Reaction mass of (1RS,3RS)-3-methyl-3-(4-methylpentyl)cyclohexyl acetate and (2RS,4aRS,8aSR)-5,5,8a-trimethyldecahydro-2-naphthalenyl acetate and (2RS,4aRS,8aRS)-5,5,8a-trimethyldecahydro-2-naphthalenyl acetate

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected March 2014; signature: May 2014

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (1RS,3RS)-3-methyl-3-(4-methylpentyl)cyclohexyl acetate and (2RS,4aRS,8aSR)-5,5,8a-trimethyldecahydro-2-naphthalenyl acetate and (2RS,4aRS,8aRS)-5,5,8a-trimethyldecahydro-2-naphthalenyl acetate

Molecular formula:

not applicable (reaction mass of constitutional isomers)

IUPAC Name:

Reaction mass of (1RS,3RS)-3-methyl-3-(4-methylpentyl)cyclohexyl acetate and (2RS,4aRS,8aSR)-5,5,8a-trimethyldecahydro-2-naphthalenyl acetate and (2RS,4aRS,8aRS)-5,5,8a-trimethyldecahydro-2-naphthalenyl acetate

Details on test material:

- Physical state: Liquid.
- Storage condition of test material: Room temperature in the dark
- Other: Colourless

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

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:

Experiment 1 (plate incorporation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method):
Salmonella strains with and without S9-mix - TA98, TA100, TA1535: 0, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Salmonella strains with S9 mix - TA1537: 0, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate
Salmonella strain without S9-mix - TA1537: 0, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
E.coli strain with and without S9-mix - WP2uvrA: 0, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Up to eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic limit of the test item following the change in test methodology.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL in solubility checks performed. Dimethyl sulphoxide was selected as the vehicle.
- Other: Formulated concentrations were adjusted by an appropriate factor to allow for the stated purity of the test substance.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

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: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (pre-incubation)

DURATION
- Exposure duration:
Experiment 1. All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required, predominantly due to interference in the base agar e.g. minor precipitation of salts/dust particles and marks on the base of the plates slightly distorting the counts.

Experiment 2. 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media Subsequently, the procedure for incubation and duration was the same as in Experiment 1.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.

Statistics:

Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls

S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	68 74 65	(69) 4.6#	9 7 15	(10) 4.2	21 33 20	(25) 7.2	20 12 19	(17) 4.4	9 7 8	(8) 1.0
	1.5 µg	67 69 63	(66) 3.1	19 9 9	(12) 5.8	24 29 33	(29) 4.5	24 16 15	(18) 4.9	8 11 15	(11) 3.5
	5 µg	67 83 86	(79) 10.2	23 8 8	(13) 8.7	27 28 27	(27) 0.6	24 16 13	(18) 5.7	20 9 12	(14) 5.7
	15 µg	74 67 75	(72) 4.4	12 8 11	(10) 2.1	29 28 32	(30) 2.1	23 15 23	(20) 4.6	8 11 5	(8) 3.0
	50 µg	64 84 80	(76) 10.6	13 15 11	(13) 2.0	16 32 21	(23) 8.2	8 11 12	(10) 2.1	3 13 4	(7) 5.5
	150 µg	64 61 67	(64) 3.0	11 16 8	(12) 4.0	15 32 23	(23) 8.5	9 25 12	(15) 8.5	7 8 13	(9) 3.2
	500 µg	64 64 55	(61) 5.2	17 16 11	(15) 3.2	23 35 35	(31) 6.9	11 19 16	(15) 4.0	8 13 3	(8) 5.0
	1500 µg	64 P 59 P 75 P	(66) 8.2	16 P 16 P 9 P	(14) 4.0	27 P 32 P 27 P	(29) 2.9	13 P 13 P 17 P	(14) 2.3	9 P 8 P 5 P	(7) 2.1
	5000 µg	53 PS 45 PS 60 PS	(53) 7.5	15 PS 11 PS 12 PS	(13) 2.1	35 P 28 P 27 P	(30) 4.4	17 P 15 P 15 P	(16) 1.2	1 PS 5 PS 8 PS	(5) 3.5
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		370 564 493	(476) 98.2	305 521 380	(402) 109.7	976 923 925	(941) 30.0	136 142 162	(147) 13.6	727 921 1066	(905) 170.1
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	72 84 80	(79) 6.1#	11 9 10	(10) 1.0	36 29 29	(31) 4.0	29 17 25	(24) 6.1	8 19 8	(12) 6.4
	1.5 µg	71 72 72	(72) 0.6	15 17 13	(15) 2.0	43 31 39	(38) 6.1	11 31 23	(22) 10.1	20 20 7	(16) 7.5
	5 µg	83 68 83	(78) 8.7	15 13 13	(14) 1.2	27 32 43	(34) 8.2	29 28 24	(27) 2.6	8 12 13	(11) 2.6
	15 µg	61 65 68	(65) 3.5	9 15 15	(13) 3.5	19 25 44	(29) 13.1	8 32 27	(22) 12.7	12 7 15	(11) 4.0
	50 µg	74 71 73	(73) 1.5	8 9 16	(11) 4.4	23 21 31	(25) 5.3	16 23 19	(19) 3.5	12 8 15	(12) 3.5
	150 µg	66 64 64	(65) 1.2	9 10 9	(9) 0.6	25 37 33	(32) 6.1	27 16 27	(23) 6.4	7 4 7	(6) 1.7
	500 µg	72 78 76	(75) 3.1	9 5 7	(7) 2.0	47 40 31	(39) 8.0	16 15 17	(16) 1.0	5 8 3	(5) 2.5
	1500 µg	35 P 34 P 37 P	(35) 1.5	3 P 2 P 6 P	(4) 2.1	28 P 31 P 32 P	(30) 2.1	9 P 12 P 24 P	(15) 7.9	1 PS 0 PS 0 PS	(0) 0.6
	5000 µg	29 PS 19 PS 24 PS	(24) 5.0	3 PS 2 PS 2 PS	(2) 0.6	25 P 35 P 19 P	(26) 8.1	8 P 8 P 7 P	(8) 0.6	0 PV 0 PV 0 PV	(0) 0.0
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		1430 1625 1514	(1523) 97.8	266 286 299	(284) 16.6	246 226 311	(261) 44.4	179 212 243	(211) 32.0	699 756 334	(596) 229.0

  

Table 2 : Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls

S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	78 64 76	(73) 7.6#	9 13 8	(10) 2.6	20 23 25	(23) 2.5	17 23 20	(20) 3.0	11 4 9	(8) 3.6
	1.5 µg	76 82 68	(75) 7.0	9 15 12	(12) 3.0	N/T		13 15 23	(17) 5.3	7 11 8	(9) 2.1
	5 µg	65 71 71	(69) 3.5	9 12 9	(10) 1.7	15 17 17	(16) 1.2	24 27 23	(25) 2.1	8 4 8	(7) 2.3
	15 µg	79 82 71	(77) 5.7	9 11 9	(10) 1.2	25 21 20	(22) 2.6	12 15 13	(13) 1.5	4 5 11	(7) 3.8
	50 µg	69 63 64	(65) 3.2	17 9 9	(12) 4.6	21 11 19	(17) 5.3	11 17 19	(16) 4.2	12 8 11	(10) 2.1
	150 µg	71 71 61	(68) 5.8	7 9 8	(8) 1.0	27 19 20	(22) 4.4	19 15 15	(16) 2.3	11 11 12	(11) 0.6
	500 µg	67 74 74	(72) 4.0	8 15 8	(10) 4.0	23 23 9	(18) 8.1	16 16 24	(19) 4.6	5 12 4	(7) 4.4
	1500 µg	75 P 72 P 74 P	(74) 1.5	4 PS 4 PS 10 PS	(6) 3.5	21 P 8 P 28 P	(19) 10.1	20 P 15 P 17 P	(17) 2.5	5 P 5 P 5 P	(5) 0.0
	5000 µg	64 PS 64 PS 57 PS	(62) 4.0	5 PS 7 PS 11 PS	(8) 3.1	19 P 24 P 20 P	(21) 2.6	15 P 15 P 9 P	(13) 3.5	4 PS 7 PS 4 PS	(5) 1.7
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		374 433 425	(411) 32.0	345 489 294	(376) 101.1	555 537 600	(564) 32.4	132 162 167	(154) 18.9	992 1155 588	(912) 291.9
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	84 72 75	(77) 6.2#	8 8 9	(8) 0.6	25 32 25	(27) 4.0	17 20 17	(18) 1.7	9 12 9	(10) 1.7
	0.5 µg	N/T		N/T		N/T		N/T		11 4 8	(8) 3.5
	1.5 µg	94 67 86	(82) 13.9	9 11 11	(10) 1.2	N/T		15 28 16	(20) 7.2	8 11 7	(9) 2.1
	5 µg	90 76 78	(81) 7.6	11 8 7	(9) 2.1	21 28 24	(24) 3.5	17 21 31	(23) 7.2	9 7 8	(8) 1.0
	15 µg	64 83 80	(76) 10.2	7 13 8	(9) 3.2	20 16 29	(22) 6.7	19 23 17	(20) 3.1	7 8 7	(7) 0.6
	50 µg	82 86 75	(81) 5.6	8 8 11	(9) 1.7	15 29 24	(23) 7.1	20 25 25	(23) 2.9	13 11 7	(10) 3.1
	150 µg	71 82 78	(77) 5.6	11 8 12	(10) 2.1	24 27 24	(25) 1.7	17 20 21	(19) 2.1	11 8 11	(10) 1.7
	500 µg	56 S 45 S 55 S	(52) 6.1	4 S 11 S 8 S	(8) 3.5	31 28 23	(27) 4.0	21 11 9	(14) 6.4	9 S 5 S 8 S	(7) 2.1
	1500 µg	49 PS 48 PS 47 PS	(48) 1.0	5 PS 5 PS 7 PS	(6) 1.2	32 P 33 P 24 P	(30) 4.9	15 P 20 P 15 P	(17) 2.9	9 PS 9 PS 5 PS	(8) 2.3
	5000 µg	56 PS 23 PS 20 PS	(33) 20.0	5 PS 5 PS 7 PS	(6) 1.2	29 P 19 P 15 P	(21) 7.2	21 P 16 P 21 P	(19) 2.9	N/T
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		1012 1005 834	(950) 100.8	207 186 195	(196) 10.5	103 130 118	(117) 13.5	102 134 118	(118) 16.0	241 242 242	(242) 0.6

 

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

4NQO  4-Nitroquinoline-1-oxide

9AA      9-Aminoacridine

BP         Benzo(a)pyrene

2AA      2-Aminoanthracene

P           Test item precipitate

S            Sparse bacterial background lawn

V           Very weak bacterial background lawn

#           Standard deviation

N/T      Not tested at this dose level

 

Table 3. Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1
Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
98		7		27		9		11
98	(88)	12	(10)	32	(33)	17	(15)	11	(10)
69		11		39		20		8
Experiment 2
Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
103		8		21		21		9
96	(107)	8	(8)	19	(21)	31	(26)	7	(8)
123		9		24		25		8

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test material was considered to be non-mutagenic in the presence and absence of S9 activation.

Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, 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 WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test substance using both the Ames plate incorporation and pre incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 0.5 and 5000 µg/plate depending on bacterial tester strain and absence or presence of S9-mix. Up to eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic limit of the test item following the change in test methodology. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test recommended in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. Following Experiment 1 and observed cytotoxicity in associated strains a subsequent Experiment 2 was conducted with the pre-incubation method wherein the same maximum dose level or the toxic limit was used in the second mutation test, depending on bacterial tester strain and absence or presence of S9-mix. A similar response in terms of test item toxicity to the bacterial tester strains was noted in the second mutation test (employing the pre-incubation modification) with weakened lawns noted in the presence of S9-mix to TA100, TA1535 and TA1537 at and above 500 µg/plate. In the absence of S9-mix weakened lawns were initially noted from 1500 µg/plate to TA1535 and at 5000 µg/plate to TA100 and TA1537. Once again, there was no visible reduction in the growth of the bacterial background lawns of TA98 and WP2uvrA at any test substance dose level, either in the presence or absence of S9-mix. Therefore these strains were tested at up to 5000 µg/plate. A test item precipitate (greasy in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies. 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 WP2uvrA- in the presence and absence of S-9 mix.