Hex-3-en-1-yl acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A with and without metabolic activation

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:

20 May - 8 Jun 2016

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:

adopted 21 Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

The pre-experiment is reported as Experiment 1.

Experiment 2:
33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA 98 and WP2 uvrA
10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for remaining strains

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 1 h
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

A test substance is considered as a mutagenic if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Mean values and standard deviations were calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1 and 2: +S9: at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: +S9: at 5000 µg/plate; Exp. 2: +S9 and -S9: starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1 and Exp. 2: +S9: at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: +S9: at 5000 µg/plate, -S9: starting at 2500 µg/plate; Exp. 2: +S9: starting at 2500 µg/plate, -S9: starting at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: +S9: at 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: No precipitation of the test substance occurred up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I (all strains were tested in the pre-experiment).

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains except WP2 uvrA. Reduced background growth was observed in Experiment 1 with metabolic activation in all strains at 5000 µg/plate. In Experiment 2 reduced background growth was noted in TA 1537 with and without metabolic activation starting at 2500 µg/plate and with metabolic activation in TA 1535 and TA 98 at 5000 µg/plate.

Table 1. Test results of Experiment 1 (plate incorporation).

With or without S9-Mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA	TA1537	TA98
-	0 (DMSO)	11 ± 1	182 ± 9	43 ± 10	8 ± 2	31 ± 6
-	0	11 ± 3	204 ± 23	35 ± 0	12 ± 3	39 ± 4
-	3	13 ± 3	169 ± 20	43 ± 6	7 ± 2	29 ± 6
-	10	12 ± 3	191 ± 12	46 ± 8	11 ± 3	25 ± 3
-	33	12 ± 4	182 ± 6	40 ± 5	11 ± 4	28 ± 4
-	100	12 ± 4	140 ± 19	52 ± 10	8 ± 5	26 ± 9
-	333	11 ± 5	158 ± 13	40 ± 9	10 ± 5	26 ± 6
-	1000	9 ± 2	89 ± 11	35 ± 8	9 ± 4	21 ± 5
-	2500	9 ± 3	78 ± 11	35 ± 7	8 ± 2	21 ± 5
-	5000	12 ± 2	63 ± 5	31 ± 6	9 ± 4	20 ± 2
Positive controls, –S9	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentration [μg/plate]	10	10	2.0 µL	50	10
	Mean No. of colonies/plate (average of 3 plates ± SD)	1114 ± 72	2068 ± 170	986 ± 80	91 ± 8	487 ± 51
+	0 (DMSO)	13 ± 1	174 ± 14	53 ± 11	16 ± 5	37 ± 6
+	0	10 ± 2	197 ± 2	52 ± 8	15 ± 1	42 ± 6
+	3	11 ± 5	167 ± 8	50 ± 10	13 ± 3	39 ± 11
+	10	11 ± 3	170 ± 16	49 ± 6	15 ± 3	41 ± 7
+	33	15 ± 1	160 ± 16	59 ± 8	13 ± 2	37 ± 3
+	100	13 ± 1	173 ± 10	52 ± 8	10 ± 5	40 ± 12
+	333	16 ± 2	173 ± 16	49 ± 12	15 ± 3	33 ± 12
+	1000	11 ± 1	175 ± 12	40 ± 8	17 ± 1	27 ± 5
+	2500	12 ± 6	146 ± 12	54 ± 8	16 ± 4	38 ± 10
+	5000	5 ± 2^M, R	59 ± 17^R	39 ± 8^R	7 ± 1^M, R	19 ± 3^M, R
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentration [μg/plate]	2.5	2.5	10	2.5	2.5
	Mean No. of colonies/plate (average of 3 plates ± SD)	379 ± 33	5047 ± 218	429 ± 36	279 ± 10	3480 ± 519

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

R: reduced background growth

 

Table 2. Test results of Experiment 2 (preincubation).

With or without S9-Mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA	TA1537	TA98
-	0 (DMSO)	12 ± 2	157 ± 7	41 ± 6	11 ± 4	19 ± 3
-	0	15 ± 4	182 ± 6	36 ± 4	14 ± 5	27 ± 4
-	10	11 ± 3	142 ± 6	-	8 ± 1	-
-	33	12 ± 2	152 ± 17	31 ± 4	9 ± 0	25 ± 5
-	100	11 ± 3	160 ± 6	38 ± 9	11 ± 5	27 ± 5
-	333	11 ± 2	133 ± 18	37 ± 8	10 ± 3	24 ± 6
-	1000	10 ± 1	69 ± 4	29 ± 3	6 ± 1	26 ± 4
-	2500	9 ± 3	69 ± 1	24 ± 1	3 ± 1^M, R	31 ± 2
-	5000	13 ± 3	68 ± 4	19 ± 4	1 ± 1^M, R	10 ± 3
Positive controls, –S9	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentration [μg/plate]	10	10	2.0 µL	50	10
	Mean No. of colonies/plate (average of 3 plates ± SD)	1034 ± 19	2017 ± 81	751 ± 41	72 ± 13	394 ± 27
+	0 (DMSO)	13 ± 3	157 ± 17	44 ± 3	16 ± 3	35 ± 9
+	0	12 ± 3	165 ± 21	50 ± 10	18 ± 1	41 ± 1
+	10	12 ± 6	140 ± 6	-	12 ± 2	-
+	33	11 ± 4	134 ± 17	51 ± 7	14 ± 3	34 ± 5
+	100	14 ± 2	146 ± 22	50 ± 6	14 ± 3	36 ± 8
+	333	14 ± 2	122 ± 7	50 ± 5	10 ± 5	36 ± 5
+	1000	14 ± 4	134 ± 6	40 ± 7	13 ± 5	32 ± 3
+	2500	13 ± 6	52 ± 12	41 ± 11	4 ± 1^M, R	25 ± 4
+	5000	8 ± 1^M, R	2 ± 2	36 ± 5	2 ± 2^M, R	5 ± 2^M, R
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentration [μg/plate]	2.5	2.5	10	2.5	2.5
	Mean No. of colonies/plate (average of 3 plates ± SD)	391 ± 33	3797 ± 405	432 ± 22	144 ± 2	5118 ± 426

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

R: reduced background growth

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation up to 5000 µg/plate.

Executive summary:

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2016). In this study the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) tested with and without metabolic activation up to 5000 µg/plate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2016). In this study the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) tested with and without metabolic activation up to 5000 µg/plate.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008.