cis-2-tert-butylcyclohexan-1-ol

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:

From 26 July 2021 to 18 November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

cis-2-tert-butylcyclohexan-1-ol

EC Number:

230-601-5

EC Name:

cis-2-tert-butylcyclohexan-1-ol

Cas Number:

7214-18-8

Molecular formula:

C10H20O

IUPAC Name:

cis-2-tert-butylcyclohexan-1-ol

Test material form:

solid

Method

Target gene:

His / trp

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat S9 liver microsomal fraction

Test concentrations with justification for top dose:

Experiment I: 0, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate
Experiment II: 0, 1.0, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other:

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION: The experimental conditions in the pre-experiment were the same as described below for the main experiment I (plate incorporation test). For the plate incorporation method, the following materials were mixed in a test tube and poured over the surface of a minimal agar plate: 100 µL of Test solution at each dose level, solvent or negative control or reference mutagen solution (positive control), 500 of µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation), 100 µL of Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain), 2000 µL of Overlay agar.
For the pre-incubation method 100 µL of the test item-preparation is pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.
NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.
DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Criteria of Validity: A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, E. coli WP2 uvrA
(pKM101))
- the negative control plates with and without S9 mix are within the historical control data range
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

valid

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:

valid

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Results from pre-experiment: As limiting toxicity of the test item was observed in both tester strains used at the maximum recommended concentration of 5000 μg/plate (with and without metabolic activation), concentrations of up to 2500 μg/plate were selected for the main experiments.

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

In experiment I toxic effects of the test item were observed in tester strains TA98 and TA1535 at concentrations of 316 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate (with metabolic activation). Toxic effects of the test item were also noted in tester strains TA100 at concentrations of 316 µg/plate and higher (with and without metabolic activation). In tester strain TA1537, toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate (with metabolic activation). Additionally, in tester strain E. coli WP2 uvrA (pKM101), toxic effects of the test item were observed at concentrations of 316 µg/plate and higher (without metabolic activation) and at a concentration of 2500 µg/plate (with metabolic activation). The reduction in the number of revertants down to a mutation factor of ≤ 0.5 and the reduction of the background lawn found in tester strain E. coli WP2 uvrA (pKM101) at a concentration of 100 µg/plate (with metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.

In experiment II toxic effects of the test item were noted in tester strain TA98 at concentrations of 316 µg/plate and higher (without and with metabolic activation). In tester strain TA100, toxic effects of the test item were observed at concentrations of 10.0 µg/plate and higher (without metabolic activation) and at concentrations of 100 µg/plate and higher (with metabolic activation). Toxic effects of the test item were also noted in tester strains TA1535, TA1537 and E. coli WP2 uvrA (pKM101) at concentrations of 316 µg/plate and higher (with and without metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met. The negative control plates with and without metabolic activation were within the historical control data range with the exception of a slightly lower spontaneous reversion frequency in tester strain TA98 in experiment I (with metabolic activation,
mean: 24). Since no technical failure occurred, the data was considered acceptable for addition to the laboratory historical database. The observed slight decrease can be attributed to biological variability and was regarded as not biologically relevant and did not influence the validity of the results.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, Verdol is considered to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

The study was conducted to determine the mutagenic potential of Verdol in Bacterial Reverse Mutation Test in accordance with the OECD Guideline 471 in compliance with GLP. Two independent experiments were performed according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101). The concentrations of up to 2500 μg/plate were selected for the main experiments based on the pre-experiment. The concentrations were 3.16, 10.0, 31.6, 100, 316, 1000, and 2500 μg/plate in experiment I and 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 μg/plate in experiment II. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). Toxic effects of the test item were noted in all tester strains used in experiment I and II. In experiment I toxic effects of the test item were observed at concentrations of 316 μg/plate and higher (with and without metabolic activation) depending on the particular tester strain. In experiment II toxic effects of the test item were noted at concentrations of 10.0 μg/plate and higher (without metabolic activation) and at concentrations of 100 μg/plate (with metabolic activation), depending on the particular tester strain. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Verdol at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. Verdol was therefore considered to be non-mutagenic under the conditions of this test.