6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-propionaldehyde

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-10-02 till 2015-11-11

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

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

Constituent 1

Reference substance name:

6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-propionaldehyde

EC Number:

251-717-2

EC Name:

6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-propionaldehyde

Cas Number:

33885-51-7

Molecular formula:

C12H18O

IUPAC Name:

3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)propanal

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, 2.1, 6.2, 19, 56, 167, 500 µg/plate (TA 1535, TA 1537, TA 98 and TA 100 with and without S9-mix)
1500 µg/plate (TA 1535 (+S9-mix))

Second bacterial reverse mutation test
0, 3.1, 7.7, 19, 48, 120, 300 µg/plate (TA 1535, TA 1537, TA 98 and TA 100 with and without S9-mix)
0, 51, 128, 320, 800, 2000, 5000 µg/plate (WP2 uvrA with and without S9-mix)

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

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium 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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Two independent tests were performed. In the first experiment of the first test and in the experiment of the second test, a stock solution of 50 mg/mL in dimethyl sulfoxide (DMSO) was prepared and in the second experiment of the first test a stock solution of 15 mg/mL in dimethyl sulfoxide (DMSO) was prepared; all resulted in a clear, colourless solution. Serial dilutions of the test substance were prepared in the solvent. In all experiments, negative controls (solvent) and positive controls were run simultaneously with the test substance.

The first test included two experiments. In the first experiment, for all Salmonella strains, both in the absence and presence of S9-mix, less than three non-toxic concentrations were obtained. Therefore, the experiment was repeated for these strains. The second test included one experiment.

In both the first and second test, negative controls (solvent) and positive controls were run simultaneously with the test substance. 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, all experiments were considered valid.

In both the first and second test, the test substance was found toxic to all strains tested, both in the absence and presence of S9-mix. In the first experiment of the first test, toxicity was observed at and above 62 μg/plate for strain TA 1535 (+S9 mix) and TA 1537 (-/+ S9-mix); at and above 185 μg/plate for strain TA 1535 (-S9 mix), TA 98 and TA 100 (both -/+ S9-mix); at and above 1667 μg/plate and at 5000 μg/plate for strain WP2 uvrA, in the absence and presence of S9-mix, respectively. In the second experiment of the first test toxicity was observed at and above 56 μg/plate for TA 1537 (-/+ S9 mix) and at and above 167 μg/plate for strain TA 1535, TA 98 and TA 100 (all -/+ S9-mix). In the experiment of the second test toxicity was observed at and above 48 μg/plate for TA 1537 (-/+ S9-mix) and TA 100 (-S9 mix); at and above 120 μg/plate for strain TA 1535, TA 98 (both -/+S9-mix) and TA 100 (+ S9-mix). A decrease of the mean number of revertants was observed with strain WP2 uvrA, but this decrease was not more than two-fold. 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 and/or pinpoint colonies.

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 (experiment 1, first test), at and above 500 μg/plate (experiment 2, first test) and at and above 800 μg/plate (second test) with the unaided eye.

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.

Applicant's summary and conclusion

Conclusions:

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). In the first test, two independent experiments were performed. In the first experiment of the first test, six concentrations of the test substance ranging from 21 to 5000 μg/plate were tested with all strains. In the second experiment of the first test, 7 concentrations of the test substance ranging from 2.1 to 1500 μg/plate were tested with strain TA 1535, in the presence of S9-mix and 6 concentrations from 2.1 to 500 μg/plate for the other strains and TA 1535 in the absence of S9-mix. In the second test, one experiment was performed where six concentrations of the test substance ranging from 3.1 to 300 μg/plate and from 51 to 5000 μg/plate were tested, for respectively the Salmonella strains and the E. coli strain. In the first experiment of the first and in the experiment of the second test, a stock solution of 50 mg/mL in dimethyl sulfoxide (DMSO) was prepared and in the second experiment of the first test a stock solution of 15 mg/mL in dimethyl sulfoxide (DMSO) was prepared; all resulted in a clear, colourless solution. In both the first and second test, negative controls (solvent) and positive controls were run simultaneously with the test substance. 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, all experiments were considered valid. In both the first and second test, the test substance was found toxic to all strains tested, both in the absence and presence of S9-mix. In the first experiment of the first test, toxicity was observed for all strains tested both in the absence and presence of S9 mix. For all Salmonella strains less than three non-toxic concentrations were tested, therefore the test was repeated. In the second experiment of the first test and in the experiment of the second test toxicity was observed for all Salmonella strains both in the absence and presence of S9 mix; for all strains at least three non-toxic concentrations and at least one toxic concentration were obtained. 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 and/or pinpoint colonies. 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.