(2α,4aα,8α)-hexahydro-1,1,5,5-tetramethyl-2H-2,4a-methanonaphthalen-8(5H)-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For this endpoint there is one study available in which the potential of the test item to induce gene mutations was investigated in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and Escherichia colistrain WP2uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation (S9). Each concentration, including the controls, was tested in triplicate.The concentrations in the pre-experiment (experiment I) were: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate. The concentrations in experiment II were for TA 100: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate and for all others: 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate.

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.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), or a tendency of higher mutation rates with increasing concentrations did not reach the threshold. The acceptance criteria were met. Therefore, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used and is considered to be non-mutagenic in the reverse mutation assay.

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:

02.09.2016 - 14.09.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

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:

rat liver S9 preparation

Test concentrations with justification for top dose:

Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate.
Experiment II for TA 100: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate and for all others: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- 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

Negative solvent / vehicle controls:

yes

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) and preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

ACCEPTANCE CRITERIA
- Regular background growth in the negative and solvent control
- The spontaneous reversion rates in the negative and solvent control must be in the range of our historical data
- The positive control substances should produce an increase above 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
- 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:

- 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:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 333 µg/plate in Experiment I, not in Experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Experiment I, without S9 at 100-5000 µg/plate and with S9 at 333-5000µg/plate. In Experiment II, without S9 at 333-5000 µg/plate and with S9 at 333-5000µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

Additional information on results:

No relevant toxic effects, evident as a dose dependent reduction of the number of revertants below 0.5 times the corresponding solvent control, occurred in the test groups with and without metabolic activation in strain TA 1537. The moderate reduction in the number of revertants in strain in experiment I without S9 mix was not dose dependent and thus judged to be based upon statistical fluctuations at such low numbers of colonies and does not represent a true toxic effect.

Summary results:

Experiment I

 

Without Metabolic Activation
Test Group	Dose level (µg/plate)	Revertant Colony Counts (mean±SD)
		TA1535	TA1537	TA98	TA100	WP2uvrA
DMSO	n.a.	8±2	17±2	21±5	176±30	35±3
Untreated	n.a.	10 ± 5	19 ± 2	23 ± 12	184 ± 10	47 ± 1
Test item	3	7 ± 3	27 ± 10	25 ± 3	177 ± 7	35 ± 7
	10	11 ± 1	24 ± 9	21 ± 7	186 ± 10	34 ± 2
	33	12 ± 1	23 ± 5	19 ± 4	157 ± 6	35 ± 2
	100	12 ± 5	9 ± 1	20 ± 4	60 ± 4	34 ± 6
	333	9 ± 2	7 ± 1	26 ± 10	45 ± 15	40 ± 6
	1000	7 ± 3	8 ± 3	30 ± 5	55 ± 6	40 ± 6
	2500	14 ± 6^P	14 ± 2^P	28 ± 4^P	47 ± 2^P	35 ± 3^P
	5000	24 ± 1^P	10 ± 3^P	27 ± 8^P	55 ± 1^P	36 ± 11^P
NaN3	10	1236 ± 41			2100 ± 87
4-NOPD	10			475 ± 65
4-NOPD	50		86 ± 10
MMS	2.0					954 ± 46
With Metabolic Activation
Test Group	Dose level (µg/plate)	Revertant Colony Counts (mean +/- SD)
		TA1535	TA1537	TA98	TA100	WP2uvrA
DMSO	n.a.	11 ± 3	27 ± 5	30 ± 7	177 ± 13	54 ± 11
Untreated	n.a.	10 ± 2	25 ± 1	42 ± 7	175 ± 10	54 ± 11
Test item	3	10 ± 5	23 ± 2	40 ± 10	139 ± 13	48 ± 8
	10	15 ± 5	31 ± 4	30 ± 8	157 ± 10	48 ± 8
	33	11 ± 6	24 ± 4	34 ± 5	163 ± 8	53 ± 6
	100	9 ± 3	30 ± 2	31 ± 3	105 ± 24	54 ± 3
	333	9 ± 4	20 ± 3	29 ± 8	51 ± 8	47 ± 5
	1000	12 ± 5	20 ± 3	27 ± 5	24 ± 7	41 ± 10
	2500	6 ± 1	25 ± 2	21 ± 1	10 ± 2	32 ± 8
	5000	10 ± 4^P	23 ± 7^P	25 ± 5^P	11 ± 2^P	34 ± 8^P
2-AA	2.5	394 ± 25	421 ± 28	4235 ± 1180	5176 ± 85
2-AA	10					438 ± 12

 

NaN3: sodium azide

2-AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

P: Precipitate

Experiment II

 

Without Metabolic Activation
Test Group	Dose level (µg/plate)	Revertant Colony Counts (mean±SD)
		TA1535	TA1537	TA98	TA100	WP2uvrA
DMSO	n.a.	12±3	12±2	22±3	145±16	37±6
Untreated	n.a.	9 ± 5	12 ± 3	38 ± 8	181 ± 21	40 ± 11
Test item	3				128 ± 3
	10	13 ± 2	13 ± 4	28 ± 6	140 ± 10	43 ± 5
	33	13 ± 3	12 ± 5	28 ± 3	94 ± 9	39 ± 8
	100	10±2	14±2	27±7	81±18	34±5
	333	10 ± 3	10 ± 5	28 ± 4	62 ± 7	35 ± 7
	1000	10 ± 4	12 ± 3	28 ± 3	46 ± 7	31 ± 8
	2500	11 ± 2	15 ± 5	26 ± 4	48 ± 5	32 ± 4
	5000	13 ± 2^P	15 ± 1^P	27 ± 5^P	25 ± 6^P M	38 ± 4^P
NaN3	10	1215 ± 100			2004 ± 55
4-NOPD	10			505 ± 28
4-NOPD	50		91 ± 2
MMS	2.0					569 ± 47
With Metabolic Activation
Test Group	Dose level (µg/plate)	Revertant Colony Counts (mean +/- SD)
		TA1535	TA1537	TA98	TA100	WP2uvrA
DMSO	n.a.	11±1	18±5	37±12	131±9	56±9
Untreated	n.a.	15 ± 5	16 ± 6	49 ± 10	189 ± 3	65 ± 6
Test item	3				110 ± 12
	10	14 ± 4	19 ± 3	41 ± 6	134 ± 21	63 ± 10
	33	14 ± 4	20 ± 4	41 ± 10	128 ± 7	50 ± 5
	100	15±1	18±5	36±3	112±15	53±3
	333	11 ± 4	25 ± 3	37 ± 5	39 ± 2	49 ± 6
	1000	15 ± 1	26 ± 1	42 ± 1	35 ± 2	44 ± 4
	2500	13 ± 4	21 ± 0	31 ± 5	21 ± 2	32 ± 1
	5000	13 ± 1	22 ± 4	30 ± 7	13 ± 2	39 ± 4
2-AA	2.5	326 ± 38	242 ± 4	3304 ± 213	4011 ± 287
2-AA	10					439 ± 23

 

NaN3: sodium azide

2-AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

P: Precipitate

M Manual count

Conclusions:

The test item is considered to be non-mutagenic.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and Escherichia coli strain WP2uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation (S9). Each concentration, including the controls, was tested in triplicate. The concentrations in the pre-experiment (experiment I) were: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate The concentrations in experiment II were for TA 100: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate and for all others: 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate.

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 μg/plate without metabolic activation and at 5000 μg/plate with metabolic activation in the first experiment and at 5000 μg/plate without metabolic activation in the second experiment.

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.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strain TA 100 in both experiments.

There was no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance with the exception of strain TA 1535 in the absence of metabolic activation in experiment I. This strain showed an increase in revertant colony numbers at the highest applied concentration, which showed precipitation too. However, the absolute numbers of colonies did not reach the threshold of 3.0.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. The acceptance criteria were met.

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in the reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The test item did not induce gene mutations by base pair changes or frameshifts in the Ames test and is considered to be non-mutagenic in the reverse mutation assay.