2-methyl-4-phenylbutan-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:

03 Aiugust - 16 November 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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-4-phenylbutan-2-ol

EC Number:

203-074-4

EC Name:

2-methyl-4-phenylbutan-2-ol

Cas Number:

103-05-9

Molecular formula:

C11H16O

IUPAC Name:

2-methyl-4-phenylbutan-2-ol

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): 2000, 600, 200, 60 and 20 µg/plate

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle:Dimethyl Phenylethyl Carbinol found to be miscible in DMSO at a concentration of 50 mg/ml. No precipitation was observed in any reaction mixture at the concentrations from 2.38 to 0.059 mg/ml corresponding to final test concentrations from 5000 to 125 µg/plate. Hence 5000 µg/plate showing no precipitation was the highest concentration selected for the preliminary cytotoxicity study

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

sodium azide

other: Without S9:3-Methylmethane sulphonate -TA100 and TA102; ICR191 - TA97a With S9: 2-Aminoanthracene - TA1535

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 mins
- Expression time: 67 hours for Experiment No. 1 and 72 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 was 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

Test results 1

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 examined

Positive controls validity:

valid

Test results 2

Species / strain:

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

Remarks:

Experiment 2

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:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: DMPEC found to be miscible in DMSO at a concentration of 50 mg/ml. No precipitation was observed in any reaction mixture at the concentrations from 2.38 to 0.059 mg/ml corresponding to final test concentrations from 5000 to 125 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, formulated in dimethyl sulphoxide, were tested for toxicity to bacterial cells.

Plate Incorporation Method
Slight cytotoxicity was observed to the bacterial background lawn at the concentrations of 5000 and 4000 µg/plate, in the absence of metabolic activation. Moderate cytotoxicity was observed to the bacterial background lawn at the concentration of 5000 µg/plate, whereas slight cytotoxicity was observed at 4000 µg/plate in the presence of metabolic activation. Hence, 4000 µg/plate was selected as the maximum test concentration for the main study in both the presence and absence of metabolic activation.

Pre Incubation Method
Severe cytotoxicity was observed to the bacterial background lawn at concentrations of 3000 to 5000 µg/plate, slight cytotoxicity was observed at the concentration of 2000 µg/plate in the absence of metabolic activation. Severe cytotoxicity was observed to the bacterial background lawn at concentrations of 5000 and 4000 µg/plate, significant (very thin lawn) cytotoxicity was observed at the concentration of 3000 µg/plate and no cytotoxicity was observed from 2000 to 125 µg/plate in the presence of metabolic activation. Hence 2000 µg/plate was selected as the maximum test concentration for the main study in both the presence and absence of metabolic activation.

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 DMPEC is non-mutagenic in Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the presence and absence of sodium phenobarbitone and β-naphthoflavone-induced rat liver S9 metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (16937), strains of S. typhimurium TA1535, TA97a, TA98, TA100, TA102 were exposed to DMPEC (99.7%) in DMSO at concentrations of 4000, 1000, 400, 100, 40 and 0 μg/plate (direct plate incorporation; experiment 1) and 2000, 600, 200, 60, 20 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).

DMPEC was tested up to the limit concentration (5000 µg/plate). 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.