Reaction product of o-cresol and camphene, obtained by addition, hydrogenation and distillation

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

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:

21 Jun - 26 Jul 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:

adopted: 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 (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 2a:
33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA1537
10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA98, TA100, TA1535 and WP2 uvrA

Experiment 2b:
10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA98
100, 333, 1000, 2000, 3000, 4000 and 5000 µg/plate without metabolic activation for TA1535

Vehicle / solvent:

- Vehicle(s/solvent used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

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)

Remarks:

Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (Experiment 1); pre-incubation (Experiment 2a and b)

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 1: +S9: 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:

no cytotoxicity, but tested up to precipitating concentrations

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 1: +S9: at 5000 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:

Exp 1: +S9: at 2500 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 333 to 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate in all experiments and at 2500 μg/plate in Experiment 1 with S9 mix. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment 1 (all strains were tested in the pre-experiment).

ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. Slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strain TA 1535, TA 98 and TA 100.

A slight dose dependent increase in the number of revertant colonies was observed in Experiment 2a without S9 mix. The threshold of thrice the number of the corresponding solvent control was neither reached nor exceeded and the result was not reproducible in Exp 2b and can therefore be judged as biologically irrelevant.

Table 1. Test results of Experiment 1 (plate incorporation method)

	Number of revertant colonies (mean of 3 plates±SD)
Without S9
Test substance (µg/plate)	TA 1535	TA 1537			TA 98	TA 100		WP2 uvrA
Ethanol	12±2	8±2 b,m			29±7	114±26		40±8
Untreated	10±5	7±1 b,m			29±9	140±27		45±7
3	10±1	7±1 b,m			27±7	117±21		45±4
10	13±3	8±3 b,m			29±8	95±5		40±4
33	11±1	7±2 b,m			34±8	70±17		46±9
100	9±1	8±2 b,m			31±6	75±6		46±1
333	10±1	8±2 b,m			33±10	63±16		36±8
1000	10±1	8±3 b,m			28±6	66±4		38±7
2500	11±1	8±2 b,m			34±4	68±6		36±1
5000	11±4 p	6±2 p,b,m			36±3 p	66±8 p		45±1 p
Positive Control	NaN3	4-NOPD			4-NOPD	NaN3		MMS
Dose (µg/plate)	10	50			10	10		2
Number of revertant colonies/plate	1166± 111	81±13 b,m			392±4	1963±80		833±56
With S9
Test substance (µg/plate)	TA 1535		TA 1537		TA 98	TA 100	WP2 uvr A
DMSO	18±4		9±2 b,m		56±4	135±5	49±7
Untreated	15±1		9±1 b,m		45±5	149±17	61±8
3	15±3		9±2 b,m		48±13	124±9	53±5
10	19±2		9±1 b,m		51±6	129±11	51±6
33	20±4		9±2 b,m		56±13	118±6	48±12
100	16±6		9±2 b,m		43±5	112±8	51±4
333	10±4		8±1 b,m		39±8	79±8	44±3
1000	12±4		9±1 b,m		44±3	78±10	51±15
2500	11±3 p,m		8±1 p,b,m		26±3 p,m	59±5 p,m	27±2 p,m
5000	7±2 p,m		9±2 p,b,m		24±5 p,m	46±4 p,m	26±2 p,m
Positive Control	2-AA		2-AA		2-AA	2-AA	2-AA
Dose (µg/plate)	2.5		2.5		2.5	2.5	10
Number of revertant colonies/plate	297±52		117±9 b,m		3492±860	3213±171	358±48
NaN3: sodium azide 2AA: 2-Aminoanthracene 4-NOPD: 4-nitro-o-phenylene-diamine MMS: methyl methane sulfonate				p: precipitate m: manual count b: extensive bacterial growth

Table 2: Test results of Experiment 2a (pre-incubation method)

	Number of revertant colonies (mean of 3 plates±SD)
Without S9
Test substance (µg/plate)	TA 1535	TA 1537			TA 98	TA 100		WP2 uvrA
Ethanol	14±3	18±6				155±11		42±8
Untreated	8±3	15±0				165±8		48±12
10	8±2					144±26		38±6
33	9±5	21±5				123±25		40±3
100	13±2	19±7				98±15		38±11
333	8±2	25±2				90±12		45±3
1000	6±1	27±5				89±11		40±4
2500	9±2	33±5				93±11		39±5
5000	10±3 p	51±6 p				96±14 p		43±3 p
Positive Control	NaN3	4-NOPD			4-NOPD	NaN3		MMS
Dose (µg/plate)	10	50			10	10		2
Number of revertant colonies/plate	1078±22	83±11				1701±99		776±5
With S9
Test substance (µg/plate)	TA 1535		TA 1537		TA 98	TA 100	WP2 uvr A
Ethanol	11±1		27±3			161±30	41±5
Untreated	7±1		20±4			177±1	59±14
10	13±2					154±25	50±7
33	13±1		28±9			157±14	54±4
100	16±1		25±2			147±5	41±5
333	10±2		24±3			107±12	52±9
1000	12±6		21±5			93±13	46±5
2500	13±2		26±6			94±14	50±6
5000	13±4 p		28±2			97±12 p	46±16 p
Positive Control	2-AA		2-AA		2-AA	2-AA	2-AA
Dose (µg/plate)	2.5		2.5		2.5	2.5	10
Number of revertant colonies/plate	384±9		144±30			3366±216	373±21
NaN3: sodium azide 2AA: 2-Aminoanthracene 4-NOPD: 4-nitro-o-phenylene-diamine MMS: methyl methane sulfonate				p: precipitate

Table 3. Results of Experiment 2b (pre-incubation method)

	Number of revertant colonies (mean of 3 plates±SD)
Without S9
Test substance (µg/plate)	TA 1537		TA 98
Ethanol	11±2		38±4
Untreated	13±3		32±6
10			39±3
33			34±7
100	11±6		40±5
333	9±5		38±12
1000	9±3		27±1
2000	12±2
2500			30±6
3000	7±3
4000	10±3
5000	13±4 p		33±7 p
Positive Control	4-NOPD		4-NOPD
Dose (µg/plate)	50		10
Number of revertant colonies/plate	119±12		374±26
With S9
Test substance (µg/plate)	TA 1537	TA 98
Ethanol		46±2
Untreated		54±9
10		49±7
33		43±12
100		48±4
333		47±7
1000		55±7
2500		42±10
5000		42±1 p
Positive Control	2-AA	2-AA
Dose (µg/plate)	2.5	2.5
Number of revertant colonies/plate		4016±153
NaN3: sodium azide 2AA: 2-Aminoanthracene 4-NOPD: 4-nitro-o-phenylene-diamine                                                               MMS: methyl methane sulfonate p: precipitate

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.

Executive summary:

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.

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 (2017). In this study 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 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) No. 1272/2008.