2-Hepten-4-one, 5-methyl-, (2E)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA 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:

16 Feb - 15 Mar 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 July 1997

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

Since the criteria of the acceptability of the assay (minimum of five analyzable doses) were not met in Experiment 2, the experiment was repeated and named as Experiment 2a.

Experiment 2a: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

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

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

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

Remarks:

+S9: 2-AA (2.5 µg/plate, TA1535, TA1537, TA98, TA100; 10 µg/plate, WP2 uvrA); -S9: sodium azide (10 µg/plate, TA1535, TA100); 4-NOPD (10 µg/plate, TA98; 50 µg/plate, TA1537); MMS (2 µL/plate, WP2 uvrA)

Details on test system and experimental conditions:

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

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

NUMBER OF REPLICATIONS: 3 replications each in 3 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 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. 2 and 2a: starting at 2500 µg/plate with and without S9 mix

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. 2 and 2a: starting at 2500 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

exp. 2 and 2a: starting at 2500 µg/plate with and without S9 mix

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. 2 and 2a: starting at 1000 µg/plate with and without S9 mix

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. 2: starting at 2500 µg/plate with and without S9 mix; exp. 2a: starting at 2500 µg/plate with S9 mix and at 5000 µg/plate without S9 mix)

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 with and without metabolic activation in Experiment 2 and 2a.

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 ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA	TA1537	TA98
-	0 (DMSO)	11 ± 4	152 ± 7	39 ± 9	10 ± 3	28 ± 5
-	0	10 ± 3	180 ± 4	46 ± 14	10 ± 3	32 ± 4
-	3	12 ± 6	147 ± 16	32 ± 7	8 ± 2	23 ± 3
-	10	12 ± 5	140 ± 7	48 ± 12	10 ± 6	23 ± 7
-	33	12 ± 2	169 ± 10	36 ± 1	10 ± 4	24 ± 6
-	100	13 ± 3	174 ± 26	41 ± 1	9 ± 3	28 ± 3
-	333	10 ± 2	190 ± 25	48 ± 8	10 ± 2	31 ± 10
-	1000	10 ± 4	147 ± 13	39 ± 7	8 ± 3	32 ± 10
-	2500	5 ± 0	94 ± 27	43 ± 4	6 ± 1	19 ± 3
-	5000	10 ± 5	178 ± 19	52 ± 8	4 ± 2	21 ± 4
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 ± SD)	1205 ± 64	2375 ± 120	1045 ± 48	91 ± 15	387 ± 7
+	0 (DMSO)	13 ± 4	125 ± 16	50 ± 11	9 ± 1	32 ± 9
+	0	14 ± 4	175 ± 26	51 ± 10	9 ± 1	42 ± 8
+	3	14 ± 4	112 ± 14	43 ± 13	11 ± 1	40 ± 3
+	10	14 ± 4	109 ± 17	48 ± 9	11 ± 6	37 ± 6
+	33	15 ± 1	122 ± 15	44 ± 1	7 ± 3	31 ± 2
+	100	13 ± 6	132 ± 16	47 ± 5	12 ± 3	33 ± 9
+	333	13 ± 1	142 ± 14	49 ± 4	10 ± 3	34 ± 2
+	1000	14 ± 5	159 ± 17	60 ± 5	7 ± 3	30 ± 10
+	2500	14 ± 3	93 ± 25	69 ± 7	5 ± 1	25 ± 6
+	5000	15 ± 1	158 ± 11	49 ± 7	9 ± 5	37 ± 15
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 ± SD)	433 ± 44	3819 ± 36	359 ± 37	198 ± 26	5032 ± 617

DMSO: dimethyl sulfoxide

NaN3: sodium azide

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

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

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 ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA	TA1537	TA98
-	0 (DMSO)	9 ± 1	128 ± 8	42 ± 4	9 ± 1	23 ± 4
-	0	7 ± 3	194 ± 15	37 ± 7	8 ± 3	24 ± 8
-	33	11 ± 3	143 ± 6	32 ± 4	8 ± 2	27 ± 4
-	100	8 ± 2	130 ± 17	39 ± 6	11 ± 5	18 ± 3
-	333	10 ± 3	117 ± 24	38 ± 12	11 ± 1	26 ± 10
-	1000	9 ± 1	74 ± 6	34 ± 4	10 ± 3	24 ± 6
-	2500	0 ± 0	1 ± 1	1 ± 1	2 ± 1	1 ± 1
-	5000	1 ± 2	0 ± 0	1 ± 1	1 ± 1	0 ± 1
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 ± SD)	1118 ± 46	1066 ± 73	639 ± 49	93 ± 14	424 ± 21
+	0 (DMSO)	12 ± 5	96 ± 7	45 ± 5	16 ± 4	35 ± 7
+	0	10 ± 3	189 ± 12	51 ± 3	11 ± 5	43 ± 9
+	33	12 ± 6	93 ± 13	53 ± 7	17 ± 4	33 ± 5
+	100	9 ± 3	103 ± 28	49 ± 9	12 ± 1	23 ± 4
+	333	13 ± 3	83 ± 22	46 ± 10	17 ± 5	29 ± 7
+	1000	7 ± 3	42 ± 14	35 ± 6	12 ± 4	34 ± 13
+	2500	1 ± 1	2 ± 2^R	1 ± 1	0 ± 0^R	4 ± 2^R
+	5000	1 ± 1	1 ± 1^R	0 ± 0	0 ± 0^R	1 ± 1^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 ± SD)	422 ± 6	1606 ± 371	501 ± 38	83 ± 11	4628 ± 345

DMSO: dimethyl sulfoxide

NaN3: sodium azide

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

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

R: reduced background growth

Conclusions:

Under the conditions of the conducted Ames 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.

Executive summary:

The mutagenicity of the test substance was studied with four mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and the Escherichia coli strain WP2 uvrA according to OECD 471. The investigations were carried out using the plate incorporation test and the pre-incubation test with and without liver homogenate (S9). In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. In conclusion, these results indicate that the test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and the Escherichia coli strain WP2 uvrA in the presence and absence of a metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

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 uvrA) tested with and without metabolic activation.

Justification for classification or non-classification

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.