(trans,trans)-5-{(4-Propyl[1,1-bicyclohexyl]-4-yl)-difluormethoxy}-1,2,3-trifluorbenzene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-12-07 to 2001-03-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed in compliance with the Good Laboratory Practice (GLP) regulations (revised in 1997, ENV/MC/CHEM(98)17).The method followed that described in the OECD Guidelines for Testing of Chemicals (Adopted: 4 April 1984) No 471 "Bacterial Reverse Mutation Test".

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

none

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(trans,trans)-5-{(4-Propyl[1,1-bicyclohexyl]-4-yl)-difluormethoxy}-1,2,3-trifluorbenzene

EC Number:

606-647-0

Cas Number:

208338-50-5

Molecular formula:

C₂₂H₂₉F₅O

IUPAC Name:

(trans,trans)-5-{(4-Propyl[1,1-bicyclohexyl]-4-yl)-difluormethoxy}-1,2,3-trifluorbenzene

Method

Target gene:

HIS operon (S. thyphimurium)
TRP operon (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

his G 428, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 6

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)

Test concentrations with justification for top dose:

The test material concentrations were used selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1. Series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate
2. Series: 50.0, 158, 500, 1580 and 5000 µg/plate

Vehicle / solvent:

Acetone

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Controls 2

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 3

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9

Controls 4

Positive controls:

yes

Positive control substance:

other: daunomycine

Controls 5

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9

Controls 6

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Metabolic acticvation:
1st Series: 10% S9 mix
2nd Series: 30% S9 mix

Rationale for test conditions:

According to Guideline

Evaluation criteria:

The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:
Mean Number of Colonies Maximal Mean Number of Colonies over the Actual(Solvent Control)
Solvent Control (Test Material)
<=10 <=9 >=30
<=30 <=19 >=40
<=80 <=29 >=80
<=200 <=49 >=120
<=500 <=79 >=200
Assessment No increase Clear increase

All further results, ranging between "no" and "clear", are assessed as "weak in-creases".Interpretations:A test material is defined as non-mutagenic in this assay if "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.). A test material is defined as mutagenic in this assay if: - a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.

Statistics:

No statistics has been applied

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation occured at 50 and 500 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Executive summary:

Purpose

The purpose of this in vitro reverse gene mutation test is to identify agents that cause mutations in bacteria cells thus providing information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

Study Design

The investigations for mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA pkM101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed for all strains. In the series with S9 mix, 10 % or 20 % S9 in the S9 mix were used in the 1st or 2nd series, respectively.

Results

The test material was dissolved in acetone and tested at concentrations ranging from 5.00 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations >=500 or 1580 µg/plate, depending upon the experimental condition tested. Toxicity to the bacteria was not observed.

Daunomycin, N-ethyl-N'-nitro-N-nitroso-guanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control compounds in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the substances used as positive controls led to a clear increase in revertant colonies, thus showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Conclusions

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.