Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-03-20 to 2006-03-19
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)
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:
(trans(trans))-4'-vinyl-4-(4-methylphenyl)bicyclohexyl
EC Number:
439-730-3
EC Name:
(trans(trans))-4'-vinyl-4-(4-methylphenyl)bicyclohexyl
Cas Number:
155041-85-3
Molecular formula:
Hill formula: C21H30 CAS formula: C21H30
IUPAC Name:
(1r,1'r,4r,4'r)-4-ethenyl-4'-(4-methylphenyl)-1,1'-bi(cyclohexane)

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 different serum concentrations were used:

- Exposure medium: RPMI- 5 (RPM 1640 with 5% heat inactivated horse serum) 470 mL RPMI 164025 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 164050 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 1640100 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin
Metabolic activation:
with and without
Metabolic activation system:
10% rat liver homogenate (S9 mix) with standard co-factors
Vehicle / solvent:
Acetone
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline N-oxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
see below
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:
cytotoxicity
Remarks:
at 2.81 and 28.1 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
It is concluded that the test item is non-mutagenic and non-clastogenic in this test system under conditions where the positive controls exerted potent mutagenic effects.
Executive summary:

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).

The test material 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 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. 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). Therefore, the study was accepted as valid. 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.

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