2-methylundecanal

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

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

Test material

Test material information

Constituent 1

Reference substance name:

2-methylundecanal

EC Number:

203-765-0

EC Name:

2-methylundecanal

Cas Number:

110-41-8

Molecular formula:

C12H24O

IUPAC Name:

2-methylundecanal

Specific details on test material used for the study:

Identification: ALDEHYDE C 12 MNA PURE
Appearance: Colourless to pale yellow liquid
Batch: VE00487208
Test item storage: At room temperature protected from light container flushed with nitrogen
Stable under storage conditions until: 09 March 2018 (expiry date)

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg bw) & ß-naphthoflavone (100 mg/kg bw)

Test concentrations with justification for top dose:

First Mutagenicity Test
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test:
Without and with S9-mix: 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30 and 35 μg/ml exposure medium.

In the absence of S9-mix, the dose levels of 25, 30 and 35 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
In the presence of S9-mix, no dose level with a cell survival below 27% was reached, therefore this experiment was rejected (see Table 6). In the repeat experiment (1A), the following dose-range was selected:
1, 5, 10, 20, 30, 35, 40, 45, 50, 55 and 60 μg/ml exposure medium.
Initially, the experiment in the presence of S9-mix was rejected twice, since no acceptable responses in the solvent controls were obtained. No data is reported.
In the presence of S9-mix, the dose levels of 40 to 60 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.05, 0.1, 0.5, 1, 5, 10, 15 and 20 μg/ml exposure medium.
With S9-mix: 1, 5, 10, 20, 30 and 35 μg/ml exposure medium.

Second Mutagenicity Test
Based on the results of the dose-range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20 and 25 μg/ml exposure medium.

Vehicle / solvent:

The vehicle for the test item was ethanol (Merck, Darmstadt, Germany).

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With Metabolic Activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

Test System: L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Rationale: Recommended test system in international guidelines (e.g. OECD, EC).
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 kept below 1 x 106 cells/ml.

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10-6).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

In addition to the criteria stated below, 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

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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.
Although the mutation frequency of one of the solvent control cultures in the first experiment in the absence of S9-mix and in the second experiment was just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges.
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 suspension growth over the two-day expression period for cultures treated with ethanol was between 15 and 18 (3 hour treatment) and 80 and 86 (24 hour treatment).
In the absence of S9-mix, the test item 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, the test item did not induce a significant increase in the mutation frequency.

Applicant's summary and conclusion

Conclusions:

In conclusion, ALDEHYDE C 12 MNA PURE is not mutagenic in the TK mutation test system under the experimental conditions described in this report.