1-(2,4-dimethylcyclohex-3-en-1-yl)propan-1-ol

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:

2015-02-06 till 2015-04-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 471 guideline study in compliance with GLP, available as unpublished report, no restrictions, fully adequate for assessment

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

Constituent 1

Reference substance name:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Test material form:

other: Solid

Details on test material:

- Name of test material (as cited in study report): FRET 11-0571
- Storage condition of test material: 2-10 °C, protected from light
- Appearance: white solid

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 experiment: 62, 185, 556, 1667 and 5000 μg/plate (all strains and species in the presence and absence of S9-mix)
First experiment (repeated): 19, 56, 167, 500, 1500 μg/plate TA 1535, TA 1537 and TA 100, absence of S9-mix)
Second experiment: 39, 78, 156, 313, 625, 1250, µg/plate (TA 1535, TA 1537, TA 100 in absence of S9-mix)
Second experiment: 78, 156, 313, 625, 1250, 2500 µg/plate (TA 1535, TA 1537, TA 100, TA98 in presence of S9-mix; TA98 in absence of S9-mix)
Second experiment: 156, 313, 625, 1250, 2500 µg/plate (E. Coli in the presence and absence of S9-mix)
Second experiment (repeated): 78, 156, 313, 625, 1250, 2500 µg/plate (E. Coli absence of S9-mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (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

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 mutagenicity study was considered valid if the mean colony counts of the solvent control values of the strains are within acceptable ranges, if the results of the positive controls meet the criteria for a positive response if no more than 5 % of the plates are lost through contamination or other unforeseen events and if at least three test concentrations are 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 does 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 demonstrated neither a dose-related increase in the mean number of revertant colonies nor a reproducible minimal two-fold increase in the mean number of revertants at any of the test concentrations.

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, 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

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

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

other: yes, except negative control for strain WP2 uvrA, in the absence of S9-mix in the second test

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Two independent tests were performed. The first test was repeated with strains TA 1535, TA 1537 and TA 100 because of toxicity observed in the first test. The second test was repeated with strain WP2 uvrA because the negative control was outside the acceptable range in the second test. 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.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In all tests, the test substance was found toxic for all strains tested, both in the absence and presence of S9-mix. In the first test, toxicity was observed for strains TA 1535, TA 1537 and TA 100 (absence of S9-mix) at and above 556 μg/plate, for strains TA 1535, TA 1537 and TA 100 (presence of S9-mix) and TA 98 and WP2 uvrA (absence and presence of S9-mix) at and above 1667 μg/plate. For strains TA 1535, TA 1537 and TA 100, in the absence of S9-mix, less than three non-toxic concentrations could be evaluated, therefore the test was repeated. In the repeated first test, toxicity was observed at and above 500 μg/plate for all strains tested. In the second test and repeated second test, toxicity was observed for strains TA 1535, TA 1537 and TA 100 (absence of S9-mix) at and above 625 μg/plate, for strains TA 1535, TA 1537, TA 100 (all presence of S9-mix) and TA 98 (absence and presence of S9-mix) at 1250 μg/plate and WP2 uvrA (absence and presence of S9-mix) at and above 1250 μg/plate. 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.

ADDITIONAL INFORMATION ON CONTROLS:
In all tests, the mean numbers of his+ and trp+ revertant colonies of the negative controls in all strains used were within the acceptable range, except for the negative control for strain WP2 uvrA, in the absence of S9-mix in the second test. Therefore, this test was repeated. In all strains the positive controls gave the expected increase in the mean numbers of revertant colonies. Therefore, all tests were considered valid.

TEST-SPECIFIC CONFOUNDING FACTORS
In the first and second test, 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 1667 μg/plate (first test), 1500 μg/plate (repeated first test) and 2500 μg/plate (second and repeated second test) with the unaided eye.

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 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, for all strains five concentrations of the test substance ranging from 62 to 5000μg/plate were tested, both in the absence and presence of S9-mix. For strains TA 1535, TA 1537 and TA 100, in the absence of S9-mix, less than three non-toxic concentrations could be evaluated, therefore the test was repeated for these strains, and five concentrations of the test substance ranging from 19 to 1500 μg/plate were tested, in the absence of S9-mix. In the second test, for strains TA 1535, TA 1537 and TA 100 (presence of S9-mix) and TA 98 (absence and presence of S9-mix), five concentrations were tested, ranging from 78 to 1250 μg/plate. For strains TA 1535, TA 1537 and TA 100 (absence of S9-mix), six concentrations were tested, ranging from 39 to 1250 μg/plate. For strain WP2uvrA (absence and presence of S9-mix) five concentrations were tested, ranging from 156 to 2500 μg/plate. Because the negative control was outside the acceptable range for strain WP2uvrA (absence of S9-mix) six concentrations were tested, ranging from 78 to 2500 μg/plate. In all tests, negative controls (vehicle) and positive controls were run simultaneously with the test substance. In all tests, the mean numbers of his+ and trp+ revertant colonies of the negative controls in all strains used were within the acceptable range, except for the negative control for strain WP2uvrA, in the absence of S9-mix in the second test. In all strains, the positive controls gave the expected increase in the mean numbers of revertant colonies. Therefore, all tests were considered valid. In the repeated first test toxicity was observed at and above 500μg/plate for all strains tested. In this test three nontoxic concentrations could be evaluated. In the second test and repeated second test, toxicity was observed for all strains tested, both in the absence and presence of S9-mix. 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 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 employed in this study.