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Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
1-bromo-4-(trans-4-ethylcyclohexyl)benzene
EC Number:
422-030-7
EC Name:
1-bromo-4-(trans-4-ethylcyclohexyl)benzene
Cas Number:
91538-82-8
Molecular formula:
Hill formula: C14H19Br CAS formula: C14H19Br
IUPAC Name:
1-bromo-4-(trans-4-ethylcyclohexyl)benzene
Test material form:
liquid: viscous

Method

Target gene:
TK gene
Species / strain
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
Controls
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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.

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