4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-17 till 2015-09-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: US Food and Drug Administration, Center for Food Safety & Applied Nutrition, Redbook 2000 Toxicological Principles for the safety of Food Ingredients. IV.C.1.a. Bacterial Reverse Mutation Test, July 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Triskelion, Utrechtseweg 48, 3704 HE Zeist, The Netherlands

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran

EC Number:

948-409-1

Cas Number:

1945993-03-2

Molecular formula:

C15H20O

IUPAC Name:

4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran

Test material form:

liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver homogenate (S9-mix)

Test concentrations with justification for top dose:

First bacterial reverse mutation test:
Experiment 1:
0, 21, 62, 185, 556, 1667, 5000 μg/plate (all strains with and without S9-mix)
Experiment 2:
0, 4.1, 12, 37, 111, 333, 1000 µg/plate ( TA 1535 (-S9-mix))
0, 1.4, 12, 37, 111, 333 µg/plate (TA 1535 (+S9-mix), TA 1537 (-S9-mix), TA 98 (+S9-mix) and TA 100 (-S9-mix))
0, 0.46, 1.4, 12, 37, 111 µg/plate (TA 1537 (+S9-mix) and TA 100 (+S9-mix))
0, 62, 185, 556, 1667, 5000 µg/plate (WP2 uvrA (+S9-mix))
Experiment 3 and 4:
0, 4.1, 12, 37, 111, 333, 1000 µg/plate ( TA 1535 (-S9-mix))

Second bacterial reverse mutation test
0, 20, 39, 78, 156, 313, 625 µg/plate (TA 1535 (-S9-mix))
0, 9.8, 20, 39, 78, 156, 313 µg/plate (TA 1535 (+S9-mix))
0, 4.9, 9.8, 20, 39, 78, 156 µg/plate (TA 1537, TA 98 (+S9-mix) and TA 100 (-S9-mix))
0, 39, 78, 156, 313, 625, 1250 µg/plate (TA 98 (-S9-mix))
0, 2.5, 4.9, 9.8, 20, 39, 78 µg/plate (TA 100 (+S9-mix))
0, 313, 625, 1250, 2500, 5000 µg/plate (WP2 uvrA)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

ethylnitrosurea

other: 2-aminoanthracene, N-ethyl-N-nitrosourea

Remarks:

In the absence of S9-mix: sodium azide: TA 1535 and TA 100; 9-aminoacridine: TA 1537; 2-nitrofluorene: TA 98; N-ethyl-N-nitrosourea: WP2uvrA. In the presence of S9-mix: 2-aminoanthracene: TA 1535, TA 98, TA 100, WP2uvrA; benzo(a)pyrene: TA 1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72 hours at ca. 37 °C

NUMBER OF REPLICATIONS: All determinations were made in triplicate.

DETERMINATION OF CYTOTOXICITY
- Toxicity was defined as a reduction (by at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth as compared to the negative (solvent) control and/or the occurrence of pinpoint colonies.

Evaluation criteria:

The study was considered valid if the mean colony counts of the vehicle control values of the strains were within the acceptable ranges, if the results of the positive controls met the criteria for a positive response, if no more than 5 % of the plates was lost through contamination or other unforeseen events and if at least three concentrations were non-toxic.

A test substance was considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates was increased in a dose-related manner or if a two-fold or greater increase was observed compared to the negative control plates. A clear positive response did not need to be verified. Marginally or weakly positive results should be verified by additional testing.

A test substance was considered to be negative in the bacterial gene mutation test if it showed neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the concentrations tested.

Positive results from the bacterial reverse mutation test indicate that a test substance induces point mutations by base pair substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that, under the test conditions used, the test substance is not mutagenic in the tested strains.Although most studies give clearly positive or negative results, in rare cases the data set may preclude making a definite judgement about the mutagenic potential of the test substance. Results may remain equivocal in this case.

Both numerical significance and biological relevance were considered together in the evaluation.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The first test included four experiments. In the first experiment, the test substance was found toxic for all Salmonella strains tested, both in the absence and presence of S9-mix. In addition, for strains TA 1535 (+S9-mix), TA 1537 (-/+ S9-mix), TA 98 (+S9-mix) and TA 100 (-/+S9-mix) less than three non-toxic concentrations could be evaluated. For strain TA 1535 (-/+ S9-mix), TA 100 (-S9-mix) and WP2UvrA (+S9-mix) the negative or positive control was outside the acceptable range. Therefore, the experiment was repeated for these strains.
In the second experiment the test substance was found toxic for all Salmonella strains tested, both in the absence and presence of S9-mix. But for all strains tested three or more non-toxic concentrations could be evaluated. For strain TA 1535 (-S9-mix), the negative control was outside the historical range. Therefore the experiment was repeated for this strain.
In the third experiment, for strain TA 1535 (- S9-mix), the negative control was outside the historical range. Therefore the experiment was repeated again. In the fourth experiment, the test substance was found toxic for strain TA 1535, in the absence of S9-mix, but at least three non-toxic concentrations could be evaluated. Based on the results of these four experiments, the first test was regarded as valid.

The second test included one experiment. In this fifth experiment, the test substance was found toxic for all Salmonella strains tested, both in the absence and presence of S9-mix, at at least one concentration, but for all strains tested three or more non-toxic concentrations could be evaluated. The mean numbers of his+ and trp+ revertant colonies of the negative controls in all strains used were within the acceptable range. In all strains, the positive controls gave the expected increase in the mean numbers of revertant colonies. Therefore, the second test was considered valid.

In all tests, toxicity was evidenced by a decrease in the mean number of revertants and/or a clearing of the background lawn of bacterial growth compared to the negative controls

In all experiments, a dose related precipitation of the test substance in the final treatment mix (top agar) was observed. Precipitation was observed in all mixtures at and above 556 μg/plate (first experiment), 333 μg/plate (second, third and fourth experiment) and 313 μg/plate (fifth experiment) with the unaided eye. In the first, second and fifth experiment, a precipitation of the test substance on the agar plates was observed. Precipitation was observed at and above 1667 μg/plate (first and second experiment) and at 5000 μg/plate (fifth experiment) with microscope only.

In both tests, in all strains tested, in both the absence and presence of S9-mix, the test substance did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the test conditions (OECD 471, GLP) the results obtained in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in the absence and presence of the S9-mix, indicate that the test substance is not mutagenic.

Executive summary:

According to OECD guideline 471 and GLP, the test substance, dissolved in DMSO, was examined for its possible mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2uvrA, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix). Two independent bacterial reverse mutation tests were performed. In the first test, four independent experiments were performed where six to ten concentrations of the test substance ranging from 4.1 to 5000 μg/plate were tested. In the second test, six concentrations of the test substance ranging from 2.5 to 1250 μg/plate were tested and five concentrations ranging from 313 to 5000 μg/plate for the Salmonella strains and WP2 UvrA strain, respectively. In all tests, negative controls (vehicle) and positive controls were run simultaneously with the test substance. In some of the experiments of the first test, the mean numbers of his+ and trp+ revertant colonies of the negative and positive controls in some of the strains appeared to be outside the acceptable range. Therefore, the experiments were repeated until the acceptance criteria were met. In the second test, the mean numbers of his+ and trp+ revertant colonies of the negative controls were within the acceptable range and the positive controls gave the expected increase in the mean numbers of revertant colonies in all strains. Therefore, both tests of this study were considered valid. In the first experiment of the first test, the test substance was found toxic to all strains tested, both in the absence and presence of S9-mix. For strains TA 1535 (+S9-mix), TA 1537 (-/+ S9-mix), TA 98 (+S9-mix) and TA 100 (-/+ S9-mix), less than three non-toxic concentrations could be evaluated, therefore the experiment was repeated for these strains (second, third and fourth experiment), which eventually resulted in a valid first test where at least three non-toxic concentrations could be evaluated. In the second test, toxicity was observed for all Salmonella strains tested, both in the absence and presence of S9-mix, but three or more non-cytotoxic concentrations could be evaluated. In all experiments, toxicity was evidenced by a decrease in the mean number of revertants and/or a clearing of the background lawn of bacterial growth compared to the negative controls. In both tests, in all strains tested, in both the absence and presence of S9-mix, the test substance did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. It is concluded that the results obtained in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and presence of the S9-mix, indicate that the test substance is not mutagenic under the conditions used in this study.