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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:
17 May 2016 to 16 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Principles of method if other than guideline:
There were no deviations from standard operating procedures that affected the integrity of the study.
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:
3-(p-cumenyl)-2-methylpropionaldehyde
EC Number:
229-695-0
EC Name:
3-(p-cumenyl)-2-methylpropionaldehyde
Cas Number:
6658-48-6
Molecular formula:
C14H20O
IUPAC Name:
3-(4-isopropylphenyl)-2-methylpropanal
Specific details on test material used for the study:
Name (as stated in the report): SILVIAL
Batch No.: SC00017272
Expiration date: 21 January 2017

Method

Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
In the first experiment, the test item was tested up to concentrations of 25 and 50 μg/ml in the absence and presence of S9-mix, respectively. In the second experiment, the test item was tested up to concentrations of 25 μg/ml in the absence of S9-mix. The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.
Vehicle / solvent:
Dimethyl sulfoxide
Controls
Untreated negative controls:
yes
Remarks:
Solvent control
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide (SeccoSolv, Merck Darmstadt, Germany).
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Test System: L5178Y/TK+/--3.7.2C mouse lymphoma cells
Source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 x 106 cells/ml.
Rationale for test conditions:
Rationale: Recommended test system in international guidelines (e.g. OECD).
Evaluation criteria:
Any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126. A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically
relevant and will be compared with the historical control data range. A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study. A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

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:
see "Any other information on results" field
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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.
The growth rate over the two-day expression period for cultures treated with DMSO was between 10 and 16 (3 hours treatment) and between 104 and 107 (24 hours treatment)

Any other information on results incl. tables

1st experiment (toxicity evaluation)

In the absence of S9-mix the dose levels of 1 to 10 μg/ml showed similar cytotoxicity. Therefore, the dose level of 1 μg/ml was not regarded relevant for mutation frequency measurement. Due to severe toxicity, the dose level of 30 μg/ml was removed during the subculture period.

In the presence of S9 -mix the dose levels of 55 and 60 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic or showed an inconsistent RSG (60 μg/ml). The dose levels of 1 to 40 μg/ml showed no cytotoxicity. Therefore, the dose levels of 1 and 5 μg/ml were not regarded relevant for mutation frequency measurement.

2nd experiment (toxicity evaluation)

In the absence of S9-mix the dose levels of 27.5 to 35 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

The dose level of 10 μg/ml showed an inconsistent RSG. Therefore, this dose level was not regarded relevant for mutation frequency measurement.

Applicant's summary and conclusion

Conclusions:
In the absence of S9-mix, SILVIAL did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.
In the presence of S9-mix, SILVIAL did not induce a significant increase in the mutation frequency.
In conclusion, SILVIAL is not mutagenic in the TK mutation test system under the experimental conditions described in this report.
Executive summary:

Evaluation of the mutagenic activity of SILVIAL in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells.

This report describes the effects of SILVIAL 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 hour 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 SC00017272 of the test item was a colourless liquid. The test item was dissolved in

dimethyl sulfoxide.

In the first experiment, the test item was tested up to concentrations of 25 and 50 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total

growth (RTG) was 4 and 11% in the absence and presence of S9-mix, respectively.

In the second experiment, the test item was tested up to concentrations of 25 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 7%.

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. 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, SILVIAL did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, SILVIAL did not induce a significant increase in the mutation frequency.

It is concluded that SILVIAL is not mutagenic in the mouse lymphoma L5178Y test system^under the experimental conditions described in this report.