### 111111111111111111111111111111111111111111111111111111111111 

## OTS0573552 One Source. One Search. One Solution. 

# HEXABROMOCYCLODODECANE (HBCD): CHROMOSOME ABERRATIONS IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES WITH COVER LETTER DATED 

# 12/12/1996

12 NOV 1996

U.S. Department of Commerce 

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# www.ntis.gov**/S_~"":\** U.S DEPARTMENTOF COMMERCE •**~'.** • Technology Administration \ • · / National Technical Information Service

.,_.,. Springfield, VA 22161CODING FORMS FOR SRC INDEXING 

### Microfiche No. OTS0573552

86970000358**ll/~2/96** 12/19/96 BD

MICROBIOLOGICAL ASSOC INC

HEXABROMOCYCLODODECANE (HBCD): CHROMOSOME ABERRATIONS IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES WITH COVER LETTER DATED**~2/~2/~996** 

### Chemical HEXABROMOCYCLODODECANE (25637-99-4)OFFIC!t OF TOXIC

**SUBS1:A.~CES** 

### CODING FORM FOR GLOBAL INDEXING RE'h 7/27/82

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## ,.CHEMICAL MANUFACTURERS AssociATION 0\\ ..,, \ t1 

## S

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### '3 \ -' December 12, 1996 uVia Certified Mail DocumentControlOffice (7407) Office ofPollutionPreventionandTaxies 

### U.S, EnvironmentalProtectionAgency RoomG-099 401M Street,SW Washington,

### DC 20460 Attention: TSCA 8(d)HealthandSafetyReportingRule <Notification) DearSirorMadam: TheChemicalManufacturersAssociationBrominatedFlameRetardantIndustry Panel (BFRIP)herebysubmitsthe followingreportonhexabromocyclndodecane (OBDPO, 

### CAS No.9233o-52-0). 

### ;;;> 

## "'> 0:. 7,

**~** ·• 

## Cf'7
- 

## ¥ 1) HEXABROMOCYCLODODECANE (HBCDl: ChromosomeAberrationsin HumanPeripheralBlood Lymphocytes, The

### BFRIPmembercompanies onwhosebehalfthisnotificationissubmittedare Jfsted inAttachment1. Thename andaddressofthelaboratory thatconductedthisstudyis

### ae follows: MicrobiologicalAssociates,Inc, 9630 MedicalCenterDrive Rockville,MD 20850 

### Ifyouhaveanyquestionsregarding

### thisTSCA

### 8(d)notification,pleasecontactmeby telephone at703/741-5637, or

### by fax at703/741-6091.

Attachment 

## 869'70000358 Sincerely,
HasmukhC. Shah,Ph.D. Manager,BFRIP



# " r..f..allestmillJiaCare• 1300WILSON BOULEVARD, ARUNGTONt VA 22209 • TELEPHONE **703~741~5000** • FAX **703~741-6000** 

## "lf"**A~~**

### AlbemarleCorporation 451 Florida Street BatonRouge, LA 70801 Ameribrom,Inc. 52

### Vanderbilt NewYork,NY 10017 GreatLakes ChemicalCorporation P.O. Box

### 2200 Highway52 NW WestLafayette, IN 47906 

## Attachment 1

### FINAL P..EPORT 

### % 

### D!:C I9 P.'l 3: I4
CHROMOSOME ABERRATIONS IN IDJMAN 

### PERIPHERAL BLOOD LYMPHOCYTES Rexabromocyclododecane Authors Ramadevi Gudi, PILD. Elizabeth 

### R Schadly, B S.

Study Completion Date

November 12, 1996 Performing Laboratory Microbiological Associates, Inc.

9630 Medical Center Drive Rockville, Maryland 20850

Laboratory Study Number G96A061.342 Sponsor Chemical Manufacturers Association 1300 Wilson Boulevard

Arlington VA 22209 Page I of34



### ~· 

### MICROBIOLOGICAL X ASSOCIATES, INC.

. ' ' 

### . . .

### STATEMENT OF COMPUANCE Study G96A06L342 was conducted in compliance with the US FDA Good Laboratory Practice Regulations as published in 21 CFR 58, the US EPA GLP Standards 40 CFR 160 and 40 CFR792, the UK GLP Compliance Progranune, the Japanese GLP Standard and the OECD Principles ofGood Laboratory Practice in all material aspects with the following exceptions:

The identity, strength, purity and composition or other characteristics to define the test or control article were not determined by the testing facility.. The stability ofthe test or control article under the test conditions was not determined by the testing facility. Analyses to determine the uniformity, concentration, or stability ofthe test or control mixtures were not pe1fonned by the testing facility.

Rarnadevi

**Gud~** PhD. Stu!iy Director

MA Study No. G96A06J.342 2 

# J//Jv/'16 Date



### ~· 

## MICROBIOLOGICAL
. 

## X. ASSOCIATES, !N.C.

# •. QUALITY ASSURANCE STATEMENT Study Titleo CHROMOSOME ABERRATIONS IN HUMAN PERIPHERJl.L BLOOD LYMPHOCYTES Study Numbero G96A061. 342 Study Director: Ramadevi Gudi, Ph.D. This study has been divided into a series of in-process phases. Using 

### a random sampling approach, Quality Assurance monitors each of these phases over a series of studies. Procedures, documentation, equipment 

### records, etc., are examine.d in order to assure that the study is 

### performed in accordance with the U.S. FDA Good Laboratory Practice Regulations (21 CFR 58), the U.S. EPA GLPs (40 

### CFR 792 and 40 

### CFR 160), 

### the UK GLP Compliance Programme, the Japanese GLP Standard, and the OECD Principles of Good Laboratory Practice and to assure that the study is conducted according to the protocol and relevant

**Standa~d** 

### Operating Procedures. The following are the inspection dates, phasP.S inspected, and report dates of QA inspections of this study. INSPECT ON 07 JUN 96, TO

**S~Y** DIR 07 JUN 96, TO MGMT 07 JUN 96 PHASE: Protocol Review INSPECT ON

**~3** JT.m 96 I TO STUDY DIR 13 JUN 96 I TO HGMT 24 JUN 96 PHASE: Colcemid treatment of the test system INSPECT ON 12 SEP 96-13 SEP 96, TO STUDY DIR 13 SEP 96, TO MGMT 17 SEP 96 PHASE: Draft Report INSPECT ON 13 NOV 96, TO STUDY DIR 13 NOV 96, TO MGM"J; 15 NOV 96 PHASE: Draft to Final Report 

### This

**repor~** 

### describes the methods and procedures used in the study and the reported results accurately :reflect the raw data of the study.



# ~1?irf~t IH54Co Diane B. Madsen QUALITY ASSURANCE DATE

3

### CHROMOSOME ABERRATIONS IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES FINAL REPORT Sponsor: Chemical Manufacturers Association 1300 Wilson Boulevard Arlington, VA 22209 Authorized Representative: Dr. Hasmukb C. Shah Performing Laboratory: Microbiological Associates, Inc. (MA) -:, 

### 9630 Medical Center Drive Rockville, Maryland 20850 

### Test Article 

### I D.: Hexabromocyclododecanc 

### MA Study No.: G96A061.342 Test Article Description: white powder Storage Conditions: room temperature, protected from light and moisture Test Article Receipt: 

### May 21, 1996 Study Initiation: June 

### 5, 1996 Laboratory Supervisor: Elizabeth 

### H. Schadly, B.S. 0


### Study Director: Rarnadevi Gudi, Ph.D. Date

if !.'
MA Study No. G96A061.342 4 I' 

## "&;,' MICROBIOLOGicAL

**~** 

## ASSOCIATES, INC.

### TABLE OF CONTENTS Page Summary
. . . . . . . . . . • . . • • . . . . • . . • . • . • . . . • • . • . . • . . • . • • . • . • . • . • . . 6 

### Purpose ...........•........................•...................•.. 8 Characterization ofT ost and Control Articles . . . . . . . . . • . . • . . . . . • . . . . . . . . . . 8 Materials and Methods ............ , .........•..................... 8 Results and Discussion ....... , ............. , . . . . . . . . . . . . . . . . • . . . . 12 Conclusion .......... , .........................•.............. 14 References . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . • . . . . . . . . . . . . . . . . . 15 Data Tables ..............•...... , .................... , ......... 16 Appendix 1: Historical Control Data .... , .... , .. , . . . . . . . . . . . . . . . . . . . . . 24 Appendix II: Study Protocol ..••.... , . , ....•.....•.•..•......... , . . . 26

MA Study No. G96A061342 

## s 

# ?.:ti• MICROBIOLOGICAL 

## 5?;:: ASSOCIATES, INC.

### SUMMARY The test article, Hexabromocyclododecane, was tested in the 

### in vitro mammalian cytogenetic test using human peripheral blood lymphocytes (HPBL) both in the absence and presence ofmetabolic activation. The assay was performed in two phases. The first phase, the initial chromosome aberration assay, was conducted to establish the dose range for testing and to evaluate the clastogenic potential ofthe test article. The second phase, the independent repeat chromosome aberration assay, was performed to confirm the test system response to the test article seen in the initial assay. Dimethylsulfoxide (DMSO) was the solvent ofchoice based on information provided by the Sponsor, the solubility ofthe test article and compatibility with the target cells. The test article was soluble in DMSO at approximately 500 mg/ml, the maximum concentration tested. In the initial chromosome aberration assay, the maximum dose tested was 2500 flg/ml. This dose was achieved using a stock concentration of250 mg/ml, and a 100 1'1 dosing aliquot added to 10 ml fresh complete medium or S9 reaction mix. Dose levels greater than 2500 Jlg/ml were insoluble in treatment medium and not tested in the assay. Visible precipitate was observed in treatment medium at dose levels 750 and 2500 Jlg/ml and was soluble but cloudy (no visible precipitate) at dose levels 75 and 250 flg/ml. The test article was soluble in treatment medium at all other dose levels tested. In the non· activated portion ofthe initial assay HPBL cells were exposed to the test article continuously for 20 hours; inthe S9-activated portion ofthe initial chromosome aberration assay, HPBL cells were exposed to the test article for 4 hours. Metaphase cells were collected for microscopic evaluation at 20 hours after the initiation oftreatment Dose levels of2500 J!g/ml in the non-activated study and 750 and 2500 flg/ml in the 89- activated study were not analyzed for chromosome aberrations due to complete mitotic inhibition. Toxicity (mitotic inhibition) of approximately 56% was observed at the highest dose level (750 flg/ml) evaluated for chromosome aberrations, in the non-activated study. 

### 1n the S9-activated study, 13% toxicity was observed at the highest dose level (250 J!g/ml) evaluated for chromosome aberrations. No statistically significant increases in chromosome aberrations were observed in either the non-activated or 59-activated test systems relative to the solvent control group regardless ofdose level (p>0.05, Fisher's exact test). Based on the results ofthe initial assay, an independent repeat chromosome aberration assay was conducted in the absence and presence ofan Aroclor-induced S9 metabolic activation system at dose levels of 10, 19, 38, 75, 150, 300, and 600 J!g/mL The test article was soluble but cloudy at dose level 75 flg/ml and was workable in treatment medium at dose levels !50 Jlg/ml and higher. The test article was soluble in treatment medium at all other concentrations tested. Inthe independent repeat assay, HPBL cells were exposed to the test article continuously for 20 or44 hours 

### inthe non-activated test system and for 4 hours in the 89-activated test system. Metaphase cells were collected for microscopic evaluation in both the non-activated and S9-activated studies at 20 and 44 hours after the initiation oftreatment. Toxicity, m,.;n.edby mitotic inhibition, was MA Study No. G96A06!.342 6 

# t.:fJ" MICROBIOLOGICAL X ASSOCIATES, INC;approximately 55% and 94% at the 20 and 44 hour ·harvests, respectively, at the highest dose levels (600 and 300 flg/ml) evaluated for chromosome aberrations in the nonactivated studies.. In the 89-activated studies, toxicity was approximately 71% and 69% at the 20 and 44 hour harvests, respectively, at the highest dose levels (300 and 600 Jlg/ml) evaluated for chromosome aberrations. Dose level 600 Jlg/ml in the non-activated 44 hour harvest and in the 89-activated 20 hour harvest was not analyzed for chromosome aberrations due to an insufficient number ofscorable metaphase cells.. No statistically significant increases in structural chromosome aberrations were observed in either the nonactivated or S9-activated studies, regardless ofdose level or harvest time (p>0.05, Fisher's exact test). No statistically significant increases in numerical chromosome aberrations were observed in either the non-activated or 89-activated studies at the 44 hour harvest time, regardless ofdose level (p>O.OS, Fisher's exact test}. Based on the findings ofthis study, Hexabromocyclododecane was concluded to be negative for the induction of structural and numerical chromosome aberrations in human peripheral blood lymphocytes.

MA Study No. G96A061342 7 

### ~·MICROBIOLOGICAL

**~** 

## ASSOCIATES, INC.PURPOSE· 

### The purpose ofthis 

### study was to evaluate the clastogenicpotential ofa test article based upon its ability to induce chromosome aberrations in hwnan peripheral lymphocytes. CHARACTERIZATION OFTEST AND CONTROL ARTICLES The test article, Hexabromocyclododecane, was received by Microbiological Associates, Inc. on May 21, 1996 and was assigned the code nwnber 96A06L The test article was characterized by the Sponsor as an off-white powder with a characteristic odor. No expiration date was provided. Upon receipt, the test article was described as a white powder and was stored at room temperature, protected from exposure to light and moisture. The solvent used to deliver Hexabromocyclododecane to the test system was DMSO (CAS No.: 67-68-5), supplied by the Fisher Scientific Company. Mitomycin C (MMC; CAS No.: 50-07-7), was obtained from the Sigma Chemical Company, and was dissolved and diluted in sterile distilled water to stock concentrations of 12.5 and 25 J.lg/ml for use as the positive control in the non-activated test system. Cyclophosphamide (CP; CAS 

### No,: 6055-19-2), was obtained from Sigma Chemical Company, and was 

### dissolved and diluted in sterile distilled water to stock concentrations of2.5 and 

### 5 mg/ml for use as the positive control in the S9-activated test system. For each positive control one dose with sufficient scorable metaphase cells was selected for analysis. The solvent for the test article was used as the solvent control at the same concentration as that found inthe test article-treated groups. RPMl-1640 complete medium (containing 15% fetal bovine serum, 2 mM L-glutarnine, I00 units penicillin and I

### 00 J.lg streptomycinlml) supplemented with 1% PHA, or S9 reaction mixture was used in the untreated controL MATERIALS 

### AND METHODS Test System Peripheral blood lymphocytes were obtained from a healthy 40 year old adult female with no recent history ofeither radiotherapy, viral infection orthe administration ofdrugs. 

### This system has been demonstrated to be sensitive to the clastogenic activity ofa variety ofchemicals (preston, eta!., 1981). Activation System Aroclor 1254-induced mt liver S-9 was used as the metabolic activation system. The S-9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal iqjection ofAroclor 1254, 500 mg/kg, five days prior to sacrifice. The S-9 was batch prepared and stored 

### at S..70°C until used. ;flach bulk preparation ofS-9 was assayed for its ability to metabolize 2-arninoanthracene imd 7,12-dimethyl-benz(a.)anthracene to forms mutagenic to 

### Salmonella 1yphimurium TAl00. MA Sttuly No.. G96A06L342 8 

# (,;'i;"MICROBIOLOGICAL 

# 'X ASSOCIATES, INC.

### Immediately prior to use, the 

## S-9 was thawed and mixed with a cofactor pool to contain 2 mM magnesium chloride, 6 mM potassium chloride, I mM glucose-6-phosphate, I mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 Jll S-9 per milliliter medium (RPMl !640 serum..free medium supplemented with I00 units penicillin and 100 Jlg streptomycin!ml, and 2 

### mM L-glutamine). SolubilityTest A solubility test was conducted using dimethylsulfoxide (DMSO) which \Vas selected by the Sponsor as the solvent of choice. The test was performed to determine the level of solubility ofthe test article in DMSO, which permitted preparation ofthe highest soluble or workable stock concentration, up to 500 mg/mL Chromosome Aberration Assays (Initial and Independent Repeat) The initial chromosome aberration assay was performed using standard procedures (Evans, 1976), by exposing duplicate cultures ofHPBL to nine concentrations ofthe test article as well as positive, solvent and untreated controls. In the initial assay, the dividing cells were harvested at approximately 20 hours nom the initiation oftreatment The independent repeat chromosome aberration assay was performed by exposing duplicate cultures ofHPBL to at least four concentrations ofthe test article, as well as positive, solvent and untreated controls. The concentrations tested were selected based on the findings ofthe initial assay. Forthe independent repeat assay, the dividing cells were harvested at two time points, 20 and 44 hours after the initiation oftreatment For the chromosome aberration assays, 0.6 ml heparinized blood was inoculated into centrifuge tubes containing 9.4 ml complete medium supplemented with 1% PHA The tubes were incubated at 37±1"C in a humidified atmosphere of5±1% CO, in air for approximately 44-48 hours. Treatment was carried outby refeeding with approximately 10 ml 

### fresh complete medium or S9 reaction mixture to which was added 100 )ll of dosing solution oftest or control article in solvent or solvent alone. An untreated control consisting ofcells in complete medium or S9 reaction raixture was also included
.. In the non-activated study, the cells were exposed for 20 or 44 hours at 37±1"C in a humidified atmosphere of5±1% C0 2 

### in air. Inthe S9-activated studies, the cells were exposed for 4 hours at 37±1'C in a humidified atmosphere of 5±1% C02 

### in 

### air After the exposure period, the treatment medium was removed, the cells washed with calcium and magnesium free-phosphate buffered saline (CMF-PBS), refed with complete medium containing I% PHA and rettrrned to the incubator for an additional 16 hours for the first harvests or 40 hours for the delayed harvest. For all treatment groups, two hours prior to the scheduled cell harvests at 

### 20 or 44hours after treatment initiation, Colcemid® was added to the cultures at a final concentration of

### OJ Jlg/ml.

MA Study No. G96A06L342 9 

### ~· 

## MICROBIOLOGICAL X 

## ASSOCIATES, INC•

. 

### . 

### 'Collection ofMetaphase Cells Two hours after the addition ofColcemid®, metaphase cells were harvested for both the activated and non-activated studies by centrifugation The cells were collected by centrifugation at approximately 1200 rpm for about 5 minutes. 1he cell pellet was resuspended 

### in 5 ml 0.075 M KCI and incubated at 37±1

# oc for 20 minutes. At the end of the KCI treatment and immediately prior to centrifuging, the cells were gently mixed and approlcimately 0.5 mi offixative (methanol:glacial acetic acid, 3:1 vivfwas added to each tube. The cells were collected by centrifugation, the supernatant aspirated, and the cells were fixed with two washes with approximately 3-5 

### ml fixative and stored in fixative overnight or longer at approximately 2·6°C. Slide Preparation To prepare slides, the ftxed cells were centrifuged at approximately 1200 rpm for 5 minutes, the supernatant lluid was aspirated, and the cells were resuspended 

### in I ml cold fresh fixative.. The cells were collected by centrifugation and the supernatant aspirated, leaving 0.1 to 0..3 

### ml fixative above the cell pellet An aliquot ofcell suspension was dropped onto a glass slide and allowed to air dry overnight. Slides were identified by the study number, dose level, activation condition, harvest time, replicate tube designation and date prepared.. The dried slides were stained with 5% Giemsa, air dried and pennanently mounted. Evaluation ofMetaphase Cells Slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells with 46 centromeres were examined under oil immersion without priorknowledge oftreatment groups. Whenever possible, a minimum of200 metaphase spreads (1 00 per duplicate treatment condition) were exantined and scored for clu·omatid-type and chromosome-type aberrations (Scott et 

### al., 1990). Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Cluomosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead were considered part ofthe incomplete exchange. Pulveri2ed chromosome(s), pulverized cells and severely damaged cells (>I0 aberrations) also were recorded. Chromatid and isochromatid gaps were recorded but not included 

### in the analysis. The 

### XX coordinates for each cell with chromosomal aberrations were recordedusing a calibrated microscope stage. The mitoticindex was recorded as thepen:entage ofcells 

### in mitosis per 500 cells counted. 

### In the delayed harvests, the percent polyploid cells was recorded per 100metaphase cells.

MA Study No. 096A061.342 10 

# s· MICROBIOLOGICAL 

## x. ASSOCIATES, INC.Controls Mitomycin C was used as the positive control in the non-activated study at final concentrations of 0.13 and 0.25 f!g/mL Cyclophosphamide was used as the positive control in the S-9 activated study at final concentrations of 25 and 50 f!g/mL For both positive controls one dose with sufficient scorable metaphase cells was selected for analysis. The solvent vehicle for the test article was used as the solvent control at the same concentration as that found in the test article-treated groups. Growth medium or 

### 89 reaction mixture was used in the untreated controL Evaluation ofTest Results The toxic effects oftreatment are based upon mitotic inhibition relative to the solvent-treated control and are presented for boththe initial and the independent repeat chromosome aberration assays The number and types of aberrations, the percentage of structurally damaged cells (percent aberrant cells) in the total population of cells examined, the percentage ofnumerically damaged cells in the total population ofcells examined, and the frequency ofstructural aberrations per cell (mean aberrations per cell) was reported for each treatment group Chromatid and isochromatid gaps are presented in the data but arc not included inthe total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell. Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test Fisher's test was used to compare pairwise the percent aberrant cells ofeach treatment group with that ofthe solvent controL In the event ofa positive Fisher'stest at any test article dose level, the Cochran-Anoitage test was used to determine dose

### responsiveness. All conclusions were based on sound scientific basis; however, as a guide to interpretation ofthe data, the test article was considered to induce a positive response when the percentages ofcells with aberrations 

### are increased in 

### a dose-responsive manner with one or more concentrations being statistically elevated relative to the solvent control group (ps;O.OS). 

### A reproducible and significant increase at 

### 11 single dose level will be considered positive.. The test article was concluded to be negative ifno statistically significant increase was observed relative to the solvent controL 

### Criteria for Determination ofa Valid Test The frequency ofcells with structural chromosome aberrations in either the untreated or solvent control must be no greater than 6%. The percentage ofcells with chromosome aberrations 

### in the positive control must be statistically increased (ps;O.OS, Fisher's exact test) relative to the solvent control or to the untreated control ifa solvent other than water was used.

MA Srudy No. G96A06L342 II 

### ~· 

## MICROBIOLOGI.CAL

**~** 

## ASSOCIATES,

**lt~C.**Record and Specimen Archives Upon completion ofthe fmal report, all raw data, reports, and stained and coded slides are maintained in the archives ofMicrobiological Associates, Inc. located in Rockville, Maryland. RESULTS AND DISCUSSION Solubility Test DMSO was selected by the Sponsor as the solvent ofchoice based on solubility ofthe test article and compatibility with the target cells> The test article was soluble in DMSO at approximately 500 mg/ml, the maximum concentration tested. Initial Assay Inthe initial chromosome aberration assay HPBL were exposed to nine concentrations oftest article ranging from 0.25 l!g/ml to 2500 11g/ml. Test article concentrations greater than 2500 l!g/ml were insoluble in treatment medium and not tested in the assay. The test article was soluble but cloudy in treatment medium 

### at dose levels 75 and 250 l!glml and workable in treatment medium 

### at dose levels 750 and 2500 l!g/mi. The test article was soluble in treatment medium at all other dose levels <ested. The osmolality and pH ofthe highest concentration tested, 2500 !'g/ml, were 419 mmollkg and approximately 8, respectively. The osmolality ofthe solvent, DMSO, was 447 mrnol/kg. Metaphase cells were collected for microscopic evaluation at 20 hours after the initiation oftreatment The activity ofHexabromocyclododecane in the induction ofchromosome abetrations in HPBL was tested following a 20 hour exposure period in the absence ofan exogenous source ofmetabolic activation. The fmdings ofthe cytogenetic analysis are presented by treatment culture in Table 1 and summarized by group in Table 3. At the highest test concentration evaluated (750 11g/ml) microscopically for chromosome aberrations, the mitotic index was reduced 

### 56% relative to the solvent control. Dose level 2500 l!g/ml 

### was not selected for analysis due to an insufficient number ofscorable cells. Dose levels 0.25, 0.75, 2.5 and 7.5 l!g/ml were tested but notrequired for analysis. The percentage of cells with structural abenations in the test article-treated groups was not significantly increased above that ofthe solvent control 

### at any dose level (p>0.05, Fisher's exact test). The percentage ofaberrant cells inthe MMC group was 8.5% 

### (p:r;O.Ol, Fisher's exact test). The activity ofHcxabromocyclododecane in the induction ofchromosome aberrations in HPBL cells was tested using a 4 hour exposure 

### and a 

### !6 hour recovery period in the presence ofan exogenous source ofmetabolic activation. The findings ofthe cytogenetic analysis are presented by treatment 

### flask inTable 2 

### and summarized by group inTable 3. Atthe highest test concentration evaluated (250 l!g/ml) for chromosome aberrations, the mitotic index 

### was reduced 13% relativeto the solverit'controL Dose levels 750

**an~··zsoo** I!Wml were not selected for analysis due to

## an insufficient number ofscorable

**c~ll~,** 

### Dose 

# ?,:;i)" MICROBIOLOGICAL



## ~ **ASSOCIA"f~,**·INC. · MA Study No. G96A061.342 12levels 0.25, 0.75, and 2.5 f!g/ml were tested but not required for analysis. The percentage ofcells with structural aberrations in the test article-treated groups was not significantly increased above that ofthe solvent control at any dose level (p>0.05, Fisher's exact test). The percentage ofaberrant cells in the CP group we.s 6.5% (pSO..OI, Fisher's exact test). Independent Repeat Assay Based on the results ofthe initial assay, dose levels of 10, 19, 38, 75, 150, 300 and 600 f!g/ml were selected for further study in both the non-activated and 89-activated portions ofthe independent repeat assay with 20 and 44 hour cell harvest times. The test article was soluble but cloudy in treatment medium at dose level 75 flg/ml, and was workable in treatment mediwn at dose levels !50 f!glml and higher. The test article was soluble in treatment mediwn at all other concentrations tested. The osmolality ofthe highest concentration (600 f!g/ml) tested was 381 mmol/kg. The osmolality ofthe solvent, DMSO, was 393 mmol!kg. The pH ofthe highest concentrations tested was approximately 8. At the time oftest article administration, due to technical oversight, the cultures were treated approximately 43 minutes to I hour 13 minutes beyond the 44-48 hour time limit; since the result ofthe study was negative, the Study Director determined this deviation to have had no significant effect upon the test system. The findings ofthe cytogenetic analysis for groups tested in the absence of an exogenous source ofmetabolic activation are presented by treatment culture in Tables 4 and 5 and summarized by group in Table 8. At the highest test concentrations evaluated for chromosome aberrations, 600 and 300 Jig/ml, the mitotic indices were reduced 55% and 94% relative to the solvent control at the 20 hour and 44 hour harvests, respectively. Dose level 600 flg/ml in the 44 hour harvest was not analyzed due to complete mitotic inhibition. Dose levels 10, 19, and 38 flg/ml in the 20 hour harvest were tested but not required for analysis. In the 44 hour harvest, dose levels I0 and 19 f!g/ml were not required for analysis. The percentage ofcells \\ith structural aberrations in the test articletreated groups was not significantly increased above that ofthe solvent control at any dose level or harvest time (p>0.05, Fisher's exact test). The percentage ofcells with numerical aberrations in the test article-treated groups 

### was not significantly increased above that of the solvent control atthe 44 hour harvest time, regardless ofdose level (p>0,05, Fisher's exact test). The percentages ofaberrant cells in the MMC groups were 7.5% and 35% (p$0.01, Fisher's exact test) for the 20 and 44 hour harvest times, respectively. 'The fmdings ofthe cytogenetic analysis for groups tested in the presence ofan exogenous source ofmetabolic activation are presented by treatment flask in Tables 6 and 7 and summarized by group in Table 8. At the highest test concentrations evaluated for chromosnme aberrations, 300 and 600 flg/ml, in the 20 

### and 44 hour harvests, respectively, the mitotic indices were reduced 71% and 69% relative to the solvent controL Dose level 600 f!g/rnl in the 20 hour harvest was not analyzed due 10 complete mitotic iultibition Dose levels 10 and 19 Jig/rnl in the 20 hour harvest and 10, )9, and 38 Jig(ml in the 44 hour harvest were tested but not required for analysis. The percentage ofcells with · structural aberrations in the test article-treated groups 

### was not statistically increased above that ofthe solvent control at any dose level or harvest time (p>O.OS, Fisher's exacttest). Thepercentage ofcells with numerical aberrations in the test article-treated groups 

### was MA Study No, G96A061.342 13 

### ~· 

## MICROBIOLOGICAL 

# X ASSOCIATES, INC. .not statistically increased above that ofthe solvent control at the 44 hour harvest time, regardless ofdose level (p>0.05, Fisher's exact test). The percentages ofaberrant cells 

### iii the CP groups were 6% and 8.5% {p$:0.01, Fisher's exact test) for the 20 and 44 hour harvests, respectively. 

### CONCLUSION The positive and negative controls fulfilled the requirements for a valid test. Under the conditions ofthe assay described in this report, Hexabromocyclododecane was concluded to be negative in the non-activated and S9-activated test systems in the in 

### vitro mammalian cytogenetics test using human peripheral blood lymphocytes.

MA Study No. G96A06!342 14 

### ~· 

## MICROBIOLO.GICAL 

# :X ASSOCIATES, INC.

### REFERENCES Evans, HJ. and M.L. O'Riordan. 1975. Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mutation Res. 31:135- 148. Evans, H..L (I976) Cytological methods for detecting chemical mutagens, 

### in: Ac Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, vol 4. Plenum Press, New York. Preston, R.J., W. Au, M.A. Bender, J.G. Brewen, A.V.. Carrano, J.A. Heddle, A.F. McFee, S. Wolffand J.S. Wassom (1981) Mammalian 

### in vivo and 

### in vitro cytogenetic assays: a report ofthe Gene-T ox Program, Mutation Research, 87:143-188. Scott, D., N.D. Danford, B.J. Dean, and D.J. Kirkland. 1990. Metaphase Chromosome Aberration Assays In Vitro. In: Basic Mutagenicity Tests: UKEMS Recommended Procedures. D.J. Kirkland (ed.) Cambridge University Press, New York, NY.

MA Study No. G96A061342 IS 

### ~·MICRO_

## B_IOLOGICAL 

# x. ASSOCIATES, INC. •TABLE 1 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAl BLOOD LYMPHOCYTES TREATED \UTH Hexi:lbromocyclododecone IN THE

**AB~NCE** OF EMOGENOUS METABOLIC ACTIVATION INITIAL ASSA'f: 20 IIOUR HARVEST
Mitotic Aberrant severely Celts Average Treatment1

·8 Culture Jndcx' scored Celts3 Damaged Aberrations 

### c•l (>)

**cells~r** Per cettP
Untreated A 1.4 100 0 0 0 0 0 0 0 0 0.000

cells B 1.6 100 1 1 1 0 6 0 0 0 0.010 DIISO 

# • 1.4 100 0 0 0 0 0 0 0 0 0.000 B 2.2 100 0 0 0 0 0 0 0 0 

### o ..ooo Hexabrcmocyclododecone 0.25 Ue/mle 0.75 pg/mle 2:u5 JIS/ml! 7.5 JJs/ml8 25 JJs/ml 

# • 1..8 100 0 0 0 0 0 0 0 0 

### o.ooo B 1.2 100 1 0 0 0 0 1 0 0 0.010 15 pgtml 

# • 1.6 100 0 0 0 0 0 0 0 0

**D~DDO** B 1.0 100 0 0 0 0 0 0 0 0 0.000 250 .us/ml A 1.2 100 0 0 0 0 0 0 0 0 

### o.ooo B 0.8 100 0 1 0 0 0 0 0 0 0.000 750 JIQ/rnl A 1.0 9 0 0 0 ;;· 0 0 0 0 0 O.OtlO B 0.6 16 0 0 0 0 0 0 0 0 

### o.aoo 2500 JJ9/rll 8 HMC, A 1.8 100 6 2 7 1 0 0 0 0 0.080 

### a:zs J.Je/ml B 1.4 100 11 1 8 6 1 0 0 0 

### a.tso

1HPBL were treated for 20 hours at 37±1•c fn 'the absence of an exogenous source of metabolic octiwticn.

**~Httottc** index = nurber mitotic figures x 100/500 cells counted. Excludine celLG wtth only

**gap~.** 'chromatid breales include chromatid and tsoehrcmatid breaks and fragments; chromatid cxchonga fisure;. CExch) inclu::te quadr·iradials, triradials ond carplex raarrnngcments. 5 Chr01n0some breaka ·inc:tude breaks and acentric fragments: die, dfcentrtc chrCUJJso:ne. 6 Severely damaged cells Includes cells with one or more r:utverized chromooanec and cella with 10 or mere

**aberrations~** 7Severely damaged cells ond pulvcri::atfans ware counted as 10 aberrations. 8 Lower dase levels

**w~re** also tested as a guard against excessive toxicity at higher do:se Levels but were not required tor analys1s. Dose level 2500 pg/ml was not

**~valuated** due to

**c~lete** mitotic inhibition_

MA Srudy No. G96A061342 16 

# ~· MICROB.IOLO.GICAL 

# X ASSOCIATES, INC. · .

. .

**~**TABLE 2 CVTOGE»ETIC Mll.lYSIS OF ffUfWl PERIPHERAl BlOOD LfHPHOCYTES TREATED WITH Hexabromocyc:loclodetane IN THE PRESENCE OF ElCOGENOUS METABOLIC ACTIVATION HUTIAL ASSA't': 20 flOUR HARVEST
Mitotic Cells Aberrant severely Average Treatmcnt1a Culture lndexl Scored ccll!:3 Damaged Aberration:. (%) CXJ

**Cells~** Per Celll ' Untreated A 1.4 100 0 0 0 0 0 0 0 0 0"000

cello B 1.6 100 0 0 0 0 0 0 0 0 0.000 OMSO A 1.4 100 0 0 0 0 0 0 0 0,000 B 1.6 100 0 0 0 0 0 0 0 0.000 Hexobromocrctododccane 0
..25 J;gtml 0.75 tJgtmla 2.5 1.19/mla 7u5 /J!I/ml A 2.2 100 0 2 0 0 0 0 0 0 0.000 B 1.6 100 0 0 0 0 0 0 0 0

**D~ODD** ··-- 25 pg/ml A 1.4 100 0 0 0 0 0 0 0 0 

### o.ooo B 1.6 100 0 0 0 0 0 0 0 0 OnOOO 75 lls/ml A 1.4 100 2 0 0 0 1 0 1 0 0.020 

# • 2.0 100 1 1 1 0 0 0 0 0 0.010 250 JJgjrnl A 1.0 100 0 0 0 0 0 0 0 0 0.000 B 1.6 100 0 0 0 0 0 0 0 0 0.000 '?SO JJg/mlB 2500 IJg/ml8 CP, A 0.4 100 7 2 10 0 0 0 0 0.110 25 Ps/mt B 0.6 100 6 1 9 0 0 1 0 0.110

**H~BL** were treated for 4 hours at 37±1°C in the presence of an exogenous source of metabolic activation. 2 Hftotic

**index~** number mitotic figures x 100/500 cells counted.

**~Exehx:ling cet·!~;**  with only 9o1ps. "Chromatid breaks include chromatid and isochrcmatid breaks and fragments; chrcrnatid exchange figures CExeh) include qundr-iradials, triredials and ccxrplex rearr-angements. 5Chromosome breaks incltx:le breaks and acentric fragments; die, dicentric chrcmo::oma. 6 Severely damaged cetls includes cells with one or more pulverized chromosomes end cells with 10 or more nberrations. 7Severely damaged cells and pulverizations were counted as 10 aberrotions. 8 Lower dose levels were tested as a guard against excessive toxicity at hfgher dose levels but not required for analysis. Dose Levels ?SO and 2500 J.lg/rol were not onnlyZed due to COillJlete mitotic inhibition.

MA Study No" G96A061.342 17



## ~·MICRO.BDOLOG
.. ICAL 

# x ASSOCIATE$,

**INP~**TABLE 3 SUHMARY INITIAL ASSAY Treatment/ X Change Aberrations Celis \.11th S9 Harvest Mftotic from control Cells Per Celt2 Aberrations1 Treatment Activation Time Index HI Scored CHean: SD) (%) Untreated 20/20 1.5 200

**0~005** :!:

**0~071** 0.5 DHSO 20/20 1.8 200

**o~ooo** ±

### a.ooo 0.0 Hexabromccyclododecane 25 J,lg/ml 20/20 1.5 17 200 0.005 ± 0.071 0.5 75 JJg/rnl 20/20 1.3 28 200 

### o ..ooo ± o.ooo 

### o.o 250 Jlg/ml 20/20 1.0 44 200

**0~000** ± 

### o.ooo o.o 750 pg/ml 20/20 0.8 56 

### as o.ooo ± 0.000 

### o.o HHCt 20{20 1.6 -7 200

**D~**115 ± 0
..439 8.5•* 0.25 J}g/ml Untreated + 4/20 1.5 200 0.000 ± 0.000 0.0 DI'ISO 4/ZO 1.5 200

**0~000** ± 0.000 0..0 Hel!abromoc:yclododecane 7.5 JJg/ml 

## • 4/20 2..0 -33 200 0.000 ± 0.000 

### o.o 25 JJ9tml 

## • 4/20 1.6 -7 200 o.ooo ± 0.000 

### o.o 75 JJgtml 

## • 4/20 1.7 ·13 200 0.015 ± 0.122 1.5 250 IJ9/Illl 

# • 4/20 1.3 13 200 

### o.ooo ± 

### o.ooo o.o CP, 

## • 4/20 0.5 67 200 0.110 ± 0.446

**6~s••** 25 J.lg/ml 1 X reduction in mitotic index relative to negative (solvent or mtreatedl controL Test article concentrations are corrpared to the solvent control and the positive control is CtJCil»lf·ed to the untr-eated control. tseverely dllJ'IIIlged .t:ells were counted as :t.., p:S0.01; Fisher's exoct test.

MA study No. G96A06J..342 10 aberrations.

18 

### ~MICROBIOLOGICAL



# ~ ASSOCIATES, INC••.·

. ' " ' . .TABLE 4 CYTOGENETIC ANALYSIS OF HUHAtl PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH Hexnbr0010cyclododecane IN lHE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION INOEPEliOEHT REPEAT ASSAY: 20 HCl.IR HARVEST
celh Severely Mitotic Aberrant Average Treatment1

**~** cutture

**lrdex~** Scored cells3 Damaged Aberrations (%) (%) Cet lsb Per CellJ • untreated A 1.4 100 0 0 0 0 0 0 0 0 0.000

cells B 1.4 100 0 0 0 0 0 0 0 0

**0~000** OHSO A 1..4 100 0 0 0 0 0 0 0 0 

### oftooo B 0.8 100 0 0 0 0 0 0 0 0 

### o ..ooo Hexabromocyctododecane 10 pg/ml 1 19 pg/mla 38 pg/mla 75 pg/ml A 1.0 100 0 0 0 0 0 0 0 0 0,000 B 0.8 100 0 0 0 0 0 0 0 0 

### o.ooo 150

**~o~s/mt** A 1..6 100 0 0 0 0 0 0 0 0 0..000 B 1.6 100 0 0 0 0 0 0 0 0 0.000 300

**~g/ml** A 1.4 100 1 0 1 0 0 0 0 0 0..010 B 0.2 100 0 0 0 0 0 0 0 0 0.000 600 pg/llll A 0.4 100 0 0 0 0 0 0 0 0 0.000 B 0.6 100 0 0 0 0 0 0 0 0 0.000 MHC A 1.8 100 8 0 4 3 0 0 1 0 0.080 0.13 pg/ml 

# • 0.4 100 7 0 5 2 0 0 0 0 0.070

1 HPBL were treated for 20 hours nt 37±1°C in the absence of an exogenous source of metabolic activation. 1 Hitotic index::: ntll"ber mitotic figures x 100/500 cells counted. 1 e.xctudtng cells with onty gaps.

**~d'lr-omatid** breaks ,nclude chranatid and isochromatid breaks and fragmentGj chromatid exchange figuren cexeh> include quadriradials, tr·tradiots and canplex rearrangements. 5 C:hrornosome breaks include breaks and acentric fraglll!nts; die, dicentric chromosome. :severely damaged cells includes cells with one or more pulveri<ted chromosomes end cells with 10 or more aberr-ations. Severely dlllllilged cells and pulverizations were counted os 10 aberrations. 8 Lower- dose levels were tested as a guard against excessive toxicity at higher dose levels but were not required for analysis.

MA Study No. G96A06l.342 19 

### ~· 

## MICROBIOLOGICAL 

# :X ASSOCIATES, INC;TABLE 5 CYTOGENETIC ANALYSIS OF HUMAN PERlPHERAl BLOOD LYMPHOCYTES TREATED WITH Hexabromocyclododecane IN THE ABSENCE Of EXOGENOUS METABOLIC ACTIVATION INDEPENDENT REPEAT

**ASS~Y:** 44 HOUR HARVEST Number of Structural

**A~rratfons** Average Mitotic Cells Cells with Aber-rations1 Chromntid·type•

**ChromosCf'llU•type~** severely Structural Treatment1·a Flask Indexa scored X Gaps Breaks Exch Breaks Die R;ng Damaged Aberrations tH.rnerical Structural Cells6 Per Celt3

·7 Untreated A 4.6 100 0 2 t t 0 t 0 0 0 0,020

cells B 5.8 tOO 0 2 0 2 0 0 0 0 0 0.020 OHSO A 5.2 tOO 0 0 0 0 0 0 0 0 0 0.000 B 4.0 tOO 0 0 0 0 0 0 0 0 0 

### o.ooo Jlcxabroro!OeyclododcctlMe: 10 JJs/mt1 19 pg/ml! 38

**~JetmL** A 4.4 tOO 0 0 0 0 0 0 0 0 0 

### o.coo B 5.6 100 0 t 0 t 0 0 0 0 0 0.010 75 J}g{ml A 5.6 too 0 0 0 0 0 0 0 0 0 

### o.ooo B 4
..4 100 0 t 0 1 0 0 0 0 0 0.010 150 pg!ml A 2.8 too 2 0 1 0 0 0 0 0 0 

### o.ooa B 3.6 100 t 1 0 t 0 0 0 0 0 0.010 300 JJs/ml A 0.2 29 

### a' 0 0 0 0 0 0 0 0 

### o.ooa B 0.4 59 o" 2 0 0 0 0 0 0 t 0.169 600 pgJml8 

# ""' A 2.4 100 1 37 t 23 27 0 0 0 0 0.500 0.13 pg/rnl B 3.8 100 2 33 3 26 22 0 0 t 0 0.490

1HPSL were tr·eated for 44 hours at 37±1°C in the absence cf an exogenous source 1Hitotic index = nunber mitotic figures x 100/500 cells counted. of metabolic activation. Jillll'2rical: includes polyploid and endoreduplicated cells.; structural: e)(cludes cells with only gaps. 4Chr'Oilatid breaks include chrOIIllltid nnd isochrc:matid breaks and fragments; chranatid exchange figures (Exch) include quadriradials. triradials art:! COI!plex rearrangeroonts. 5Chranosome breaks include breaks and acentric fragroonts; die, dtcentric chrOillOsome. 6Severaly dlll!Uilged cells includec cella with one or more pulverized chromosomeo. and cells with 10 or more aberrations. 7Severely damaged cells and pulverizations were counted as 10 aberrations. 8 Lower dose levels were tested as a guard against excessive toxicity at higher dose levels but were not required for

**analysis~** Dose level 600 pgjml was not evaluated due to complete mitotic inhibition. 9 Peroentage based on e total of 56 cells scored for numerical oberraticns. 1aPcrccntage

**~cd** on a total of 75 c:ct Ls scored 'for numrical eberrations.

MA Study No. G96A061.342 20 

# (.tfMICROBIOLOGICAL•.. .

**~** 

## ASSOCIATES; INC.
.-·TABLE 6 CYTOGENETIC ANALYSIS OF HUHAtl PERIPHERAL BLOOO LYMPHOCYTES TREATED WITH Hexabromocyclododecone IN TilE PRESENCE OF EXOGENCXJS HETABOLtC ACTIVATION IHDEPEHDEHT REPEAT ASSAY: 2.0 HOUR HARVEST Tetol

**l!~r** of stru::turol Aberrations Mitotic Cells Aberrant Chromatid·tvwf Ch r'Dti\OStxne'"tvoe5 severely Average Treatment1 8 culture

**Index~** Seor'ed Cetts3 Gaps BreaKs E>ch BreaKs Djc Ring Damaged Aberrations

**(~)** 

# ,., **Cells~** Per Cell37
Untreated A 1.2 100 0 0 0 0 0 0 0 0 

### c.ooo

celts B 1.8 100 0 0 0 0 0 0 0 0 0.000 OMSO A 1.6 100 0 0 0 0 0 0 0 0 0..000 B 1.2 100 0 0 0 0 0 0 0 0

**D~ODO** Hcxabroroocyclododecane 10 pe/mla 19 pg/mt6 38 JJS/tll A 1.4 100 0 0 0 0 0 0 0 0 0"000 B 1 2 100 0 0 0 0 0 0 0 0 0.000 75 pg/ml A 1.0 100 0 0 0 0 0 

# " 0 0 

### o.aoo B 1..6 100 0 0 0 0 0 0 0 0 0..000 150 pg/ml A 1.B 100 0 0 0 0 0 0 0 0 0.000 B 1.6 100 0 0 0 0 0 0 0 0 0.000 300 pg/ml A 0.6 100 0 0 0 0 0 0 0 0 0.000 6 0.2 100 0 0 0 0 0 0 0 0 0.000 600 vstmln CP A 0.4 100 6 0 5 2 0 0 0 0 0.070 25 t.r91ml B 0.6 100 6 0 3 3 0 0 0 0 0.060

1 HPBL were treated for 4 hours at 37±1ac in the presence oi an exogenous source of rootobol ic activation.

**~itotic** index 

### =nl.liber mitotic figUres x 1001500 cells counted. 3E11cludlng celts with only

**gaps~** 4Chromntid breaks include chromatid and isochrctMtid breaks and fragments: cbranatid exchange figUres ;nctude

quadriradials, triraclials and

**cC~qJlex** rearrangements. 5 Chr011'1CSO!Tle breaks include breaks nnd acentric fragJTJ(!nts: die, dic:entric chnxoosome. 6 Scverely damaged cells includes cells with one or more pulverized chromcsaues e.nd cells with 'severely damaged cells and pulverizations were counted os 10 aberrations. 10 or more aberrations. 8 Lower dose levels were tested as a guard against excessive toxicity at higher

**do~e** levels but were not required for analysis.. Dose leveL 600 pg/mt was not analyZed due to an insufficient m.mber of sc:orablc cell&.

MA Study No. G96A06!.342 21 

### ~· 

## MiCROBIOLOGICAL

**~** 

## ASSQCIATES,JNC. ·TABLE 7 t'fTOOEIIETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYHPIIOCYTES TREATED UrTH llexabromocyclododecane IN TilE PRESEHCE OF EXQGENOOS HETABOUC ACTIVATrON INDEPENDENT REPEAT ASSAY: 44 IIOUR HARVEST Nurber of Structural Aberrations Average i Hltotfc tells Cells with Aberrations3

**chrcmatid·type~** ChrCifiiOSO!!'!e•type5 Severely Structural Treatment18 Flask lndex2 Scored 

# ' Gaps arenks EXch Breaks Die Ring

**Domag<y~** Aberrations Nt.rnerical Structural Cells Per cellJ 1
Untreated A 7,.6 100 0 0 0 0 0 0 0 0 0 

### o.ooo

cells B 4.2 100 0 0 0 0 0 0 0 0 0 

### o.ooo DHSO A 6.4 100 0 0 0 0 0 0 0 0 0 O.O!lO B 8.,0 100 0 1 0 0 0 1 0 0 0 0.010 Hexabromocyclododecane 10 ,ug/mt8 75 pg/ml A 3.8 100 0 0 0 0 0 0 0 0 0 0.000 B 5.8 100 0 0 0 0 0 0 0 0 0 0.000 150 f.Jg/ml A 4.0 100 3 0 1 0 0 0 0 0 0.010 B 2.6 100 0 0 0 0 1 0 0 0 0.010 300 f.Jg/ml A 3.6 100 2 0 0 1 0 0 0 0.020 B 3.0 100 1 0 0 0 0 0 0 0..010 600 J.19/ml A 1..2 100 4 1 5 0 0 0 0 B 0.050 B 3.2 100 2 0 2 0 1 0 0 0 0.030 CP A 5.2 100 0 9 1 10 4 0 0 0 0.150 25 pg/ml B 4.0 100 0 

### a 0 10 1 0 0 0 

### o. 120 :HPBL were treated for 4 hours at 37±1°C in the presence of an exogenous source of metabolic activation,

**~Mitotic** in:::lex = nl.ll't:ler mitotic figures x 100/500 ceLls counted.

**~lfllf!ricat:** includes polyploid ard endoreduplicated cetls.; Structural: excludes cells with only gaps.

**~Chromatid** breaks lnclude chromatid and isoehromatid breaks and frag;nents; chr01l'13tid exchange figures include quadriradials, trlradials and cocrplex

**rearrangements~** 'cttromosome breaks include breaks and acentric fragments; die, dicentric chrancsaoo. feverely damaged cells incll.Kfes cells with one or more pulverized chromas;:omes and cells with 10 or more aberrations, Severely damaged cells and pulverizations were counted as 10 aberrations. 2Lower dose levels were tasted as a guard against excessive toxicity at higher dose levels but were not required for analysis.

MA Study No. G96A061.342 22 

### ~·MICROBIOLOGICAL 

# 'X ASSOCIATES, ·INC. . ..._TABLE 8 SUMMARY INDEPENDENT REPEAT ASSAY Treatment/ ); Change' Total Aberrations Cells With Aberrations 59 Harvest Mitotic fr-om Control Cells. Per Cellz 

# • Treatment Activation Time lrdex Hl Scored (Mean ± SO)**N1.100rical~** Structural CHrs.) (Hean) Untreated 20/20 L4 200 OnOOD ± 0.000 0.0 DHSO 20/20 1"1 200**0~000** ± 

### o.ooo 0.0 Hexabromocyclododecane 75 pg/ml 20/20 0.9 18 200 o.ooo ± o.ooo 

### o.o 150 pg/rnt 20/20 1.6 -45 200 0.000 ± o.ooo 0.0 300 pg/ml 20/20 0.8 27 200 0.005 ± 0.071 0.5 600 pg/ml 20/20 0.5 

### ss 200 o.ooo ± 0.000 0.0 MHC 20/20 L1 21 200 0.075 ± 0
..264 7.5*"" o. 13 IJg/ml Untreated 44/44 5.2 200 o.ozo ± 0.140 0.0 2.0 DHSO 1,/•f"'· 4.6 200 o.ooo ± o.ooo 0.0 

### o.o Hexabromocyctododecane 38 JJg/ml**~** 44/44 5.0 

# _, 200 0.005 :t**0~071** 0.0 0.5 75 /Js/mt 44/44 5.0 ·9 200 0
..005 ± 0.071 0.0 0.5 150 pg/ml 44/44 3.2 30 200 0.005 :t 0.071

### ~:~~ o..s 300 pg/ml 44/44 0.3 93 88 0
..**11~** ± L066 1
.. , HHC 44/44 3.1 38 200 O.tt95 ± 0.802 1
..5 35.0*• o.13 J.ls/mt Untreated + 4/20 1.5 200 

### o.ooo ± o.ooo 

### o.o DHSO + 4/20 1.4 200 

### o.ooo :t 

### o.ooo o.o**Hexebromocyclododecan~** 38 pg/mt + 4/20 1.3 7 200 0.000 ± 

### o.ooo o.o 7S pgfmt + 4/20 1.3 7 200 0•.000 ± 0.000 

### o.o 150 pg/rol + 4/20 1.8 ·22 200**0~000** ± 0.000 0.0 300 pgtml + 4/20 0.4 71 200 

### o.ooo ± 

### o.ooo o.o CP + 4/20 0.5 67 200 0.065 ± 0.267 6.0"'* 25 pgfmt Untreated + 4/44 5.9 200 0.000 ± 0.000 0.0 0.0 OHSO + t./44 7.2 200 

### o.oos ± 0.071 0.0 0.5 Hexnbromoeyclododccane 7S pg/mt + 4/44 4.8 33 200 0.000 ± 

### o.ooo o.o o.o 150 IJ9/111l + 4/44 3.3 54 200 0.010 ± o.100 1.5 1.0 300 JJ9/flll 

## • 4/44 3.3 54 200 0.015 ± 0.122 1.0 1.5 600 IJg/ml + 4/44 2.2 69 200 0.040 :t 0.242 1.0 3.0 CP + 4/44 4.6 22 200 0.135 ± 0.508 0"0 85** 25 pgtml**·~** reduction tn mttottc tndei**r~(attve** to neget1ve**(solv~nt** or untreated) control. Test arttcle concentrations are CO!Il'ared to the**solv~nt** control and the positive r:ontr-ol is compared to the untreated fDntrol. severely damaged cells were counted as 10 aberrations.**~.,** pS0.01; Ficher'& exact tct:t.**~ata** not cotler:ted fer ZO hour harvest tfme. 5 Percantago bas;ed on a total of 131 cellG &cored for ntiOOtical nberrotlonG.

MA Study No. G96A06t:l42 23

## ~· MICROBIOLOGICAL 

# x ASSOCIATES, INC.

### APPENDIX I 

### Historical Control Data

MA Study No, G96A061.342 24 

## ~·MICROBIOLOGICAL 

## X ASSOCIATES, INC.

### IN VITRO MAMMALIAN CYTOGENETIC TEST USING HUMAN PERIPHERAL LYMPHOCYTES

Historical

Values
Mean Standard Deviation Range

Historical Values
Mean Standard Deviation Range HISTORICAL CONTROL VALUES 1993-1995 NON-ACTIVATED TEST SYSTEM Aberrant Cells Untreated Solvent

Control Control'

0.2% 0.3% 0.5% 0.6% 0.0% to 2.5% 0.0% to 4.5%

S-9 ACTIVATED TEST SYSTEM Aberrant Cells Untreated Solvent

Control Control'

0.3% 0.3% 0.5% 0.5% 0.0% to 2.0% 0.0% to 3.0% Positive Control'

19.9% 13.9% 6.5% to 87.5%

Positive Control'

15.0% 5.8% 7.0% to 34.0%

1

### Solvents include water, saline, dimethylsulfoxide, ethanol, acetone, lactic acid buffer, :r,lacebo, 0.1

### OSN HCl, AD284 diluent, and 45% hydroxypropyl+cyclodextrin -Positive control for non-activated studies, mitomycin C, (MMC) 'Positive control for S-9 activated studies, cyclophosphamide, (CP)

MA Study No. G96A06U42 25 

### ~· 

## MICROBIOLOGICAL 

## X ASSOCIATE;S,. INC.

### APPENDIX II 

### Study Protocol

MA Study No. G96A061.342 26 

## (fZI\IIICROBIOLOGICAL ·.

.. X ASS()C,IATES,JI\I9,o

··. • - .. 'c::



# );::./t~~;;.,i&f:3~.~\{J-~.,.::~~/;t:

# Received

# byRA/OA

**~**

# -7-_it... MA Study Nwnber: _G96A061.3.12 

### Chromosome Aberrations in Human

**P~ripheral** 

### Blood Lymphocytes 1.0 PURPOSE

2.0

3.0 

### The purpose ofthis study is to test the

### clastogenic potential ofa test article based 

### upon its 

### ability to induce chromosome aberrations in human peripheral blood lymphocytes 

### (HPBL). - • . •·. • SPONSOR 2.1 Name:

**Chemi~::~l** 

### M•nuf•cturers Association 2.2 

### Address: 1300 Wilson Boulevard Arlington, VA 22209 2.3 

### Rcpzesentativc: 

### Dr. Hasmukh C. 

### Shah _, 

### IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 TestArticle:

**Huabromocyclododec::~ne** 

### 3.2 Controls: Untreated: Unrreated cells Solvent: TestArticle Solvent Positive: Mitomycin C (MMC), Cyclophosphamide (CP) 3.3 Determination ofPurity 

### --· The Sponsorwill be 

### directly nesponsible for detennination and 

### documentation of the analytical purity and composition ofthe test article. 3.4 TestArticle Retention Sample The n:tention of' a n:serve sample oflhe test article will be 

### the nesponsibility of the 

### Microbiological Associates, 

### Inc. 

### 4.0 TESTING FACILITY AND KEY PERSONNEL



## -- . 4.1 Name: 4.2 Addrr:ss:
4.3 Study Director: PniCGCOI No. SPGT342 01103/96 MA

### Study No. G%A061342 

### Genetic Toxicology 

### Division Microbiological Associates, Inc. 

### 9630 Medical Center Drive Rockville, MD 

### 20850 Ramadevi Gudi, Ph.D. 

# ...

**~··**

# -
. "

loiS 

## ?:;,'

## MICROBIOLOGICAL . :>?;: 

## ASSOCIATES, INC. ·..

### 4.4 Associate Study Director: DonaldPutman, Ph.D.
5.0 

### TEST SCHEDULE 5.1 

### Proposed Experimental Initiation

**~ate:** 

## 6/11/'lf. 5.2 Proposed Experimental Completion Date:

# 8;/Jo/"'I, 5.3 .Proposed 

### Rcpan Date:

## qjJJ 

# /'1b



# .. 6.0 TEST SYSTEM 

### Peripheral blood lymphocytes will 

### be obtained from healthy adults without six months ClCJlOSUte 

### to either radiotherapy, viral infections or the administration ofdrugs. 

### This system has been demonstrated to 

### be sensitive to 

### the clastogcnic activity ofa variety 

### of chemicals (Preston eta!., 1981). 7.0 EXPERIMENTAL 

### DESIGN AND 

### METHODOLOGY The 

### initial assay.will be perfonned by exposing lymphocytes to 

### a minimum of

### nine conceotrations of

### the test 

### article as 

### well as positive, untrcaled and solvent controls in duplicate 

### cultures (Evans 

### and O'Riordan, 1975). In the non-activated test system, ClCJlOSUte 

### will 

### be continuous up

### to 

### the time ofcell collection; in 

### the S9-activa1ed system, 

### exposure willbe for

### 4hours. The dividing cells willbe

### harvested ata 

### singletime pain!, apprcximately 20

### hours fromthe initiation oftrealment Thebighest scorable dose level with 

### at least 50"/o 

### mitotic inhibition and three lower doses will be evaluated microscopically for 

### strUctural chromosome aberrations. Inthe independent

# repeat assay, 

### a minimum offour concentrations will be 

### selected 

### based on the fuu:lings ofthe initial 

### assay. Theexposurecondilionswill 

### remain 

### the samebut twO

### harvestS will be

### carriedout, approximately 20

### hours

## aftCrthe initiation oftreatmentand 24 

### hours later(44

### ho.urs after initiation oftreatment). The clastogenicpatcntial ofthe test 

### axticlc will be 

### measured by 

### ilsabililytoincreasestructuralchromosomeaberrationsin

### adose-responsive ll!lllllm

### whee comparedto

### the solventcontrolgroup. The test

### article willalso 

### be assessed 

### for itsability 10 

### induce numerical chromosome aberrations. 

### 7.1 Solubility Dctennination 

### Unlessthe Sponsorhasindicated

### the test

### articlesolvent,

### asolubilitydetetmination 

### willbeconducted 10dctennioc solvent and 

### themaximum

### solubleconccnllalionup to

### amaxilnumofSOO 

### mglml. 

### Vebicles compaliblewith

### this testsystem,

### inorder ofprcf:rence, include but 

### arc not limiled to distilled 'Wlller 

### (CAS m2-18-S), 

### dimethylsulfoxide (CAS 67-68-5), ethanol (CAS 64-17-S), and 

### acetone (CAS 67- 64-1). The 

### vebicle 

# wm be 

### the solvent, selcc1ed in order ofpreference, that permits 

### preparation of

### the bighest stock concentration, up 10 SOO 

### mg/mL

2orl


### MA StudyNo.G96A061.342 28 

## ~MicRoBIOLOGICAL 

## X 

## ASSOCIATES, INC.
. ' 

### . .

### . . 

### .

. .



### : :

### 7.2 Dose Levels For the initial cytogenetics assay, 

### HPBL cells will be 

### exposed to at least nine concentrationsoftest

### anicle at

### half-log dilutions. The highestconcentrationtested will be 

### 5 mg/ml for freely soluble test anicles, or the ma.ximum concentration resulting in 

### a workable suspension for poorly soluble test articles. The pH will be measured at the highesttest article treatmentconditionand will be adjusted, if necessary, in order to liUlintain a neutral 

### pH in the treatment medium. The osmolality ofthe highesttreatment condition will also 

### be 

### measured. Ifexcessive precipitation oflite test 

### article-solvent solution occurs upon addition to treatment medium, or ifthe osmolality ofthe treatment medium 

### is excessive. the Sponsor will be 

### consulted. For the independent repeat assay, 

### HPBL cells will be 

### exposed to at 

### least four concentrations oflest article at no greater 

### than half-fold dilutions, the highest concentrationbeingthat

### whichis

### estimated to causeatleast

## soo;.,mitoticinhibition {regardless ofsolubility) relative to 

### the solventcontrol. In the absence ofat 

### least Sj)"lo mitoticinhibitiontheconcentration

### resultingin

### minimumprecipitationin test 

### medium will be the highest concentration tested. 

### Minimumprecipitation will be determined by direct visual 

### inspection. If 

### excessive precipitation of the test 

### article·sol'[enl solution O<:CIL"S 

### upon addition to 

### treatment medium, or 

### If the osmolality ofthe test article·solvent solution in treatment medium exceeds lite osmolalityof thetreatmentmedium-solventsolutionbyapproximately 

### I00mOslkg or 

### greater, the Sponsor will 

### be 

### consulted. The pH 

### and osmolality will be monitored as in lite initial 

### assay. 73 Frequency 

### and 

### Route ofAdministration Target cells will be

### treated 

### for approximately 20

### or44

### hours in the 

### non-activated system,

### depending !!pon

### harvesttime, and for4 hours in

### the S9- activatedsystem. 

### Treatmentwillbeachievedby

### incorporationofthe test

### article-solventmixtureinto the medium. This 

### technique has 

### demonstrated to be 

### an effective method of detaction ofchemical clastogens in this test

### system {Evans, 

### 1976). 7.4 Metabolic Activation System Aroclor 12.54-inducedratliverS9 will be

### used asthemetabolic activationsystem. 

### The 

### S9 will be prepared from male 

### Sprague-Dawley rats 

### induced with a single intraperitoneal injectionofAroclor 1254, 500 

### mglkg, live days

### priorto 

### sacrifice. The S9 will be 

### batch prepared 

### and 

### stored frozen at 

### approximately 

### ·7!1'C vntil

.. used. ••• ---



### Immediately prior to 

### use, 

### the S9 

# wm be thawed 

### and mixed with cofiu:tars to 

### contain 2 

### mM 

### magnesium chloride {MgCI,), 6 mM

### potassium chloride (KCI), 1 

### mM 

### gluco!!C-6-phosphate, 1 mM 

### nicotinamide adenine dinucleotide phosphnte (NADP) and 20 fll S9 

### per ml serum-free 

### medium.

0111131!16 MAStudy No. 096A061.34Z 3

## ora 

## i.:SM!CROBI.OLOGICAL 

# 'X: ASSOCIATES,

**~NC.**

### 7.5 Controls 7.5.1 Untreated Controls Untreated cellswill be

### used as the untteated control. 7.5.2 SolventControl Thesolventvehicle for the leSt

### articlewill be used

### as the solventcontroL For solvents other than water or mediwn, the fllllli 

### concentration in treatment medillltlwill notexceed 1%.
• 7 .5.3 Positive ContrOls Mitomycin C (MMC) 

### will be

### used at

### a 

### concentration within 0.10.0.25 j1g/ml as 

### the positive control in 

### the non-activated stUdy. Cyclophosphamide (CP) will be 

### used at a concentration within 25·100 

### llelml as the 

### positive contrOl in

### the 59-activated study. 7.6 I'Rparalion ofTargetCells Peripheral blood cells will be culllllCd 

### in complete medium (RPMI-1640 containing 15% fetal bovine 

### scnun. 2mM 

### L-glutamine, 100 units penicillin 

### and 100 ug

### strcptomycirllml)colllaining 1%phytoht'lll388lutinin(PHA)byadding0.6 mlheparinizedbloodto

### acentrifugetube

### conlaining9.4ml

### completemediumwith 1% PHA. The tubes 

### wUI be 

### incubated upright at 

### 37±l'C in a humidified atmosphere of5:1:1% CO, in air

### for

### 44-48 holllS. 7.7 ldentificauon ofTest System **~** 

# ··-.....

Using a

### permaoent'~ldng pen,

### thecultu."C flasks/testtubes,

### etc. used in

### the 

### test 

### system will be 

### idcnulied by 

### the study number,

**th~** 

### treatment condition and test 

### pbase. 7.8 Treat:meiltofTSigei Cells All treatments 

### will be 

### carried out in

### duplicate. About44-48 hauls 

### after culture 

### initia!Um, tubes 

# wm be 

### rcfed (centrifugation at 

### approximately 1200 zpm 

### for appnncimately S minllle$)

### with 10ml ofcomplete mediumforthenoo-adiwted Cl<JIO&We

### or 10 ml

### of59rcactionlllixturco forthe activatedexposureto

### which

### wi'll be added 

### 100 j1l 

### ofdosing solution oftestorcontrol article 

### insolventorsolvent alone. Largervolumes ofdosingsolu1ionmay be

### used if water

### ormedium

### isused 

### oothesolvent. An

### untreatedcontrol consistingofcells 

### incompletemediumor89 t=lionmilltute will also be

### included. 

### to the non-activated study, 

### the

### cells will be

### exposed 

### for 20 hours (harvesll) or for44holliS (bar\'esl2) at 

### 37±1°C in 

### a humidified atmosphereofS±1% CO. 

### in air. Exposure 

### willcontinue until collectioo ofmetaphase cells. Pro!O<OINo. SPGI'342 01103196 4ol'8
MA Study No. G%A061.342 

# '?.::!: MICROBIOLOGicAL 

# 5?: **ASSO~Tal. IN~**  

## . \:-·

.

**~** ' .

### In the 59-activated study, the cells y,ill be e.'<pOsed 

### for 4 hours at 37±t•C in a humidified atmosphere of5±1% C_O, in air. After the

**e~posure** 

### period. the cells will be 

### centrifuged at approximately 1200 rpm for approximately 5

### minutes. the treatment medium \\ill be aspirated, the cells washed with phosphate buffered saline, refed v.ith complete medium containing I% 

### PHA. and returned to the incubator for an additional 16 

### hours (harvest 

### I) or 40 

### hours (harvest 2). 7.9 

### Collection ofMetaphase Cells Cells will be 

### collected approximately 20 hours and 44 hours after initiation of treatment. These times 

### are selected to 

### assure analysis offirst division metaphase cells after initiation of test article treatment 

### as well 

### as to allow for analysis of delayed cells. Two 

### hours prior to harvest, Colcemid® 1.1.ill be 

### added to the cultures at a final concentration of 0.1 J1glml. The cells will be 

### collected by centrifugation(approximately 1200 rpm for 5

### minutes), treated with0.075M

### KCI, washedwithtwochangesoffiX3tive(methanol:glacialaceticacid, 3:1 v/v), capped andstored overnightorlongeratapproximately

### 2-6'C. Toprepare slides,the cells will be 

### collected by centri!iJgation, resuspended in fresh fL"<l11ive. One to two drops offtJCCd cells will be

### dropped onto 

### awetslide and air-dried The slide will be identifiedbythe MA

### study number, treatmentcondition and date. Atleasttwo slides will_bepicpared from each treatment tube. 

### The slides will be stained with Giemsa and permanently mounted.. 7.10 Scoring for Metaphase Aberrations Prior to 

### analysis, slides from the initial and 

### independent repeat studies will be scanned forscorable metapbases. In

### eachstudy, wheneverpossible,the high

### dose will be selected to yield approximately 

### SO% orgreater mitotic inhibition. Slides from the 

### highest scorable dose level and three 

### lowerdose levels will be selected " for analysis.
- Slideswill be

### coded using random numbers byanindividual notinvolvedwiththe scoringprocess. Metaphase cellswith 46 

### centromcres will be

### e.'Wilinedunder

### oil immersion without prior knowledge oftreatment groups. Whenever possible, 

### a •mlnlmum of200 

### mCIISpbase spreads (I00 

### per duplicate flask) 

### will be 

### examined

_and scored for chromatid-type and chromosome-type aberrations (Scott 

## era!.;-·-·-..

1990). Chromatid-typeaberrationsincludechromatidandisochromatidbteaksand eKchange figures sueb as quadrir.ulials (symmctrical and 

### asymmetrical interchanges), triradials, 

### and complex rearrangements. Chromosome·lype aberrations include chromosome breaks and exchange figures such as dicentrics · and rings. 

### fragments (chromatid 

### or acentric) observed in the absence of any exchange

### figur)! willbe

### scoredasa break(chromatidorchromosome). Fragments observed with an exchange figure will 

### notbe scored 

### as an aberration but will be considered 

### part of the incomplete exchange. 

### Pulverized chromosome(s), pulverized cells and 

### severely damaged cells (:1:10 aberrations) will also be recorded.. Chromatidimd isochromatid gaps

### will be

### recorded

### butnot included in the analysis. The 

### XY

### coordinatesforeachcellwithchromosomal aberrations

### will be 

### recorded using 

### a calibrated 

### microscope stage. In the delayed barv=t, 

### the Protocol No. SPGT34: 01/UJ/96
MA Study No. 0%A061.342 SoU
31 

## ?.:;,.· MICROBIOLOGICAL 

# 'X: ASSOCIATES, INC.

### percent polyploid cells will be cwaluated per 100 metaphase cells. The mitotic 

### irule:< will be recorded

### as the perceniage of cells in mitosis per 500 cells counted. 8,0 CRITERIA FOR DETER.'I.!INATION OF A VALID TEST 

### 8.1 Untreated and Solvent Controls The frequency ofcells with structural 

### and numerical chromosome aberrations in the untreated or solvent control must be 

### no greater 

### than 6%. 

### 8.2 Positive Controls The percentage ofcells with aberrations must be statistically increased 

### (¢0.05, F.isher's exact test) relative to the solvent control orto the untreated control ifa 

### solvent other 

### than water is 

### used. 

### 9.0 EVALUATION OF TEST RESULTS The toxic effects of treatment are based upon mitotic inhibition relative to the solvent•treatedcontrolandwillbepresentedforboththe initialandtheindependentrepeal chromosome aberration assays. The number and types 

### ofabenations, the percentage of structurally damaged cells (percent abemml 

### cells) in the total population of cells examined, 

### the percentage ofnumerically damaged cells in the total population ofcells examined, andthe frequency ofstructural aberrations 

### percell (mean abenations per

### cell) willbereportedforeachtreatmentgroup. Chromatid

### andisoc:hromatidgaps arepresented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency ofstructural aberrations 

### percell. Statistical analysis ofthe p;rcentaberrantcellswill be performedusing theFisher'sexact test. Fisher's test will be used 

### to compare pairwise the percent aberrant cells ofeach treatmcot group with that ofthe solvent controL In the eventofa positive Fisher's test at any test article dose level, the Cochran·Armitage trend test 

### will be used 

### to measure dose-responsiveness.



# -

# ....

**~**

## ...... All 

### com:lwsioos will be baoed 

### on sound scientific

### judgement; however, as a 

### guide to interpretation ofthe data, 

### the test

### article will be considered to 

### induce apositiveresponse when the pe:centage ofcells with aberrations is 

### increased in a

### dose·rcsponsive manner 

### withoneor more concentrationsbeing statisticallyelevatedrelative

### tothe solventcontrol group (p:;;O.OS). A reproducible and significant increase at 

### a single dose 

### level will be 

### considered positive. Testarticles notdemonstrGtiug a

### S'"wttL-ti=lly

**~i~.ifi!!!!!!t**

### incmasc in abenalions will be concluded to be negative. 

### 10.0 REPORT 

### A report of

### the

### iesults of

### this study

### Win be 

### prepared by the 

### Testing Laboratory

### and will IICCurately describe all methods 

### used for gener2tion 

### arid analysis ofthe data.
Protocol No. SPCTJ.U 01/03196 

### MA 

### Study No. GIJ6ACl61.:142 6oU



### ~· 

## MICROBI.OLOGICAL• X ASSOCIATES; INC; •

### Results presented will include, but not

## h<: limited to: cells used test conditions: composition of medium. CO.'! concentration, incubation time, 

### conccnttation of test article and rationale for selection ofconcentration. dUiation of treatment, dUiation of treatment with and concentration of Colcemid®, type 

### of metabolic activation system used, positive, solvent and untreated controls number ofcell cultures number ofmetaphases analyzed (data given separately for each culture) • mitotic index • criteria for scoring abenations type 

### and number ofaberrations. given separately for each treated and.control cullure • historical control data 1LO RECORDS AND ARCHIVES Upon completion ofthe final report, all raw data and reportS will be maintained by the QualityAssuranceUnitofMicrobiologicalAssociates, Inc.,Rockville, MD,inaccordance with the relevant Good Laboratory Practice Regulations. 12.0 

### REGULATORY **~QUIREMENTS/GOOD** 

### LABORATORY PRACOCE This 

### protocol has been written to 

### comply with EU 

### Legislation L 383 

### a/148, 8.10 

### Mutagenicity (In Vitro 

### MammalianCytogeneticTest), December, 1992; OECD Guideline 473 (Genetic Toxicology: In Vitro 

### Mammalian Cytogenetic Test), May, 1983;

### and EPA HealthEffectsTesting Guidelines, Subpart798.5375 

### (In Vitro

### MammalianCytogenetics), Fed.Register, vol. 50, 

### September, 1985 wilhrevisions Fed. Register,vol. 52, May, 1987. This study will be performed in compliance wilh the provisions oflhe Good Laboratory PracticeRegulations for EPATSCA Regulations, 40 

### CFR792 

### and OECD ISBN 922·84· !2367-9. .. 

### Will this study be submitted to a regulatory agency? Yes Ifso, 10

### which ogeney 

### or agencie;? Ell and 

### EPA-TSCA Unless llml!lgements are made to the c:onttary, 

### unused dosing solutions will be disposed ofioltowiog administration to 

### the testsystemand

### all residual test 

### articlewill bedisposed offollowing fmalization ofthe 

### report. 13.0 

### REFERENCES 

### Evans, 

### HJ. and M.L. O'Riordan. !975. Human peripheral blood lytnphocytes for the analysis ofchromosome aberrations in mutagen testS. 

### Mutation Res. 31;135·148. 

### Evans, 

### HJ. 1976. Cytological methods for detecting chemical mutagens. In; Chemical Mutagens, Principles and Methods for their Detection, vol. 4. A. 

### Hollaender (ed). Plenum Press, 

### New York, NY. Protocol No. SPGT341 01103/96
MA 

### Study No. 

### G96A061.342 7 orB
33 

### ~·MICROBIOLOGICAL 

### X ASSOCIATES, INC.

### Preston, RJ.,W. Au, M.A. Bender,J.G. Brewen, A.V. Carrano, J.A. Heddle. A.

### F. McFee. S. Wolff, J.S. Wassom. 1981. 

# Mammalian in vivo and 

### in ·vitro cytogenetic assays: a report cf

### the Gene-Tox Program. Mutation 

### Res.. 87:143-188. Scott, D., N.D. Danford, B.J. Dean and DJ. Kirkland. 1990. Metaphnse Chromosome Aberration Assays In Vitro. In: Basic Mutagenicity Tests: UKEMS Recommended Procedures. DJ Kirkland (ed). Cambridge University Press, New Yorl:, NY. 14.0 APPROVAL
SPONSOR REPRESENTATIVE 

### J/4 S(\1\.1

# kh Sh,.b (Print or TYJli' Name)

# ~tt1R..dka: 

# fru.d. MASTIJDY DIRECTOR**~--** 2 

### '1- 

## "1(, DATE

DATE

Pracocol No. SPCT34l Ol/ll3196 SorB

## ~MI.C.RO.B ..IOLO_GICAL 

## % ASSOCIATE!;, INC.
. MA 

### Study No. G96A06J.J.l2 34-

.-.-· :- ·: <:"-

# • Reproduced 

# by

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