2-methyl-1-phenylpropan-2-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:

05 August - 08 December 2017

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-1-phenylpropan-2-ol

EC Number:

202-896-0

EC Name:

2-methyl-1-phenylpropan-2-ol

Cas Number:

100-86-7

Molecular formula:

C10H14O

IUPAC Name:

2-methyl-1-phenylpropan-2-ol

Test material form:

other: Colourless to slight yellow liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Yinghai (Cangzhou) Aroma Chemical Company Ltd.;CP008-170101
- Expiration date of the lot/batch: 30-06-2018
- Purity: 99.7%

Method

Species / strain

Species / strain / cell type:

other: S. typhimurium TA1535, TA97a, TA98, TA100, TA102

Metabolic activation:

with and without

Metabolic activation system:

Sodium phenobarbitone and β-naphthoflavone-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary test: 5000, 4000, 3000, 2000, 1000, 500, 250 and 125 µg/plate
Plate incorporation method (Experiment 1): 4000, 1000, 400, 100 and 40 µg/plate
Pre-incubation method (Experiment 2): 4000, 1000, 400, 100 and 40 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO

- Justification for choice of solvent/vehicle: Dimethyl Benzyl Carbinol was found to be miscible in DMSO at a concentration of 50 mg/ml. No precipitation was observed in the final reaction mixture at the concentrations of 2.38 mg/ml, corresponding to final test concentrations of 5000 g/plate. 5000 µg/plate showing no precipitation was the highest concentration selected for the preliminary cytotoxicity study.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

other: TCR191, CAS No. 17070-45-0

Controls 2

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene, CAS No. 613-13-8

Controls 3

Positive controls:

yes

Positive control substance:

other: 2-Aminofluorene, CAS No. 153-78-6

Controls 4

Positive controls:

yes

Positive control substance:

other: Danthron, CAS No. 117-10-2

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 20 mins
- Expression time: 48 hours for Experiment No. 1 and 70 hours for Experiment No. 2.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn reduction

Evaluation criteria:

1. Criteria for a Positive Response
A test item is considered to be positive (mutagenic), if it induces a concentration dependent increase and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate, in at least one strain with or without metabolic activation system, which is at least 2 fold (3 fold for TA1535) of that observed in the corresponding concurrent vehicle control.
2. Criteria for a Negative Response
A test item for which the results do not meet the above criteria is considered non-mutagenic in this test. In order a substance considered to be negative, if the revertant colonies cannot be greater than 2 (or 3 for strain TA 1535), or less than 0.5 in at least 5 doses for all strains tested. However, reproducibility of negative results will be confirmed by repeat experimentation.
3. Criteria for an Equivocal Response
Occasionally, a test item cannot be judged to be positive or negative (e.g., concentration dependent increases that fail to reach 2 fold (3 fold for TA1535) control values, or  2 fold (3 fold for TA1535) increases that do not appear to be concentration dependent). In these rare instances, the results may be classified as equivocal. Equivocal or weak positive results may indicate the need to repeat the test, possibly with a modified study plan such as appropriate spacing of dose levels.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA1535, TA97a, TA98, TA100, TA102

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Dimethyl Benzyl Carbinol was found to be miscible in DMSO at a concentration of 50 mg/ml. No precipitation was observed in the final reaction mixture at the concentrations of 2.38 mg/ml, corresponding to final test concentrations of 5000 µg/plate. (Appendix 4).

RANGE-FINDING/SCREENING STUDIES:
Before commencing the study, the test item was assessed for cytotoxicity to the tester bacteria using tester strain TA100. Eight concentrations (5000, 4000, 3000, 2000, 1000, 500, 250 and 125 µg/plate), of the test item, dissolved in DMSO, were tested for toxicity to bacterial cells.

Plate Incorporation Method
Dimethyl Benzyl Carbinol was found to be slightly cytotoxic to the bacterial cells at the concentrations of 5000 and 4000 µg/plate for plate incorporation method. Hence, 4000 µg/plate showing slight toxicity was selected as the maximum test concentration for main study for plate Incorporation method.

Pre Incubation Method
Moderate cytotoxicity observed at the concentration of 5000 µg/plate and slight toxicity observed at the concentrations of 4000 µg/plate both in presence and absence of metabolic activation system for pre incubation method. Hence, 4000 µg/plate showing slight toxicity was selected as the maximum test concentration for main study for pre-incubation method.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data: Concurrent positive controls demonstrated sensitivity of the assay with and without metabolic activation (Appendix 6).

- Negative (solvent/vehicle) historical control data: Plate counts for the spontaneous histidine revertant colonies in the vehicle control groups were found to be within the frequency ranges expected from the laboratory historical control data at INTOX PVT. LTD (Appendix 6).

Applicant's summary and conclusion

Conclusions:

Under the conditions described for this study, it is concluded that Dimethyl Benzyl Carbinol is non-mutagenic in Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102.

Executive summary:

In a reverse gene mutation assay in bacteria (16940), strains of S. typhimurium TA1535, TA97a, TA98, TA100, TA102 were exposed to Dimethyl Benzyl Carbinol (99.7%) in DMSO at concentrations of 4000, 1000, 400, 100, 40 µg/plate and 0 μg/plate (direct plate incorporation; experiment 1) and 2000, 600, 200, 60, 20 µg/plate and 0 μg/plate (20 minute pre-incubation; experiment 2) in the presence and absence of mammalian metabolic activation (Sodium phenobarbitone and β-naphthoflavone-induced rat liver S9).

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.