4'-tert-butyl-2',6'-dimethyl-3',5'-dinitroacetophenone

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:

15 December 2016 - 07 March 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:

4'-tert-butyl-2',6'-dimethyl-3',5'-dinitroacetophenone

EC Number:

201-328-9

EC Name:

4'-tert-butyl-2',6'-dimethyl-3',5'-dinitroacetophenone

Cas Number:

81-14-1

Molecular formula:

C14H18N2O5

IUPAC Name:

1-(4-tert-butyl-2,6-dimethyl-3,5-dinitrophenyl)ethanone

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: International Flavors & Fragrances; 0008085963
- Expiration date of the lot/batch: 20 August 2018
- Purity: 99.8%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40 °C on the day of each experiment. No correction for purity was required. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period.

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:

Phenobarbital/β-naphthaflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation; + and -S9): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate

Experiment 1 (pre-incubation; + and -S9): 15, 50, 150, 500, 1500, 5000 μg/plate

Vehicle / solvent:

The test item was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation);
Experiment 2: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: Approximately 48 hours for both experiments

NUMBER OF REPLICATIONS: Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the growth of the bacterial background lawn

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS.
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response.
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Species / strain:

other: S. typhimurium strains TA1535, TA1537, TA98 and TA100; E. coli WP2uvrA

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:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

other: S. typhimurium strains TA1535, TA1537, TA98 and TA100; E. coli WP2uvrA

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:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Manual counts were performed at 5000 μg/plate because of test item precipitation (Experiment 1). Manual counts were performed at and above 1500 μg/plate because of test item precipitation. (Experiment 2).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data: The historical ranges of the positive control reference items for 2015 and 2016 are presented in Appendix 1.
- Negative (solvent/vehicle) historical control data: Combined historical negative and solvent control ranges for 2015 and 2016 are presented in Appendix 1.

Applicant's summary and conclusion

Conclusions:

In a reverse bacterial mutation assay (Ames test), musk ketone was negative with and without metabolic activatiion.

Executive summary:

In a reverse gene mutation assay in bacteria (QC11WT), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A were exposed to musk ketone (99.8%) in DMSO at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000μg/plate (plate incorporation; experiment 1) and 15, 50, 150, 500, 1500, 5000 μg/plate (20 minute pre-incubation; experiment 2) in the presence and absence of mammalian metabolic activation (Phenobarbital/β-naphthoflavone-induced rat liver S9).

Musk ketone 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.