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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 

National Technical Information Service 
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CODING 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 

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CHEMICAL MANUFACTURERS AssociATION 0 \\ ..,, \ t

1 

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0 nf_(; \ '3 \ -' 

December 12, 1996 uVia Certified Mail 

Document Control Office (7407) 

Office of Pollution Prevention and Taxies 

U.S, Environmental Protection Agency 

RoomG-099 

401 M Street, SW 

Washington, DC 20460 

Attention: TSCA 8(d) Health and Safety Reporting Rule <Notification) 

Dear Sir or Madam: 

The Chemical Manufacturers Association Brominated Flame Retardant Industry 

Panel (BFRIP) hereby submits the following report on hexabromocyclndodecane (OBDPO, 

CAS No. 9233o-52-0). ;;;> "'> 0:. 7, ~ ·• Cf '7 - ¥ 

1) HEXABROMOCYCLODODECANE (HBCDl: Chromosome Aberrations in 

Human Peripheral Blood Lymphocytes, 

The BFRIP member companies on whose behalf this notification is submitted are 

Jfsted in Attachment 1. 

The name and address of the laboratory that conducted this study is ae follows: 

Microbiological Associates, Inc, 

9630 Medical Center Drive 

Rockville, MD 20850 

If you have any questions regarding this TSCA 8(d) notification, please contact me by 

telephone at 703/741-5637, or by fax at 703/741-6091. 

Attachment 

869'70000358 

Sincerely, 

Hasmukh C. Shah, Ph.D. 

Manager, BFRIP 

" r..f..a llestmillJiaCare• 

1300 WILSON BOULEVARD, ARUNGTONt VA 22209 • TELEPHONE 703~741~5000 • FAX 703~741-6000 "lf" A~~ 
Albemarle Corporation 

451 Florida Street 

Baton Rouge, LA 70801 

Ameribrom, Inc. 

52 Vanderbilt 

NewYork,NY 10017 

Great Lakes Chemical Corporation 

P.O. Box 2200 

Highway 52 NW 

West Lafayette, IN 47906 

Attachment 1 
FINAL P..EPORT 

% D!:C I 9 P.'l 3: I 4 

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 of 34 

~· 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 CFR 792, the UK GLP Compliance Progranune, the Japanese GLP Standard and 

the OECD Principles of Good 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 of the test or control article under the test 

conditions was not determined by the testing facility. 

Analyses to determine the uniformity, concentration, or 

stability of the 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 I H54Co 

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 of T 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 of metabolic 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 of the 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 of choice based on information provided 

by the Sponsor, the solubility of the 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 of 250 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 of the initial assay HPBL cells were exposed to the test article 

continuously for 20 hours; in the S9-activated portion of the 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 of treatment Dose 

levels of 2500 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 of dose level (p>0.05, Fisher's 

exact test). 

Based on the results of the initial assay, an independent repeat chromosome aberration 

assay was conducted in the absence and presence of an 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. In the independent repeat assay, HPBL cells 

were exposed to the test article continuously for 20 or 44 hours in the 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 of treatment. Toxicity, m,.;n.ed by 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 of scorable metaphase cells.. No statistically 

significant increases in structural chromosome aberrations were observed in either the nonactivated or S9-activated studies, regardless of dose 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 of dose level (p>O.OS, Fisher's exact test}. 

Based on the findings of this 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 of this study was to evaluate the clastogenic potential of a test article 

based upon its ability to induce chromosome aberrations in hwnan peripheral lymphocytes. 

CHARACTERIZATION OF TEST 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 

of 2.5 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 in the test article-treated groups. RPMl-1640 complete 

medium (containing 15% fetal bovine serum, 2 mM L-glutarnine, I 00 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 of either radiotherapy, viral infection or the administration of drugs. 

This system has been demonstrated to be sensitive to the clastogenic activity of a variety 

of chemicals (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 of Aroclor 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 of S-9 was assayed for 

its ability to metabolize 2-arninoanthracene imd 7,12-dimethyl-benz(a.)anthracene to forms 

mutagenic to Salmonella 1yphimurium TAl 00. 

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 I 00 units 

penicillin and 100 Jlg streptomycin!ml, and 2 mM L-glutamine). 

Solubility Test 

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 of the test article in DMSO, which permitted preparation of the 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 of HPBL to nine concentrations of the 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 of treatment 

The independent repeat chromosome aberration assay was performed by exposing 

duplicate cultures of HPBL to at least four concentrations of the test article, as well as 

positive, solvent and untreated controls. The concentrations tested were selected based on 

the findings of the initial assay. For the independent repeat assay, the dividing cells were 

harvested at two time points, 20 and 44 hours after the initiation of treatment 

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 of 5±1% CO, in air for 

approximately 44-48 hours. Treatment was carried out by refeeding with approximately 

10 ml fresh complete medium or S9 reaction mixture to which was added 100 )ll of 

dosing solution of test or control article in solvent or solvent alone. An untreated control 

consisting of cells 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 of 5±1% C02 in air. In the 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 44 hours 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 of Metaphase Cells 

Two hours after the addition of Colcemid®, 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 of fixative (methanol:glacial acetic acid, 3:1 vivf was 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 of cell 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 of Metaphase 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 prior knowledge of treatment groups. Whenever possible, a minimum of 200 

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 of the incomplete exchange. Pulveri2ed chromosome(s), pulverized 

cells and severely damaged cells (>I 0 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 recorded using a calibrated microscope stage. 

The mitotic index was recorded as the pen:entage of cells in mitosis per 500 cells counted. 

In the delayed harvests, the percent polyploid cells was recorded per 100 metaphase 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 of Test Results 

The toxic effects of treatment are based upon mitotic inhibition relative to the 

solvent-treated control and are presented for both the 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 of numerically damaged cells in the total population of cells 

examined, and the frequency of structural 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 in the 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 of each 

treatment group with that of the solvent controL In the event of a positive Fisher's test at 

any test article dose level, the Cochran-Anoitage test was used to determine doseresponsiveness. 

All conclusions were based on sound scientific basis; however, as a guide to 

interpretation of the data, the test article was considered to induce a positive response 

when the percentages of cells 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 if no statistically 

significant increase was observed relative to the solvent controL 

Criteria for Determination of a Valid Test 

The frequency of cells with structural chromosome aberrations in either the untreated 

or solvent control must be no greater than 6%. The percentage of cells 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 if a solvent other than water 

was used. 

MA Srudy No. G96A06L342 II ~· MICROBIOLOGI. CAL ~ ASSOCIATES, lt~C. 
Record and Specimen Archives 

Upon completion of the fmal report, all raw data, reports, and stained and coded slides 

are maintained in the archives of Microbiological Associates, Inc. located in Rockville, 

Maryland. 

RESULTS AND DISCUSSION 

Solubility Test 

DMSO was selected by the Sponsor as the solvent of choice based on solubility of the 

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 

In the initial chromosome aberration assay HPBL were exposed to nine concentrations 

of test 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 of the 

highest concentration tested, 2500 !'g/ml, were 419 mmollkg and approximately 8, 

respectively. The osmolality of the solvent, DMSO, was 447 mrnol/kg. Metaphase cells 

were collected for microscopic evaluation at 20 hours after the initiation of treatment 

The activity of Hexabromocyclododecane in the induction of chromosome abetrations 

in HPBL was tested following a 20 hour exposure period in the absence of an exogenous 

source of metabolic activation. The fmdings of the 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 of scorable cells. Dose levels 

0.25, 0.75, 2.5 and 7.5 l!g/ml were tested but not required for analysis. The percentage of 

cells with structural abenations in the test article-treated groups was not significantly 

increased above that of the solvent control at any dose level (p>0.05, Fisher's exact test). 

The percentage of aberrant cells in the MMC group was 8.5% (p:r;O.Ol, Fisher's exact 

test). 

The activity of Hcxabromocyclododecane in the induction of chromosome aberrations 

in HPBL cells was tested using a 4 hour exposure and a ! 6 hour recovery period in the 

presence of an exogenous source of metabolic activation. The findings of the cytogenetic 

analysis are presented by treatment flask in Table 2 and summarized by group in Table 3. 

At the highest test concentration evaluated (250 l!g/ml) for chromosome aberrations, the 

mitotic index was reduced 13% relative to the solverit 'controL Dose levels 750 an~··zsoo 

I!Wml were not selected for analysis due to an insufficient number of scorable c~ll~, Dose 

?,:;i)" MICROBIOLOGICAL 

MA ~ ASSOCIA"f~, · INC. · Study No. G96A061.342 12 
levels 0.25, 0.75, and 2.5 f!g/ml were tested but not required for analysis. The percentage 

of cells with structural aberrations in the test article-treated groups was not significantly 

increased above that of the solvent control at any dose level (p>0.05, Fisher's exact test). 

The percentage of aberrant cells in the CP group we.s 6.5% (pSO .. OI, Fisher's exact test). 

Independent Repeat Assay 

Based on the results of the 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 of the 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 of the 

highest concentration (600 f!g/ml) tested was 381 mmol/kg. The osmolality of the 

solvent, DMSO, was 393 mmol!kg. The pH of the highest concentrations tested was 

approximately 8. At the time of test 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 of the study was negative, the Study Director determined 

this deviation to have had no significant effect upon the test system. 

The findings of the cytogenetic analysis for groups tested in the absence of an 

exogenous source of metabolic 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 I 0 and 19 f!g/ml were not 

required for analysis. The percentage of cells \\ith structural aberrations in the test articletreated groups was not significantly increased above that of the solvent control at any dose 

level or harvest time (p>0.05, Fisher's exact test). The percentage of cells with numerical 

aberrations in the test article-treated groups was not significantly increased above that of 

the solvent control at the 44 hour harvest time, regardless of dose level (p>0,05, Fisher's 

exact test). The percentages of aberrant 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 of the cytogenetic analysis for groups tested in the presence of an 

exogenous source of metabolic 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 of cells with · 

structural aberrations in the test article-treated groups was not statistically increased above 

that of the solvent control at any dose level or harvest time (p>O.OS, Fisher's exact test). 

The percentage of cells with numerical aberrations in the test article-treated groups was 

MA Study No, G96A06 1.342 13 ~· MICROBIOLOGICAL X ASSOCIATES, INC . . 
not statistically increased above that of the solvent control at the 44 hour harvest time, 

regardless of dose level (p>0.05, Fisher's exact test). The percentages of aberrant 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 of the 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 (I 976) 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. Wolff and J.S. Wassom (1981) Mammalian in vivo and in vitro cytogenetic assays: 

a report of the 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 Celts severely 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 

1

HPBL 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~ 7

Severely damaged cells ond pulvcri::atfans ware counted as 10 aberrations. 

8Lower 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 Treatmcnt1 a 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/ml a 

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/ml B 

2500 IJg/ml 8 

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. 5

Chromosome 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. 7

Severely 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:yc l ododecane 

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 Mitotic Aberrant Severely Average 

Treatment1 ~ cut ture lrdex~ Scored cells3 Damaged Aberrations 

(%) (%) Cet lsb Per Cell J • 

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/ml a 

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 Celt 3

·

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!Oeyc l ododcctlMe: 

10 JJs/mt 1 

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 

1

HPSL were tr·eated for 44 hours at 37±1°C in the absence cf an exogenous source 1

Hitotic index = nunber mitotic figures x 100/500 cells counted. 

of metabolic activation. 

Jillll'2rical: includes polyploid and endoredupl icated cells.; structural: e)(cludes cells with only gaps. 4

Chr'Oilatid breaks include chrOIIllltid nnd isochrc:matid breaks and fragments; chranatid exchange figures (Exch) include 

quadriradials. triradials art:! COI!plex rearrangeroonts. 5

Chranosome breaks include breaks and acentric fragroonts; die, dtcentric chrOillOsome. 6

Severaly dlll!Uilged cells includec cella with one or more pulverized chromosomeo. and cells with 10 or more aberrations. 7

Severely 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. 1

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

MA Study No. G96A06 1.342 20 (.tf MICROBIOLOGICAL •. . . ~ 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/ml a 

19 pg/mt 6 

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 vstml n 

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. 3

E11cludlng celts with only gaps~ 4

Chromntid 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 

Treatment1 8 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 

Hexabromocyc lododecane 

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. 2

Lower 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.) (He an) 

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 

(fZ I\IIICROBIOLOGICAL · . .. X ASS()C,IATES,JI\I9,o 

··. • - .. 'c:: );::./t~~;;.,i&f:3~.~\{J-~.,.::~~/;t: 
Received by RA/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 of this study is to test the clastogenic potential of a 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 Test Article: Huabromocyclododec::~ne 

3.2 Controls: Untreated: Unrreated cells 

Solvent: Test Article Solvent 

Positive: Mitomycin C (MMC), Cyclophosphamide (CP) 

3.3 Determination of Purity 

--· The Sponsor will be directly nesponsible for detennination and documentation of 

the analytical purity and composition of the test article. 

3.4 Test Article Retention Sample 

The n:tention of' a n:serve sample of lhe 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: Donald Putman, 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: q jJJ /'1 b .. 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 of drugs. This 

system has been demonstrated to be sensitive to the clastogcnic activity of a 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 of cell collection; in the S9-activa1ed system, 

exposure will be for 4 hours. The dividing cells will be harvested at a single time pain!, 

apprcximately 20 hours from the initiation of trealment The bighest scorable dose level 

with at least 50"/o mitotic inhibition and three lower doses will be evaluated 

microscopically for strUctural chromosome aberrations. In the independent repeat assay, 

a minimum of four concentrations will be selected based on the fuu:lings of the initial 

assay. The exposure condilions will remain the same but twO harvestS will be carried out, 

approximately 20 hours aftCr the initiation of treatment and 24 hours later (44 ho.urs after 

initiation of treatment). The clastogenic patcntial of the test axticlc will be measured by 

ils abilily to increase structural chromosome aberrations in a dose-responsive ll!lllllm whee 

compared to the solvent controlgroup. The test article will also be assessed for its ability 

10 induce numerical chromosome aberrations. 

7.1 Solubility Dctennination 

Unless the Sponsor has indicated the test article solvent, a solubility detetmination 

will be conducted 10 dctennioc solvent and the maximum soluble conccnllalion up 

to a maxilnum of SOO mglml. Vebicles compalible with this test system, in order 

of prcf: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 of preference, that 

permits preparation of the bighest stock concentration, up 10 SOO mg/mL 

2orl 

MA Study No. 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 

concentrations of test anicle at half-log dilutions. The highest concentration tested 

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 highest test article treatment condition and will be adjusted, if 

necessary, in order to liUlintain a neutral pH in the treatment medium. The 

osmolality of the highest treatment condition will also be measured. If excessive 

precipitation of lite test article-solvent solution occurs upon addition to treatment 

medium, or if the osmolality of the treatment medium is excessive. the Sponsor 

will be consulted. 

For the independent repeat assay, HPBL cells will be exposed to at least four 

concentrations of lest article at no greater than half-fold dilutions, the highest 

concentration being that which is estimated to cause at least soo;., mitotic inhibition 

{regardless of solubility) relative to the solvent control. In the absence of at least 

Sj)"lo mitotic inhibition the concentration resulting in minimum precipitation in test 

medium will be the highest concentration tested. Minimum precipitation 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 of the test article·solvent solution in treatment medium exceeds lite 

osmolality of the treatment medium-solvent solution by approximately I 00 mOslkg 

or greater, the Sponsor will be consulted. The pH and osmolality will be 

monitored as in lite initial assay. 

73 Frequency and Route of Administration 

Target cells will be treated for approximately 20 or 44 hours in the non-activated 

system, depending !!pon harvest time, and for 4 hours in the S9- activated system. 

Treatment will be achieved by incorporation of the test article-solvent mixture into 

the medium. This technique has demonstrated to be an effective method of 

detaction of chemical clastogens in this test system {Evans, 1976). 

7.4 Metabolic Activation System 

Aroclor 12.54-induced rat liver S9 will be used as the metabolic activation system. 

The S9 will be prepared from male Sprague-Dawley rats induced with a single 

intraperitoneal injection of Aroclor 1254, 500 mglkg, live days prior to 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 

MA Study No. 096A061.34Z 

3 ora i.:S M!CROBI.OLOGICAL 'X: ASSOCIATES, ~NC. 
7.5 Controls 

7.5.1 Untreated Controls 

Untreated cells will be used as the untteated control. 

7.5.2 Solvent Control 

The solvent vehicle for the leSt article will be used as the solvent controL 

For solvents other than water or mediwn, the fllllli concentration in 

treatment medillltl will not exceed 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 of Target Cells 

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) by adding 0.6 

ml heparinized blood to a centrifuge tube conlaining 9.4 ml complete medium with 

1% PHA. The tubes wUI be incubated upright at 37±l'C in a humidified 

atmosphere of 5:1:1% CO, in air for 44-48 holllS. 

7.7 ldentificauon of Test System 

~ ··-..... 

Using a permaoent'~ldng pen, the cultu."C flasks/test tubes, etc. used in the test 

system will be idcnulied by the study number, th~ treatment condition and test 

pbase. 

7.8 Treat:meilt of TSigei Cells 

All treatments will be carried out in duplicate. About 44-48 hauls after culture 

initia!Um, tubes wm be rcfed (centrifugation at approximately 1200 zpm for 

appnncimately S minllle$) with 10 ml of complete medium for the noo-adiwted 

Cl<JIO&We or 10 ml of 59 rcactionlllixturco for the activated exposure to which wi'll 

be added 100 j1l of dosing solution of test or control article in solvent or solvent 

alone. Larger volumes of dosing solu1ion may be used if water or medium is used 

oo the solvent. An untreated control consisting of cells in complete medium or 89 

t=lion milltute will also be included. 

to the non-activated study, the cells will be exposed for 20 hours (harvesll) or 

for 44 holliS (bar\'esl2) at 37±1°C in a humidified atmosphere of S±1% CO. in 

air. Exposure will continue until collectioo of metaphase cells. 

Pro!O<OI No. 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 of 5±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 of Metaphase Cells 

Cells will be collected approximately 20 hours and 44 hours after initiation of 

treatment. These times are selected to assure analysis of first 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 with 0.075M KCI, 

washed with two changes offiX3tive (methanol:glacial acetic acid, 3:1 v/v), capped 

and stored overnight or longer at approximately 2-6'C. To prepare slides, the cells 

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

drops of ftJCCd cells will be dropped onto a wet slide and air-dried The slide will 

be identified by the MA study number, treatment condition and date. At least two 

slides will_be picpared 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 for scorable metapbases. In each study, whenever possible, the high dose 

will be selected to yield approximately SO% or greater mitotic inhibition. Slides 

from the highest scorable dose level and three lower dose levels will be selected 

" for analysis. -

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

scoring process. Metaphase cells with 46 centromcres will be e.'Wilined under oil 

immersion without prior knowledge of treatment groups. Whenever possible, a 

• mlnlmum of200 mCIISpbase spreads (I 00 per duplicate flask) will be examined _ and scored for chromatid-type and chromosome-type aberrations (Scott era!.;-·-·-.. 1990). Chromatid-type aberrations include chromatid and isochromatid bteaks and 

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)! will be scored as a break (chromatid or chromosome). Fragments 

observed with an exchange figure will not be 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.. Chromatid imd isochromatid gaps will be recorded but not included in 

the analysis. The XY coordinates for each cell with chromosomal 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 of cells with structural and numerical chromosome aberrations in 

the untreated or solvent control must be no greater than 6%. 

8.2 Positive Controls 

The percentage of cells with aberrations must be statistically increased (¢0.05, 

F.isher's exact test) relative to the solvent control or to the untreated control if a 

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•treated control and will be presented for both the initial and the independent repeal 

chromosome aberration assays. The number and types of abenations, the percentage of 

structurally damaged cells (percent abemml cells) in the total population of cells 

examined, the percentage of numerically damaged cells in the total population of cells 

examined, and the frequency of structural aberrations per cell (mean abenations per cell) 

will be reported for each treatment group. Chromatid and isoc:hromatid gaps are presented 

in the data but are not included in the total percentage of cells with one or more 

aberrations or in the frequency of structural aberrations per cell. 

Statistical analysis of the p;rcent aberrant cells will be performed using the Fisher's exact 

test. Fisher's test will be used to compare pairwise the percent aberrant cells of each 

treatmcot group with that of the solvent controL In the event of a 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 of the data, the test article will be considered to induce a positive response 

when the pe:centage of cells with aberrations is increased in a dose·rcsponsive manner 

with one or more concentrations being statistically elevated relative to the solvent control 

group (p:;;O.OS). A reproducible and significant increase at a single dose level will be 

considered positive. Test articles not demonstrGtiug 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 of the 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 of concentration. dUiation of 

treatment, dUiation of treatment with and concentration of Colcemid®, type of 

metabolic activation system used, positive, solvent and untreated controls 

number of cell cultures 

number of metaphases analyzed (data given separately for each culture) 

• mitotic index 

• criteria for scoring abenations 

type and number of aberrations. given separately for each treated and.control cullure 

• historical control data 

1 LO RECORDS AND ARCHIVES 

Upon completion of the final report, all raw data and reportS will be maintained by the 

Quality Assurance Unit of Microbiological Associates, Inc., Rockville, MD, in accordance 

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 Mammalian Cytogenetic Test), December, 1992; OECD Guideline 

473 (Genetic Toxicology: In Vitro Mammalian Cytogenetic Test), May, 1983; and EPA 

Health Effects Testing Guidelines, Subpart 798.5375 (In Vitro Mammalian Cytogenetics), 

Fed. Register, vol. 50, September, 1985 wilh revisions Fed. Register, vol. 52, May, 1987. 

This study will be performed in compliance wilh the provisions of lhe Good Laboratory 

Practice Regulations for EPA TSCA Regulations, 40 CFR 792 and OECD ISBN 922·84· 

!2367-9. .. 

Will this study be submitted to a regulatory agency? Yes 

If so, 10 which ogeney or agencie;? Ell and EPA-TSCA 

Unless llml!lgements are made to the c:onttary, unused dosing solutions will be disposed 

of ioltowiog administration to the test system and all residual test article will be disposed 

of following fmalization of the report. 

13.0 REFERENCES 

Evans, HJ. and M.L. O'Riordan. !975. Human peripheral blood lytnphocytes for the 

analysis of chromosome 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-

.-.-· :- ·: 

<:"-


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