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# • 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 

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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(•**

HEXABR0r>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 I7, 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.

ABL: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



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## 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, Louisiana70898 Telex 784583

### ETC BTR Telephone (504) 389·7600 8000GSRI 

### Awnue Baton 

### Rouge, Louisiana70820I. 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 8SUMMARY 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 Bottoms 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



## ~ "':·.: "' .... "
..·. .

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## ;"~ " ··.

## ~.~~~;=~~}:.":~~:~£.:>.**·~~·~:_. ·~·** **~·~**

# ·- 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 attachrnent. 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.obser** ved, 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
. 

# ¢1/;s-:;::· Date:

•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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