method of 

preparation) from the freezer, thaw at room temperature and vortex prior to aliquoting. 

2. Preparation of the standard curve requires dilution of the 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 protein value is used to prepare a standard curve 

in PBS with0.5% Triton X-100. The protein values for the standard curve should be 

between 0.25 to 10 μg/100μl (i.e. 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, 10μg/100μl). 

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Sponsor Study No. 211-TBA-S Final 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 SAMPLE PREPARATION

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 (85-95oC) is added and then sonified. In both cases, the samples are stored 

within 24 hours at -70oC. 

 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- 

 95o

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% 

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Sponsor Study No. 211-TBA-S Final 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 REAGENT PREPARATION

1. Volumes Required- The volumes of reagents are 100μl/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. Blotto- PBS + 5% powdered milk. Step 4 uses BLOTTO without Triton X100, in steps 7 and 9 , BLOTTO contains 0.5% TritonX-100. Before the assay is started, 

the total volume of BLOTTO needed for the entire assay should be determined (total 

volume should be made fresh on the day of the assay). The larger volume allows a good 

mix for the addition of the Triton X-100 detergent. 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 

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Sponsor Study No. 211-TBA-S Final Protocol 

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

4.4 ASSAY PROCEDURE

1. Coat Immulon-2 flat bottom plates with αGFAP, 1.0μg total immunoglobulin 

 protein / 100μl PBS / well. (25μl of αGFAP, Dako Z0334, in 10ml of PBS is the quantity 

needed per plate). 

 2. Incubate the coated plate at 37o

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 4oC. 

 3. Empty plate, tap on absorbent paper to remove excess (this procedure is important to 

eliminate the possibility of any reagent carry-over between steps). Wash plates 4X with PBS 

(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 αGFAP (Chemicon) at a 

 dilution of 1:500 plus alkaline phosphatase conjugated anti-mouse IgG 

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Sponsor Study No. 211-TBA-S Final 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 CALCULATIONS

 1. Plates are read on a Molecular Devices Spectra 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 plate in the assay. The template includes the position of the 

standard curve and value for each point, and the name, number, and dilution factor of the 

unknown samples. The dilution factor for the samples is determined by the amount of total 

protein loaded into the wells (e.g. 10μg of total 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.) 

 

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 GFAP STANDARD CURVE

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: 4-Param eter C orr. C oeff:

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

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Sponsor Study No. 211-TBA-S Final Protocol 

 3. The above standard curve is a typical curve generated from the rat hippocampus standard. 

The OD values for unknown samples need to fall on the straight portion of the curve, 

between STD 3 and STD 5. 

 4. The SoftMax Pro program automatically subtracts the blank from each OD reading (plate 

blank information is given in the table above), means the two readings for each sample, and 

multiplies the value obtained from the curve for this OD by the dilution factor for the 

unknown sample. 

 5. The report generated by the 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 hippocampus samples that has been imported into 

an Excel spreadsheet. These samples were diluted to 2.5μg of total protein/100μl, therefore 

the dilution factor for all ten samples is 400(this 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. 

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Sponsor Study No. 211-TBA-S Final 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 Protein: Comparison of Slot-Immunobinding Assay 

with a Novel Sandwich ELISA, O’Callaghan, J.P., Neurotoxicology and Teratology, Vol. 13, pp. 

275-281, Pergamon Press plc. 1991 

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

Brain Tissue Preparation Standard Operating Procedure 

1 SCOPE OF APPLICATION

 The following is an SOP to be used for preparing rat or mouse brain tissue to be 

 assayed for total protein concentration and the concentration of glial fibrillary 

 acidic protein (GFAP). 

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Sponsor Study No. 211-TBA-S Final Protocol 

2 PREREQUISITES

2.1 EQUIPMENT and SUPPLIES

 

2.1.1 EQUIPMENT

Ultrasonic Cell Disruptor 

 Pipettes 

 Hot/Stir Plate 

 Plate Reader 

2.1.2 CHEMICAL REAGENTS

Sodium Dodecyl Sulfate, Bio-Rad #161-0302

2.1.3 SUPPLIES

Microfuge tubes 

 Pipet tips 

 

2.2 TRAINING REQUIREMENTS

 Ability to dissect discrete regions of the rat brain. 

3 CAUTIONARY NOTES OR SPECIAL CONSIDERATIONS

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

4 PROCEDURE

4.1 REAGENT PREPARATION

1% SDS

 A stock solution of 10% SDS is prepared w/v, 100 gms of SDS in 1 litre of deionized water. 

The 1% SDS is a dilution of the stock- 10ml of 10X stock in 90ml of deionized water. 

 

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Sponsor Study No. 211-TBA-S Final Protocol 

4.2 TISSUE PREPARATION

1. Control and Test substance exposed animals are sacrificed, brains then are rapidly removed 

from the skull case with the aid of pointed scissors (to open the brain case) and curved blunt 

forceps (to remove the brain). 

2. The brain is placed on a cold (approximately 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, hippocampus, 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 immersed in 10 volumes of hot 1% SDS (85-95o

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 sonification with an ultrasonification microprobe 

(Heat Systems, Farmingdale, NY). The samples then are stored within 24 hours at -70oC 

prior to assay. 

 

Sample Storage/Handling and Shipping Standard Operating Procedure 

1 SCOPE OF APPLICATION

The following is an SOP to be used for storing/handling and shipping of brain tissue 

homogenates that serve as standards and unknowns for determining total protein concentration 

and the concentration of glial fibrillary acidic protein (GFAP) in each homogenate. 

 

2 PREREQUISITES 

2.1 EQUIPMENT and SUPPLIES

 

2.1.1 EQUIPMENT

-70 oC Freezer 

 

2.1.2 CHEMICAL REAGENTS

2.1.3 SUPPLIES

2.2 TRAINING REQUIREMENTS

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 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 STORAGE/HANDLING

Homogenates will be stored frozen at -70oC prior to shipping to CDC-NIOSH and shipped 

frozen on dry ice. 

4.2 SHIPPING

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

BCA PROTEIN ASSAY Standard Operating Procedure 

1 SCOPE OF APPLICATION

The following is an SOP to be used in the determination of total protein in brain tissue 

by the Pierce BCA method. BSA (Bovine Serum Albumin) is used as the standard and 

samples are prepared by sonification in 1% (w/v) SDS. 

2 PREREQUISITES

 

2.1 EQUIPMENT and SUPPLIES

 

2.1.1 EQUIPMENT

Plate Reader 

 Pipettes 

 Incubator 

 Vortex-Mixer 

2.1.2 CHEMICAL REAGENTS

BCA Protein Kit, Pierce #23223 

 Bovine Serum Albumin (BSA), Sigma A7888 

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Sponsor Study No. 211-TBA-S Final Protocol 

 Sodium Dodecyl Sulfate, Bio-Rad #161-0302 

 

2.1.3 SUPPLIES

Microtubes 1.2ml 

 96 well microtiter plates 

 Pipette Tips 

2.2 TRAINING REQUIREMENTS

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

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 REAGENT PREPARATION

1. 1% SDS 

 A stock solution of 10% SDS is prepared w/v, 100 gms of SDS in 1 litre of deionized water. 

The 1% SDS is a dilution of the stock- 10ml of 10X stock in 90 ml deionized water. 

2. BCA Reagent

 This reagent is made according to the directions provided by Pierce, 50ml of solution A and 1ml 

of solution B. Thoroughly mix while adding solution B. 

4.2 STANDARD PREPARATION

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

1. Prepare 1mg/ml BSA in 1% SDS. Weigh out 0.100 gms of dry BSA on an analytical 

balance. Add the BSA to a 100 ml volumetric flask then gradually add 1% SDS to the flask. 

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Sponsor Study No. 211-TBA-S Final 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 standard(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. 

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Sponsor Study No. 211-TBA-S Final 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 SAMPLE PREPARATION

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 10μl 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 PROTEIN ASSAY

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 mix the plate using the automatic mix button on the 

plate reader or vortex gently prior to placing the plate into the reader. 

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 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 wavelength of 562nm. “Pop” any bubbles with a needle or a pipette tip 

prior to reading the plate. 

4.5 CALCULATIONS

1. Software programs coupled to specific plate readers are used to calculate protein values 

based on comparing OD readings of the unknowns with those obtained from the standard curve. 

 OD values and standard protein values are plotted in a linear fashion on both axes. The 

following is a typical standard curve for the BCA Protein Assay when bovine serum albumin is 

used for the standard. The table gives the actual values for each point on the standard curve and 

their corresponding OD readings. 

BCA Total Protein Standard Curve

Curve Fit: Linear Corr. Coeff:

A = 0.0240 B = 0.0395 

y = A + B * x

0.999

 1 10 Linear scale of μg

0

0.4

OD

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Sponsor Study No. 211-TBA-S Final 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 the standard curve and the protein concentration of 

each unknown expressed as mg/ml or μg/μl. The table below gives the calculated protein 

values for unknown hippocampus samples in mg/ml or μg/μl. These values include the 

subtraction of the blank value and a dilution factor of 2. 

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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 tube 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 REFERENCES

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

 SoftMax Pro User’s Manual, Molecular Devices Corporation. Sunnyvale, CA.

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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 Barbara A. Litzenberger, B.S.,

M.T. (ASCP)

DIRECTOR, TOXICOLOGY

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

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

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

PATHOLOGIST Katharine M. Whitney, D.V.M.,

Ph.D., Diplomate A.C.V.P.

STUDY MONITOR 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 Saladdin, 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.