### CODING FORMS FOR SRC INDEXING Microfiche No. OTS0559170

88970000235 8EHQ-0797-13985

02/12196 07/31197 8E

ELF ATOCHEM NORTH AMERICA INC

CTRE INTL DE TOXICOLOGIE

INITIAL SUBMISSION: IN VITRO MAMMALIAN CELL GENE MUTATION TEST IN L5178Y TK

### +1- MOUSE LYMPHOMA CELLS OF N-PROPYL BROMIDE, WITH COVER LEITER DATED 7/28/1997 Chemical 

### Category N-PROPYL BROMIDE

# eiFatochem**/~** July 28, 1997 Elf Atochem North America, Inc:. 2000 Markel Street Philadelphia. PA 19103-3222 Tel.: 215.419.7000

UPS NEXT DAY DELIVERY
Document Control Office (7407) Office ofPollution Prevention andToxies Environmental Protection Agency 401 lII. St., S.W. Washington, D.C. 20460 Subject: TSCA Section See) Submission DearSirlMadam: 

# 1IIIII1IIIIII 8EHC-97-13985 

# Contains 

# No CBi

ElfAtochemNorth America, Inc. (ElfAtochem) has received the final report ofagene mutationtest and is submitting this reportto the Environmental Protection Agency (EPA) pursuantto Toxic Substances ControlAct (TSCA) Section 8(e). This study provides infonnation on n-propyl bromide, CAS number 106-94-5, and does not involve effects in humans. Nothing in this letterorthe enclosed study report is considered confidential business information ofElfAtochem. The in vitropotential oftest substance n-propyl bromideto induce mutations atthe TK locus in LSI78Y mouse lymphomacells was evaluated in this study. Thetestsubstance induced a reproducible and signficant increase in the mutation frequency and the numberofsmall colonies atdose levels between 1000 and 1500J..'g/mlwithout activation and at levels between 1500 and 2000 J1.g/ml with activation, giving a positiveresponse underthe conditions ofthis test. This new information adds to the existing genotoxicity database forthis material which includes: positive and negative responses in the Ames Salmonellaassay, negative results in the mouse micronucleus assay andtherat dominant lethal assay, and weakpositive results in a study for chromosomal aberrations. Results fromthis study have been incorporated into the ElfAtochem Material Safety Data Sheetforthetestmaterial. Based on EPA guidance we believethatthese datameetEPA criteriaforreponingunderTSCA Section 8(e). f,!1rtherquestions regardingthis submission may be directedto me at(215) 419-5890. Sincerely.

# ~~ DebraRandall, DABT Product Safety Manager 

# 11111111111111 889?0000235

## , CIlf/Study No. 13293 MLY/n-PROPYL BROMIDEIElfAtochem



### SPONSOR ElfAtochem S.A. Direction Securite Environnemem

- Produit La 

### Defense 10

- Cedex. 42

92091 Paris-la-Defense France



### STUDYTITLE IN VITRO 

### MAMMALIAN CELL GENE**~ATlONTEST** 

### IN L5178Y TK+

- MOUSE LYMPHOMA CELLS TESTSUBSTANCE 

### n-PROPYLBROMIDE

STUDYDIRECTOR

Brigitte Molinier



### STUDYCOMPLETIONDATE 12thFebruary 

### 1996



### PERFORMINGLABORATORY Centre International deToxicologie 

### (C.LT.) Miserey

- 27005 

### Evreux

- France



### LABORATORYSTUDYNUMBER 13293MLY CONFIDENTIAL

### CITIStudy No. 13293 MLYin-PROPYL BROMIDElElfAtochem I • 

### CONTENTS STATEMENT OFTHE STUDY DIRECTOR STATEMENT OF QUALITY ASSURANCE UNIT SUMMARY 1. 

### INTRODUCTION 2. 

### MATERIALS AND METHODS 2.1. TEST AND CONTROL SUBSTANCES 2.1.1 Identification 2.1.1.1 Test substance 2.1.1.2 

### Vehicle 2.1.2 Preparation 2.1.3 Positive controls 2.2. 

### TEST SYSTEM 2.2.1 Cells 2.2.2 Metabolic activation system 2.3. EXPERIMENTAL DESIGN 2.3.1 Treatment 2.3.2 Preliminary toxicity test 2.3.3 Mutagenicity experiments 2.4. 

### EVALUATION OFTHE 

### RESULTS 2.5. ARCHIVES 2.6. CHRONOLOGY OFTHE STUDY 3. RESULTS (tables 1 to 5) 

### 3.1. PRELIMll'rARYTOXICITY TEST 

### (table 1) 3.2. MDTAGENICITY EXPERIMENTS (tables 2 to 5) 

### 4. CONCLUSION 5. REFERENCES Table 1: Preliminary toxicity test Table 2: FIrstexperimentwithout59 mix 

### Table 3: FIrStexperimentwith S9 mix Table 4: Secondexperimentwithout S9 mix 

### Table 5: Secondexperimentwith 59 mix 

### 2

4 

### 5 6 

### 8 8 8 8 8 8 9 

### 9

..**~.:**

### .. 9



# ·'9

9 10 

### 10 10 10 11 

### 12 13 14 14 14 

### 15 15 16 17 

### 18 19 

## 4

0I CITlStudy No. 13293 MLYin-PROPYL BRO:MlDElElfAtochem
APPENDICES 1. Analytical certificate ofthe test substance and test anicle description 2. Historical data 3. Protocol and amendment 

### 3
21 22 

### 25 27 to 36

### aT/Study No. 13293 MLYin-PROPYL BROMIDEIElfAtochem
STATEMENT 

### OFTHE STUDY DIRECTOR 4



### The study was performed in compliance with the principles ofGood Laboratory Practice Regulations:

· Decret N° 

### 90-206 du 7 

### mars 1990 concernant les Bonnes Pratiques de Laboratoire

(Ministere de I'Industrie et de l'

### Amenagementdu Territoire),

· US Environmental Protection Agency, Federal Register, 40 CFR Part 792; Toxic

Substances Control Act; Good Laboratory Practice Standards. November 29, 1983 (and subsequent amendments).

· O.E.C.D. Principles ofGoodLaboratory Practice. C(81)30(final) Annex 2. May 12, 1981. I 

### declare that this report constitutes a troe and faithful record ofthe procedures undertaken and the results obtainedduring the performance ofthe study. This study was performed at the Centre International de Toxicologie (C.I.T.), Miserey, 27005 Evreux. France.



# -- B. Molinier Date: 12.2.96 Study Director DoctorofToxicology Head ofGeneticToxicology, 

### CIT/Study No. 13293 MLY/n-PROPYL BROMIDElElfAtochem
STATEl\1ENT QF QUALITY ASSURANCE UNIT



### Dates (day/month/year) Type ofinspections

------------------------------------------------------ Inspections Report to Report to Study Director 

### C*) Management 

### C
..) Protocol 25.7.95 1.8.95 1.8.95 Study 11.10.95 16.10.95 18.10.95 Study 2.11.95 3.11.95 6.11.95 Report 24.1.96 26.1.96 28.1.96 

### 5

The inspections were perfonned in compliance with C.LT. Quality Assurance Unit procedures and the Good Laboratory Practice Regulations.
. 

### C"') The dates mentioned correspond to the dates of signature of audit reports by Study Director and Management.

L. Valene-Talbi Date: 12.2.96 DoctorofBiochemistry Head ofQuality Assurance Unit and Scientific Archives

# . ·CH/Study No. 13293 MLY/n-PROPYLBROMIDElElfAtochem


### SUMMARY 6

The objective of this study was to evaluate the potential ofthe test substance n-PROPYL BROMIDE to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells. Methods After a preliminary toxicity test, n-PROPYL BROMIDE was tested in two independent experiments, with or without a metabolic activation system. the S9 mix, prepared from a livermicrosomal fraction (S9 fraction) ofrats induced with Aroclor 1254. Approximately 0.5.106 

### celIslml in 20 ml 

### culture medium with 5% horse serum were exposed to the test or comrol substances, in the presence or absence of 59 mix (final concentration ofS9 fraction 2%), for three hours at 37cC. 

### Cytotoxicity was then determined using cloning efficiency**(CE~** 

### before expression ofthe murant phenotype. Cells viability (using cloning efficiency CEo,) and number of mutain clones (differentiating small and large colonies) were checked after the expression ofthe mutant phenotype. The teSt substance was dissolved in dimethylsulfoxide (DMSO). The dose-levels for the positive controls were as follows:

without S9 mix: 25 J.lg/ml 

### ofmethylmethane sulfonate

· with 59 mix: 3 Ilglml ofcyclophosphamide. 

### Results The cloning efficiencies CEo and**C~** 

### and mutation frequencies ofthe vehicle and positive contrOls were within the range ofourhistorical data. The top dose-level was selected according to the criteria specified in the international regulations. Since the test substance was toxic, the top dose-level 

### was based on the level of toxicity: a toxic dose-level giving 10-20% relative survival assessed by relative cloning effiency determined on day /) 

### post-treatment**(RCE~.** 

### Without 59 mix, 

### the selected treatment-levels were:

first experiment: 125,250,500, 1000 and 1500 !J.g/ml,



### · second experiment: 250, 500, 1000, 1250 and 1500 l-lglml. The test substance induced a reproducible and significant increase in the mutation frequency together with an increase in the number ofsmall colonies at dose-levels between 1000 and 1500 llglml. With 59 mix, the selected treatment-levels were:

· first experiment: 125,250.500. 1000, 1500 and 2000 l-lgimI..

second experiment: 500, 1000, 1500, 2000 and 2500 !J.g/mL (At 2500 IlgimI.. all 

### the cells were deadthree hours aftertreatment). The test substance did not induce any significantincrease in the 

### mutation frequency 

### iii. the 

### :first experiment. In the second one, it induced a significant increase in the mutation frequency together with an increase 

### in the number of small colonies at 

### 1500 and 2000

### p.gIml.

### CITIStudy No. 13293 MLYIn-PROPYL BROMIDElElfAtochem
Conclusion 7



### Under our experimental conditions. the test substance n-PROPYL BROMIDE showed mutagenic activity in this mouse lymphoma assay. especially without S9 mix.

### .CIT/Study No. 13293 MLYIn-PROPYLBROMIDEIElfAtochem
1. 

### L."ITRODUCTION 

### This study was pertormed at the request ofElfAtochem S.A.• Paris-la-Defense. France. 

### 8

The objective of this study was to evaluate the potential of the test substance to induce mutation at the TK 

### (thymidine kinase) locus in LS178Y mouse lymphomacells. This in vitro 

### mammalian cell gene mutation test is able to identify substances that cause base-pair mutations. frameshift mutations. small deletions. large deletions and rearrangements of the relevant**chro~osomes.** 

### Mutagenic**sUbst~ces** 

### can induce forward mutation from the parental type (TK+ 

### J to the mutant form (TK- ") 

### permitting mutant cells to grow and form colonies in selective medium. while non-mutant cells cannot. In addition. twO 

### types of colonies are 

### produced: small. slow growing colvni(.S mainly produced by chromosome rearrangements and large colonies mainly produced by point mutations. This study was designed in accordance with the following guidelines:

· O.E.C.D. guideline No. 476, 4th April 1984 and revised Draft document of December

1994,

· EE.C. Directive No. 87/302lE.E.C.. 18thNovember 1987.

· U.S.AJE.P.A./T.S.C.A. Federal Register. Vol. 50, No. 188. Subpart F. 27th September

1985.
2- 

### MATERIALS A!\T]) METHODS 2-1. 

### TEST 

### AJ.'\'l>**CO~-rROL** 

### SUBSTAJ.'i'CES 

### 2.1.1 Identification 2.1.1.1 Test substance The test substance, n-PROPYL BROMIDE, used in the study was supplied by Elf Atochem. Documentation suppliedby the Sponsoridentified the test substance as follows:

· denomination:

- protocol and labelling: n-PROPYLBROMIDE

· batch number:

- protocol: 3-337-02

- labelling: 3-337-02 CAL4318194

· description: colourless liquid

· container: one glass flask

· date ofreceipt: 4.10.94

· storage conditions: atroom temperature andkeptfrom light

· purity: 99.3%. Data relating to the characterization ofthe test substance are documented in 

### an analytical 

### certificare andatestarticle description (presentedin 

### section 6) 

### providedbythe Sponsor. 2.1.1.2 Vehicle The vehicle was 

### dimethylsulfoxide (DMSO, batch 

### No. 313Kl9492750. Merck.**Clev~ot,** 

### 77500 Chelles. France).CIT/Study No. 13293 MLYIn-PROPYL BROMIDEIElfAtochem 

### 9
2.1.2 Preparation T,e test substance was dissolved in the vehicle at concentrations between 300 and 500 mglml. The preparations were made immediately before use. 2.1.3 Positive controls Two known mutagens. dissolved in distilled water, were used to check the sensitivity of the test system:

. wirhout S9 mix: methylmethane sulfonate (M:.\ilS), used at a final concentration of

25 Ilglml,

. wirh S9 mix: cyclophosphamide (CPA), used at a final concentration of3 lJg/ml.


### 2.2. TEST SYSTEM 2.2.1 Cells L5178Y cells are an established cell line recommended by international regulations for 

### in vitro mammalian cell gene mutation rest. They have demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a stable spontaneous mutant**frequenc)(:·~The** average cell cycle time is 12-14 hours and rhe TK phenotypic ex.pression time is two 

### Oays." Regularchecks demonstrated the absence ofmycoplasma contamination. L5178Y cells, originally obtained through ATCC, were kindly suppliedby Dr. OudelkbimDiot (Vitry-sur-Seine, France). The cells were stored in a cryoprotective medium (10% horse 

### serum and 10% dimethylsulfoxide (DMSO» at -80°C. 2.2.2 Metabolic activation system The S9 mix consists of induced enzymatic systems contained in rat liver microsomal fraction (59 fraction) and

## me cofactors necessary for their function. 59 fraction was purchased from Moltox (Molecuiar- Toxicology, INC, Annapolis, 

### MD 21401, U.S.A.) and obtained from the liver ofrats treated with Aroelor 1254 (500 mg/kg) by intraperitoneal route. The 59 fraction 

### was preserved 

### in sterile tubes within a liquid nitrogencontainer, until use. The S9 mix 

### was prepared at +4°C immediately before 

### use and maintained at this temperaru.rc until added to culture medium. The S9 

### mix containedper5 

### ml: 1 

### ml Glucose-6-Phosphate (180 mg/ml). 1 ml NADP (25 mglml), 1 

### ml KO (150 mM). 2 

### ml S9 fraction (final concentration of 59 fraction 

### in the 59 mix: 40%. vlv). (batch Nos. 588 and 0616. protein concentrarion: 37 and 38 

### gil. respectively).,C1TlStudy No. 13293 MLYIn-PROPYL BROMJDElElfAtochcm


### %.3. 

### EXPERIMENTAL OESIGN 10



### 2.3.1 TlUtment The day before treatment. the cells were seeded in 50 ml of RPMI 1640 medium containing 10% horse serum. L-Glutamine (2 roM), penicillin (100 U/ml). streptomycin (100 ",glmI) and sodium pyruvate (200**~ml).** 

### The cells were then incubated at 37"C in a humiclified atmosphere of

### 5%**COI9S~** air. After 24-hour incubation. culture medium was removed and cells 

### were counted. Appro;lf,imately 0.5 Icf cells1m1 ill 20 

### m1 culture medium with 5% horse 

### serum. were**C;lf,~** to the test or control substances. in the presence or absence of 59 mix (final concentration of59 fr.K:tion: 2e;t,). for

### tM:c hours at 37"C. 

### The treatment medium was then removed and the cells were counted and seeded as fonows: Cytotoxicity plates 1.6 cells/well (one 96-well platclculture 

### = two plates/dose-level) to determine cytoto;lf,icity using cloning efficiency 

### (CE.,). After 

### 12

# = I days ofincubation at 37"C. the 

### clones were counted. To enable the uj)l'l:SSion ofthe mutant phenotype. 2.1rY cellslml were reincubated ati1':'C. and replated 

### on day I at 2.lrY cellslml. After two days. the cells were seeded as follows:
. Viability

# *'" 1.6 c:dlsIwe11 (one 96--well plalClcu1ture

# =two plates/dose-level) to derme the number of viable 

### cdls (CE..). After 12

# =I days of incubation at 37°C. the dorIeS WC'R counted.

- MugntRiata 

### 2000 cd1sIwdI (two 96-wcL p1aIesIculture 

### = four plates/dose-level) to select the 

## TFr« (U'ifluorolhymid resistmt) mutant cells (for determination of 

# cr->. AAu 12

# =I days**o~** iDcubalioo at 37"C 

### in the presence of 4 

### J.I8 TFTtmI ofcu1aft medium.

# me cloacs were 

### counted. differentiating small 

### and Ja."'J,'C 

### coIoaic:s:
• size 

### ofsmall coIoaies. 

### <**2S~** of

# me diameterofthe well.

e Si2c 

### of

## IarJCc:oIoDies.. 

### > 2S4lt of

# me diameterofthe well.**2.3.~** Prelimia.vy toxicity u:st To aucss 

### of

# me u:st subswx:c. at least six dose-levels of D-PROPYL 

### BROMIDE (ODe 

### cuItun:IcSose-leveI) were tested both 

### with and without metabolic**IOI~_** 

### 1.3..3 MurapNcit)'

### experimems b t"O 

### .Ddepe:deDt

### experimealu. at 

### least 

### four**c.tosc-~** 

### ofD-PROPYL BROMIDE (two cukuraIdoIe-leYe1) 

### were IaIeCS bodl 

### wilh

### aDdwimow """RboIic activation. 

### In eacb ap':I"iIDeDl.

# mefoDowiD&CODtrOIs were 

### iDclDdedusiDg atk'astduplicate cultures:



### . vebide

### CODa"Ols: cuIIurcs 

### aeaIed with die 

### vebide.



## . poua'veccaaoIs: cuIDIIa

### aaaedwitb:



### •MMS. iA

# me of59 mix., 

### •CIA.. III tbe pi 

### eace ofS9 

### mix..

### 'PT/Study No. 13293 MLYIn-PROPYLBROMIDElElfAtochem 11


### 2.4. EVALUATION OFTHE RESULTS Treatment ofresults Data from cytotoxicity plates (empty wells) are used to calculate CEo (cloning efficiency after treatment) and RCEo (survival relative to vehicle controls after treatment). These values give a measure of the toxicity of the treatment with the test substance. CEo is calculated from the zero term ofthe Poisson distribution: [ empty wells ] -Ln total wells number ofce!lslwell (- 1.6)


### CEo treated CEo vehicle control 

### x 100

number ofcells/well (- 1.6)**C~=** 

### Data from viability plates (empty wells) are used to calculate CE., (cloning efficienc/M-the' end ofthe expression period) and RCE., (viability relative to vehicle controls at the end of the expression period). These values indicate the viability ofthe cell populations at the end ofthe expression period. CE:z is calculated from the zero term ofthe PoissC'n distribution: [ empty wells ] -Ln 

### total wells**RC~= C~**  

### treated 

## CE:2 vehicle control x 

### 100



### numberofcells/well (- 2000) CE 

# = lIIIIQDt 

### Data

### fromthe mutantplates (empty wells) 

### are used to calculate 

### CE_ t 

### (cloning efficiency 

### in selective medium). This value is 

### an indicator ofthe absolute mutant frequency. 

### CEmutaDl 

### is calculated 

### from the 

### zero term ofthe Poisson distribution (only mutant clones 

### are able to grow 

### in

### TFrcontaining medium): [ empty wells ] 

### -Ln - total wells



### MF= The 

### relative mutantfrequency (MF) is 

### calculatedas: CEIIIlIlmR

### x 10 6

### CITIStudy No. 13293 MLYIn-PROPYL BROMIDElElfAtochem 12
For each experimental point, the scored values (empty wells), the number of small colonies, CEo' RCEo ' CE:!,**RC~** 

### and MF will be 

### presented in a table. Acceptance criteria This study was considered valid since the following criteriawere fulfilled:

· the cloning efficiency ofthe vehicle controls were between 0.6-1.4 for CEo and between

0.7-1.3 for CE."

· the mutation frequency ofthe vehicle controls were between 60-200.10-6,



### · the mutation frequency of the positive controls was higher than that of the vehicle

controls and within the range ofour historical data. Evaluation criteria A reproducible two-fold increase in the mutant frequency when compared with the vehicle controls, at any dose-level and/or evidence a dose-relationship were considered as a pOSitive result. Reference to historical data, or other considerations ofbiological relevance might be 

### also taken into account in the evaluation ofthe dataobtained.


### 2.5. ARCHIVES The study documentation and materials. namely:

· protocol and possible amendments.

· raw data,

· correspondence,

· final report and possible amendments. · a sample ofthe test substance,
are stored in 

### the archives ofC.I.T. (Miserey. 27005 Evreux, France), for five years after the end ofthe experimental study. At the end ofthis period, the study documentation will be 

### returned to the Sponsor.

### CITIStudy No. 13293 MLYin-PROPYL BROMIDElElfAtochem


### 2.6. CHRONOLOGY OFTHE STUDY The chronology ofthe study is summarized as 

### follows: 13

Procedure Protocol approved by:



### · Study Director

· Sponsor Preliminary toxicity tl;sl with and without S9 mix

· treatment First experiment without S9 mix.

· treatment with S9 mix

· treatment Second experiment without and with 59 mix



### · treatment Date 5.7.95 24.7.95
13.9.95
11.10.95 17.10.95
31.10.95

### 'CITIStudy No. 13293 MLYin-PROPYL BROMIDElElfAtochem
3. RESULTS (tables 1 to 5) 3.1. PRELIMINARY TOXICITY TEST (table I) 14



### The test substance was freely soluble in the vehicle (DMSO) at 500 mglml. Thereafter, a moderate to strong emulsion was observed in the culture medium at the final dose-levels of 2500 to 5000 Ilglml. Consequently, the maximum dose-level was 2500 Ilglml. The treatment-levels were: 10, 25, 100, 250, 1000 and 2500 Ilglml. Marked cy[Qtoxicity of the test substance was recorded at 2500 Ilglml: all the cells were dead after three hours oftreatment both with and without S9 mix. At the lower dose-levels, a moderate reduction of the cloning efficiency CEo oftreated cultures when compared to control cultures was observed. 3.2. MUTAGENICITY EXPERIMENTS (tables 2 to 5) The cloning efficiencies CEo and**C~** 

### and the mutation frequencies ofthe vehicle and.p'ositive controls were as specified in acceptance criteria and within the range ofour historical
. data.
. The top dose-level was selected according to the criteria specified in the international regulations. Since the test substance was toxic, the tOP dose-level was based on the level of toxicity: a toxic dose-level giving 10-20% relative survival assessed by relative cloning effiency determined on day 0 post-treatment (RCEo). Without S9 mix, the selectedtreatment-levels were:

· first experiment: 125,250.500, 1000 and 1500 Ilg/ml,



### · secondexperiment: 250, 500, 1000, 1250 and 1500 ,.:.glml. At 1500 Ilglm.l, 

### 21 and 33% 

### RCEo were noted when compared to control cultures for the first and secondexperiments, respectively. At 1250

### J.lglml (second experiment), RCEo was 46%. At lowerdose-levels. no significantdifferences between treated. and control were noted. The number ofviable cells two days 

### after treatment (assessed 

### by the cloning efficiencies**~)** 

### was considered equivalentto 

### mat ofthe control cultures. The test substance induced a reproducible and significant increase 

### in the mutation frequency togetha with an increase 

### in the numberofsmall colonies at 

### dose-Ie
..**~ls** 

### between 1000 and 1500 JIg/mi. 

### With S9 

### mix. the selected treatment-levels 

### were:

· firstexperiment: 125,250,500. 1000, 1500 and2000

### J.18Iml,

· second experiment: 500.1000.1500.2000 and2500 

### p.gImJ.CIT/Study No. 13293 MLY/n-PROPYL BROMIDElElfAtochem 

### 15
No significant cytotoxicity was observed in the first experiment. only a moderate reduction of the RCEo (59%) was noted at 2000 IJglml. In the second experiment. cytotoxicity was more marked: At 2500 IJglml. all the cells were dead three hours after treatment. At 2000 IJglml. RCE

### owas 9%. At 1500 IJg/ml. RCE

### owas 36%. At lower dose-levels. no marked differences between treated and control were noted. The number of viable cells two days after treatment (assessed by the cloning efficiencies**CE~)** was considered equivalent to that ofthe control cultures. The test substance did not induce any significant increase in the mutation frequency in the**firs~** experiment. In the second one. it induced a significant increase in the mutation frequency together with an increase in the numberofsmall colonies at 1500 and 2000 IJglml.

4. CONCLUSION Under our experimental conditions. the test substance n-PROPYL BROMIDE

### s~6wed' mutagenic activity in this mouse lymphoma assay. especially without 59 mix.


### 5. REFERENCES Nestrnann. E.R.; Brillinger, R.L.; Gilman. l.P.W.; Rudd. C.l. and Swierenga, S.H.H.: Recommended protocols based on a survey of current practice in genotoxicity testing Laboratories: II. Mutation in Chinese hamster ovary. LS178Y Chinese hamster lung and L5178 mouse lymphomacells. Mutation Research. 246. 255-284 (1991). Ames. B. N.; Mc Cann. D. and 

### Yamasaki. E.: Methods for detecting carcinogens and mutagens with the 

### Salmonella Mammalian-microsome mutagenicity test. Mutation Research.

# n. 347-364 (1975).

### . CIT/Study No. 13293 MLY/n-PROPYL BROMIDElElfAtochem


### Table I: Preliminary toxicity test 16

Doses Empty Total CEo RCEo (J,Ig1ml) wells wells % 0 96 2.85 100 without 10 7 96 1.64 57 S9mix 25 5 96 1.85 65 100 2 96 2.42 85 250 5 96 1.85 65 1000 10 96 1.41 50 2500 96

0 2 96 2.42 100 with 10 4 96 1.99 82 S9mix 25 4 96 1.99 82 100 4 96 1.99 82 250 5 96 1.85 76 1000 8 96 1.55 64 2SOO 96
• : no seeding ofCEo performed since all cdls died after 3 hours ofexposure to the testsubstance 

## o: vehicle control (DMSO) CEo: cloning efficiency RCEo : relative cloning efficiency<:;IT/Study No. 13293 MLY/n-PROPYL BROMIDEIElfAtochem 17
Table 2: First experiment without S9 mix
Cytotoxicity: CEo Viable cells: CE:! Mutation frequency: MF Doses Empty TOlll.l CEo RCEo Empty Total CE:! RCE:! Nb. Empty TOlal MF R (J.lglml) wells wells % wells wells % SC wells wells :H 96 18 96 5 77 96 0 0.66 100 0.92 100 4 78 96 122 1.0 33 96 26 96 6 76 96 6 76 96 20 96 29 96 6 73 96 125 0.94 142 0.77 84 7 75 96 156 1.3 23 96 27 96 6 78 96 7 76 96 31 96 22 96 7 72 96 250 0.87**I~"** 0.78 85 4 75 96 158**,,~I.3** .J_ 

# ....,..

## - 17 96 33 96 5 78 96 10 75 96**~.,** 96 30 96 8 72 96 .J_ 500 0.72 109 0.85 93 7 80 96 143 1.2 29 96 19 96 2 73 96 5 76 96 38 96 29 96 8 73 96 1000 0.74 112 0.73 79 12 62 96 250 2.1 21 96 31 96 15 66 96 12 66 96 71 96 27 96 29 41 96 1500 0.14 21 0.79 86 29 53 96 514 4.2 83 96 27 96 43 37 96 32 39 96 35 96 55 96 23 52 96 MMs 0.64 97 0.41 44 27 44 96 856 7.0

25 34 96 45 96 30 49 96 27 46 96 

### CEo and**~:** cloning efficiency RCE(o and 2): relative dolling efficiency SC: small colonies R: Ratio betwcco MutationFrequency oftR:alCd c:ellsIMuwion Frequency ofCOIltrol c:e11s

### 'CIT/Study No. 13293 MLY/n-PROPYL BROMIDEIElfAtochem 18


### Table 3: First experiment with S9 mix Cytotoxicity: CEo"\ Viable cells: CE:!. Mutation frequency: MF Doscs Empty Total CEo RCEo Empty Total CE:!. RCE:!. Nb. Empty Total MF R (l-lglrnl) wells wells % wells wells % SC wells wells 24 96 33 96 5 76 96 0 0.79 100 0.79 100 5 77 96 162 1.0 30 96 21 96 6 74 96 3 70 96 17 96 29 96 5 76 96 125 1.03 130 0.82 103 7 78 96 135 0.8 20 96 23 96 6 75 96 6 79 96 26 96 25 96 8 76 96 250 0.83 105 0.74 93 5 75 96 176 1.1 "\0,.-. 25 96 34 96 6 72 96 " 

## ....- 3 73 96 28 96 23 96 7 65 96 500 0.75 94 0.96 122 3 75 96 154 1.0 30 96 18 96 6 73 96 8 72 96 96 20 96 5 70 96 1000 0.5:5 69 0.87 109 1l 66 96 191 1.2 36 96 28 96 9 72 96 10 68 96 39 96 28 96 9 73 96 1500 0.67 84 0.70 88 9 69 96 245 1.5 27 96 35 96 8 68 96 8 63 96 41 96 27 96 9 76 96 2000 0.47 

### S9 0.73 92 9 74 96 149 0.9 50 96 33 96 2 84 96 8 75 96 52 96 5S 96 36 40 96 CPA 0.36 

### 4S 0.33 42 35 38 96 1284 7.9

3 56 96 58 96 31 44 96 34 42 96 CEo and CE2: cloniDgcfficieocy RCE(o aDd 2): relative cloningcfficicocy SC: small

### c:oloaics R: RabobctweeDMuwionFR:quc:ocyofIn:ated "CDsIMlDIioaFrequeoc:y ofcoatrolc:eUsCI:r/Study No. 13293 MLYin-PROPYL BROMIDEIElfAtochem
Table 4: Secondex.periment without 59 mix 19

Cytotoxicily: CEo Viable cells: CEl Mutation frequency: MF Doses Empty Total 

## ceo RCEo Empty Total CE2 ReEl Nb. Empty Total MF R (l1g1ml) wells wells % wells wells % SC wells wells 30 96 32 96 4 83 96 0 0.80 100 0.80 100 6 76 96 147 1.0**::!~** 96 21 96 5 71 96 8 73 96 35 \/6 21 96 2 78 96 250 0.74 92 0.92 114 0 75 96 122 0.8 24- 96 23 96 4 

### n 96 5 77 96 12 96 27 96 2 76 96 500 1.14 142 0.85 106 2 82 96 133**~:o.9.** 19 96 22 96 6 74 96 4 74 96 34 96 28 96 5 73 96 1000 0.64 80 0.87 lOS 8 62 96 239 1.6 35 96 20 96 10 60 96 11 59 96 46 96 26 96 14 62 96 1250 0.37 46 0.84 105 12 67 96 275 1.9 60 96 24 96 16 56 96 18 57 96 56 96 18 96 22 53 96 ISoo 0.26 33 0.88 109 28 42 96 424 2.9 70 96 29 96 30 46 96 33 41 96 39 96 83 96 22 54 96 MMS 0.71 88 0.22 27 23 S9 96 1467 10.0

2S 23 96 S3 96 32 4S 96 32 46 96 CEo aod eEl: clollingef6cieDCy RCE(o aod 2): relativecloning ef6c:ieDCy SC: small colODies R.: Ratio betweenMUWiou FreqacDcy ofaearcdcdIsIMuwioDFtequeucy ofCODuolccUs

### · CITIStudy No. 13293 MLYIn-PROPYL BROMIDElElfAtochem
Table 5: Second experiment with 

### S9 mix 

### 20

Cytotoxicity: CEo Viable cells: CE:! Mumtion frequency: MF Doses Empty Tot:l1 CEo RCEo Empty Tot:l1 CE:! RCE:! Nb. Empty Total MF R (lJg!ml) wells wells % wells wells % SC wells wells 27 96 29 96 78 96 0 0.83 lOO 0.83 100 6 75 96 123 1.0**2~** 96 22 96 79 96 2 81 96 

# -., 96 19 96 5 71 96 .J_ 500 0.60 72 1.03 124 3 77 96 125 1.0 4" 96 18 96 9 71 96 2 78 96**~1** 96 33 96 4 67 96 ",

# .. 1000 0.60 73 0.78**9~** 7 79 96 200·''- '1.6 

## -., 96 22 96 6 69 96 

## ,,- 7 66 96 55 96 21 96 23 56 96 1500 0-30 36 0.85 103 19 54 96 316 2.6 64 96 28 96 15 57 96 11 57 96 86 96 39 96 29 46 96 2000 0.07 9 0.63 76 631 5.1 85 96 31 96 20 40 96 17 44 96 60 96 49 96 54 19 96 

### CPA 0-31 38 0.43 52 49 28 96 1433 11.6

3 56 96 48 96 38 37 96 43 29 96 CEo andCEl: cloning efficiency RCECo and 2): relative cloning efficiency SC: small colonies R: Ratio between Mutation**~uCDC)'** oftreated cel1sIMutaIionFrequency ofcontrol cells -: due to the toxicity ofthe teSt substance. therewere notenough cells to allow seedingofthis duplicateCITIStudy No. 13293 MLYIn-PROPYL BROl'vlIDEIElfAtochem

APPEl'o'DICES 

### 21

### CITIStudy No. 13293 MLYIn-PROPYL BROMIDElElfAtochem

1_ Analytical certificate ofthe test substance and test anicle 

### description 

### 22

### CIT/Sludy**~o.** 

### 13293 MLYin-PROPYL BROMIDEIElfAlochem


## elfatochem

Port·d
...Bauc:n=c. 

### 23



### Pore de 30uc. le**3~-OS-1994 Da~e**  

### de l'expedition ;:)es::.i::ataire**n-08-S~** CAL kg

Let: 2IID 3-337-02

## --**~-~-=_=-=-=-=-~-3_~-=-=-**

=:::z:a.=.:::1=

### ===::;r ;:top 

### Alcool ChJ..o::::ure 3 7 '0 

### Lou=ds 59.3**O.O~** 0.07 % O.:!.6 % 0.35 %**O.O~** l:



### Le Resoonsabl.e Laboratoire

- !Un!NAUD

### 'C1·TlSludy 

### No. 13293 

### MLY/n-PROPYl.. BROMIOElElfAJochem 24

TOXICOLOCY D(P
.**..\RT;\IE~T** CO"'t:rDEi\l'l" L 7**Se;'l~** 199" 

# elf atochem s.a. u defense: 10. cede:c 42 92091 Paris-la-Defense, France 

### TE. :T,'RTICLEDESCRiPTION



### n-PROPYL BROMIDE rSTRl,;CTtiRAL fOR."'IULA 

# l**BrCH~·CH2·CH~**



# ---------

# I:::::,.~ I**C~mi~3.l ~~**  

### C

...\$**tl\I~~ ;'~O<C<:~l2:** **:~r::'lula ~oI«ubt**  

### wftIht

..rit1'**O~Sin** 

### anc:**~:(!'::** 

## Ear**Alodl~m** fi4ing**~mbcr :'l·?~opyl**  bromide Bromo- I ·prop.anc 106·94.$ BrC;H7 12;**~.~%** ElfAlochem. Por.:-<lc-Bouc.**~lc:h ~·337-02** **C~ 4~1S/94**  ,.HYSICAL

_ ...**~[) CH£~IC."L**  PROPERTIES**A;'~c S~r~~~vi;,** **~'cl:i~ ~;n;** **9011i~** Xlin: va:>o:**~s~te ~DOin:**  SoJu:lililY 

### C01oriess 10 dar**~lIow** liquid I**~5~** al 2O-C .11O'"C il-C ;.16 at 20ec <21-(: (cIoseC! cup) 02S % in**~er** at 2O-c SOluble in OMSO soluble in ahylic alc0hoi TOXICOLOGICAL I:"FOfl\.(ATIONS AND USE SAFETY**LD~** :> 2000 mgI1r;,.. IrriuN 10 lhe skin and eyes. STORAGE**A~O** DISPOSAL**Sl~ E..~da:c**  

### In darIc and 

### ac room cernperuure December 1995 IncInetuioa

### .CITIStudy No. 13293 MLYin-PROPYL BROMIDElElfAtochem



### 2. Historical data 

### 25

### ,CIT/Study No. 13293 MLY/n-PROPYL BROMIDElElfAtochem

l\lLY 7.2.95 

### Historical data 

### Mouse lymphoma assay 

### 26

without 59 mi.'l: with 59 mi.'l: \'ehicle 1\11\15: 2S**~glml** vehicle CPA: 30**~~m1** CEO CE2 MF CEO CE2 MF CEO CE2 MF CEO CE2 MF assay No. 1 0.58 1.07 126 0.49 1.02 458 0.62 1.12 131 0.39 0.99 457 2 0.65 0.74 135 0.36 0.43 945 0.75 0.91 182 0.4 0.97
. 442 3 0.78 1.27 109 1.02 0.60 754 0.93 0.58 235 0.76 0.61
. 610 4 0.62 1.29 93 0.55 0.67 413 0.78 0.96 128 0.62 0.76 453 5 0.91 0.90 90 0.84 0.46 806 0.58 O.SO 105 0,35 0.4 812 6 0.69 1.73 81 0.71 0.96 399 ,'" '

# .

..

## --- 7 0.95 0.95 8S 1.13 0.73 511 8 0.50 0.55 207 0.51 0.46 625 0.87 0.93 131 0.53 0.6 638 9 0.63 0.57 236 0.59 0.57 70s 0.65 1.48 99 0.42 0.57 642 10 1.39 1.21 65 0.76 0.84 396 1.59 1.73 48 0.56 0.73 384 11 1.43 1.04 73 1.35 0.62 445 0.82 UP- 45 0.59 0.95 386 12 0.73 I.73 40 0.95 0.74 387 0.66 

### O.SS 59 0.5 0.53 380 13 0.58 0.82 107 0.34 0.32 650 0.74 0.88 98 0.56 0.7 224 14 0.82 0.90 49 0.45 0.78 330 0.57 0.80 161 0.4 0.48 S6S 15 0.63 0.51 169 0.45 0.34 884 0.76 0.74 193 0.17 0.24 1804 16 0.60 0.68 60 0.21 0.36 452 0.67 0.85 139 0.17 0.23 1513 17 0.65 0.93 184 0.63 0.45 775 0.43 0.74 177 0.04 0.16 1520 18 0.61 0.55 202 0.50 0.47 800 0.64 0.73 177 0.09 0.16 1974 19 0.82 0.79 121 0.65 0.48 771 0.68 0.66 134 0.25 0.38 1057 20 0.65 0.65 94 0.70 0.48 S6S 0.63 0.59 129 0.1 0.17 1819 21 0.56 0.83 77 057 0.65 4S4 0.75 0.62 'S4 0.48 0.41 758 lI1iniInum 0.50 0.51 40 0.21 0.32 330 0.43 0.58 4S 0.04 0.16 224 maximum 1.43 1.'73 236 1.35 1.02 94S 1.59 1.82 23S 0.76 0.99 1974 CEO: Cytotoxicity: cloning effac:iency at the end ofthe tre:atmeDt period CE2: Viability: cloniDg effic:ieDcy atthe time ofplatiDg ill selective CODditioas MF: ltIutmt frequeacy: Dumberofmutmt cells dividc.d by the Dumber

## orviable cells Vebic:Ie: DMSO. c:uIIun: medium

### ordistilled

# -= . MMS: merhylmetbaoe sulfOQate CPA: c:yelopbospbamidc

# · . CIT/Study No. 13293 MLY/n-PROPYL BROMIDE/ElfAtochem



### 3. Protocol and amendment 27

# CIT
centre international de tOxicologie MIS::lE':' ':!P 563 27005 EVREUx C:D:X FRANCE Tel. 32 29 2625 TclcCQPIE 32 67 87 OS 

### Miserey, 5thJuly 1995 

### "-PROPYLBROMIDE IN VITRO 

### MAMMALIAN CELL GENE MUTAnON

## TEST TKIL5178Y MOUSE LYMPHOMA 

### 28

Protocol from

Sponsor Address

Study Monitor Study Director Study Number Centre International de Toxicologie Miserey BP 563

- 27005 Evreux Cedex France ElfAtochem S.A. La 

### Defense 10, cedex 42 92091 Paris-la-Defense France
J.F. Regnier
B. Molinier
13293MLY

### CITIStudy No. 13293 MLYin-PROPYL BROMIDElElfAtochem
INTRODUCTION 2 29

The objective of this study is to evaluate the potential of the test substance to induce a gene mutation at the TK (thymidine kinase) locus in L5178Y Mouse lymphoma cells. The purpose of this ill vitro 

### mammalian cell gene mutation test is to identify substances that cause base-pair mutations. frameshift mutations, small deletions. large deletions and rearrangements ofthe relevant chromosomes. Mutagenic substances can induce a forward mutation from the parental type (TK+1 -) 

### to the mutant form (TK-1 -) 

### in the cells which are then able to grow and form colonies on a selective medium. while non-mutated cells cannol. In addition. two types of colonies are produced: small. slow growing colonies mainly produced by chromosome rearrangements and large colonies mainly produced by point mutations. -. This protocol complies with:

O.E.C.D. guideline No. 476. 4th April 1984 and revised Draft document of

### Dece~r . 1994. E.E.C. Directive No. 871302lE.E.C.. 18th November 1987. U.S.AJE.P.A.ff.S.C.A. Federal Register. Vol. 

### SO, No. 188. Subpart F. 27th September 1985. The study will be 

### conducted in compliance with the following Good 

### Laboratory Practice regulations:

· Deeret N° 90-206 du 7 mars 1990 concernant les Bonnes Pratiques de Laboratoire

(Ministere de I'Industrie etde I'

### Amenagementdu Territoire).

· US Environmental Protection Agency. Federal Register. 40 CFR 

### Part 792; Toxic

Substances Control Act: Good 

### Laboratory Practice Standards. November 29. 1983 (and subsequent amendments).

· O.E.C.D. Principles of Good 

### Laboratory Practice. C(SI)30(final) Annex 2. 

### May 12.

1981.CITIStudy No. 13293 MLYIn-PROPYL BROMIDElElfAtochem
MATERIAlS AND METHODS 

### 1. TEST AND CONTROL SUBSTANCES 1.1 Identification 

### 3 30

1.1.1 Test substance

· Denomination: n-PROPYL BROMIDE

· Batch No.: 3-337-02

· Description: colourless to clear yellow liquid

· Storage conditions: in dark and at room temperature

· Physico-chemical properties: i.e. purity, composition, stability and expiry date which refer to the batch to be used and handling conditions will be indicated in the test substance dOlta sheet (to be completed by the Sponsor). An analytical certificate will also be provided by the Sponsor.

· Required amount: 5 g 

### 1.1.2 Vehicle The vehicle will be selected according to the results ofsolubility tests performed before the preliminary toxicity test (I). 

### 1.2 Preparation The test substance will be dissolved in the vehicle to provide approximately 100 to 400 fold the final dose, depending on the vehicle. The preparations wili be made immediately before use. 

### 1.3 Positive controls Two known mutagens, dissolved in distilled water, will be used to check the sensitivity of the test system:

· without 59 mix: methylmethane sulfonate (MM5), used at 25 J,lglml,

· with S9 mix: cyclophosphamide (CPA), used at 3 J,lglml. 

### 2.. TESTSYSTEM 2.1 Cells 178Y cells are an established cell line recommended by international regulations for 

### in

_. 1'0 TTkl.mmaJian cell gene mutation test. They have a high cloning efficiency (> 50%), an cell cycle time of 12-14 houlS and a TKphenotypic expression time oftwo days. 

### are checlc:edperiodically for mycoplasmaconwninations. are stored in a cryoprotective medium containing 10% horse serum and 10% ylsulfoxide (DMSO) at-SOOC.



### obe specified.. 

### dated andsigned 

### by the

### Spoasor

### CIT/Study No. 13293 MLYin-PROPYL BROMIDElElfAtochem 4 31
2.2 

### Metabolic activation system: 

### 59 mix The S9 mix consists of induced enzymatic systems contained in rat liver microsomal fractions (59) and the cofactors necessary for their function. S9 will be prepare(; at C.I.T. from the liverofrats treated with Aroclor 1254 (500 mglkg) by the intrnperitone:1 route. The S9 fraction is preserved in sterile tubes within a liquid nitrogen container. until use. The S9 mix 

### will be prepared at +4°C immediately before use and will 

### be maintained at +4°C tt:roughout the experiment. The S9 mix will contain per 

### 5 ml:

I 

### ml Glucose-6-Phosphate (180 

### mg/ml). · I ml NADP (25 mg/ml). · I 

### ml KCI (150 

### mM). · 2 

### ml S9 (therefore final**concent~tion** 

### ofS9 in the S9 mix will be 40% (v/v». 

### 3. EXPERIMENTAL DESIGN 3.1 Rationale for dose selection The top dose will be selected according to the following criteria specified in international regulations:

· pH shifts ofno more than one pH unitwhen compared to vehicle control medium.

· osmolality increase of no more than 100 mosmlkg H2

### 0 when compared to vehicle

control culture medium.

· for non-toxic freely soluble testsubstances. the top dose will be 10 

### mM or 5000 

### pg/ml.

· for poorly soluble test substances. the top dose will be a dose above the limit of

solubility in the final culture medium at 

### the end ofthe treatmentperiod.

· for toxic test substances. the top dose will be a toxic dose giving 10-20% relative

survival relative cloning efficiency determined on day 

### 0 post-treatment. However. this 

### top dose will not exceed the limit ofsolubility in the final culture medium 

### at the end of the treatment period. 3.2 

### Treatment The day before treatment. the cells will be seeded in 50 ml of RPM! 1640 medium containing 10% horse serum. L-Glutamine (2 mM). 

### penicillin (100 U/ml). streptomycin (100 pg/ml) and sodium pyruvate (200 pg/ml) in flasks. The flasks will then be placed at 37°C in a humidified abnosphere of5% 

### CO/9S% air for 24 hou[S. The culture medium will then be removed and cells will be counted. Approximately 05 

## uf cells will be exposed to the test orcontrol substances. with or without 

### 59 mix. for three hours at 37"C in the culture medium with S% horse serum (containing 2% 

### 59. for treatment with 

### 59 mix).

### CITIStudy No. 13293 MLYIn-PROPYL BROMIDEIE!fAtochem 5 32
The treatment medium will then be 

### removed and the cells will be counted and seeded as follows: 1.6 cells/well (one 96-well plate/culture 

### = two plates/dose) to detennine cytotoxicity I.:sing cloning efficiency (CEo). After 12:: 1 days ofincubation at 37°C. the clones will be counted. 2.10S 

### cells/mJ will be reincubated at 37°C into flasks for two days to enable the expression ofthe mutantphenotype. They will be 

### replated on day 1 at 2. lOs 

### cells/ml. Afterthese two days. the cells will be 

### seeded as follows:

· 1.6 cells/well (one 96-well plate/culture 

# =two plates/dose) to define the number of

viable cells**(C~).** 

### After 12 ± 

### I days ofincubation at 3rc. the clones will be 

### counted.

· 2000 cells/well (two 96-well plates/culture 

# = four plates/dose) to select the TFTR



### (trifluorothymidine resistant) mutant cells. After 12 ± 

### 1 days ofincubation at 3rC in the presence of 4 J.Ig TFT/ml of culture medium. the clones will be

### coli~.· differentiating small and large colonies:

- size ofsmall colonies < 25% diameterofone well,
• size oflarge colonies> 25% diameterofone well. 3.2.1 Preliminary toxicity test 

### To assess the toxicity of the test substance to the cells. at least six doses of the test substance (one culture/dose) will be 

### tested on the LSI78Y cells. with or without metabolic activation. 3.2.2 Mutagenicity tests 

### In two independent tests. at least four doses ofthe test substance «(\:.'0 

### cultures/dose) will be 

### tested on the LS178Y cells. with orwithout metabolic activation. During each test, the following controls will be 

### made using at leastduplicate**cul~res:**



### · vehicle control: culture treated with the vehicle.,

· positive control: culture treated with:

- MMS. without S9 mix.

- CPA. with 59 mix.CITlStudy No. 13293 MLYIn-PROPYL BROMIDElElfAtochem


### 4. EVALUATION OFTHE RESULTS 6 33

Treatment ofresults During each test, for each experimental point, the number ofclones corresponding to the cloning efficiencies CEo and**C~,** and the number of 

### TFTR mutant clones (small and large) will be scored. Individual results corresponding to cytotoxicity**(CE~,** viable cells**(C~),** mutation frequency (MF) will be presented in a table. For each dose, the cloning efficiencies will be calculated from the zero term of the Poisson distribution 

### as follows: empty wells -Ln total wells CE= numberofcells per well The cytotoxicity ofthe test substance, 

## CEo (%), will be expressed as a percentage related to that ofthe vehicle controls. The mutation frequency (MF) will be calculated 

### as follows: CE mutant x 106 MF= 

## CE:! with number ofcells per well

# = 1.6 for

## CEo and

## CE:z and 2000 for MF Acceptance criteria This study will be considered valid ifthe following criteria

### are funy met:

· the cloning efficiency of the controls should be between 

### 60 and 140% for CEo and

between 70 and 130% for CE..,

· the mutation frequency ofthe-controls should

### be between 60-200.10-6,

· the mutation frequency ofthe positive controls should be higher than the controls and

within the range ofourhistorical data. Evaluation criteria Biologic:l1 relevance of the results will be considered first. In addition, the following criteria may 

### be used as an aid for determining a positive 

### response: · a dose-related increase in mutantfrequency, and/or, · a reproducible increase: in the mutant frequency (i.e. at least a doubling when compared

to that ofthe controls) for at least one ofthe doses. 

## 5. PROCEDURES The 

### procedures used 

### during the stUdy will be 

### those documented in 

### the relevant 

### C:I.T. procedures manual.CITIStudy No. 13293 MLYIn-PROPYL BROMlDElElfAtochem


### 6. AMENDMENTS 

### TO THE PROTOCOL 7 34

Ifnecessary, amendments to the protocol will be made after agreement between the Study Director and the Study Monitor. 

### 7. REPORTING The Study Directorwill conr<lct the Study Monitor when necessary. The final report, in English, will contain**al~ata** collected throughout the study. Number of copies ofthe final report:

# s< + 1 unbound) (1) Proposed issue ofthe draft report: one mon after the end ofthe study. 

### S. QUALITY ASSURANCE UNIT The Quality Assurance Unit will conduct inspections according to Good Laboratory Practice Regulations (as specified on page 2). The dates on which the findings of these inspections are reported to the Study

### Di;~or and 

### C.LT. Management will be specified in the final report. 

### 9. ARCHIVES The study archives:

· protocol and possible amendments

· raw data

· correspondence

· final report and possible amendments

· sample ofthe test substance will be stored on the premises ofC.lT.• 27005 Miserey. Evreux. 

### France.. for five years after the end ofthe experimental study. At the end ofthis period, the study archives will be returned to the Sponsor. 

### 10. TI1\-IE SCHEDULE Beginning ofthe study: 2nd halfofAugust 1995. End ofthe study: 2nd halfofOctober 1995.

(I) Exceptfor special 

### requests made by the SponsorCIT/Study 

### No. 13293 MLYIn-PROPYL BROMIDElElfAtochern

Protocol approved by: 

### 8 35

J.F. Le Bigot orS. de Jouffrey C.I.T. Scientific Management Date: 5.7.95

B. Molinier C.I.T. Study Director Date: 5.7.95 J.F. 

### egmer Study Monitor /' Date: 

# i (,1 ( 

# CJ1 

# 1':)

# C I ·'4:ITlStudy No. 13293 

### MI..YIn-PROPYL BROMIDEIElfAtochem 

## AMENDMENTTO PROTOCOL STUDYNo.: 

### 13293 MLY SPONSOR: ElfAtochem lEST SUBSTANCE: Q-PROPYL BROMIDE 

### AMENDrvfENTNo.: 01



### Date ofapplication: 13.9.95

MATERIALS AND METHODS 2. TEST SYSTEM 2.2 

### Metabolic activation system: S9 mix 36



### Page III

S9 fraction 

### will be purchased from Moltox (MolecularToxicology, INC, Annapolis, MD 21401, 

### U.S.A).



### Scientificmanagement J.F 

### LeBigotorS. deJouffrey StudyDirector B. MoliDier 

### Date: 29.1.96

. Signature: - . 

### '-;'
..> () I)

# \~ ...) 

# -- :./ StudyMonitor J.FRegnier 

### Date: 

# G

# (f?1. 

# /56'

# ~-tfj~

# ~S~