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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutagenicity (Ames test): negative, with and without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 (OECD TG 471) (Huntingdon, 1989).

Bacterial mutagenicity (Ames test): negative,with and without metabolic activation in E. coli WPuvrA (OECD TG 471) (Envigo, 2016).

Link to relevant study records

Reference

Reference 1

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

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

Qualifier:

according to guideline

Guideline:

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

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

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.

Target gene:

histidine locus

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.

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

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.

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

6 June 1989 to 19 June 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The restriction is that no fifth strain to detect cross-linking mutagens was used in the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

no data

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: April 1990
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at 4°C in the dark
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle: soluble in ethanol
- 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:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:

Target gene:

histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254- -induced rat liver metabolic activation

Test concentrations with justification for top dose:

Dose range finding test: 5, 50, 500 and 5000 µg/plate
Main test: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537, without metabolic activation

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

TA100; TA1535, without metabolic activation

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Nitrofluorene

Remarks:

TA98, without metabolic activation

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoantracene

Remarks:

TA98, TA100, TA1535 and TA1537, with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in histidine deficient agar
- Cell density at seeding (if applicable):

ACTIVATION: S9 mix contained S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM).

DURATION
- Exposure duration: 3 days at 37°C, with and without metabolic activation, first and second mutation test
- Expression time (cells in growth medium): no data

NUMBER OF REPLICATIONS: triplicate cultures

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

OTHER EXAMINATIONS: None

Rationale for test conditions:

No toxicity was observed in the preliminary study. Therefore, 5000 µg/plate was the highest dose tested in the main study.

Evaluation criteria:

If treatment with a test material produced an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix was considered positive for mutagenic activity.

Statistics:

Not used.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no data

RANGE-FINDING/SCREENING STUDIES: No toxic or mutagenic potential was observed in any of the test strain at any of the test concentrations.

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

Table 1: Test 1, mean number of revertants

Concentration µg/plate	TA 1535	TA 1535		TA 1537	TA 1537	TA 98	TA 98	TA 100	TA 100
	+MA		-MA	+MA	-MA	+MA	-MA	+MA	-MA
5000 µg	11		12	9	7	15	13	69	122
1500 µg	12		9	10	10	18	19	78	119
500 µg	8		7	9	9	19	17	82	110
150 µg	13		8	10	9	21	18	95	118
50 µg	12		13	10	9	21	17	115	116
0 µg	15		8	8	7	20	18	114	144
ethanol	12		11	9	11	21	18	119	115
ENNG 5.0			120
ENNG 3.0									350
9 AC 80.0				104
NF 1.0						350
AA 2.0	101			40
AA 0.5						126
AA 1.0								618

Table 2: Test 2, mean number of revertants

Concentration µg/plate	TA 1535	TA 1535		TA 1537	TA 1537	TA 98	TA 98	TA 100	TA 100
	+MA		-MA	+MA	-MA	+MA	-MA	+MA	-MA
5000 µg	5		5	6	4	14	18	104	103
1500 µg	4		47	5	7	19	14	108	91
500 µg	4		7	4	4	18	16	114	93
150 µg	7		4	4	6	23	14	68	78
50 µg	7		8	2	4	17	16	123	87
0 µg	7		7	7	8	15	18	141	86
ethanol	9		12	3	3	13	10	81	73
ENNG 5.0			201
ENNG 3.0									386
9 AC 80.0					685
NF 1.0							82
AA 2.0	142			103
AA 0.5						307
AA 1.0								460

Conclusions:

2,4,6,8-Tetramethyl nonyl acetate has been tested in a valid bacterial reverse mutation assay, according to the OECD TG 471 with acceptable restrictions, and under GLP, using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537. No increase in the number of revertants was observed in any 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

2,4,6,8-Tetramethyl nonyl acetate (complex mixture of isomers), a structural analogue of 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 Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 (Huntingdon, 1989). The restrictions were that fifth strain to detect cross-linking mutations was not used in the study. No increase in the number of revertants was observed in any test strain, with or without metabolic activation. Appropriate positive, negative and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

To fulfil the requirements of the guideline, a second study in bacteria was conducted with the registered (target) substance in order to assess its mutagenic activity in a fifth strain E. coli WPuvrA.

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 (Envigo, 2016). 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.

Justification for classification or non-classification

Based on the weight of evidence for Acetic acid, C11-14-isoalkyl esters, C13-rich, no classification is required for genetic toxicity according to Regulation (EC) No. 1272/2008.