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 of Pollution Prevention and Toxies

Environmental Protection Agency

401 lII. St., S.W.

Washington, D.C. 20460

Subject: TSCA Section See) Submission

Dear SirlMadam:

1IIIII1IIIIII

8EHC-97-13985

Contains No CBi

ElfAtochem North America, Inc. (ElfAtochem) has received the final report ofa gene mutation test and is

submitting this report to the Environmental Protection Agency (EPA) pursuant to Toxic Substances Control Act

(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 letter or the enclosed study report is considered confidential business information ofElfAtochem.

The in vitro potential oftest substance n-propyl bromide to induce mutations at the TK locus in LSI78Y mouse

lymphoma cells was evaluated in this study. The test substance induced a reproducible and signficant increase in

the mutation frequency and the number ofsmall colonies at dose levels between 1000 and 1500 J..'g/ml without

activation and at levels between 1500 and 2000 J1.g/ml with activation, giving a positive response under the

conditions ofthis test.

This new information adds to the existing genotoxicity data base for this material which includes: positive and

negative responses in the Ames Salmonella assay, negative results in the mouse micronucleus assay and the rat

dominant lethal assay, and weak positive results in a study for chromosomal aberrations. Results from this study

have been incorporated into the ElfAtochem Material Safety Data Sheet for the test material.

Based on EPA guidance we believe that these data meet EPA criteria for reponing under TSCA Section 8(e).

f,!1rther questions regarding this submission may be directed to me at (215) 419-5890.

Sincerely.

Debra

~~

Randall, DABT

Product Safety Manager

11111111111111

889?0000235
, CIlf/Study No. 13293 MLY/n-PROPYL BROMIDEIElf Atochem

SPONSOR

Elf Atochem S.A.

Direction Securite Environnemem - Produit

La Defense 10 - Cedex. 42

92091 Paris-la-Defense

France

STUDY TITLE

IN VITRO MAMMALIAN CELL GENE

~ATlONTEST

IN L5178Y TK+ - MOUSE LYMPHOMA CELLS

TEST SUBSTANCE

n-PROPYL BROMIDE

STUDY DIRECTOR

Brigitte Molinier

STUDY COMPLETION DATE

12th February 1996

PERFORMING LABORATORY

Centre International de Toxicologie (C.LT.)

Miserey - 27005 Evreux - France

LABORATORY STUDY NUMBER

13293MLY

CONFIDENTIAL
CITIStudy No. 13293 MLYin-PROPYL BROMIDElElf Atochem I •

CONTENTS

STATEMENT OF THE 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 OF THE RESULTS

2.5. ARCHIVES

2.6. CHRONOLOGY OF THE STUDY

3. RESULTS (tables 1 to 5)

3.1. PRELIMll'rARY TOXICITY TEST (table 1)

3.2. MDTAGENICITY EXPERIMENTS (tables 2 to 5)

4. CONCLUSION

5. REFERENCES

Table 1: Preliminary toxicity test

Table 2: FIrst experiment without 59 mix

Table 3: FIrSt experiment with S9 mix

Table 4: Second experiment without S9 mix

Table 5: Second experiment with 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

40
I CITlStudy No. 13293 MLYin-PROPYL BRO:MlDElElf Atochem

APPENDICES

1. Analytical certificate of the 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 BROMIDEIElf Atochem

STATEMENT OF THE STUDY DIRECTOR

4

The study was performed in compliance with the principles of Good Laboratory Practice

Regulations:

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

(Ministere de I'Industrie et de l'Amenagement du 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(81)30(final) Annex 2. May 12, 1981.

I declare that this report constitutes a troe and faithful record of the procedures undertaken

and the results obtained during the performance of the 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

Doctor ofToxicology

Head of Genetic Toxicology
,

CIT/Study No. 13293 MLY/n-PROPYL BROMIDElElf Atochem

STATEl\1ENT QF QUALITY ASSURANCE UNIT

Dates (day/month/year)

Type of inspections ------------------------------------------------------

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

Doctor ofBiochemistry

Head of Quality Assurance Unit

and Scientific Archives
.

·CH/Study No. 13293 MLY/n-PROPYL BROMIDElElf Atochem

SUMMARY

6

The objective of this study was to evaluate the potential of the 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

liver microsomal fraction (S9 fraction) of rats 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 of S9 fraction 2%), for three hours at 37c C.

Cytotoxicity was then determined using cloning efficiency (CE~ before expression of the

murant phenotype. Cells viability (using cloning efficiency CEo,) and number of mutain

clones (differentiating small and large colonies) were checked after the expression of the

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 of methylmethane sulfonate

· with 59 mix: 3 Ilglml of cyclophosphamide.

Results

The cloning efficiencies CEo and C~ and mutation frequencies of the vehicle and positive

contrOls were within the range of our 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 /) 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 dead three hours after treatment).

The test substance did not induce any significant increase 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 BROMIDElElf Atochem

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-PROPYL BROMIDEIElf Atochem

1. L."ITRODUCTION

This study was pertormed at the request of Elf Atochem 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 lymphoma cells.

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.. 18th November 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 supplied by the Sponsor identified the test substance as follows:

· denomination:

- protocol and labelling: n-PROPYL BROMIDE

· batch number:

- protocol: 3-337-02

- labelling: 3-337-02 CAL 4318194

· description: colourless liquid

· container: one glass flask

· date ofreceipt: 4.10.94

· storage conditions: at room temperature and kept from light

· purity: 99.3%.

Data relating to the characterization of the test substance are documented in an analytical

certificare and a test article description (presented in section 6) provided by the 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 BROMIDEIElf Atochem 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 of 3 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."

Regular checks demonstrated the absence of mycoplasma contamination.

L5178Y cells, originally obtained through ATCC, were kindly supplied by 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 of rats treated with Aroelor 1254 (500 mg/kg)

by intraperitoneal route.

The 59 fraction was preserved in sterile tubes within a liquid nitrogen container, 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 contained per 5 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 BROMJDElElf Atochcm

%.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 of 59 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 of incubation at

37"C. the clones were counted.

To enable the uj)l'l:SSion of the 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 of cu1aft medium. me cloacs were counted. differentiating small and

Ja."'J,'C coIoaic:s:

• size ofsmall coIoaies. < 2S~ ofme diameter ofthe well.

e Si2c ofIarJC c:oIoDies.. > 2S4lt ofme diameter of the 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-~ of D-PROPYL BROMIDE (two

cukuraIdoIe-leYe1) were IaIeCS bodl wilh aDd wimow """RboIic activation.

In eacb ap':I"iIDeDl. me foDowiD& CODtrOIs were iDclDded usiDg at k'ast duplicate cultures:

. vebide CODa"Ols: cuIIurcs aeaIed with die vebide.

. poua've ccaaoIs: cuIDIIa aaaed witb:

•MMS. iA me of59 mix.,

•CIA.. III tbe pi eace ofS9 mix..
'PT/Study No. 13293 MLYIn-PROPYL BROMIDElElf Atochem 11

2.4. EVALUATION OF THE RESULTS

Treatment of results

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 of the Poisson distribution:

[

empty wells ] -Ln

total wells

number of ce!lslwell (- 1.6)

CEo treated

CEo vehicle control

x 100

number of cells/well (- 1.6) C~=

Data from viability plates (empty wells) are used to calculate CE., (cloning efficienc/M-the'

end of the expression period) and RCE., (viability relative to vehicle controls at the end of

the expression period). These values indicate the viability of the cell populations at the end

of the expression period. CE:z is calculated from the zero term of the PoissC'n distribution:

[

empty wells ] -Ln

total wells

RC~=

C~ treated

CE:2 vehicle control

x 100

number of cells/well (- 2000)

CElIIIIQDt=

Data from the mutant plates (empty wells) are used to calculate CE_t (cloning efficiency

in selective medium). This value is an indicator of the absolute mutant frequency. CEmutaDl

is calculated from the zero term of the Poisson distribution (only mutant clones are able to

grow in TFr containing medium):

[

empty wells ] -Ln - total wells

MF=

The relative mutant frequency (MF) is calculated as:

CEIIIlIlmR x 106
CITIStudy No. 13293 MLYIn-PROPYL BROMIDElElf Atochem 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 criteria were fulfilled:

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

0.7-1.3 for CE."

· the mutation frequency of the 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 of our 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 of biological relevance

might be also taken into account in the evaluation of the data obtained.

2.5. ARCHIVES

The study documentation and materials. namely:

· protocol and possible amendments.

· raw data,

· correspondence,

· final report and possible amendments.

· a sample of the test substance,

are stored in the archives of C.I.T. (Miserey. 27005 Evreux, France), for five years after

the end of the experimental study. At the end of this period, the study documentation will

be returned to the Sponsor.
CITIStudy No. 13293 MLYin-PROPYL BROMIDElElf Atochem

2.6. CHRONOLOGY OF THE STUDY

The chronology of the 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 BROMIDElElf Atochem

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 of treatment both with and without S9 mix. At the lower dose-levels,

a moderate reduction of the cloning efficiency CEo of treated 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 of the vehicle and.p'ositive controls were as specified in acceptance criteria and within the range of our 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 selected treatment-levels were:

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

· second experiment: 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 second experiments, respectively.

At 1250 J.lglml (second experiment), RCEo was 46%.

At lower dose-levels. no significant differences between treated. and control were noted.

The number of viable cells two days after treatment (assessed by the cloning efficiencies

~) was considered equivalent to mat ofthe control cultures.

The test substance induced a reproducible and significant increase in the mutation

frequency togetha with an increase in the number ofsmall colonies at dose-Ie..~ls between

1000 and 1500 JIg/mi.

With S9 mix. the selected treatment-levels were:

· first experiment: 125,250,500. 1000, 1500 and 2000 J.18Iml,

· second experiment: 500.1000.1500.2000 and 2500 p.gImJ.
CIT/Study No. 13293 MLY/n-PROPYL BROMIDElElf Atochem 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. RCEowas 9%.

At 1500 IJg/ml. RCEowas 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 of the 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 number of small 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 lymphoma cells. 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 BROMIDElElf Atochem

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 of exposure to the test substance

o: vehicle control (DMSO)

CEo: cloning efficiency

RCEo : relative cloning efficiency
<:;IT/Study No. 13293 MLY/n-PROPYL BROMIDEIElf Atochem 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~".J_ 0.78 85 4 75 96 158 ,,~I.3

....,..- 17 96 33 96 5 78 96

10 75 96

~.,

.J_ 96 30 96 8 72 96

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 Mutation Frequency oftR:alCd c:ellsIMuwion Frequency ofCOIltrol c:e11s
'CIT/Study No. 13293 MLY/n-PROPYL BROMIDEIElf Atochem 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: cloniDg cfficieocy

RCE(o aDd 2): relative cloning cfficicocy

SC: small c:oloaics

R: Rabo bctweeD Muwion FR:quc:ocy ofIn:ated"CDsIMlDIioa Frequeoc:y ofcoatrol c:eUs
CI:r/Study No. 13293 MLYin-PROPYL BROMIDEIElf Atochem

Table 4: Second ex.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: clolling ef6cieDCy

RCE(o aod 2): relative cloning ef6c:ieDCy

SC: small colODies

R.: Ratio between MUWiou FreqacDcy ofaearcd cdIsIMuwioD Ftequeucy ofCODuol ccUs
· CITIStudy No. 13293 MLYIn-PROPYL BROMIDElElf Atochem

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

-., .J_ 96 19 96 5 71 96

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 and CEl: cloning efficiency

RCECo and 2): relative cloning efficiency

SC: small colonies

R: Ratio between Mutation ~uCDC)' of treated cel1sIMutaIion Frequency of control cells

-: due to the toxicity of the teSt substance. there were not enough cells to allow seeding ofthis duplicate
CITIStudy No. 13293 MLYIn-PROPYL BROl'vlIDEIElf Atochem

APPEl'o'DICES

21
CITIStudy No. 13293 MLYIn-PROPYL BROMIDElElf Atochem

1_ Analytical certificate of the test substance and test anicle description

22
CIT/Sludy ~o. 13293 MLYin-PROPYL BROMIDEIElf Alochem

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: 2II D 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.. BROMIOElElf AJochem 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,'RTICLE DESCRiPTION

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;

~.~%

Elf Alochem. 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 BROMIDElElf Atochem

2. Historical data

25
,CIT/Study No. 13293 MLY/n-PROPYL BROMIDElElf Atochem

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 of the tre:atmeDt period

CE2: Viability: cloniDg effic:ieDcy at the time of platiDg ill selective CODditioas

MF: ltIutmt frequeacy: Dumber of mutmt cells dividc.d by the Dumber or viable cells

Vebic:Ie: DMSO. c:uIIun: medium or distilled -= . MMS: merhylmetbaoe sulfOQate

CPA: c:yelopbospbamidc
· . CIT/Study No. 13293 MLY/n-PROPYL BROMIDE/Elf Atochem

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, 5th July 1995

"-PROPYL BROMIDE

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

Elf Atochem S.A.

La Defense 10, cedex 42

92091 Paris-la-Defense

France

J.F. Regnier

B. Molinier

13293MLY
CITIStudy No. 13293 MLYin-PROPYL BROMIDElElf Atochem

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 of the 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 et de I'Amenagement du 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 BROMIDElElf Atochem

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 of solubility 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.. TEST SYSTEM

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 TK phenotypic expression time of two days.

are checlc:ed periodically for mycoplasma conwninations.

are stored in a cryoprotective medium containing 10% horse serum and 10%

ylsulfoxide (DMSO) at -SOOC.

o be specified.. dated and signed by the Spoasor
CIT/Study No. 13293 MLYin-PROPYL BROMIDElElf Atochem 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 liver of rats 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 of S9 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 of no more than one pH unit when compared to vehicle control medium.

· osmolality increase of no more than 100 mosmlkg H20 when compared to vehicle

control culture medium.

· for non-toxic freely soluble test substances. 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 treatment period.

· 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 of 5% 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 or control 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!f Atochem 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 of incubation 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 of the mutant phenotype. They will be replated on day 1 at 2. lOs cells/ml.

After these 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 of incubation 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 of incubation 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% diameter of one well,

• size of large colonies> 25% diameter of one 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 of the test substance «(\:.'0 cultures/dose) will

be tested on the LS178Y cells. with or without metabolic activation.

During each test, the following controls will be made using at least duplicate 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 BROMIDElElf Atochem

4. EVALUATION OF THE RESULTS

6 33

Treatment of results

During each test, for each experimental point, the number of clones 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=

number of cells per well

The cytotoxicity of the test substance, CEo (%), will be expressed as a percentage related

to that of the vehicle controls.

The mutation frequency (MF) will be calculated as follows:

CE mutant x 106

MF=

CE:!

with number of cells per well =1.6 for CEo and CE:z and 2000 for MF

Acceptance criteria

This study will be considered valid if the 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 of the positive controls should be higher than the controls and

within the range of our historical 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 mutant frequency,

and/or,

· a reproducible increase: in the mutant frequency (i.e. at least a doubling when compared

to that of the controls) for at least one of the 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 BROMlDElElf Atochem

6. AMENDMENTS TO THE PROTOCOL

7 34

If necessary, amendments to the protocol will be made after agreement between the Study

Director and the Study Monitor.

7. REPORTING

The Study Director will conr<lct the Study Monitor when necessary.

The final report, in English, will contain al~ata collected throughout the study.

Number of copies of the final report:s< + 1 unbound) (1)

Proposed issue of the draft report: one mon after the end of the 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 of the test substance

will be stored on the premises of C.lT.• 27005 Miserey. Evreux. France.. for five years

after the end of the experimental study. At the end of this period, the study archives will

be returned to the Sponsor.

10. TI1\-IE SCHEDULE

Beginning of the study: 2nd half of August 1995.

End of the study: 2nd half of October 1995.

(I) Except for special requests made by the Sponsor
CIT/Study No. 13293 MLYIn-PROPYL BROMIDElElf Atochern

Protocol approved by:

8 35

J.F. Le Bigot or S. 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 BROMIDEIElf Atochem

AMENDMENT TO PROTOCOL

STUDY No.: 13293 MLY

SPONSOR: ElfAtochem

lEST SUBSTANCE: Q-PROPYL BROMIDE

AMENDrvfENT No.: 01

Date of application: 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 (Molecular Toxicology, INC, Annapolis, MD 21401,

U.S.A).

Scientific management

J.F Le Bigot or S. de Jouffrey

Study Director

B. MoliDier

Date: 29.1.96

. Signature: - .

'-;'..> () I)\~ ...)

-- :./

Study Monitor

J.FRegnier

Date: G(f?1. /56' ~-tfj~

~S~