2-(1-methylethoxy)ethyl acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Jul - 23 Aug 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(1-methylethoxy)ethyl acetate

EC Number:

242-901-3

EC Name:

2-(1-methylethoxy)ethyl acetate

Cas Number:

19234-20-9

Molecular formula:

C7H14O3

IUPAC Name:

2-(propan-2-yloxy)ethyl acetate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 57033
- Expiration date of the lot/batch: 25 November 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark.

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
First and second experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: the test item was fully miscible in sterile distilled water at 50 mg/mL.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG, 2, 3 and 5 µg/plate, -S9, WP2uvrA, TA 100 and TA1535, respectively); 9-Aminoacridine (9AA, 80 µg/plate, -S9, TA 1537), 4-Nitroquinoline-1-oxide (4NQO, 0.2 µg/plate, -S9, TA 98)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA, 1, 2 and 10 µg/plate, +S9, TA 100, TA 1535 and TA 1537 and WP2uvrA, respectively); Benzo(a)pyrene (BP, 5 µg/plate, +S9, TA 98)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection 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 concentration.
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 judgement about test item activity. Results of this type will be reported as equivocal.

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Test Results of Experiment 1 (plate incorporation).

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	0	115 ± 7.6	17 ± 9.2	29 ± 5.3	25 ± 4.0	12 ± 7.0
–	50	111 ± 11.5	17 ± 7.6	26 ± 6.1	23 ± 3.5	15 ± 2.6
–	150	115 ± 11.0	20 ± 6.1	31 ± 0.0	21 ± 2.1	12 ± 3.6
–	500	121 ± 14.6	14 ± 4.2	21 ± 0.0	18 ± 2.1	8 ± 4.0
–	1500	110 ± 7.5	26 ± 4.2	28 ± 8.0	25 ± 3.5	13 ± 1.5
–	5000	116 ± 4.7	19 ± 9.3	25 ± 7.8	25 ± 10	8 ± 4.5
Positive controls, –S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentrations (μg/plate)	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	475 ± 3.1	222 ± 28.4	690 ± 69.2	232 ± 25.2	763 ± 17.3
+	0	107 ± 15.1	14 ± 4.9	37 ± 10.1	19 ± 2.9	15 ± 3.5
+	50	103 ± 7.0	8 ± 0.6	43 ± 1.7	20 ± 1.2	10 ± 4.0
+	150	106 ± 1.7	10 ± 2.1	41 ± 7.4	25 ± 4.7	12 ± 3.6
+	500	109 ± 9.8	10 ± 1.2	42 ± 2.1	27 ± 7.1	14 ± 2.9
+	1500	93 ± 4.6	13 ± 4.0	35 ± 9.3	27 ± 8.4	14 ± 10.3
+	5000	87 ± 10.4	9 ± 1.5	34 ± 1.2	27 ± 3.8	18 ± 4.2
Positive controls, + S9	Name	2AA	2AA	2AA	BP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	1760 ± 16.6	278 ± 13.3	345 ± 48.8	166 ± 6.4	145 ± 62.2

ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine

4NGO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

Table 2: Test Results of Experiment 2 (preincubation).

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	0	91 ± 15.5	14 ± 4.0	31±2.5	30 ± 8.9	14 ± 3.1
–	50	84 ± 7.2	12 ± 1	33 ± 9.5	24 ± 8.1	10 ± 5.7
–	150	87 ± 1.0	16 ± 7.0	29 ± 9.2	29 ± 8.6	16 ± 8.3
–	500	83 ± 4.6	17 ± 2.3	27 ± 4.5	23 ± 2.5	8 ± 4.5
–	1500	83 ± 6.7	16 ± 1.0	27 ± 3.2	25 ± 4.7	9 ± 5.1
–	5000	98 ± 6.9	13 ± 2.5	28 ± 5.8	28 ± 7.5	13 ± 4.0
Positive controls, –S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentrations (μg/plate)	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	509 ± 120.5	256 ± 45.4	790 ± 12.5	191 ± 9.8	677 ± 25.1
±	0	97 ± 16.3	15 ± 2.3	37 ± 5.5	39 ± 6.5	11 ± 1.7
±	50	103 ± 4.0	15 ± 4.7	30 ± 9.5	24 ± 6.9	15 ± 1.7
±	150	102 ± 10.7	14 ± 3.1	33 ± 6.1	38 ± 6.7	13 ± 1.5
±	500	98 ± 15.9	16 ± 2.3	33 ± 8.2	37 ± 5.7	15 ± 2.5
±	1500	98 ± 22.9	13 ± 2.1	40 ± 7.6	28 ± 4.5	21 ± 4.9**
±	5000	109 ± 6.2	14 ± 1.2	32 ± 7.2	33 ± 9.7	17 ± 5.9
Positive controls, ± S9	Name	2AA	2AA	2AA	BP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	2092 ± 559.3	160 ± 20.2	339 ± 22.2	197 ± 20.0	188 ± 53.6

ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine

4NGO: 4-NItroquinoline-1-oxide

9AA: 9-Aminoacridine

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

** p≤0.01

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative