Cyclohexanone, 2-methyl-4-(1,1,2-trimethylpropyl)-, cis-

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:

14 April - 28 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Protocol for the study was developed in accordance with the OECD guideline on the bacterial reverse mutation test. Study is performed by a GLP accredited laboratory.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Reaction mass of (2R,4R)-4-(2,3-dimethylbutan-2-yl)-2-methylcyclohexanone and (2S,4S)-4-(2,3-dimethylbutan-2-yl)-2-methylcyclohexanone

EC Number:

938-347-3

Cas Number:

28068-91-9

Molecular formula:

C13H24O

IUPAC Name:

Reaction mass of (2R,4R)-4-(2,3-dimethylbutan-2-yl)-2-methylcyclohexanone and (2S,4S)-4-(2,3-dimethylbutan-2-yl)-2-methylcyclohexanone

Details on test material:

- Name of test material (as cited in study report): Iriswood
- Substance type: pure active substance
- Physical state: undercooled liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Bacterial strains were exposed to the test system at 0.06, 0.19, 0.56, 1.67, and 5 µl/plate. The lower concentrations were prepared by 1:3 serial
dilutions of the highest concentration.

Vehicle / solvent:

96% ethanol

Controls

Untreated negative controls:

yes

Remarks:

vehicle of test substance

Negative solvent / vehicle controls:

yes

Remarks:

ethanol 96%

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see details of test system and conditions.

Details on test system and experimental conditions:

The bacterial strains used for the study were grown from controlled Working Banks obtained from Master Banks (generated in Vivotecnia) in nutrient broth supplemented with the corresponding antibiotics when required.
Inoculums were liquid grown overnight until the late exponential-early stationary phase of growth was reached (approximately 1.2-1.4 OD at 660nm). This OD indicates that bacteria are growing in the late exponential or early stationary phase of growth (approximately 1-2x109 CFU/mL).

Evaluation criteria:

The Samonella typhimurium and the Escherichia coli reverse mutation assays are considered acceptable if:
1) the vehicle control is within historical range in each strain.
2) the responses of the positive controls are in historical range.

Statistics:

The test substance was considered mutagenic if for any strain the total number of revertants in tester strain TA98, TA100 ,WP2(pKM101) is greater than two times the concurrent control and the total number of revertants in tester strains TA1535, TA1537 is greater than three times the concurrent control.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Species / strain:

S. typhimurium TA 98

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

Test results 4

Species / strain:

S. typhimurium TA 100

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

Test results 5

Species / strain:

E. coli WP2 uvr A pKM 101

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

Additional information on results:

No cytotoxicity was observed at the maximum test substance concentration (Table 2).
None of the concentrations showed an increase in the colony count (R value) either with or without S9 metabolic activation.
No dose-response for the test substance was observed in any of the bacterial strains tested.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2 Mean results of the test substance cytotoxicity assay (10 -12 April 2012) with S. typhimurium TA100. The test substance was dissolved in 96% ethanol.

µl/plate	revertants /plate	s.d.	R
0 (solvent)	84	5.6
0.06	72	8.3	0.9
0.19	66	1.7	0.8
0.56	68	6.4	0.8
1.67	77	3.6	0.9
5.00	71	5.6	0.8

Table 2a Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli strain. Mean number of revertant colonies per three replicate plate (± standard deviation) without metabolic activation - direct incorporation.

	TA98				TA100				TA1535				TA1537				WP2uvrA
	revertants / plate	SD	R		revertants / plate	SD	R		revertants / plate	SD	R		revertants / plate	SD	R		revertants / plate	SD	R
solvent control	19	5.3			90	9.0			19	4.9			6	3.2			239	36.8
positive control	395	44.3	20.8		826	37.7	9.1		859	61.3	46.0		182	17.8	28.7		1994	204.0	8.4
5	16	4.2	0.9		70	8.7	0.8		22	8.7	1.2		7	2.5	1.2		261	42.7	1.1
1.67	21	8.3	1.1		82	4.6	0.9		18	8.5	1.0		6	2.1	1.0		301	31.5	1.3
0.56	23	4.5	1.2		85	10.4	0.9		20	6.1	1.1		8	2.6	1.3		266	40.0	1.1
0.19	18	1.7	0.9		78	14.0	0.9		16	3.6	0.9		7	3.8	1.1		272	19.6	1.1
0.06	19	5.8	1.0		88	9.0	1.0		17	1.5	0.9		9	1.5	1.4		275	33.7	1.2

Table 2b Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli strain. Mean number of revertant colonies per three replicate plate (± standard deviation) without metabolic activation - pre-incubation.

	TA98				TA100				TA1535				TA1537				WP2uvrA
	revertants / plate	SD	R		revertants / plate	SD	R		revertants / plate	SD	R		revertants / plate	SD	R		revertants / plate	SD	R
solvent control	22	1.7			81	9.5			26	2.0			5	1.5			244	37.2
positive control	335	21.2	15.2		877	40.1	10.8		910	51.9	35.0		170	18.6	31.9		2162	121.7	8.9
5	21	2.3	1.0		77	3.1	0.9		14	2.3	0.5		8	0.6	1.4		260	42.7	1.1
1.67	20	4.7	0.9		69	6.6	0.9		15	1.5	0.6		4	0.6	0.8		310	38.1	1.3
0.56	17	6.7	0.8		75	11.4	0.9		17	3.5	0.7		9	3.5	1.6		296	20.4	1.2
0.19	19	5.7	0.8		78	7.4	1.0		23	7.5	0.9		4	0.6	0.8		280	17.5	1.1
0.06	20	2.1	0.9		78	14.0	1.0		16	3.5	0.6		6	2.1	1.1		238	24.7	1.0

Table 3a Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli strain. Mean number of revertant colonies per three replicate plate (± standard deviation) with metabolic activation - direct incorporation.

	TA98				TA100				TA1535				TA1537				WP2uvrA
	revertants / plate	SD	R		revertants / plate	SD	R		revertants / plate	SD	R		revertants / plate	SD	R		revertants / plate	SD	R
solvent control	18	2.6			73	5.7			19	4.0			7	2.3			265	19.4
positive control	727	19.7	40.4		1751	79.8	23.9		354	41.2	18.3		208	15.3	31.2		1952	16.9	7.4
5	15	2.5	0.9		73	4.5	1.0		19	0.0	1.0		8	3.5	1.2		254	62.6	1.0
1.67	22	2.1	1.2		65	9.7	0.9		18	2.5	0.9		6	1.2	1.0		267	16.7	1.0
0.56	22	2.5	1.2		70	7.5	1.0		20	3.5	1.1		5	1.2	0.8		217	37.4	0.8
0.19	27	5.0	1.5		74	5.9	1.0		17	2.0	0.9		6	0.6	0.9		260	35.0	1.0
0.06	19	3.2	1.1		80	20.5	1.1		15	3.2	0.8		6	1.2	1.0		256	31.7	1.0

Table 3b Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli strain. Mean number of revertant colonies per three replicate plate (± standard deviation) without metabolic activation - pre-incubation.

	TA98				TA100				TA1535				TA1537				WP2uvrA
	revertants / plate	SD	R		revertants / plate	SD	R		revertants / plate	SD	R		revertants / plate	SD	R		revertants / plate	SD	R
solvent control	23	3.2			82	18.2			20	7.0			6	4.5			238	28.0
positive control	615	23.9	26.3		1477	56.0	18.1		455	66.2	22.8		167	10.7	26.3		2164	131.9	9.1
5	18	6.0	0.8		106	12.4	1.3		21	7.2	1.1		6	2.0	0.9		260	29.4	1.1
1.67	17	1.2	0.7		89	13.0	1.1		20	6.1	1.0		8	4.6	1.3		296	50.5	1.2
0.56	16	5.9	0.7		76	4.0	0.9		18	6.8	0.9		7	1.5	1.2		297	23.1	1.2
0.19	16	1.2	0.7		75	3.8	0.9		20	3.1	1.0		6	1.0	0.9		272	28.0	1.1
0.06	18	3.8	0.8		100	7.5	1.2		17	6.2	0.9		9	2.0	1.4		274	16.1	1.1

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Based on the results, it is concluded that the test substance is not mutagenic in the Salmonella typhimurium and the Escherichia coli reverse mutation assay.

Executive summary:

The bacterial reverse mutation test (Ames test) assesses the mutagenic or promutagenic potential of the test substance in the several bacterial strains. The test was performed in accordance with OECD Guideline 471. No cytotoxic activity was observed at a test item concentration of 5μL/plate. Five test substance doses ranging from 5.00 and 0.06 μL/plate were assayed. None of the concentrations showed an increase in the colony counting either with or without S9 metabolic activation regardless of the exposure procedure. No dose response for the test substance was observed in any of the tested bacterial strains. Based on the results obtained in this study, it can be concluded that the test item does not induce point mutations or frame-shifts in the genome of the bacterial strains with or without metabolic activation regardless of the procedure. Therefore, the test substance is considered to be NON MUTAGENIC / NON PRO-MUTAGENIC under the experimental conditions assayed.