Ethanol, 2-(ethenylthio)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: No mutagenic effect was detected.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-03-08 to 2016-03-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

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
- Lot/batch No.of test material: 230-2015
- Expiration date of the lot/batch: 17 Nov 2017

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat livers induced with phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
In agreement with the recommendations of current guidelines 5 mg/plate were selected as maximum test dose at least in the 1st Experiment.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

with metabolic activation, strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

without metabolic activation, strains: TA 1535, TA 100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NOPD)

Remarks:

without metabolic activation, strain: TA 98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation, strain: TA 1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation, strain: E. coli WP2 uvrA

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: about 20 minutes
- Exposure duration: 48 – 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn

Evaluation criteria:

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- Fresh bacterial culture containing approximately 10E^9 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Statistics:

No statistical analysis performed.

Key result

Species / strain:

other: Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate or preincubation test up to the highest required concentration.

Experiment 1 (standard plate test):

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA1535	TA100	TA1537	TA98	WP2 uvrA
	Results without S9
DMSO	9.7 ± 3.1	92.7 ± 3.5	6 ± 2.6	24.3 ± 5	24.3 ± 3.8
33	11.7 ± 1.5	99.7 ± 11	7.7 ± 2.5	26.3 ± 1.5	25 ± 11.5
100	11.3 ± 6.7	97.7 ± 13.6	9.3 ± 2.5	19.3 ± 4.9	26.7 ± 8.5
333	12 ± 1	109.7 ± 15.6	9.3 ± 2.1	30.3 ± 4.6	23.7 ± 3.2
1000	14.3 ± 3.5	90 ± 3.6	13 ± 5.3	28 ± 8.2	25.7 ± 2.5
2500	8.7 ± 4	101.3 ± 4.7	7 ± 3.6	28.7 ± 6.4	22.3 ± 6.5
5000	15.3 ± 3.8	118.7 ± 25.1	6.3 ± 1.2	28.7 ± 2.3	31.7 ± 1.2
MNNG (5.0)	5349.3 ± 493.9	5244 ± 266.4
AAC (100)			342.7 ± 263.6
NOPD (10)				902 ± 60.1
4-NQO (5)					1366.3 ± 42.6
	Results with S9
DMSO	12 ± 2.6	101.3 ± 12	9.7 ± 1.2	31.7 ± 6	34.7 ± 6
33	14 ± 1.7	104.7 ± 13.7	9.3 ± 4.5	36.3 ± 2.1	32 ± 7.2
100	16.7 ± 9.9	116 ± 12.5	9 ± 2.6	27.3 ± 2.5	21 ± 5.6
333	12.7 ± 5.5	104 ± 13	11.3 ± 4.2	32.3 ± 5.1	23 ± 3.5
1000	11 ± 6	103.7 ± 4.5	12.3 ± 2.1	33.3 ± 6.4	33 ± 10.4
2500	15.3 ± 5.5	86 ± 14.8	12.7 ± 1.5	35.3 ± 1.5	43.3 ± 12.7
5000	15.3 ± 3.5	138.3 ± 14.6	12 ± 3	31.3 ± 6.7	35 ± 11.5
2-AA (2.5)	221.7 ± 22	2170.3 ± 95.7	149.7 ± 8	1595 ± 182.6
2-AA (60.0)					95.7 ± 8.3

 

Experiment 2 (preincubation test):

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA1535	TA100	TA1537	TA98	WP2 uvrA
	Results without S9
DMSO	9 ± 2.6	90.3 ± 10.1	9.7 ± 2.9	17.7 ± 3.2	23.7 ± 2.1
33	6 ± 2	83 ± 5	5 ± 1	16 ± 4.6	20.7 ± 3.2
100	10.7 ± 2.9	90.3 ± 15.9	5.3 ± 1.5	20.3 ± 1.5	21.7 ± 5.1
333	8 ± 2.6	100.3 ± 9.1	11.3 ± 4.5	20 ± 3	23.3 ± 5.5
1000	10.7 ± 5.5	87.7 ± 2.5	6 ± 2	17.7 ± 2.5	25 ± 8.7
2500	6.7 ± 4.5	111 ± 3.6	10 ± 2.6	24.3 ± 3.2	27.7 ± 6.5
5000	11 ± 1.7	120 ± 13.1	8.7 ± 3.5	13.7 ± 7.1	21.7 ± 5.5
MNNG (5.0)	4131 ± 382.4	3850.7 ± 46.8
AAC (100)			1227.7 ± 53.5
NOPD (10)				965 ± 9.5
4-NQO (5)					651 ± 14.4
	Results with S9
DMSO	9.3 ± 2.1	99.7 ± 9.1	10.7 ± 0.6	27.3 ± 8.1	27.3 ± 4
33	7.3 ± 3.1	91 ± 6.1	12.3 ± 2.3	23 ± 2.6	30.7 ± 8
100	9.3 ± 1.5	91.3 ± 9.5	13.7 ± 3.1	36 ± 3.6	26.7 ± 2.1
333	7.3 ± 3.1	92.3 ± 15.5	10 ± 2	25.7 ± 5.9	27 ± 8.7
1000	10.3 ± 1.5	92.3 ± 20.6	8 ± 2.6	30.3 ± 3.1	27.3 ± 1.2
2500	6.7 ± 3.5	112.7 ± 10	13.7 ± 1.2	31.7 ± 2.9	28 ± 5.6
5000	10.3 ± 2.9	130.7 ± 2.5	11.3 ± 3.5	25.7 ± 7.8	28.3 ± 9.9
2-AA (2.5)	107.7 ± 11.9	1173 ± 15.7	79.3 ± 17.7	903.3 ± 181
2-AA (60.0)					93.3 ± 16.2

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames

The study tested the mutagenicity of the test substance. The study was conducted according to OECD TG 471. The standard plate and preincubation tests were performed. Both were done with and without the addition of a metabolizing system obtained from rat liver (S-9 mix) using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. No cytotoxicity was observed independent on strain and experimental conditions. All tests and strains showed a negative effect for genotoxicity. Therefore the test substance was considered non mutagenic in this study under the corresponding conditions.

Justification for classification or non-classification

Genetic toxicity

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218. As a result the substance is considered to be not classified as mutagenic.