Acetic acid, C11-14-isoalkyl esters, C13-rich

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 July 2016 to 25 July 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only single strain was tested

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

Only single strain was tested to fulfil the criteria for Bacterial mutagenicity

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acetic acid, C11-14-isoalkyl esters, C13-rich

EC Number:

283-740-9

EC Name:

Acetic acid, C11-14-isoalkyl esters, C13-rich

Cas Number:

84712-50-5

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

Acetic acid, C11-14-isoalkyl esters, C13-rich

Test material form:

liquid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark
- Stability under test conditions: Not specified, assumed to be stable
- Solubility and stability of the test substance in the solvent/vehicle: The test material showed to be fully miscible in acetone at 100 mg/ml during solubility checks.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was accurately weighed and approximate half-log dilutions were prepared in acetone.
- Preliminary purification step (if any): No correction was made for purity.
- Final dilution of a dissolved solid, stock liquid or gel: Acetone was toxic to the bacterial cells at 0.1 ml after employing the pre-incubation modification. Therefore, all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. Analysis for concentration, homogeneity and stability of the test item formulations was not performed.

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

The S9 microsomal fraction was pre-pared using standardized in-house procedures

Test concentrations with justification for top dose:

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 15, 50, 150, 500, 1500 and 5000 µg/plate
5000 µg/plate was the maximum recommended dose level.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/ml, but was fully miscible in acetone at 100 mg/ml during solubility tests. Therefore, acetone was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

WP2uvrA, without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoantracene

Remarks:

WP2uvrA, with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) Experiment 1, preincubation method was used for Experiment 2
- Cell density at seeding (if applicable): an overnight sub-culture of the coded stock culture was prepared in nutrient broth and incubated at 37°C for approximately 10 hours.

ACTIVATION: The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of test. The S9-mix contained 5.0 ml S9, 1.0 ml of 1.65 M KCl/ 0.4 M MgCl2, 2.5 ml of 0.1 M glucose-6-phosphate, 2.0 ml of 0.1 M NADP, 25.0 ml of 0.2 M sodium phosphate buffer (pH 7.4), 14.5 ml of sterile distilled water.

DURATION
- Plate incorporation: Experiment 1
- Preincubation period: Experiment 2
- Exposure duration: Experiment 1, 48 hours at 37°C, with and without metabolic activation; Experiment 2, 20 min at 37°C, with and without metabolic activation
- Expression time (cells in growth medium): Experiment 2, 8 hours at 37°C, with and without metabolic activation

NUMBER OF REPLICATIONS: triplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: number of revertant colonies (cloning efficiency)


OTHER EXAMINATIONS:
- Precipitation

Rationale for test conditions:

Acetone was toxic to the bacterial cells at 0.1 ml after employing the pre-incubation modification. Therefore, all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. Analysis for concentration, homogeneity and stability of the test item formulations was not performed. 5000 µg/plate was the maximum recommended dose level.

Evaluation criteria:

The result was considered positive when one or all of the following was observed:
- A dose-related increase in mutant frequency over the dose range tested;
- A reproducible increase at one or more concentrations;
- Biological relevance against in-house historical control data;
- Statistical analysis of data as determined by UKEMS;
- Fold increase greater than two times the concurrent solvent control for the tester strain.

Statistics:

Dunnetts Regression Analysis (* = p < 0.05) was used to confirm statistical significance 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

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES: No data

HISTORICAL CONTROL DATA
- Positive historical control data: within the ranges of historical control data
- Negative (solvent/vehicle) historical control data: within the ranges of historical control data

Any other information on results incl. tables

Table 1: Mean number of revertants, Experiment 1 and 2, with and without metabolic activation

Concentration μL/plate	WP2 uvrA Experiment 1		WP2 uvrA Experiment 2
	+MA	-MA	+MA	-MA
Acetone	33	22	32	20
1.5	24	22
5	30	25
15	30	22	30	23
50	29	24	34	26
150	29	22	29	25
500	26	26	33	20
1500	30	23	30	26
5000	28	22	29	22
ENNG 2µg		880		1090
2AA 10 µg	300		226

Applicant's summary and conclusion

Conclusions:

Acetic acid, C11-14-isoalkyl esters, C13-rich has been tested in a valid bacterial reverse mutation assay, according to the OECD TG 471 with acceptable restrictions, and under GLP, using E. coli WPuvrA. No increase in the number of revertants was observed in the test strain, with or without metabolic activation. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.