1-bromo-4-(trans-4-ethylcyclohexyl)benzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

GLP compliant OECD 471 study: negative with and without metabolic activation

GLP compliant OECD 476 study: negative with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 08 - Jun 09, 2006

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

TK gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Growth media
Three media, supplementing RPMI 1640-medium with Glutamax 1 with diffe¬rent serum concentrations were used:

Exposure medium:
RPMI- 5 (RPM 1640 with 5% heat inactivated horse serum) 470 mL RPMI 1640
25 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin

Culture medium:
RPMI-10 (RPMI 1640 with 10% heat-inactivated horse serum) 445 mL RPMI 1640
50 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin

Survivor- and selection medium:
RPMI-20 (RPMI 1640 with 20% heat-inactivated horse serum) 395 mL RPMI 1640
100 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9 mix

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 50 ... 2810 µg/ml
Concentration range in the main test (with metabolic activation): 88.9 ... 1580 µg/ml
Concentration range in the main test (without metabolic activation): 50 ... 2810 µg/ml
Concentration range in the main test (without metabolic activation): 28.1 ... 2810 µg/ml

Vehicle / solvent:

acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9,10-dimethylbenzanthracene

Remarks:

DMBA with S9, NQO without S9

Details on test system and experimental conditions:

Exposure period (without metabolic activation): 3 hours and 24 hours
Exposure period (with metabolic activation): 3 hours

Expression time:
2 days

Selection time:
10-14 days

Evaluation criteria:

The effects of the test material upon the mutation frequency are defined as
• "No effect" or "no increase" in the mutation frequency if the mean frequency of the parallel incubations of a given test material concentration is less than 2.0-fold above the mean of the actual negative controls or the mean mutation falls within the historical range of the negative controls.
• "Clear effect" or "clear increase" in the mutation frequency if the test material induces at least a 3.0-fold increase above the mean of the actual negative controls and the mean mutation frequency for a given test material concentration is at least 1.5-fold above the highest value of the historical negative controls.
• All other results are defined as a "weak effect" or a "weak increase" of the mutation frequency.

Test materials are assessed as negative or non-mutagenic in this test system if
• the assay is considered valid and
• no effect (no increase in the mutation frequency) occurs in the two experimental series performed or
• a weak effect (weak increase) occurs in one series and no effect (no increase) in the other series of experiments.

Test materials are assessed as positive or mutagenic in this test system if
• the assay is considered valid and
• a clear effect (clear increase in the mutation frequency) occurs at similar concentrations of the test material in the two experimental series performed, or
• a clear effect (clear increase) occurs in one series and a weak effect (weak increase) in the other series of experiments at identical concentrations, or
• weak effects (weak increases) occur dose-dependently (over at least two test material concentrations) and reproducibly at identical concentrations in the two experimental series performed.

In all other cases, further decisions for testing strategies should be made following the scientific evaluation of all existing data including those of non-toxicological investigations.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Observations:
Precipitation of the test material in the cell culture medium occurred at concentrations >= 500 µg/mL. The test material did not lead to a relevant fluctuation in both, the pH and the osmolarity of the cell culture medium. Toxicity to the cells was observed at concentrations >= 158µg/mL.

No relevant increases in mutant frequency were observed following treatment with the test item in the two experimental series in the absence and presence of metabolic activation.

Conclusions:

It is concluded that the test item is non-mutagenic in this test system under conditions where the positive controls exerted potent mutagenic effects.

Executive summary:

Purpose

The objective of this study was to evaluate the mutagenic activity of the test material by examining its ability to induce TK mutations in L5178Y cells in the absence and presence of a rat liver metabolizing system (S9 mix).

Study Design

The test item was assayed for its ability to induce mutations at th TK locus (5-trifluorothymidine resistance) in L5178Y mouse lymphoma cells using a fluctuation protocol acording to OECD Guideline 476. The GLP compliant study consisted of two independent experimental series, each conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). Acetone was used as the solvent. The exposure times were 3 and 24 hours in the absence and 3 hours in the presence of S9 mix, respectively.

Results

Various test material concentrations ranging from 28.1 to 2810 µg/mL were tested in the absence or presence of S9 mix. Precipitation of the test material in the incubation medium was observed at concentrations  >= 281 µg/mL. Clear cytotoxic effects, i.e. a relevant decrease in either the % relative survival or % total growth of the test cells, occurred at concentrations >= 158 or >= 2810 µg/mL, depending upon the experimental condition used. The concentrations for the determination of viability and mutagenicity (5-trifluorothymidine (TFT) resistance) were selected 2 days after treatment.

Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline N-oxide (without S9 mix) and 7,12-dimethylbenz[a]anthracene (with S9 mix). The relation of small to large colony mutant frequencies for the negative and positive controls were in the range acceptable for this cell line and assay (Applegate et al. 1990, Moore et al. 1985). Therefore, the study was accepted as valid.

No relevant increases in mutant frequency were observed following treatment with the test material in the two experimental series in the absence and presence of metabolic activation. The relation of small to large colony mutant frequencies was not altered due to treatment with the test material.

Conclusions

Based on the results it is concluded that the test material is non-mutagenic in this test system under conditions where the positive controls exerted potent mutagenic effects.

Reference 2

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:

Jan 25 - Mar 16, 2006

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

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

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9mixType and composition of metabolic activation system:
- concentration or volume of S9 mix and S9 in the final culture medium: 10% (1. experiment), 30% (2. experiment)

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 5 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 5 ... 5000 µg/plate

Vehicle / solvent:

Solvent: acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin Sodium azide Cumene hydroperoxide 9-Aminoacridine 4-Nitroquinolin-N-oxide 2-Aminoanthracene Benzoja]pyrene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: two

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 to 3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of background lawn of non-revertant bacteria

Rationale for test conditions:

according to Guideline

Evaluation criteria:

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:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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:

no cytotoxicity, but tested up to precipitating concentrations

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 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Precipitation of the test material on the agar plates occurred at concentrations 500 µg/plate. Toxicity to the bacteria was not observed.

Conclusions:

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

Executive summary:

Study Design

The procedures used in this study were in accordance with OECD Guideline 471 (adopted 1997), EEC Annex V Test B 13B 14 (2000), UKEMS Guidelines (1990) and ICH Harmonised Tripartite Guideline (1997).
The test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed.

Results

The test item  was dissolved in acetone and tested at concentrations ranging from 1.58 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations >= 500 µg/plate. Toxicity to the bacteria was not observed.

Daunomycin, sodium azide, 9-aminoacridine, 4-nitroquinolin-N-oxide and cu¬mene hydroperoxide served as strain specific positive control test materials 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 test materials 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.

In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain.

Thus, the test item was not mutagenic under the described experimental conditions.

Conclusion

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the provided information there is no need to classify the test item according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.