111111111111111111111111111111111111111111111111111111111111 • 

OTS0522234 N 

One Source. One Search. One Solution. 

GENETIC TOXICOLOGY RAT HEPATOCYTE PRIMARY 

CULTURE/DNA REPAIR TEST ON 

HEXABROMOCYCLODODECANE WITH COVER LETTER 

DATED 030890 

01 JUL 1985 

U.S. Department of Commerce 

National Technical Information Service 
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/'i~"":\ U ..S. DEPARTMENT OF COMMERCE 

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CODING FORM FOR SRC INDEXING REVISED 10/15186 

OTS0522234 

GEtJt::TIC TO>: ICOLOG"{ RAT HEPA10CY1E t=·RIMARV CUL TURE./DI~H REI=·Al R 

TEST ON HEXABROrlOCYCLODODECANE WITH COVER LE:IIER DATED 

(),3~)89(• 

HE XABR0r>10CYCLODODECANE \31 '34 -55 ·-6) 
~- CONTAINS NO CS\ 

' ETHYL CORPORATION 

HEALTH and ENVIRONMENT DEPARTMENT 

ETHYL TOWER, 451 FLORIDA 

BATON ROUGE, LOUISIANA 70801 

March 8, 1990 

CERTIFIED MAll RETURN RECEIPT REOUESTEQ P 131 520 103 

Document Processing Center (TS-790) Office of Toxic Substances 

Environmental Protection Agency 401 H Street S. W. 

Washington, OC 20460 'i?f.-'10 0000 1~3 

Attention: S{d) Reporting 

Dear Sir: 

RE: [OPTS-82034; FRL-3666-9] Chemical Name CAS Reoistrv # 

Hexabromocyclododecane 3194-55-6 

"" 0 

;; .... 

:;..::: 

·--

?: 

9 

'" t."l 

In accordance with 40 CFR 716.35{aj{4), Ethyl Corporation hereby submits this list of studies previously sent to federal 

agencies without confidentiality claims. 

~ 

: 

~ 

Chemical Name Hexabromocyclododecane CAS number 3194-55-6 

The following studies were sent on Hay 23, 1988 to: 

Hr. Robert Brink 

Executive Secretary TSCA Interagency Testing Committee {TS-792) U.S. Environmental Protection Agency 401 M Street, S .. W. 

Washington D.C. 20460 

!. Mutagenicity Test of GLS-S6-41A 

Lab GSRI 

Date of Study November I 7, 1978 

2. Primary Dermal Irritation {Rabbit) Dermal corrosion {Rabbit) Ocular Irritation {Rabbit) Oral LD50 (Rabbit) 

lab-Consumer Product Testing Company Inc. 

Date of Study October 17, 1978 
Inhalation LCSO {Rat) 

Dermal LOSO {Rabbit) 

lab Consumer Product Testing 

Date of Study November 7, 1978 

Submitting Company: 

Submitting Official: 

Ethyl Corporation 451 Florida Blvd. 

Baton Rouge, La 70801 

Hs. Anne B. Lemmon 

Information Specialist Health & Environment Dept. 451 Florida Blvd. 

Baton Rouge, La. 70801 

All technical questions concerning this submission should be 

directed to Dr. L. L. Wen, Regulatory Affairs at (504) 3BB-7650. 

AB L: pm 

Sincerely, 

ETHYL CORPORATION 

,. I' r- LL, ..... ,,j" ) .,\..c.·..............-~·--

Anne B. Lemmon 

Infotmation Specialist 
/i{X!NTAINS NO C£1 

ETHYL CDQPOQ!illON 

TECHNICAL CENTER 

TOXICOLOGY AND INDUSTRIAL HYGIENE DEPARTMENT 

July 1, 1985 

Genetic Toxicology 

Rat Hepatocyte Primary Culture/DNA 

Repair Test 

HPC 024 • it083 

HBCD Bottoms 

& EPA-OTB 

IHIW~III!DIIII~~mo 001Jb3bbb3U 

¥-b-'iDOcooi b~ 

Q. YDu.1.llo/lancL 

D. E. Johnsplj, Study Director 

Associate D¥rector 

Toxicology and Industrial 

Hygiene Department 

A. Mulholland 

Corporate Genetic Toxicologist 

Toxicology and Industrial 

Hygiene Department 

P. 0. Box 14799 

Baton Rouge, Louisiana 70898 

Telex 784583 ETC BTR 

Telephone (504) 389·7600 

8000 GSRI Awnue 

Baton Rouge, Louisiana 70820 
I. 

II. 

III. 

rv. 

v. 

VI. 

VII. 

TABLE OF CONTENT 

Summary ........ n ............................................. . 

Study Description .................................. . 

Protocol ........................................................... 

Results and Discussion 

Conclusion .. ................................................. .. 

Data ......... " ................................................ . 

Quality Assurance Statement 

Page 

l 

2 

3-5 

6 

6 

7 

8 
SUMMARY 

Test article, HBCD Bottoms, was received as a brittle black 

solid. Freshly isolated rat hepatocytes were treated with 20 ul of 

HBCD Bottoms at 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500 and 1000 

ugjwell in 2 mL of media. In addition, a acet.one group, an 

untreated control, and 2-Acetoamidofluorene (2AAF) at a concentration of 10-6M, were evaluated concurrently. Cytotoxicity by HBCD 

Bottoms was produced at 1000 ug/well. The highest dose scored for 

HBCD Bottoms was 1000 ug/well. Unscheduled DNA repair synthesis was 

quantified by a net nuclear increase of black silver grains. This 

value was determined by taking a nuclear count and the average of 

three adjacent cytoplasmic counts. 

The results for test article HBCD Bottoms were positive in the 

Rat Hepatocyte Primary Culture/DNA Repair Test under the conditions 

of this assay. These results are based upon the ability of HBCD 

Bottoms to produce a mean grain count of five or greater than the 

vehicle control mean grain count and a statistically significant 

difference between the HBCD Bottoms treated cells and the vehicle 

control in the number of cells with net nuclear grain counts greater 

than zero. A dose-related increase in net nuclear counts and cells 

greater than zero was demonstrated by HBCD Bottoms. 

-1-
Compound Name: 

G.T. il: 

Date Recejved: 

Recejved From: 

Solvent: 

Doses Tested: 

STUDY DESCRIPTION 

lll'ICD Bot toms 

083 

3/4/85 

R. zussman 

Acetone 

o.os, 0.1, o.s, 1 1 5, 10 1 50, 100, 500 and 1000 

ug/well 

Assay Performed by: R. o. Johannessen 

Study InHiated: 4/10/84 

Study Completed: 5/31/85 

-2-
PROTOCOL - ETTOX 028 

Rat Hepatocyte Primarv Culture/DNA Repair Test 

Sponsor: 

Testing Facility: 

Test Substance: 

Studv No .. : 

PUrDose of 

the Study 

Study Director: 

Q .A. U •• 

Responsible 

Personnel: 

Date of 

Performance 

Good Laboratorv 

Practices 

Statement: 

Tentative Date. 

of Submission of 

FLnal Report: 

Records 

~laintained: 

Ethyl Corporation 

0000 GSRl Avenua 

Baton Rouge, Louisiana 70808 

Genetic-Toxicology Laboratory 

Ethyl Technical Center 

P.O. Box 14799 

Baton Rouge, La 

1+ Oc'D 'lloffooiiJ 

DJ..Lf- Of?] 

To evaluate the ability of a chemical to interact 

with DNA by exposing rat liver cells to the 

chemical and tritiated thymidine. The amount 

of radioaCtivity incorporated into the nucleus 

of exposed cells is measured and compared to 

unexposed cells to determine the extent of 

repair occurring in the DNA. · 

D. E. Johnson 

Beverly Pancamo 

The proposed date of study initiation is generally 

four weeks from the receipt of the test article 

and signed protocol. 

This study will be conducted in complianca with 

the Good Laboratory Practices Regulations as 

stated in the Federal Register, Vol. 43, No. 247 

Friday, December 22, 1978. 

Approximate date for submission of final report 

to the sponsor is eight weeks after study 

initiation. , .,. ~~Y .. ""·:· , ••. 

All correspondence pertinent to the study including protocol, amendments to the protocol, raw 

data, test chemical weight and volume, dispensation reports, quality assurance reports and the 

final report will be maintained in the Genetic 

Toxicology Laboratory - Ethyl Technical Center. 

. ' 

. ' ~ . 

050583 
\ 

Rnt..r Qata: 

Vehicle: 

Special Handlinq. 

Instructions! 

Rationale for 

Control Article: 

Positive Control 

Article: 

Special Handling 

Instructions: 

Analysis of 

Purity: 

Rationale for 

Positive Control 

Article: 

Species: 

Strain: 

Supplier: 

~: 

Weight at 

Initiation: 

, Housing: 

Justification for 

Selection of the 

Test System: 

Maintained in a Genetic Toxicology Laboratory 

!lotebook 

NEGATIVE CONTROL ARTTCLE 

DMSO - spectrophotometric grade, or other 

other suitable solvent 

Standard precautions for solvents 

Solubilitv and or suspendability o£ the test 

article. -

POSITIVE CONTROL ARTICLE 

2-Acetamidofluorene 

Special p~ecautions for carcinogens 

' 

Responsibility of the manufacturer 

2-Acetamidofluorene has been shmm to induce 

unscheduled DNA synthesis (repair) in rat 

hepatocyte primary cultures. 

'rEST SYSTEH 

Rat 

Fischer-344 

Charl~s River Breeding Laboratories, Wilmington, 

flassachusetts, or any U.S.D.A. acceptable source. 

Uale 

150-300 grams (adult rats) 

. -

LSD, School of Veterinary !ledicine, Daten Rouge, 

Louisiana 

Rat Hepatocytes in Primary Culture have been 

shown to incorporate [3H] Thymidine into DNA 

due to unscheduled DNA synthesis induced by 

known car~inogens. 

-4-
Isolation of Adult 

Rat Heoatocytes: 

Perfusion r-1edia: 

Surgical and 

Pe!:fusion 

Procedure: 

Cell Harvesting: 

Dosacre Selection: 

.,,Test Protocol: 

' ' 

:-·---

' Data Quantification: 

.:~:~: 

... ... ~ ... 

....... 

HETHODS 

The protocol is a modification of the method 

described by Nilliams (1978). 

!!edium A. 0. 5 mil EGTA prepared as described 

in SOP ~ ETTOX 029. 

Hediurn B. Collagenase, 100 units/mL prepared 

as described in SOP ~ ETTOX 029. 

Hale Fisher rats ·are anesthet.ized with SoC.ium 

Nembutal. by intraperitoneal injection. Follo,»-

ing anesthetization, the liver is pex:·fused and 

removed following the procedure outlined in 

SOP ~ ETTOX 029. 

The liver is excised and the hepatocytes are 

harvested accordinq t<> ··SOP il ETTOX 029. · ·-

The highest. dose will be a 5% solution or i:P.e highest 

solmle concentration of the test article in tr..e acorooriate 

solvent.. The remaining nine doses will be half-lo~r dilutions fron tr.e 5% solutions. 

Hepatocytes are treated idt.h the· test article, 

fixed onto microscooe coverslides, stained, 

dipped, and developed according to SOP ~ ETTOX 

029 ~ "':·.: "' .... " .. · . . t ;"~ " ··. ~.~~~;=~~}:.":~~:~£.:>. ·~~·~:_. ·~· ~·~ ·- Unscheduled DNA repair synthesis, evidenced by 

a net increase in black silver grains in the 

nucleus, is quantified by determining nuclear 

and background grain counts of 25 cells/slide 

or as many cells as possible (up to 25) in the 

presence of toxicity using an automatic colony 

count.er with a microscope a ttachrnent. The test. 

compound is reported as positive or equivocal 

if there is a dose-related increase of mean net 

nuclear grain counts, a mean net nuclear grain 

count of 5 over background is co~sistently.observed, and/or there is a stailldcally significant -~-- .. dose related increase over background of cells 

with net nuclear counts greater than zero. The 

Chi square test will be used to statiStically eval1..-

Sponsor: 

_ ate t.he results. . ... If ·fl /. .. . Co.p, .. Study Director: S .Y.- £~~V""'~ 

Date: 

t I . Date: ¢1/;s-:;::· •t. ~ "'. 
.. 

RESULTS AND DISCUSSION 

The vehicle and positive control net nuclear grain counts were 

acceptable. Color and appearance of the test artir::le did not change 

from the time of receipt to the time of use. V.BCD Bottoms caused an 

increase in mean net nuclear counts over acetone at the top five 

dose levels counted (1000, 500, 100, 50 and 5 ug/well,l. A Chi 

square analysis was done to compare treated cells to untreated 

controls. The number of cells with net nuclear grain counts greater 

than zero of the top four scored dose levels of HBCD Bottoms was 

significantly increased statistically over acetone. 

Data are given on the next page. 

CONCLUSION 

HBCD Bottoms was genetically active in the Rat Hepatocyte 

Primary Culture/DNA Repair Test according to Protocol - Ettox 028. 

-6-
DATA 

Net Nuclear Grains Treatment Concentration/Well :!:S.D. 

Acetone 0.47 + 0.28 

2AAF 1o-6M 48.4 :!:10.0* 

HBCD Bottoms 1000 ug 5.25 + 4.32 

HBCD Bottoms 100 ug 4. 21 + o. 80 

HBCD Bottoms 50 ug 1.26 + 0.86 

HBCD Bottoms 10 ug 0.70 + 0.63 

HBCD Bottoms 5 ug 0.30 + 0.19 

HBCD Bottoms 1 ug O.ll + 0.15 

HBCD Bottoms 0.5 ug 0.11 + 0.16 

HBCD Bottoms 0.1 ug 0. 32 + 0.17 

HBCD Bottoms 0.05 ug 0.63 + o. 47 

*Posltive Finding 

Due to toxicity, only 1 slide (25 cells) were counted. 

-7-
E T H Y L C 0 R P 0 R A T I 0 N 

QUALITY ASSURANCE UNIT STATEMENT 

This study was performed in accordance with the Good'·Laboratory 

Practices Regulations for nonclinical laboratory studies developed 

by the u. s. Food and orug Administration and Environmental 

Protection Agency. 

study No. HPC 024- t083 

The following inspections were performed: 

Interval Date 

Reporting Phase 6/14/85 

Results of the above inspections were submitted to the Study 

Director and Management during the course of the study. 

(Date) 

-8-



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