1-bromo-3,4,5-trifluorobenzene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-bromo-3,4,5-trifluorobenzene

EC Number:

418-480-9

EC Name:

1-bromo-3,4,5-trifluorobenzene

Cas Number:

138526-69-9

Molecular formula:

Hill formula: C6H2BrF3 CAS formula: C6H2BrF3

IUPAC Name:

5-bromo-1,2,3-trifluorobenzene

Test material form:

liquid

Method

Target gene:

HIS operon (S. thyphimurium)

Species / strain

Species / strain 1

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 2

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 3

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 4

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 5

Species / strain / cell type:

S. typhimurium TA 1538

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : S9 was prepared in-house on 31/5/95. It was prepared from the livers of male Sprague-Dawley rats weighing - 200g. These had each received a single i.p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 preparation.

- concentration or volume of S9 mix and S9 in the final culture medium: 10% liver S9 in standard co-factors

Test concentrations with justification for top dose:

Preliminary Toxicity Study
For the selection of appropriate dose levels for use in the main study, a preliminary test with TA 100 was carried out to determine the toxicity of the test material to the tester organisms. The dose levels were 0, 50, 150, 500, 1500 or 5000 µg/plate.

Main study
Experiment 1: Six concentrations (5, 15, 50, 150, 500 or 1500 µg/plate) of the test material were assayed in triplicate against each tester strain.
Experiment 2: The second experiment was performed using methodology as described for experiment 1, using fresh bacterial cultures, test material and control solutions in triplicate.
Metabolic activation: 10% liver S9 in standard co-factors

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix, TA100, TA1535

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix, TA98

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

without S9 mix, TA1535

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix, TA1537

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix, all strains

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: The plates were incubated at 37°C for approximately 48 hours and the number of revertant colonies counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: revertant colonies and examined for a thinning of the background lawn

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results or equivocal results are obtained, then a third experiment may be used to confirm the correct response.
To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.

Statistics:

no

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

visible reduction in the growth of the bacterial lawn at 500 µg/plate (without S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

visible reduction in the growth of the bacterial lawn at and above 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

visible reduction in the growth of the bacterial lawn at and above 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

visible reduction in the growth of the bacterial lawn at and above 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

visible reduction in the growth of the bacterial lawn at and above 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: no

RANGE-FINDING/SCREENING STUDIES
The dose range of the test material used in the preliminary toxicity study was 0, 50, 150, 500, 1500 and 5000 µg/plate. The test material exhibited toxicityat and above 1500 µg/plate in the strain of Salmonella used (TA100).

Ames test:
- Signs of toxicity
The test material caused a visible reduction in the growth of the bacterial lawn at and above 1500 µg/plate in all of the tester strains and 500 µg/plate in Salmonella strain TA 98 (without S9-mix only)
- Individual plate counts: see attachment
- Mean number of revertant colonies per plate and standard deviation: see attachment

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

The test item was tested in an assay performed according to OECD TG 471 with GLP compliance. Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA98 and TA 100 were treated with the test material using the Ames plate incorporation method at six dose levels, in triplicate, both with and without the addition of S9 mix. The dose range was determined in a preliminary toxicity assay and was 5 - 1500 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh chemical formulations. An extra dose level was incorporated into each experiment to allow for the toxicity of the test material to all of the tester strains.

The vehicle (dimethyl sulphoxide) control plates produced counts of revertant colonies within the normal range. All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the metabolising system. The test material caused a visible reduction in the growth of the bacterial lawn at >= 1500 µg/plate in all of the tester strains and at 500 µg/plate in TA 98 (without S9 -mix only). No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. The test material was found to be non-mutagenic under the conditions of this test.