Reaction mass of (3R)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]-1-butanol and (3S)-3-[(1R)-4-methyl-3-cyclohexen-1-yl]-1-butanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD 471, Bowles 2007

Link to relevant study records

Reference

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:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, performed under GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate.
Experiment 1 (range-finding test): 0, 1.5, 5, 15, 50, 150, 500 and 1500 ug/plate.
Experiment 2 (main test): 0, 5, 15, 50, 150, 500 and 1500 ug/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): 48 hours at 37 degrees C

NUMBER OF REPLICATIONS: triplicate

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Species / strain:

E. coli WP2 uvr A

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

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Small but statistically significant increases in revertant colony frequency were observed in TA 100 (with and without S9-mix) and TA1537 (without S9) in the range-finding test only. The increases were all within the range specified by the Standard Test Method, did not exceed 2 fold over the concurrent solvent control and did not exhibit a dose response relationship. Therefore, these increases were considered to be of no toxicological relevance.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Range finding test results

Range-finding test with and without S9
		Number of revertants (mean number of colonies per plate)
S9 Mix	Test substance concentration (ug/plate)	TA100	Mean	TA1535	Mean	WP2uvrA-	Mean	TA98	Mean	TA1537	Mean
-	0	81	78	23	20	35	27	22	44	10	8
		73		18		20		80		9
		79		18		25		29		5
-	1.5	76	79	26	25	14	22	22	22	5	6
		69		26		29		19		7
		93		23		22		25		5
-	5	85	86	14	19	18	21	24	24	9	9
		85		21		24		23		10
		87		21		20		25		9
-	15	92	97	25	24	30	30	30	24	11	8
		93		26		C		19		8
		107		20		29		24		5
-	50	76	84	19	25	26	28	20	21	9	8
		78		32		24		22		11
		97		25		33		21		5
-	150	96	91	19	16	23	24	13	17	9	11
		96		14		24		18		9
		81		16		26		19		15
-	500	97	100	25 T	23	18	20	19	18	19 T	16
		101		12 T		15		18		16 T
		101		32 T		27		16		12 T
-	1500	0T	0	0 T	0	0 T	0	0 T	0	0 T	0
		0T		0 T		0 T		0 T		0 T
		0T		0 T		0 T		0 T		0 T
+	0	63	66	11	11	27	30	23	20	9	9
		66		11		31		20		10
		68		10		31		18		8
+	1.5	68	71	5	6	33	28	25	20	9	10
		73		5		26		15		7
		71		8		25		19		15
+	5	91	86	8	7	27	29	18	22	10	10
		84		3		26		15		10
		82		10		35		33		9
+	15	80	87	3	4	16	24	21	21	18	15
		90		5		36		22		13
		91		4		20		20		15
+	50	73	66	9	7	27	33	23	22	18	11
		65		3		32		23		9
		59		9		40		21		7
+	150	62	71	5	8	31	35	16	20	14	11
		79		9		31		26		8
		73		11		44		19		10
+	500	58	69	4	5	32	26	20	14	4	6
		C		8		24		9		8
		80		3		23		14		7
+	1500	0 T	0	0 T	0	0 T	0	0 T	0	0 T	0
		0 T		0 T		0 T		0 T		0 T
		0 T		0 T		0 T		0 T		0 T
Positive controls		ENNG		ENNG		ENNG		4NQO		9AA
Concentration (ug/plate)		3		5		2		0.2		80
without S9		416	453	101	108	473	457	607	585	2251	1923
		487		101		505		564		1914
		457		122		392		585		1605
Positive controls		2AA		2AA		2AA		BP		2AA
Concentration (ug/plate)		1		2		10		5		2
With S9		1519	1591	227	210	477	472	232	204	415	303
		1677		191		431		169		227
		1576		211		507		210		267

Table 2. Main test results

Main test with and without S9
		Number of revertants (mean number of colonies per plate)
S9 Mix	Test substance concentration (ug/plate)	TA100	Mean	TA1535	Mean	WP2uvrA-	Mean	TA98	Mean	TA1537	Mean
-	0	97	91	15	20	39	32	16	15	9	14
		88		22		33		11		9
		88		22		25		18		24
-	5	94	88	25	23	31	26	16	16	9	13
		84		18		19		13		9
		85		26		27		20		21
-	15	94	87	20	22	20	26	23	16	7	8
		87		28		28		14		7
		80		17		31		11		9
-	50	100	90	17	18	11	16	10	12	11	13
		97		18		22		8		19
		73		19		16		18		8
-	150	88	83	14	20	18	24	26	20	21	15
		69		23		27		14		8
		93		23		27		19		15
-	500	77	69	13	12	15	20	11	11	6 T	7
		63		7		22		10		7 T
		66		16		22		11		8 T
-	1500	0 T	0	0 T	0	0 T	0	0 T	0	0 T	0
		0 T		0 T		0 T		0 T		0 T
		0 T		0 T		0 T		0 T		0 T
+	0	81	75	7	10	25	29	37	28	3	7
		64		9		36		22		8
		79		13		26		25		10
+	5	68	75	14	12	28	23	28	22	8	6
		69		13		25		22		5
		87		9		15		17		6
+	15	87	78	10	9	34	31	21	21	11	10
		66		8		26		18		4
		82		10		33		24		14
+	50	55	66	11	10	25	28	17	20	8	8
		73		11		32		19		7
		71		8		28		23		8
+	150	77	76	10	9	26	28	19	25	11	7
		84		10		27		30		6
		67		7		30		25		4
+	500	64	60	6 T	10	25	26	24	25	19	10
		53		9 T		22		24		4
		64		14 T		31		27		8
+	1500	0 T	0	0 T	0	0 T	0	0 T	0	0 T	0
		0 T		0 T		0 T		0 T		0 T
		0 T		0 T		0 T		0 T		0 T
Positive controls		ENNG		ENNG		ENNG		4NQO		9AA
Concentration (ug/plate)		3		5		2		0.2		80
without S9		708	703	242	248	528	560	492	522	703	684
		694		269		580		587		638
		706		233		572		576		712
Positive controls		2AA		2AA		2AA		BP		2AA
Concentration (ug/plate)		1		2		10		5		2
With S9		687	653	242	243	539	523	231	240	510	492
		633		239		522		244		459
		639		249		507		244		506

T: Partial or complete absence of bacterial background lawn.

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this test the test material was considered to be non-mutagenic.

Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B13/14 and the EPA (TSCA) OPPTS harmonised guidelines under GLP conditions, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA in both the presence and absence of S-9 mix. A preliminary toxicity test and range-finding test was performed to determine the test concentrations for main study. The test material caused a visible reduction in the growth of the bacterial background lawn to the tester strains used (TA100 and WP2uvrA-), initially at and above 500 ug/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate or its toxic limit depending on strain type. In the main study, the plate incorporation method was used and the test substance was evaluated at a concentration of up to 1500 ug/plate. Positive controls appropriate for each strain, in the presence and absence of S9 -mix, were included. The test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The sensitivity of the test system and the metabolic activity of the S9-mix were demonstrated by the increases in the numbers of revertant colonies induced by the positive control substances. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2 uvrA in the presence and absence of S-9 mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

OECD 471, 2007 - The study was performed to the requirements of OECD Guideline 471, EU Method B13/14 and the EPA (TSCA) OPPTS harmonised guidelines under GLP conditions, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA in both the presence and absence of S-9 mix. A preliminary toxicity test and range-finding test was performed to determine the test concentrations for main study. The test material caused a visible reduction in the growth of the bacterial background lawn to the tester strains used (TA100 and WP2uvrA-), initially at and above 500 ug/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate or its toxic limit depending on strain type. In the main study, the plate incorporation method was used and the test substance was evaluated at a concentration of up to 1500 ug/plate. Positive controls appropriate for each strain, in the presence and absence of S9 -mix, were included. The test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The sensitivity of the test system and the metabolic activity of the S9-mix were demonstrated by the increases in the numbers of revertant colonies induced by the positive control substances. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2 uvrA in the presence and absence of S-9 mix.

Justification for selection of genetic toxicity endpoint
Study selected is an in vitro study (Klimisch 1)

Justification for classification or non-classification

The substance does not meet classification criteria under EU Directive 67/548/EEC for mutagenicity

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity