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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 February 2016 - 05 April 2016
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: In Vitro Mammalian Cell Gene Mutation Test With L5178y Mouse Lymphoma Cells

Test material

Constituent 1
Chemical structure
Reference substance name:
4-formyl-2-methoxyphenyl isobutyrate
EC Number:
243-956-6
EC Name:
4-formyl-2-methoxyphenyl isobutyrate
Cas Number:
20665-85-4
Molecular formula:
C12H14O4
IUPAC Name:
4-formyl-2-methoxyphenyl 2-methylpropanoate
Test material form:
other: liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Isobutavan
- Physical state: Almost colourless to pale yellow liquid
- Lot/batch No.: SC00016272
- Expiration date of the lot/batch: 11 December 2017
- Storage condition of test material: At room temperature protected from light

Method

Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
In the first experiment, ISOBUTAVAN was tested up to concentrations of 490 and 1000 μg/ml in the absence and presence of S9-mix, respectively.
In the second experiment, the test item was tested up to concentrations of 450 μg/ml in the absence of S9-mix.
Vehicle / solvent:
The test item was dissolved in dimethyl sulfoxide (DMSO, Merck Darmstadt, Germany).
Controls
Untreated negative controls:
yes
Remarks:
Solvent
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Remarks:
with and without S9 activation
Details on test system and experimental conditions:
Dose range finding test:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 17 to 1600 μg/ml in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, an increase above the positive threshold of MF(controls) + 126 (GEF= 237 x 10-6) was observed at the toxic top dose of 490 μg/ml. The increase observed was at a RTG of 10% and according to the guideline a result would not be considered positive if the increase in MF occurred only at or below 10% RTG. The increase observed in the next dose level of 436 μg/ml with a RTG of 32%, was a 1.9-fold increase in the mutation frequency and was not above the GEF.

After the prolonged treatment period (24 hour), an increase in the mutation frequency above the critical value of 170 per 106 survivors was only observed at the toxic top dose of 450 μg/ml with a RTG of 12%. The mutation frequency at this concentration was not above the GEF and there was no concentration related increase observed. Therefore the result is considered to be negative and of no biological relevance.

In the presence of S9-mix, ISOBUTAVAN did not induce a significant increase in the mutation frequency. Since the meaningful increase (above the positive threshold, GEF) in the mutation frequency at the TK locus in the absence of S9-mix is only observed at the top concentration with a RTG of 10% after the 3 hour treatment and the increase was above the critical value of 170 per 106 survivors after the prolonged treatment period but not above the GEF, the biological relevance of these increases is doubtful. Therefore the test results for the 3hr test in the absence of S9-mix is considered equivocal under the experimental conditions described in the report. The test item was not mutagenic in the current test in the presence of S9-mix.

Applicant's summary and conclusion

Conclusions:
The test item was not mutagenic in the current test in the presence of S9-mix.
Executive summary:

Evaluation of the mutagenic activity of ISOBUTAVAN in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. This report describes the effects of ISOBUTAVAN on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hours treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The study procedures described in this report were based on the most recent OECD guideline. Batch SC00016272 of ISOBUTAVAN was an almost colourless to pale yellow liquid. The test item was dissolved in dimethyl sulfoxide. In the first experiment, ISOBUTAVAN was tested up to concentrations of 490 and 1000 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. The relative total growth (RTG) was 10% in the absence of S9-mix. In the presence of S9-mix, no cytotoxicity was observed, however ISOBUTAVAN was tested up to the precipitating dose level of 1000 μg/ml in compliance with the guideline. In the absence of S9-mix, an increase above the positive threshold of MF(controls) + 126 (GEF= 237 x 10-6) was observed at the toxic top dose of 490 μg/ml with a RTG of 10%. The mutation frequency observed in the second highest dose level of 436 μg/ml (RTG of 32%) was not above the GEF and therefore considered negative. In the presence of S9-mix, none of the tested concentrations reached a mutation frequency of MF(controls) + 126. In the second experiment, the test item was tested up to concentrations of 450 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 12%. After the prolonged treatment period, an increase in the mutation frequency above the critical value of 170 per 106 survivors was only observed at the toxic top dose of 450 μg/ml. The increase observed in the first experiment (short treatment) was at a RTG of 10% and according to the guideline a result would not be considered positive if the increase in MF occurred only at or below 10% RTG. After the prolonged treatment period (24 hour), an increase in the mutation frequency above the critical value of 170 per 106 survivors was only observed at the toxic top dose of 450 μg/ml with a RTG of 12%. The mutation frequency at this concentration was not above the GEF and there was no concentration related increase observed. Therefore the result is considered to be negative and of no biological relevance. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Since the meaningful increase (above the positive threshold, GEF) in the mutation frequency at the TK locus in the absence of S9-mix is only observed at the top concentration with a RTG of 10% after the 3 hour treatment and the increase was above the critical value of 170 per 106 survivors after the prolonged treatment period but not above the GEF, the biological relevance of this increase is doubtful. Therefore the test results for the 3hr test in the absence of S9-mix are considered equivocal under the experimental conditions described in the report. The test item was not mutagenic in the current test in the presence of S9-mix.

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