(R)-6-(isopropyl)-3-methylcyclohex-2-en-1-one

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 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:

31 May - 05 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

/ During the study, 2-Aminoanthracene was used as sole indicator of the efficacy of the S9-mix

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

yes

Remarks:

/ During the study, 2-Aminoanthracene was used as sole indicator of the efficacy of the S9-mix

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 July 1992/ 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, tryp operon

Species / strain / cell type:

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

Remarks:

and 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 and β-naphthoflavone

Test concentrations with justification for top dose:

First experiment (dose-finding study):
all strains: 3 - 5000 μg/plate with and without metabolic activation (tested up to the limit concentration)

Second experiment:
TA 1535 and TA 1537: 10 - 5000 μg/plate with and without metabolic activation (tested up to the limit concentration)
TA 98, TA 100, WP 2 uvrA: 3 - 5000 μg/plate with and without metabolic activation (tested up to the limit concentration)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubility properties and its relative nontoxicity for bacteria.

Untreated negative controls:

yes

Remarks:

untreated

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: plate incorporation test (experiment I = pre-experiment) and preincubation (experiment II = main assay)

DURATION
- Preincubation period: 1 h (experiment I)
- Exposure duration: 48 h (exp. I and II)

NUMBER OF REPLICATIONS: The dose-finding assay and main assay were carried out in triplicate plating for each dose level.

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

Evaluation criteria:

Acceptance criteria
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of historical data
- the positive control substances induce a duplication (TA 98, TA 100, and WP2 uvrA) or triplication (TA 1535 and TA 1537) of the colony count compared to the corresponding solvent control
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5

Evaluation criteria
When the test substance shows a biologically relevant increase in the number of revertant colonies to at least twice (TA 98, TA 100, and WP2 uvrA) or three times (TA 1535 and TA 1537) as many as that of the solvent control, the response is judged to be positive. TA 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, if 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:

- S9: reduced background growth at ≥ 2500 µg/ plate; + S9: reduced background growth at ≥ 2500 µg/ plate and reduced number of revertant colonies 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:

- S9: reduced background growth at ≥ 2500 µg/ plate and reduced number of revertant colonies at ≥ 5000 µg/ plate; + S9: reduced background growth at ≥ 1000 µg/ plate and reduced number of revertant colonies at ≥ 2500 µg/ plate

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:

- S9: reduced background growth at ≥ 1000 µg/ plate and reduced number of revertant colonies at ≥ 5000 µg/ plate; + S9: reduced background growth at ≥ 1000 µg/ plate and reduced number of revertant colonies at ≥ 2500 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:

- S9: reduced background growth at ≥ 1000 µg/ plate and reduced number of revertant colonies at ≥ 2500 µg/ plate; + S9: reduced background growth and reduced number of revertant colonies 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:

- S9: reduced background growth at ≥ 2500 µg/ plate and reduced number of revertant colonies at ≥ 5000 µg/ plate; + S9: reduced background growth and reduced number of revertant colonies at ≥ 2500 µg/ plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
yes (for further details please refer to "Any other information on results incl. tables", Table 1)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
yes (for further details please refer to "Any other information on results incl. tables", Table 3)

Table 2: Summary of test results (experiment 1; dose-finding assay)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Frameshift type		Base-pair substitution type
		TA1537	TA98	TA100	TA1535	WP2 uvrA
–	Solvent control (DMSO)	7 ± 2	33 ± 6	169 ± 16	13 ± 3	44 ± 7
	Untreated control	12 ± 3	28 ± 2	173 ± 10	8 ± 5	42 ± 1
	3	8 ± 2	31 ± 8	149 ± 7	10 ± 5	36 ± 1
	10	9 ± 3	29 ± 4	167 ± 5	13 ± 3	38 ± 2
	33	7 ± 2	30 ± 5	185 ± 5	13 ± 2	39 ± 15
	100	9 ± 1	34 ± 3	160 ± 24	13 ± 3	43 ± 9
	333	10 ± 6	31 ± 5	159 ± 21	12 ± 2	38 ± 10
	1000	8 ± 2	39 ± 7	127 ± 32	15 ± 3	42 ± 3
	2500	4 ± 1	25 ± 9	65 ± 5	13 ± 6	41 ± 1
	5000	3 ± 1	11 ± 2	51 ± 4	11 ± 3	45 ± 6
	Positive controls (unit/plate)	4-NOPD (50 µg)	4-NOPD (10 µg)	SA (10 µg)	SA (10 µg)	MMS (2 µl)
	Mean No. of colonies/plate (average of 3 plates)	77 ± 3	427 ± 30	1999 ± 68	1253 ± 30	908 ± 42
+	Solvent control (DMSO)	14 ± 0	40 ± 6	178 ± 15	10 ± 4	52 ± 5
	Untreated control	19 ± 5	45 ± 2	195 ± 20	11 ± 4	49 ± 2
	3	15 ± 3	40 ± 7	169 ± 21	13 ± 3	53 ± 7
	10	16 ± 3	30 ± 5	186 ± 14	11 ± 5	52 ± 11
	33	13 ± 3	37 ± 5	164 ± 11	12 ± 5	49 ± 3
	100	16 ± 5	47 ± 7	171 ± 2	12 ± 4	53 ± 1
	333	15 ± 6	40 ± 7	164 ± 9	9 ± 3	48 ± 11
	1000	11 ± 4	37 ± 10	144 ± 9	12 ± 4	36 ± 12
	2500	14 ± 4	17 ± 1	58 ± 13	13 ± 1	23 ± 4
	5000	5 ± 2	6 ± 2	10 ± 3	6 ± 2	19 ± 3
	Positive controls (µg/plate)	2AA (2.5)	2AA (2.5)	2AA (2.5)	2AA (2.5)	2AA (10)
	Mean No. of colonies/plate (average of 3 plates)	224 ± 27	4313 ± 457	5077 ± 151	396 ± 13	478 ± 13

4-NOPD = 4-nitro-o-phenylene-diamine

SA = sodium azide

MMS = methyl methane sulfonate

2AA = 2-aminoanthracene

SD = standard deviation

 

Table 3:Summary of test results (experiment 2; main assay) 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Frameshift type		Base-pair substitution type
		TA1537	TA98	TA100	TA1535	WP2 uvrA
–	Solvent control (DMSO)	11 ± 1	28 ± 6	147 ± 6	8 ± 2	32 ± 6
	Untreated control	11 ± 1	30 ± 6	188 ± 12	10 ± 3	34 ± 2
	3	-	29 ± 7	138 ± 4	-	29 ± 5
	10	8 ± 1	26 ± 10	137 ± 11	13 ± 3	35 ± 2
	33	10 ± 1	31 ± 5	138 ± 19	10 ± 1	33 ± 0
	100	8 ± 3	30 ± 3	132 ± 23	11 ± 1	31 ± 3
	333	13 ± 3	38 ± 4	105 ± 6	9 ± 2	37 ± 5
	1000	13 ± 1	37 ± 9	81 ± 4	16 ± 4	36 ± 7
	2500	12 ± 3	29 ± 3	16 ± 1	13 ± 3	26 ± 6
	5000	4 ± 4	7 ± 4	0 ± 1	12 ± 4	12 ± 4
	Positive controls (unit/plate)	4-NOPD (50 µg)	4-NOPD (10 µg)	SA (10 µg)	SA (10 µg)	MMS (2 µl)
	Mean No. of colonies/plate (average of 3 plates)	87 ± 12	578 ± 35	1967 ± 33	1199 ± 32	443 ± 23
+	Solvent control (DMSO)	16 ± 0	35 ± 6	123 ± 19	12 ± 4	43 ± 6
	Untreated control	11 ± 3	34 ± 3	179 ± 14	13 ± 3	41 ± 5
	3		33 ± 3	120 ± 13		50 ± 7
	10	12 ± 4	44 ± 8	127 ± 16	11 ± 3	41 ± 1
	33	16 ± 3	38 ± 13	121 ± 9	10 ± 1	41 ± 6
	100	14 ± 2	39 ± 6	117 ± 13	8 ± 3	39 ± 3
	333	14 ± 3	42 ± 6	98 ± 3	9 ± 2	34 ± 6
	1000	15 ± 4	24 ± 2	46 ± 5	10 ± 3	40 ± 2
	2500	0 ± 0	15 ± 4	2 ± 2	7 ± 1	8 ± 3
	5000	0 ± 0	0 ± 0	0 ± 0	3 ± 3	0 ± 1
	Positive controls (µg/plate)	2AA (2.5)	2AA (2.5)	2AA (2.5)	2AA (2.5)	2AA (10)
	Mean No. of colonies/plate (average of 3 plates)	179 ± 19	4913 ± 498	3863 ± 364	378 ± 43	355 ± 22

 4-NOPD = 4-nitro-o-phenylene-diamine

SA = sodium azide

MMS = methyl methane sulfonate

2AA = 2-aminoanthracene

SD = standard deviation 

Conclusions:

Under the conditions of the Ames Assay the substance was not mutagenic in any of the five strains (TA 100, TA 1535, TA 98, TA 1537 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 uvrA) tested with and without metabolic activation.

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 uvrA) tested with and without metabolic activation.

Justification for classification or non-classification

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. Based on available data on genetic toxicity,the test item is not classifiedaccording to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.