1-bromo-3,4,5-trifluorobenzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: In a bacterial reverse mutation test (plate incorporation test) according to OECD TG 471 the test item was not mutagenic in the absence and presence of a rat liver metabolizing system (S9 mix) (reference 7.6.1-1).

Gene mutation in mammalian cells: In a study performed according to OECD TG 476, the test item was assessed for its ability to induce mutation at the tk locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The test item was not mutagenic in this test system both in the absence or presence of S9 mix (reference 7.6.1 -2).

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

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

Target gene:

HIS operon (S. thyphimurium)

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 / 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 / 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 / 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 / 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

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

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

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

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

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

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998 - 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

07-1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y TK +/- mouse lymphoma cells obtained from the American Type Culture Collection

For cell lines:
- Absence of Mycoplasma contamination: yes
Each batch of frozen cells was purged of TK- mutants and checked for spontaneous mutant frequency.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: cells diluted in RPMI 10 medium and incubated in a humidified atmosphere of 5% v/v CO2 in air at 37°C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats, induced with Aroclor 1254. The S-9 was obtained from Molecular Toxicology Incorporated, USA.
- concentration or volume of S9 mix and S9 in the final culture medium :
- quality controls of S9: Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Test concentrations with justification for top dose:

Experiment 1: 0, 18.75, 37.5, 75, 150, 175, 200, 225, 250 and 300 µg/mL (+/- S9 mix)
Experiment 2: 0, 12.5, 25, 50, 100, 150, 200, 250 and 300 µg/mL (-S9 mix); 0, 37.5, 75, 150, 162.5, 175, 187.5, 200, 212.5 and 225 µg/mL (+S9 mix)
Experiment 3: 0, 75, 150, 162.5, 175, 187.5, 200 and 212.5 µg/mL (+S9 mix)

Vehicle / solvent:

DMSO
Preliminary solubility data indicated that the test item was soluble in sterile anhydrous analytical grade DMSO in the region of 1215 to 5000 µg/mL and the solubility limit in tissue culture medium was in the region of 1250 μg!mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix

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

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 3

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours or 24 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Cultures were maintained in flasks for a period of 2 days during which the TK- mutation would be expressed.
- Selection time: TFT (trifluorothymidine, 300 µg/mL) was diluted 100-fold into cell suspensions to give a final concentration of 3 µg/mL. Plates were incubated at 37°C in a humidified incubator gassed with 5% v/v CO2 in air until scorable (12 days) and wells containing clones were identified as above and counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS)

Evaluation criteria:

The assay was considered valid if all the following criteria were met:
-the mutant frequencies in the negative (solvent) control cultures fell within the normal range (above 60 mutants per 10E+6 viable cells but not more than three times the historical mean value)
-at least one concentration of each of the positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value)
-the plating efficiencies of the negative controls from the mutation experiments were between the range of 60 – 140 % on day 0 and 70 – 130 % on day 2.

The test article was considered to be mutagenic if all the following criteria were met:
-the assay was valid (see above)
-the mutant frequency at one or more doses was significantly greater than that of the negative control (p < 0.05)
-there was a significant dose-relationship as indicated by the linear trend analysis (p < 0. 05)

Statistics:

All calculations such as determination of survival or viability, determination of mutant frequency or assessment of statistical significance of mutant frequency were performed by computer using validated software. The control log mutant frequency
(LMF) was compared with the LMF from each treatment concentration based on Dunnett's test for multiple comparisons, and secondly the data was checked for a linear trend in mutant frequency with treatment concentration using weighted
regression. The test for linear trend is one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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: Upon addition of the test article to the cultures, precipitate was observed at the top two doses tested in the absence and presence of S-9 (250 and 300 µg/mL) . However, no precipitate was observed following completion of the 3 hour treatment incubation period. In the second experiment and the absencen of S-9 no precipitate was observed upon addition of the test compound to the cultures or following completion of the 24 hour treatment incubation period. In the presence of S-9, precipitate was observed, at the top two doses tested (212.5 and 225 µg/mL) only upon addition of the test article to the cultures.

RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity range-finding experiment, 3 hour treatment, ten doses of test item were tested separated by two fold intervals and ranging from 9.766 to 5000 µg/mL. Upon addition of the test article to the cultures, precipitate was observed at the top three doses (1250 to 5000 µg/mL) in the absence and presence of S-9. However following the 3 hour treatment incubation period, the precipitate was observed only at the top dose (5000 µg/mL) in the absence and presence of S-9 and was removed during post-treatment cell washing. Extreme toxicity (< 10% relative survival) was observed at the top five dose levels tested in the absence and presence of S-9 (312.5 to 5000 µg/mL). The top dose which showed greater than 10% relative survival in the absence and presence of S-9 was 156.25 µg/mL, which yielded 58.20% and 34.85% relative survival, respectively.
In the cytotoxicity range-finding experiment, 24 hour treatment, ten doses of test item were tested in the absence of S-9, separated by two fold intervals and ranging from 0.625 to 320 µg/mL. No precipitate was observed upon addition of substance to the cultures or following completion of the treatment incubation period. Extreme toxicity (< 10% relative survival) was observed at the top dose tested. The top dose that showed greater than 10% relative survival was 160 µg/mL, which yielded 66.42% relative survival.

STUDY RESULTS
- Results from cytotoxicity measurements: Two days after treatments, the top three doses tested in the absence and presence of S-9, were considered to be too toxic for selection to determine viability and 5-TFT resistance (toxicity was indicated by a depression in Day 2 cell counts).

- Genotoxicity results: tables see attachment
In the presence of S-9 statistically significant increases in mutant frequency were observed at the top four doses tested in Experiment 1 and a linear trend was obtained. It should be noted that the statistically significant increases in mutant frequency observed in Experiment 1 were small for doses 75 to 175 µg/mL (< 2.4-fold). A larger increase in mutant frequency was observed at 200 µg/mL but this was a highly toxic dose (12.91 % relative survival, 0.07 relative total growth). When tested up to toxic doses in Experiments 2 and 3, no statistically significant increases in mutant frequency were observed at any dose tested and a linear trend was not obtained. Hence the statistically significant increases in mutant frequency observed in Experiment 1 are considered likely to be due to a combination of chance and high toxicity. Reproducible, statistically significant and dose-related increases in mutant frequency were not obtained in this study, in three independent experiments, therefore the substance did not fulfil the criteria for a test article to be considered mutagenic in this test system.
In addition, for the negative and positive controls and doses of test article where a statistically significant increase in mutant frequency was observed, the number of wells containing small colonies and the number containing large colonies were scored. Thus the small and large colony mutant frequencies could be estimated and the proportion of small mutant colonies could be calculated. For the negative controls, the proportion of small colony mutants in the absence and presence of S-9 ranged from 52% to 63 % in Experiments 1, 2 and 3. Marked increases in the number of both small and large colony mutants were observed following treatment with the positive control chemicals NQO and BP. At the doses of test item where a statistically significant increase in mutant frequency was obtained, increases in both small and large colony mutant frequencies were observed. The 25 µg/mL dose tested in Experiment 2, in the absence of S-9 was excluded from analysis due to marked heterogeneity between replicates in the Day 2 viability data. The exclusion of this dose does not prejudice the validity of the study in any way.

In the absence of S-9, no statistically significant increases in mutant frequency were observed following treatment with test item at any dose level tested, in Experiment 1 or 2.

Conclusions:

In this GLP study performed according to OECD TG 476, the test item was assessed for its ability to induce mutation at the tk locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. Based on the obtained results and under the conditions employed in this study, the test item was not mutagenic in this test system both in the absence or presence of S9 mix.

Executive summary:

In this GLP study performed according to OECD TG 476, the test item was assessed for its ability to induce mutation at the tk locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finding experiment, followed by two independent experiments conducted in the absence of metabolic activation and three independent experiments conducted in the presence of metabolic activation. A three hour treatment incubation period was used for all experiments performed in the presence of S9 mix. In the absence of S9 mix, the range-finder used 3 and 24 hour treatment incubation periods, experiment 1 used a 3 hour treatment incubation and experiment 2 used a 24 hour treatment incubation.

In the absence of S9 mix, no statistically significant increases in mutant frequency were observed following treatment with the test item at any dose level tested, in experiment 1 or 2. In the presence of S9 mix, in experiment 1 small but statistically significant increases in mutant frequency were observed following treatment with doses from 75 – 175 µg/mL. A larger increase in mutant frequency was observed at 200 μg/mL but this was a highly toxic dose (12.91 % relative survival, 0.07 relative total growth). However, when tested up to toxic doses in experiments 2 and 3, no statistically significant increases in mutant frequency were observed at any dose tested and a linear trend was not obtained. Hence reproducible, statistically significant and dose-related increases in mutant frequency were not obtained in this study and the test item did not fulfil the criteria for a test article to be considered mutagenic in this test system.

Based on the obtained results and under the conditions employed in this study, the test item was not mutagenic in this test system both in the absence or presence of S9 mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test

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 (reference 7.6.1 -1).

Gene mutation in mammalian cells

The test item was tested according to OECD TG 476 for its ability to induce mutation at the tk locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finding experiment, followed by two independent experiments conducted in the absence of metabolic activation and three independent experiments conducted in the presence of metabolic activation. A three hour treatment incubation period was used for all experiments performed in the presence of S9 mix. In the absence of S9 mix, the range-finder used 3 and 24 hour treatment incubation periods, experiment 1 used a 3 hour treatment incubation and experiment 2 used a 24 hour treatment incubation.

In the absence of S9 mix, no statistically significant increases in mutant frequency were observed following treatment with the test item at any dose level tested, in experiment 1 or 2. In the presence of S9 mix, in experiment 1 small but statistically significant increases in mutant frequency were observed following treatment with doses from 75 – 175 µg/mL. A larger increase in mutant frequency was observed at 200 μg/mL but this was a highly toxic dose (12.91 % relative survival, 0.07 relative total growth). However, when tested up to toxic doses in experiments 2 and 3, no statistically significant increases in mutant frequency were observed at any dose tested and a linear trend was not obtained. Hence reproducible, statistically significant and dose-related increases in mutant frequency were not obtained in this study and the test item did not fulfil the criteria for a test article to be considered mutagenic in this test system.

Based on the obtained results and under the conditions employed in this study, the test item was not mutagenic in this test system both in the absence or presence of S9 mix (reference 7.6.1 -2).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.