Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-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 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:

08 Mar - 05 Apr 2017

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:

Commission Regulation (EC) No. 440/2008 B13/14, dated May 30, 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, TA 100 and E. coli WP2

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/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate with and without metabolic activation
The pre-experiment is reported as Experiment I.

Experiment II:
TA100: 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate with and without metabolic activation
All remaining strains: 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 low toxicity to 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: in agar (plate incorporation) (Experiment I); pre-incubation (Experiment II)

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

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

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

Evaluation criteria:

A test item is considered as a mutagen 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. I and II: at 5000 µg/plate +S9

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. I: at 5000 µg/plate +/- S9; Exp. II: at and above 1000 µg/plate -S9, at and above 2500 µg/plate +S9

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. I: at and above 2500 µg/plate + S9; Exp. II: at and above 2500 µg/plate -S9, at and above 1000 µg/plate +S9

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. I: at and above 1000 µg/plate +/- S9; Exp. II: at and above 100 µg/plate -S9, at and above 333 µg/plate +S9

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 2. Results of Experiment I (plate incorporation)

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		12 ± 4	11 ± 1	26 ± 6	183 ± 26	52 ± 5
	Untreated		14 ± 4	11 ± 1	33 ± 3	212 ± 10	47 ± 10
	Test substance	3 µg	11 ± 1	9 ± 5	27 ± 10	181 ± 6	43 ± 10
		10 µg	10 ± 4	11 ± 4	31 ± 1	201 ± 19	44 ± 5
		33 µg	9 ± 2	11 ± 4	20 ± 2	193 ± 7	46 ± 10
		100 µg	13 ± 3	10 ± 4	29 ± 4	184 ± 11	45 ± 5
		333 µg	9 ± 3	8 ± 3	31 ± 10	86 ± 13	51 ± 11
		1000 µg	11 ± 1	6 ± 1	24 ± 6	69 ± 7	41 ± 6
		2500 µg	12 ± 2^P	5 ± 2^P	27 ± 6^P	22 ± 2^P M	48 ± 3^P
		5000 µg	13 ± 3^P	1 ± 1^P M	15 ± 5^P	4 ± 1^P M	35 ± 6^P
	NaN3	10 µg	1411 ± 69			2412 ± 96
	4-NOPD	10 µg			496 ± 42
	4-NOPD	50 µg		96 ± 8
	MMS	2.0 µL					913 ± 66
With Activation	DMSO		13 ± 4	13 ± 6	31 ± 11	191 ± 12	50 ± 15
	Untreated		10 ± 5	14 ± 5	41 ± 7	212 ± 21	61 ± 13
	Test substance	3 µg	14 ± 4	9 ± 2	34 ± 7	170 ± 22	53 ± 16
		10 µg	14 ± 5	14 ± 3	35 ± 7	166 ± 15	60 ± 3
		33 µg	12 ± 4	12 ± 6	33 ± 8	176 ± 30	47 ± 13
		100 µg	15 ± 2	12 ± 5	28 ± 2	169 ± 3	52 ± 4
		333 µg	13 ± 2	13 ± 2	41 ± 1	176 ± 4	58 ± 6
		1000 µg	8 ± 4	7 ± 4	36 ± 3	33 ± 9	43 ± 6
		2500 µg	9 ± 1^P	7 ± 2^P	13 ± 1^P	5 ± 1^P	44 ± 6^P
		5000 µg	3 ± 1^P	4 ± 1^P M	3 ± 1^P M	0 ± 1^P	23 ± 2^P
	2-AA	2.5 µg	455 ± 19	184 ± 21	4445 ± 599	4796 ± 494
	2-AA	10.0 µg					386 ± 28

NaN3: sodium azide

2-AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

P: Precipitate

M: Manual count

Table 3. Results of Experiment II (pre-incubation)

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		13 ± 3	10 ± 1	21 ± 4	154 ± 12	39 ± 8
	Untreated		11 ± 3	8 ± 1	33 ± 7	217 ± 10	41 ± 6
	Test substance	1 µg				164 ± 21
		3 µg				149 ± 22
		10 µg	11 ± 1	11 ± 2	34 ± 6	167 ± 6	37 ± 5
		33 µg	11 ± 1	9 ± 4	24 ± 4	157 ± 8	43 ± 7
		100 µg	11 ± 4	11 ± 1	26 ± 5	58 ± 4	43 ± 2
		333 µg	11 ± 2	7 ± 2	25 ± 6	56 ± 2	35 ± 2
		1000 µg	8 ± 3	2 ± 1^R	14 ± 2	46 ± 4^P R	31 ± 8
		2500 µg	11 ± 1^P M	0 ± 0^P R	13 ± 5^P R	18 ± 2^P M R	26 ± 4^P
		5000 µg	6 ± 2^P M	0 ± 0^P R	4 ± 2^P M R		20 ± 6^P
	NaN3	10 µg	1342 ± 46			1884 ± 81
	4-NOPD	10 µg			311 ± 21
	4-NOPD	50 µg		115 ± 27
	MMS	2.0 µL					736 ± 46
With Activation	DMSO		13 ± 2	18 ± 2	36 ± 6	111 ± 27	34 ± 3
	Untreated		13 ± 4	16 ± 2	46 ± 12	102 ± 55	50 ± 8
	Test substance	1 µg				91 ± 3
		3 µg				92 ± 17
		10 µg	14 ± 3	19 ± 3	37 ± 4	88 ± 30	41 ± 1
		33 µg	13 ± 1	16 ± 6	35 ± 2	98 ± 43	49 ± 3
		100 µg	16 ± 4	19 ± 3	31 ± 8	57 ± 13	36 ± 0
		333 µg	15 ± 5	17 ± 3	41 ± 4	38 ± 2	35 ± 3
		1000 µg	8 ± 1	13 ± 1	2 ± 1^R	0 ± 0^R	35 ± 6
		2500 µg	8 ± 2^P	6 ± 1^P R	0 ± 0^P R	0 ± 0^P R	26 ± 3^P
		5000 µg	4 ± 1^P M	1 ± 1^P R	0 ± 0^P R		25 ± 1^P
	2-AA	2.5 µg	350 ± 16	223 ± 19	3553 ± 920	2194 ± 573
	2-AA	10.0 µg					246 ± 72

NaN3: sodium azide

2-AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

P: Precipitate

R: Reduced background growth

M: Manual count

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five bacterial strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation up to cytotoxic or precipitating concentrations of 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 (2018). 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 cytotoxic or precipitating concentrations of 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.