Reaction mass of (3E)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-1-en-1-yl)butan-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)butan-2-one

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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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: July 2012; signature: November 2012

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (3E)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-1-en-1-yl)butan-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)butan-2-one

EC Number:

700-956-5

Molecular formula:

not applicable (reaction mass of constitutional isomers)

IUPAC Name:

Reaction mass of (3E)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-1-en-1-yl)butan-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)butan-2-one

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Physical state: liquid
- Storage condition of test material: Approximately 4°C in the dark under nitrogen.
- Other: Pale yellow

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:

- Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
- Experiment 1 range-finding test (plate incorporation method): 0 (solvent control), 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
- Experiment 2 (pre-incubation method):
Salmonella strains and E.coli strain with and without S9-mix: 0 (solvent control), 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Up to sevent 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: Acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at 100 mg/ml in solubility checks performed. Acetone was therefore selected as the vehicle.

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.

Experiment 2. Measured aliquots (0.1 ml) of one of the bacterial cultures followed by 0.5 ml of S9-mix or phosphate buffer and 0.05 ml of the vehicle or test item formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten amino acid supplemented, top agar. 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).
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:

cytotoxicity

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: Range-finding test: 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 (Acetone)		95 (100) 114 12.7# 90		17 (15) 17 2.9 12		33 (36) 41 4.2 35		32 (26) 27 6.6 19		15 (11) 7 4.0 12
		5 µg		96 (94) 92 2.1 95		19 (17) 18 2.1 15		33 (36) 39 3.1 37		26 (21) 17 4.7 19		17 (13) 12 3.2 11
		15 µg		91 (101) 101 10.5 112		12 (11) 13 2.1 9		35 (37) 37 1.5 38		22 (24) 24 2.0 26		9 (9) 8 0.6 9
		50 µg		81 (90) 79 17.9 111		19 (16) 18 4.4 11		38 (31) 24 7.0 31		25 (21) 20 3.6 18		11 (13) 11 2.9 16
		150 µg		91 (90) 82 7.1 96		11 (14) 15 3.1 17		29 (28) 28 0.6 28		19 (17) 16 2.1    15		14 (11) 12 3.6 7
		500 µg		90 (102) 108 10.7 109		14 (14) 13 0.6 14		37 (32) 31 5.0 27		17 (20) 21 2.3 21		8 (9) 11 2.1 7
		1500 µg		96 TP (100) 89 TP 14.0 116 TP		17 TP (17) 18 TP 1.5 15 TP		29 P (30) 29 P 1.7 32 P		35 P (24) 11 P 12.1 25 P		7 TP (7) 7 TP 0.6 6 TP
		5000 µg		84 TP (56) 59 TP 30.1 24 TP		6 TP (9) 7 TP 3.8 13 TP		28 TP (36) 35 TP 8.0 44 TP		16 TP (17) 19 TP 1.5 17 TP		6 TP (6) 5 TP 0.6 6 TP
Positive controls S9-Mix (-)		Name		ENNG		ENNG		ENNG		4NQO		9AA
		Dose		3 µg		5 µg		2 µg		0.2 µg		80 µg
		Level No. of Revertants		851 (702) 616 129.5 639		626 (555) 462 84.2 577		1155 (1031) 1068 146.6 869		180 (190) 184 14.6 207		1096 (846) 696 217.7 747
S9-Mix (+)	Dose Level Per Plate		Number of revertants (mean) +/- SD
			Base-pair substitution strains						Frameshift strains
			TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)		116 (106) 102 8.4# 101		16 (15) 10 5.0 20		38 (35) 36 3.6 31		37 (35) 39 5.9 28		11 (10) 12 2.1 8
	5 µg		110 (105) 103 4.4 102		13 (13) 11 1.5 14		30 (30) 30 0.6 31		28 (27) 26 1.2 26		7 (8) 10 2.1 6
	15 µg		86 (100) 111 12.8 103		16 (14) 8 4.9 17		38 (36) 34 2.0 36		16 (20) 16 6.4 27		12 (12) 12 0.6 11
	50 µg		101 (98) 103 7.6 89		9 (11) 13 2.1 12		33 (35) 44 8.6 27		29 (26) 26 2.5 24		9 (9) 7 2.0 11
	150 µg		103 (110) 123 11.0 105		13 (12) 13 1.2 11		35 (36) 35 1.2 37		22 (25) 30 4.6 22		12 (11) 12 1.7 9
	500 µg		122 (101) 71 26.8 111		14 (13) 9 3.2 15		42 (38) 40 4.7 33		32 (32) 32 0.6 33		6 (9) 10 2.3 10
	1500 µg		119 TP (110) 111 TP 9.5 100 TP		11 TP (13) 17 TP 3.8 10 TP		29 P (36) 39 P 5.8 39 P		32 P (32) 29 P 3.5 36 P		10 P (10) 10 P 0.0 10 P
	5000 µg		94 TP (91) 95 TP 6.7 83 TP		10 TP (12) 13 TP 1.7 13 TP		38 TP (37) 38 TP 2.3 34 TP		30 P (31) 34 P 2.6 29 P		18 TP (11) 8 TP 5.8 8 TP
Positive controls S9-Mix (+)	Name		2AA		2AA		2AA		BP		2AA
	Dose		1 µg		2 µg		10 µg		5 µg		2 µg
	Level No. of Revertants		1376     (1533) 1484     186.4 1739		274       (259) 249       13.4 253		334       (377) 440       55.6 358		269       (283) 285       12.7 294		301       (243) 182       59.5 245

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-AminoacridineENNG:

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

T: Partial absence of bacterial background lawn

P: Precipitate

#: Standard deviation

 

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 (Acetone)		115 (120) 123 4.6# 123	21 (22) 24 2.1 20			24 (33) 42 9.0 33		19 (25) 21 8.7 35		10 (11) 16 4.2 8
	5 µg		104 (116) 105 19.9 139	16 (18) 18 2.5 21			28 (27) 25 1.7 28		18 (16) 16 2.5 13		4 (7) 9 2.5 7
	15 µg		134 (107) 68 34.6 119	18 (20) 20 2.0 22			25 (31) 36 5.7 33		15 (21) 20 6.0 27		9 (12) 16 3.8 10
	50 µg		100 (99) 98 1.2 100	17 (17) 18 0.6 17			25 (28) 31 3.1 29		19 (18) 17 1.2 19		10 (12) 15 2.6 11
	150 µg		99 (106) 108 6.7 112	17 (20) 19 3.6 24			25 (31) 37 6.0 31		13 (19) 27 7.4    16		10 (11) 15 4.0 7
	500 µg		123 (121) 132 11.6 109	16 (17) 20 2.3 16			19 (25) 29 5.5 28		25 (25) 26 1.0 24		13 T (10) 10 T 3.5 6 T
	1500 µg		124 TP (110) 108 TP 13.6 97 TP	22 TP (15) 9 TP 6.5 15 TP			46 P (33) 27 P 11.3 26 P		15 TP (15) 17 TP 2.0 13 TP		10 TP (10) 10 TP 0.6 11 TP
	5000 µg		118 TP (100) 98 TP 16.6 85 TP	16 TP (12) 9 TP 3.5 12 TP			30 TP (26) 28 TP 4.7 21 TP		18 TP (16) 16 TP 2.5 13 TP		3 TP (4) 5 TP 1.2 3 TP
Positive controls S9-Mix (-)	Name		ENNG	ENNG			ENNG		4NQO		9AA
	Dose		3 µg	5 µg			2 µg		0.2 µg		80 µg
	Level No. of Revertants		589 (607) 627 19.1 605	581 (544) 526 32.0 525			593 (649) 695 51.8 660		129 (137) 147 9.3 134		344 (507) 646 152.5 532
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100			TA1535	WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)	77 (85) 72 17.8# 105			20 (14) 13 6.0 8	30 (32) 32 2.0 34		22 (26) 31 4.6 25		8 (12) 14 3.2 13
	5 µg	92 (86) 90 8.7 76			16 (12) 9 3.8 10	36 (38) 44 4.9 35		25 (27) 30 2.9 25		13 (12) 7 4.2 15
	15 µg	86 (87) 82 5.6 93			11 (9) 9 1.5 8	31 (31) 31 0.6 30		26 (23) 26 4.6 18		18 (13) 12 4.2 10
	50 µg	95 (96) 96 1.5 98			10 (9) 9 1.0 8	32 (31) 25 5.1 35		24 (30) 31 6.0 36		10 (13) 16 3.1 12
	150 µg	101 (87) 89 14.6 72			11 (14) 15 2.3 15	26 (32) 32 5.5 37		26 (26) 21 5.0 31		13 (12) 10 2.1 14
	500 µg	74 (72) 63 8.6 80			11 (12) 11 2.3 15	34 (39) 40 4.2 42		21 (22) 32 9.1 14		16 T (11) 10 T 4.6 7 T
	1500 µg	81 TP (78) 75 TP 3.1 77 TP			13 TP (11) 8 TP 2.5 11 TP	29 P (32) 34 P 2.9 34 P		25 P (23) 16 P 6.2 28 P		12 TP (10) 8 TP 2.0 10 TP
	5000 µg	61 TP (68) 69 TP 7.0 75 TP			9 TP (9) 9 TP 0.6 8 TP	31 TP (35) 33 TP 5.9 42 TP		24 TP (19) 17 TP 4.0 17 TP		8 TP (10) 14 TP 3.5 8 TP
Positive controls S9-Mix (+)	Name	2AA			2AA	2AA		BP		2AA
	Dose	1 µg			2 µg	10 µg		5 µg		2 µg
	Level No. of Revertants	849 (736) 653 101.2 707			201 (193) 179 12.2 199	346 (325) 334 26.1 296		227 (252) 255 24.1 275		167 (174) 185 9.6 170

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-AminoacridineENNG:

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

T: Partial absence of bacterial background lawn

P: Precipitate

#: Standard deviation

 

Table 3. Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding test: EXP1
Number of revertants (mean number of colonies per plate)
Base-pair substitution type			Frameshift type
TA100	TA1535	WP2uvrA	TA98	TA1537
83 94 (91) 97	17 13 (19) 27	28 29 (32) 38	15 25 (22) 26	7 4 (7) 10
Main test: EXP2
Number of revertants (mean number of colonies per plate)
Base-pair substitution type			Frameshift type
TA100	TA1535	WP2uvrA	TA98	TA1537
125 109 (114) 108	25 20 (21) 17	33 40 (36) 35	26 22 (24) 25	9 8 (9) 10

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
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 OPPT 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 seven 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 the range-finding test was determined in a preliminary toxicity assay and was 5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range, fresh cultures of the bacterial strains and fresh test item formulations. Additional dose levels and an expanded dose range were selected in both experiments in order to achieve four non-toxic dose levels and the toxic limit of the test item. The vehicle (acetone) 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. In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains (except TA98 dosed in the presence of S9-mix), initially from 1500 μg/plate in both the presence and absence of S9-mix. In the main test (pre-incubation method) the test item induced toxicity to all of the tester strains with Salmonella strain TA1537 exhibiting weakened lawns from 500 μg/plate in both the absence and presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 μg/plate. A test item precipitate (globular in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. 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.