DuPont-4436

Page 1 of 27 Study Title AAWG #873: In Vitro Chromosome Aberration Test in Human Peripheral Blood Lymphocytes
Laboratory Project ID: DuPont-4436

T EST G UIDELINES : U.S. EPA Health Effects Test Guidelines OPPTS 870.375 (1998) OECD Guideline for Testing of Chemicals Section 4: Health Effects, No. 473 (Adopted 1997)

A UTHOR : Patrick T. Curry, Ph.D.
S TUDY C OMPLETED ON : November 20, 2000
T ESTING F ACILITY : DuPont Pharmaceuticals Company Safety Assessment Section Stine-Haskell Research Center P.O. Box 30, Elkton Road Newark, Delaware 19714-0030
S PONSOR R EPRESENTATIVE : E.I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine P.O. Box 50, Elkton Road Newark, Delaware 19714-0050
W ORK R EQUEST N UMBER : 13384

S ERVICE C ODE N UMBER : 544
D U P ONT P HARMACEUTICALS C OMPANY S TUDY N O .: THA00-07-28AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 2 - CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study.AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 3 - TABLE OF CONTENTS Page
CERTIFICATION.................................................................................................................. .........2 LIST OF TABLES ................................................................................................................. .........4 STUDY INFORMATION.............................................................................................................. .5 STUDY PERSONNEL................................................................................................................ ....6 SUMMARY ........................................................................................................................ ............7 INTRODUCTION................................................................................................................... ........8 MATERIALS AND METHODS ....................................................................................................8 A. Study Protocol............................................................................................................... .......8 B. Test Material................................................................................................................ ........9 1. Test Substance and Solvent Selection............................................................................. 9 2. Negative and Vehicle Control Substances ...................................................................... 9 3. Positive Control Substance.............................................................................................. 9 4. Stability and Concentration Verification....................................................................... 10 C. Test System and Test System Justification........................................................................10 D. Preparation of the Target Cells ..........................................................................................10 E. Metabolic Activation System.............................................................................................10 F. Method of Administration of the Test and Control Substances.........................................11 G. Test and Control Substance Treatment..............................................................................11 1. Treatment Medium........................................................................................................ 11 2. Treatment of the Target Cells........................................................................................ 11 H. Cell Harvest ................................................................................................................. ......11 I. Initial Cytotoxicity/Chromosome Aberration Assay .........................................................12 J. Chromosome Aberration Assay.........................................................................................12 K. Evaluation of Results and Statistical Analysis ..................................................................13 L. Data Acceptability Criteria ................................................................................................13 1. Acceptable Controls ...................................................................................................... 13 2. Acceptable High Concentration .................................................................................... 13 3. Number of Doses........................................................................................................... 13 M. Classification Guidelines ...................................................................................................1 3 RESULTS AND DISCUSSION ...................................................................................................14 A. Test Substance Solubility...................................................................................................1 4 B. Preliminary Cytotoxicity Assay.........................................................................................14AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 4 - C. Chromosome Aberation Assay ..........................................................................................15 CONCLUSIONS.................................................................................................................... .......16 RECORDS AND SAMPLE STORAGE.......................................................................................16 REFERENCES..................................................................................................................... .........17 TABLES......................................................................................................................... ...............18 APPENDIX - Historical Control................................................................................................. .26

LIST OF TABLES Page

Table 1 Toxicity Assessment of #873: 3.0-Hour Treatment – Trial 1 .......................................... 21 Table 2 Toxicity Assessment of #873: 3.0-Hour Treatment - Trial 2 ........................................... 22 Table 3 Individual Culture Analysis of Chromosome Aberrations in Human Peripheral Blood: 3.0-Hour Treatment without Metabolic Activation .................................. 23 Table 4 Individual Culture Analysis of Chromosome Aberrations in Human Peripheral Blood: 3.0-Hour Treatment with Aroclor ® 1254-Induced S9 .............................. 24 Table 5 Summary of Chromosome Aberrations of #873: 3.0-Hour Treatment............................ 25AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 5 - STUDY INFORMATION 9th Collective Nomenclature: 1-Propene, 2-bromo-3,3,3-trifluoroSynonyms/Codes: • 2-Bromo-3,3,3-trifluoropropene

• 2-Bromo-1,1,1-trifluoroprop-2-ene

• AAWG #873

• Q66-16 (Synquest Laboratories) (Lot No.)

• 1300-B-05 (ID Code)

• H-24499
Haskell Number: 24499 CAS Registry Number: 1514-82-5 Composition: 2-Bromo-3,3,3-trifluoroprop-1-ene Known Impurities: Not supplied by the sponsor Physical Characteristics: Colorless liquid Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Sponsor: Advanced Agent Working Group Halon Alternatives Research Corporation Arlington, VA 22201 Study Initiated/Completed: July 28, 2000 / (see report cover page) In-Life Initiated/Completed: August 2, 2000 / September 1, 2000AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 6 - STUDY PERSONNEL The following individuals participated in the conduct of this study: DuPont Pharmaceuticals Company Study Director: Patrick T. Curry, Ph.D. Technicians: Kapal Dewan, M.S. Louis Nimeroff, B.S. Director of Genetic Toxicology: Ronald D. Snyder, Ph.D.

E.I. du Pont de Nemours and Company Management: Carolyn S. VanPelt, D.V.M., Ph.D. Study Monitor: E. Maria Donner, Ph.D. Report Preparation: Brenda TiffinAAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 7 - SUMMARY The test substance, #873, was evaluated for in vitro clastogenic (chromosome-damaging) potential in human peripheral blood lymphocytes in the absence and presence of an exogenous metabolic activation system (Aroclor ® -induced rat liver S9). An initial cytotoxicity/chromosome aberration assay was conducted to establish the dose range for testing. Dimethyl sulfoxide (DMSO) was selected to be the solvent based on information provided by the sponsor, the solubility of the test substance, and target cell compatibility. The test substance was	
	
			 		
	
	

	
	
 In the initial cytotoxicity/chromosome aberration assay, the following concentrations were 
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 and positive controls were shared with another in vitro chromosome aberrations study (DuPont-4437). The test substance was miscible in the treatment medium at the top 2 concentrations and soluble in the treatment medium at all other tested concentration levels in the non-activated and activated studies. Upon centrifugation of the top 2 concentrations during the removal of the test substance, an organic phase was noted at the bottom of the centrifuge tube. The organic phase was removed with a pipette. Selection of concentration levels for the chromosome aberration assay was based on the reduction of mitotic index relative to the solvent control. The cells were treated for 3 hours in the non-activated and in the S9 activated assay. One independent trial was conducted. Metaphase cells were harvested approximately 22 hours after the initiation of the treatment. The test substance was soluble at all concentrations tested. In the initial cytotoxicity/chromosome aberration assay (Trial 1) none of the tested concentrations were selected for analysis due to either excessive toxicity or insufficient toxicity. The assay was repeated and the concentration levels were reduced with smaller intervals. The following concentrations were tested in the repeat assay: 0.088, 0.175, 0.350, 0.450, 0.500, 	!		%& 		)*(		

	


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 controls were sub-optimal and the second trial was rejected. The assay was repeated at the same concentration levels with cells from a different donor (Trial 2). A reduction of the mitotic index,
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	 non-activated system and a reduction of the mitotic index of 45.4% was observed at the 0.350 	 oncentration in the S9-activated system. No statistically significant increases in the percent of aberrant cells were observed at any concentrations in any of the testing conditions. The negative and positive control substances met the requirements for a valid test. Under the conditions of this study, #873 was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9 activated in vitro mammalian chromosome aberration test in human blood peripheral lymphocytes.AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 8 - INTRODUCTION The objectives of this study were to evaluate the clastogenic (chromosome-damaging) ability of the test substance, #873, in human peripheral blood lymphocytes (HPBL) in vitro , in the presence and absence of an exogenous S9 metabolic activation system. Chromosome aberrations are a consequence of failure or error in repair mechanisms such that breaks either do not rejoin or rejoin inappropriately. The chromosome aberration test in human lymphocytes is an in vitro test which detects structural damage to chromosomes (or the mitotic apparatus) induced by genotoxic agents. This chromosomal damage can be directly visualized using light microscopy. Structural chromosome damage detected in this assay is observed as chromosome breaks or rearrangements. This system has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals. (1,2) Numerical damage, observed as polyploidy (or endoreduplication) and indicative of damage to the mitotic spindle apparatus, was also assessed. Lymphocyte cells in circulating blood do not usually divide but can be stimulated to divide in culture by treatment with phytohemagglutinin (PHA). At predetermined intervals after exposure to the test substance, the cells were treated with Colcemid ® to arrest them in metaphase, harvested, treated with a hypotonic buffer, stained, and analyzed microscopically for the presence of chromosome aberrations and numerical chromosome changes. Many mutagenic chemicals do not act directly on DNA but do so after being enzymatically converted to active intermediates by the liver. Human lymphocytes have only a limited capacity to metabolize test substances, so an exogenous metabolic activation system (Aroclor ® -induced rat liver S9 homogenate) was included in a series of treatments to allow detection of clastogenic metabolic intermediates.

MATERIALS AND METHODS

A. Study Protocol This study was conducted using the Good Laboratory Practice (GLP) Regulations for Nonclinical Laboratory Studies as a guideline, and applicable DuPont Pharmaceuticals Company Standard Operating Procedures (SOPs) were utilized. The study was designed to comply with the following guidelines, but does not fulfill GLP compliance requirements:

 U.S. EPA, Office of Prevention, Pesticides and Toxic Substances (OPPTS) Guidelines (Subpart H, 40 CFR Part 799.9510 [1989])

 Guidelines of the Organisation for Economic Cooperation and Development (OECD Guidelines for Testing of Chemicals, No. 473 [adopted 1997])AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 9 - B. Test Material
1. Test Substance and Solvent Selection The test substance, #873, is a clear liquid. Additional information regarding the test substance can be found on the study information page of this report. Test substance characterization (such as determination of the identity, stability, purity, and strength) was the responsibility of the Sponsor. The Sponsor did not provide the expiration date of the test substance. The unique identification number of the test substance was H-24499. Information on the stability, purity, composition, contaminants, synonyms, CAS registry number, basic physical properties, hazards, storage conditions, and hazardous material classification that was available are kept in the study notebook. The test substance was assumed to be stable during the study and no evidence of instability was observed. The test substance was stored refrigerated, tightly capped, and protected from light. Based on the solubility test, dimethyl sulfoxide (DMSO, CAS 67-68-5, manufacturer) was chosen as the solvent. Unused dosing solutions were disposed of following administration to the test system, and all remaining test substance will be disposed of after finalization of the study report.
2. Negative and Vehicle Control Substances Cells exposed to DMSO served as the vehicle control. Negative (untreated) controls were also run concurrently for comparison. Negative control cultures were exposed to the treatment medium only. Vehicle controls were exposed to the treatment medium plus DMSO (1% of the volume of the treatment medium).
Under the conditions of the in vitro chromosome aberration assay in human peripheral blood lymphocyte (HPBL), the final concentration of the solvent did not exceed 1% of the volume of the treatment medium. After the addition of the dosing solution the treatment medium was observed for precipitation (with the naked eye).
3. Positive Control Substance The positive controls were mitomycin-C (MMC, CAS No. 50-07-7, Lot # 69H2512, Sigma Chemical Company, St. Louis, MO) for the non-activated system and cyclophosphamide (CP, CAS No. 6055-19-2, Lot # 108H0568, Sigma Chemical Company, St. Louis, MO) for the S9 activated system. Both positive controls were dissolved in sterile water and delivered to the treatment medium in a volume of 10% (v/v). The final concentrations of MMC were 0.1 and 0.5 			+
	

	+	01	*			! 				+		
 concentrations of each positive control was selected for inclusion in the analysis of clastogenicity. The exposure period for each of the positive control substances was 3 hours. Exposure to the positive control substances was included in the preliminary cytotoxicity portion of the study. The positive control was not expected to contain any contaminants that would interfere with the conduct and validity of the study, and was assumed to be stable under the conditions of administration. Concentration verification of the positive control substances was not conducted.AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 10 - 4. Stability and Concentration Verification All solutions of the test substance were prepared immediately prior to treatment of the cultures. Treatment and control dosing solutions were not analyzed for concentration, uniformity, or stability. Treatment media were not assayed for stability or concentration of the test or control substances since this assessment was not considered necessary to achieve the objectives of the study. No evidence of instability was observed.

C. Test System and Test System Justification For each assay whole venous blood was drawn aseptically from selected healthy, non-smoking, male donors less than 50 years old without previous chemotherapy or radiotherapy; and without recent (within the last 6 months) viral disease, use of prescription drugs or X-ray exposure. The blood was collected, in-house, into heparinized containers just prior to use. Different donors were used for trials 1, 2, and 3. HPBL cells, exposed to test substances in vitro , are routinely used to detect clastogenic activity. (1)

D. Preparation of the Target Cells HPBL cultures were initiated in labeled, sterile 15 mL centrifuge tubes by inoculating 0.8 mL heparinized blood in 9.2 mL RPMI 1640 medium supplemented with approximately 15% fetal(
	
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	,&	- and 2% phytohemagglutinin-M (PHA-M, v/v, Murex reagent grade, reconstituted per supplier ’ s directions). The labels (for the centrifuge tubes and for the slides made from the contents of the centrifuge tubes) included the study number, dose level, replicate indicator (A or B), metabolic activation system (+/-S9), and exposure period (3 hr). Cultures were incubated with loose caps at 37 o ± 2 o C in a humidified atmosphere of 5% ± 1.5% CO 2 in air.

E. Metabolic Activation System The metabolic activation system was the liver (1 g wet liver: 3.0 mL 0.154 M KCl) microsome 9000 x g supernatant fraction (S9) obtained from young male Sprague-Dawley rats injected i.p. with Aroclor ® five days before sacrifice. (3) The S9 homogenate was obtained from a commercial supplier (MOLTOX TM , Boone, North Carolina) and stored at 5	4! o C until needed. The energy producing system consisted of NADP and isocitric acid. The S9 and the energy producing components were formulated as described in the following Table: Component Final Concentration (in culture) NADP (sodium salt) 1.5 mg/mL (1.8 mM) Isocitric acid 2.7 mg/mL (10.5 mM) Homogenate (S9 fraction) # 	,#/- The S9 metabolic activation system was prepared immediately before use and kept on ice until used. The metabolic activity of the S9 was demonstrated by the response of the CP-treated cultures.AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 11 - F. Method of Administration of the Test and Control Substances The test substance, and the negative, vehicle, and positive control substances, were administered to whole human peripheral blood lymphocyte (HPBL) cultures by addition to the culture medium. The negative controls, vehicle controls, and positive controls for this study were shared with another in vitro chromosome aberration study. (4)

G. Test and Control Substance Treatment
1. Treatment Medium The treatment medium for each culture consisted of approximately 16 mL RPMI 1640 medium that had been supplemented with L-glutamine (2 mM), penicillin (50 IU/mL), streptomycin (50 μg/mL), and HEPES buffer (25 mM). In treatments with activation, S9 and cofactor
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 acid (2.7 mg/mL), and NADP (sodium salt) 1.5 mg/mL. The 16 mL volume of the treatment was used to reduce the headspace of the cultures and prevent evaporation of the potentially volatile test substance.
2. Treatment of the Target Cells Approximately forty eight hours after HPBL culture initiation the whole blood cultures were centrifuged and the culture medium was discarded and replaced with a 16 mL volume of	
	
		

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	 headspace in the centrifuge tube. Duplicate HPBL cultures were exposed to 8 concentrations of the test substance and the positive and vehicle control substances. The treatment medium was serum-free RPMI 1640 medium for the approximately 3 hour exposure without metabolic activation, and serum-free RPMI 1640 medium plus S9 reaction mixture for the approximately 3-hour exposure with metabolic activation. All treatments were conducted at approximately 37 ° C. After the 3-hour exposures the cells were pelleted by centrifugation, the cultures were rinsed with Hank's Balanced Salt Solution (HBSS). Complete RPMI 1640 culture medium (containing 2% PHA-M) was added to each culture, and incubation continued until cell harvest.

H. Cell Harvest Approximately 20 hours after the initiation of exposure to the test substance, Colcemid ® was	,+
	
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 ® addition, i.e., approximately 22 hours (~1.5 times the normal cell cycle) after treatment initiation, the cells were harvested at a single time point. The cultures were pelleted by centrifugation and the medium was removed. The cells were swollen in hypotonic buffer (0.075 M KCl at approximately 37 ° C), fixed in at least 3 changes of methanol:glacial acetic acid (3:1, v/v), and stored refrigerated overnight or longer. To prepare slides, the cells were collected by centrifugation and re-suspended in fresh fixative. At least 2 slides per culture were prepared by applying an aliquot of the fixed cells onto clean microscope slides and air drying them. TheAAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 12 - slides were stained by Giemsa and permanently mounted. The slides were identified by the study number, dose level, replicate indicator (A or B), metabolic activation system (+/-S9), and exposure period (3 hr). At least 1000 cells per culture were counted to establish a mitotic index (MI, percentage of cells in metaphase) for each culture. All slides were coded before chromosome aberration analysis to control bias.

I. Initial Cytotoxicity/Chromosome Aberration Assay An initial cytotoxicity assay, both in the absence and presence of a S9 metabolic activation system, was conducted to determine the concentration range to be selected for the chromosome aberration assays. Ideally, the highest test concentration selected for the chromosome aberration treatments should be clearly cytotoxic (e.g., causing significant cell-cycle delay demonstrated by an approximate 50% decrease in mitotic index), while allowing sufficient metaphases for a reliable analysis. (5) The mitotic index was calculated by counting the number of metaphases per 1000 cells. In the absence of demonstrable cytotoxicity, the test substance was evaluated up to the limit of solubility in the treatment medium. At least two additional lower concentrations were selected to extend into the noncytotoxic range. Osmolality measurements were conducted on an aliquot of the culture medium and each concentration level in culture medium. The pH of the culture medium was only to be assessed if there is a visually observable color change (due to the pH indicator) in the test medium at the beginning or the end of the exposure period.

J. Chromosome Aberration Assay Whenever possible, the highest concentration that was selected for the chromosome aberration assay induced approximately a 50% reduction of the mitotic index relative to the vehicle control. (6) At least 2 additional dose levels, demonstrating limited toxicity or no toxicity were also evaluated. Metaphase cells were selected for scoring based on good chromosome morphology and staining characteristics. Well-spread metaphases were evaluated for structural chromosome aberrations at 1000x magnification using oil immersion. Only metaphase cells with 46 centromeres were analyzed for structural aberrations. At least 200 cells per dose level (100 from each replicate culture), when available, were analyzed for aberrations. At least 100 metaphase cells were scored for numerical chromosome aberrations (polyploidy and endoreduplication cells). Cell cultures treated with the negative, solvent, or positive control substances were analysed similarly. The aberrations observed were tabulated and categorized as chromatid- or chromosome-type aberrations. Gaps were also recorded.AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 13 - K. Evaluation of Results and Statistical Analysis The data were summarized in tables containing mitotic index, percent polyploidy/ endoreduplication (numerical aberrations), number of cells analyzed, types of structural aberrations, frequencies of structural aberrations per cell, and the percentage of cells with structural aberrations. Chromatid and chromosome gaps were listed but were not added to the totals for structural aberration evaluation (gaps are not considered true structural damage). The experimental unit is the cell; therefore the percentage of cells with structural aberrations was the basis for evaluation. Statistical analysis consisted of a Cochran-Armitage test for dose responsiveness (significance was judged at p ≤ 0.01); and Fisher ’ s Exact Test to compare the percentage of cells with structural aberrations (or the percentage of cells with more than one aberration, if required) in the test substance treated groups with the vehicle control response. (7)
For Fisher ’ s Exact Test, statistical significance was judged at the 5% (p < 0.05) level. Chromatid and isochromatid gaps were excluded from the statistical evaluation; they are listed separately in the appropriate appendices.

L. Data Acceptability Criteria The assay was considered acceptable for evaluation of test results because all of the following criteria were satisfied. (For the trials that were rejected due to insufficient or excessive toxicity, or sub-optimal mitotic indices, the data were not used. However, all rejected data are retained in the study records).
1. Acceptable Controls The vehicle and negative (untreated) controls had to contain less than approximately 5% cells with structural aberrations. The positive control response had to be significantly greater (p 50.01, Fisher ’ s exact test) than the vehicle control response.
2. Acceptable High Concentration The assay analysis had to include the highest concentration possible if the results were negative and there was no significant reduction in mitotic index (at least 50% reduction relative to the vehicle controls).
3. Number of Doses Each assay treatment had to include at least 3 analyzable concentration levels.

M. Classification Guidelines The statistical analysis was used as a guide to determine whether or not the test substance induced a positive (i.e., clastogenic) response. A response would have been considered positive for the induction of chromosome aberrations if a statistically significant increase (p 5 0.05,AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 14 - Fisher ’ s exact test) in the percentage of cells with structural aberrations was seen in one or more treatment groups relative to the vehicle control response. Interpretation of the statistical analysis did also rely on additional considerations including the magnitude of the observed test substance response relative to the vehicle control response and the presence of a dose responsive trend. The test substance was classified as negative (i.e., not clastogenic) if there is was no statistically significant increase in the percentage of cells with structural aberrations in any treatment group relative to the vehicle control group. The test substance response may have been classified equivocal if results observed in any of the assay would have resulted in statistically significant elevations in structural chromosome aberrations at more than one test concentration level without demonstrating a dose-responsive trend. In the event of an equivocal response the Sponsor would have been consulted to determine if a repeat test was necessary.

RESULTS AND DISCUSSION

A. Test Substance Solubility A solubility determination was not conducted. Dimethyl sulfoxide (DMSO) was selected to be the solvent based on information provided by the sponsor, the solubility of the test substance, and target cell compatibility. The test substance was completely soluble in DMSO at 500 		
	
	

	
	
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 substance, test substance and solvent were mixed in sealed containers with as little headspace as
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B. Preliminary Cytotoxicity Assay The target concentrations that were tested in a preliminary cytotoxicity portion of the assay (Trial #-	*		 	.	#%	$#	%$	#&	&		 		'	
	

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		 medium was 341 mOsm/kg (vehicle in medium was 443 mOsm/kg). This difference in osmolality is less than 150 mOsm/kg and is considered to be of no significance for the outcome of the study. There was no color change in the test medium, so the pH was not assessed. Upon centrifugation of the top 2 concentrations during the removal of the test substance, an organic phase was noted at the bottom of the centrifuge tube. The organic phase was removed with a pipette. There were no cytogenetically analyzable test concentration levels in the initial cytotoxicity assay due to either excessive toxicity or insufficient toxicity (Table 1). This portionAAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 15 - of the assay was repeated and the concentration levels were reduced, with smaller intervals, to ensure that there would be a sufficient number for analysis. The following concentrations were tested in the repeat assay: 0.088, 0.175, 0.350, 0.450, 0.500, 	!		%& 		7			

		

	


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(	 vehicle controls were sub-optimal (data will not be reported and will be retained in study records). The assay was repeated again at the same concentration levels with cells from a different donor (Trial 2). The cells were treated for 3 hours in the non-activated and in the S9 activated assay. One independent trial was conducted. Metaphase cells were harvested approximately 22 hours after the initiation of the treatment. The test substance was soluble at all concentrations tested. A reduction of the mitotic index (relative to the vehicle control) of 38.1%*
	
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C. Chromosome Aberation Assay One independent trial of chromosome aberration analyses was evaluated without and with metabolic activation (Tables 3 and 4). In the non-activated test, chromosome aberrations were evaluated at dose levels of 0.18, 0.35, 	  		7		
	
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 each trial. Post-treatment harvest times of 22 hours were selected. Results for the non-activated individual cultures 3-hour exposure are presented in Table 3 and summarized by group in Table 5. At the highest concentration of #873 evaluated for clastogenicity in the non-activated 
	  	

	



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(			' observed mitotic inhibition, relative to the solvent control, at this concentration was less than 50%, however it was selected as the top concentration for analysis for several reasons. All other concentrations were well in excess of 50% mitotic inhibition, the interval between this
				

	
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 inhibition was decreased 45.4% relative to the solvent control. The percentage of cells with structural and numerical chromosome aberation was not significantly increased above that in the solvent control (p > 0.05). As expected, the positive indicators, MMC and CP, induced statistically significant increases in the percent of abnormal cells.

CONCLUSIONS Under the conditions of this study, no evidence of in vitro clastogenic activity was detected in any assay with the test substance. Based on the findings, #873 was concluded to be negative forAAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 16 - the induction of structural and numerical chromosome aberrations in human lymphocytes in both metabolically activated and non-activated systems.

RECORDS AND SAMPLE STORAGE Laboratory-specific or site-specific data, such as personnel files and equipment records will be retained by the testing facility. At the request of the Sponsor, raw data, slides, and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware.AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 17 - REFERENCES

1. Preston, R.J., Au, W, Bender, M.A., Brewen, J.G., Carrano, A.V., Heddle, J.A., McFee, A.F., Wolff, S. and Wassom, J.S. (1981). Mammalian in vivo cytogenetics assays: A report of the US EPA ’ s Gene-Tox Program. Mutation Res. 87:143-188.
2. Evans, H.J. (1976). Cytological Methods for Detecting Chemical Mutagens. In: Chemical Mutagens, Principles and Methods for their Detection, Vol. 4, Hollaender, A. (ed.), Plenum Press, New York and London.
3. Maron, D.M. and Ames, B.N. (1983). Revised methods for the Salmonella mutagenicity test. Mutation Res. 113:173-215.
4. DuPont Haskell Laboratory Report Number DuPont-4437.
5. Galloway, S.M., Aardema, M.J., Ishidate, M., Jr., Ivett, J.L., Kirkland, D.J., Morita, T., Mosesso, P., and Sofuni, T. (1994). Report from the working group on in vitro tests for chromosomal aberrations. Mutation Res., 312(3):241-261.
6. Organization for the Economic Cooperation and Development (OECD). Genetic toxicology: in vitro mammalian chromosomal aberration test. OECD Guideline for Testing Chemicals, updated and adopted July 21, 1997; No. 473.
7. Thakur, A.J., Berry, K.J., and Mielke, P.W., Jr. (1985) A FORTRAN program for testing trend and homogeneity in proportions, Computer Programs in Biomedicine, 19:229-233.
8. Hilliard, C.A., Armstrong, M.J., Bradt, C.I. et al., Chromosomal aberrations in vitro related to cytotoxicity of nonmutagenic chemicals and metabolic poisons. Env. Mol. Mutagenesis, 31:316-326.AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 18 - TABLESAAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 19 - TABLES

EXPLANATORY NOTES
ABBREVIATIONS:
Definitions of Chromosome Aberrations for Giemsa Stained Cells

SIMPLE
TB Chromatid Break: An achromatic region in one chromatid, larger than the width of a chromatid. The associated fragment may be partially or completely displaced.
SB Chromosome Break: Chromosome has a clear break, forming an abnormal (deleted) chromosome with an acentric fragment that is dislocated. This classification now includes the acentric fragment (AF). An AF is different from a SB only in that it can not be related to any specific chromosome.
DM “ Double Minute ” Fragment: These are small double dots, which may represent terminal or interstitial deletions, or even small rings. These possible origins are not distinguishable.
COMPLEX
ID Interstitial Deletion: Length of chromatid “ cut out ” from midregion of a chromatid, resulting in a small fragment or ring lying beside a shortened chromatid or a gap in the chromatid. TR Triradial: An exchange between two chromosomes, or one chromosome and an acentric fragment, which results in a three-armed configuration. QR Quadiradial: An exchange like a triradial, but resulting in a four-armed configuration. CR Complex Rearrangement: An exchange among more than two chromosomes or fragments, caused by the induction of several breaks. D Dicentric: An exchange between two chromosomes which results in a chromosome with two centromeres. This is often associated with an acentric fragment in which case it is classified as DF. DF Dicentric with fragment TC Tricentric: An exchange involving three chromosomes and resulting in a chromosome with three centromeres. Often associated with two to three AF. Such exchanges can involve many chromosomes and are named as follows: QC Quadricentric: Four centromeres, up to four AFAAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 20 - COMPLEX PC Pentacentric: Five centromeres, up to five AF HC Hexacentric: Six centromeres, up to six AF R Ring: A chromosome which forms a circle containing a centromere. This is often associated with an acentric fragment, in which case it is classed as RF. RC Ring Chromatid: Single chromatid ring (acentric). RF Ring with associated acentric fragment.
CI Chromosome Intrachange: Exchange within a chromosome; e.g., a ring that does not include the entire chromosome. T Translocation: Obvious transfer of material between two chromosomes resulting in two abnormal chromosomes. When identifiable, scored as "T" not "2AB".
AB Abnormal: Abnormal monocentric chromosome. This is a chromosome whose morphology is abnormal for the karyotype, and often the result of a translocation, pericentric inversion, etc. Classification used if abnormality cannot be ascribed to, e.g., a reciprocal translocation.
OTHER
GT Greater than Ten: Greater than 10 aberrations: A cell which contains more than 10 aberrations. Heavily damaged cells will be analyzed to identify the types of aberrations because multiple fragments, such as those found associated with a tricentric, do not count as independent aberrations. PP Polyploid Cell: A cell containing multiple copies of the haploid number (n) of chromosomes. E Endoreduplication: A failure of chromosomes to separate, resulting in a 4n cell.
NOT COMPUTED TG Chromatid Gap: ("tid gap"). An achromatic (unstained) region in one chromatid, the size of which is equal to or smaller than the width of a chromatid. These are noted but not usually included in final totals of aberrations as they may not all be true breaks. SG Chromosome Gap: ("isochromatid gap, IG"). Same as chromatid gap but at the same locus in both sister chromatids.
UC Uncoiled Chromosome: Failure of chromatin packing. Probably not a true aberration.AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 21 - Table 1 Toxicity Assessment of #873: 3.0-Hour Treatment – Trial 1 a
Culture b Treatment A B Mitotic Index (%) c Percent MI Change (Relative to Vehicle) d
A. WITHOUT METABOLIC ACTIVATION Negative Control e 30 38 3.4 NA Vehicle Control f 36 38 3.7 NA   41 36 3.9 5.4% .  46 43 4.5 21.6% #%  26 34 3.0 -18.9% $#  29 26 2.8 -24.3% %$  02 0 0.1 -97.3% #&  0 0 0 -100% &  0 0 0 -100%   0 0 0 -100%

B. WITH METABOLIC ACTIVATION Negative Control e 24 28 2.6 NA Vehicle Control f 40 32 3.6 NA   25 25 2.5 -30.6% .  33 40 3.7 2.0% #%  48 43 4.6 28.0% $#  44 28 3.6 0% %$  9 1 5 38.9% #&  0 0 0 -100% &  0 0 0 -100%   0 0 0 -100% a 19 hr recovery period. b Metaphase cells/1000 cells. c MI = (# metaphases/1000 cells)*100. d Percent change = ([Treatment MI

– Control MI]/Control MI)*100 e Negative Control is untreated exposed to treatment medium only. f Vehicle Control is dimethyl sulfoxide (1% of the volume of the treatment medium).AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 22 - Table 2 Toxicity Assessment of #873: 3.0-Hour Treatment - Trial 2 a
Culture b Treatment A B Mitotic Index (%) c Percent MI Change (Relative to Vehicle) d
A. WITHOUT METABOLIC ACTIVATION Negative Control e 42 39 4.1 --- Vehicle Control f 39 44 4.2 --- "  25 39 3.2 -23.8% #.  41 37 3.9 -7.1% $  42 34 3.8 -9.5%    29 23 2.6 -38.1%   18 15 1.7 -59.5%   ND ND -- -- .  ND ND -- -- %$  ND ND -- --

B. WITH METABOLIC ACTIVATION Negative Control e 28 21 2.5 --- Vehicle Control f 19 25 2.2 --- "  40 26 3.3 0% #.  17 14 1.6 -27.3% $  10 13 1.2 -45.4%    4 8 0.6 -72.7%   ND ND -- --   ND ND -- -- .  ND ND -- -- %$  ND ND -- -- a 19 hr recovery period. b Metaphase cells/1000 cells. c MI = (# metaphases/1000 cells)*100. d Percent change = ([Treatment MI

– Control MI]/Control MI)*100 e Negative Control is untreated exposed to treatment medium only. f Vehicle Control is dimethyl sulfoxide (1% of the volume of the treatment medium).AAWG #873:In VitroChromosome Aberrations inHuman Peripheral Blood LymphocytesDuPont-4436- 23 -Table 3Individual Culture Analysis of Chromosome Aberrations in Human Peripheral Blood: 3.0-Hour Treatment withoutMetabolic ActivationaNumber And Type Of AberrationsNot Computed Simple Complex OtherCellsTreatment Scored TG IG UC TB SB ID TR QR CR DI R CI IB GTNegative Control A100 1B100 1 1A+B200 1 1 1Vehicle Control10l/mLA100 1B1001A+B200 1 1MMC0.5g/mLA100 2 2 3 1 1B100 3 1 1 5 1A+B200 3 1 2 3 8 1 1 1#8730.18l/mLA93 1B91 1A+B184 1 10.35l/mLA100B100 2 1 2A+B200 2 1 20.45l/mLA94B77 1 1 3A+B171 1 1 3a 19 hr recovery period.b MMC is mitomycin CNote: Negative Control is treatment medium, Vehicle control is dimethyl sulfoxide (1% of the volume of the treatment medium)AAWG #873:In VitroChromosome Aberrations inHuman Peripheral Blood LymphocytesDuPont-4436- 24 -Table 4Individual Culture Analysis of Chromosome Aberrations in Human Peripheral Blood: 3.0-Hour Treatment with Aroclor®1254-Induced S9aNumber And Type Of AberrationsNot Computed Simple Complex OtherCellsTreatment Scored TG IG UC TB SB ID TR QR CR DI R CI IB GTNegative Control A 100B 100 1A+B 200 1Vehicle Control 10l/mL A 100 11B 100 2A+B 200 31CPb5l/mL A 100 1 4 1 2B 100 3 5 2A+B 200 4 9 3 2#873 0.09l/mL A 100 3B 100 1 2A+B 200 3 1 20.18l/mL A 100 3 1 1B 100 1 2A+B 200 3 1 3 10.35l/mL A 100 1 1B 100 1A+B 200 1 1 1a 19 hr recovery period.b CP is cyclophosphamideNote: Negative Control is treatment medium, Vehicle control is dimethyl sulfoxide (1% of the volume of the treatment medium)AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 25 - Table 5 Summary of Chromosome Aberrations of #873: 3.0-Hour Treatment a
Cells % Cells With % Cells With % of cells with Treatment Scored Aberrations >1 Aberrations Polyploidy and Endoreduplication
A. WITHOUT METABOLIC ACTIVATION Negative Control 200 0.6% 0.0% 1.0%
Vehicle Control 10 l/mL 200 1.0% 0.0% 0.5%
MMC b 0.5 g/mL 200 7.0% * 0.0% 0.5%
Test Substance 0.18 l/mL 184 0.0% 0.0% 1.0%
0.35 l/mL 200 1.0% 0.0% 1.0%
0.45 l/mL 171 1.2% 0.6% 0.5%

B. WITH METABOLIC ACTIVATION Negative Control 200 0.5% 0.0% 0.5%
Vehicle Control 10 l/mL 200 0.5% 0.0% 1.0%
MMC b 5 g/mL 200 7.0% * 0.0% 0.0%
Test Substance 0.09 l/mL 200 1.0% 0.5% 0.5%
0.18 l/mL 200 2.0% 0.0% 2.5%
0.35 l/mL 200 0.5% 0.0% 0.0% a 19 hr recovery period. b MMC is mitomycin C *Significantly greater than the vehicle control, p ≤ 0.01 Note: Negative Control is treatment medium, Vehicle control is dimethyl sulfoxide (1% of the volume of the treatment medium)AAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 26 - APPENDIX - HISTORICAL CONTROLAAWG #873: In Vitro Chromosome Aberrations in Human Peripheral Blood Lymphocytes DuPont-4436

- 27 - Historical Control Data a Controls % of Cells % of Cells % Polyploidy and Treatment With Aberrations With >1 Aberrations Endoreduplicated Cells

A. WITHOUT METABOLIC ACTIVATION

Pooled MIN 0.0 0.0 0.0 Negative MAX 0.5 0.0 0.5 and Vehicle AVG 0.1 0.0 0.1 Controls SD( ± ) 0.2 0.0 0.2 N8 8 8
Positive - MIN 2.5 0.0 0.0 MMC MAX 10.0 2.0 1.5 3-Hour AVG 6.8 0.6 0.3 SD( ± ) 2.9 0.7 0.6 N8 8 8

B. WITH METABOLIC ACTIVATION
Pooled MIN 0.0 0.0 0.0 Negative MAX 0.5 0.0 1.0 and Vehicle AVG 0.1 0.0 0.2 Controls SD( ± ) 0.2 0.0 0.4 N8 8 8
Positive - MIN 3.8 0.0 0.0 CP MAX 8.0 1.0 1.0 3-Hour AVG 6.0 0.5 0.5 SD( ± ) 1.6 0.4 0.3 N8 8 8 a Data are based on 2 studies reported during the year 2000.
ABBREVIATIONS: MMC = mitomycin C CP = cyclophosphamide N = Number of cultures evaluated