(1'R,2R)-2-Methyl-4-(2',2',3'-tri-nal and methylcyclopent-3'-en-1'-yl)-4-pent-enal

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:

11-01-1995 to 28-03-1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. Relevant validity criteria were met with acceptable deviations which were in accordance with the guidelines at the time of the study.

Remarks:

study did not include TA102 or WP2uvrA strains

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

study did not include TA102 or WP2uvrA strains ; relevant validity criteria were met with acceptable deviations which were in accordance with the guidelines at the time of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see above

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: January 1994 ; signature: March 1994

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

422-120-6

EC Name:

-

Cas Number:

166432-53-7

Molecular formula:

C14H22O

IUPAC Name:

2-methyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)pent-4-enal

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: room temperature in the dark
- Other: colourless liquid

Method

Target gene:

histidine or tryptophan locus

Species / strain

Species / strain / cell type:

other: TA98, TA100, TA1535, TA1537 and TA1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat liver S9
- source of S9: Purchased Aro. S9/24/11/94 prepared in house (dates within full study report)
- method of preparation of S9 mix: Documented in the full study report. Stored at -196ºC
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Concurrent positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory

Test concentrations with justification for top dose:

Preliminary toxicity test (TA100): 0, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1 (plate incorporation method):
Without S9: All strains between dose range: 0, 0.05, 0.15, 0.5, 1.5, 5, 50, 150, 500, 1500, 5000 µg/plate
With S9 (10%) : All strains between dose range: 0, 1.5, 5, 50, 150, 500, 1500, 5000 µg/plate
Experiment 2 (plate incorporation method):
Without S9: All strains between dose range: 0, 0.05, 0.15, 0.5, 1.5, 5, 50, 150, 500, 1500, 5000 µg/plate
With S9 (10%) : All strains between dose range: 0, 0.5, 1.5, 5, 50, 150, 500, 1500, 5000 µg/plate
Testing was conducted in up to eight dose levels and/or to ensure a minimum of three or four non-toxic dose levels was achieved and testing up to the maximum recommended dose level of 5000 µg/plate, depending on strain specific cytotoxicity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was fully miscible in acetone at the required concentrations in solubility checks performed. Acetone was 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

other: 2-Aminoanthracene (2AA) ; 4-nitro-o-phenylenediamine (4NOPD)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (plate incorporation)

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) of the background lawn. 0.1 mL aliquots of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 mL of molten. trace histidine supplemented. top agar at 45°C. These sets comprised two test tubes for each bacterial tester strain. 0.1 mL of the appropriately diluted test material or vehicle control was also added to each of the two tubes followed by either 0.5 mL of the S9 liver microsome mix or 0.5 mL of pH 7.4 buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner agar plates Cone tube per platel. This procedure was repeated. In triplicate. for each bacterial strain and for each concentration of test material. Positive controls were similarly prepared with and without S9 activation system, as applicable.

Experiment 2. 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) of the background lawn.

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.
2. A reproducible increase at one or more concentrations.
3. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met. To be considered negative the number of induced evertants compared to spontaneous revertants should be less than two-fold at each dose level employed. the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was both toxicity and the maximum recommended dose depending on bacterial strain type and presence/absence of S9 mix.

In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.

To be considered negative the number of induced evertants compared to spontaneous revertants should be less than two-fold at each dose level employed. the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was both toxicity and the maximum recommended dose depending on bacterial strain type and presence/absence of S9 mix.

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:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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		TA1538		TA98		TA1537
	Solvent Control (acetone)	139 151 128	(139) 11.5#	18 15 18	(18) 1.7	20 30 19	(23) 6.1	18 18 18	(18) 0.0	18 22 23	(21) 2.6
	0.05 µg	N/T		N/T		N/T		N/T		17 17 12	(15) 2.9
	0.15 µg	N/T		N/T		N/T		15 22 20	(19) 3.6	20 17 22	(20) 2.5
	0.50 µg	N/T		20 22 15	(19) 3.6	N/T		17 19 23	(20) 3.1	20 13 29	(20) 8.0
	1.50 µg	N/T		25 17 15	(19) 5.3	N/T		15 28 20	(21) 6.6	17 8 13	(13) 4.5
	5 µg	N/T		19 20 10	(16) 5.5	N/T		19 25 14	(19) 5.5	17 17 13	(16) 2.3
	15 µg	N/T		14 13 32	(20) 10.7	38 30 22	(30) 8.0	20 20 5	(18) 2.9	28 15 8	(17) 10.1
	50 µg	128 121 125	(125) 3.5	18 10 15	(14) 4.0	32 33 24	(30) 4.9	18V 17V 15V	(17) 1.5	10S 12S 9S	(10) 1.5
	150 µg	132 94 122	(116) 19.7	14 10 13	(12) 2.1	27 25 32	(28) 3.6	13V 17V 15V	(15) 2.0	9V 14V 9V	(11) 2.9
	500 µg	116 95 128	(113) 16.7	12S 8S 12S	(11) 2.3	13 29 24	(22) 8.2	13V 17V 14V	(15) 2.1	N/T
	1500 µg	102 91 125	(106) 17.3	N/T		23S 20S 20S	(21) 1.7	N/T		N/T
	5000 µg	49V 48V 32V	(43) 9.5	N/T		15V 19V 22V	(19) 3.5	N/T		N/T
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		4NOPD		4NQO		9AA
		3 µg		5 µg		5 µg		0.2 µg		80 µg
		466 427 430	(441) 21.7	174 169 183	(175) 7.1	384 364 343	(364) 3.5	165 160 191	(172) 16.6	470 281 344	(365) 96.2
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		TA1538		TA98		TA1537
	Solvent Control (acetone)	133 102 113	(116) 15.7#	24 33 20	(26) 6.7	17 17 28	(21) 6.4	24 24 25	(24) 0.6	17 13 9	(13) 4.0
	1.5 µg	N/T		N/T		30 23 27	(27) 3.5	N/T		12 5 14	(10) 4.7
	5 µg	N/T		N/T		18 39 25	(23) 6.1	22 28 20	(23) 4.2	12 15 9	(12) 3.0
	15 µg	N/T		N/T		20 30 19	(27) 6.9	30 35 20	(28) 7.6	9 10 15	(11) 3.2
	50 µg	118 113 137	(123) 12.7	32 29 18	(26) 7.4	35 23 23	(27) 6.9	33 23 24	(27) 5.5	5S 10S 8S	(8) 2.5
	150 µg	156 128 125	(136) 17.1	33 28 25	(29) 4.0	30 30 24	(28) 3.5	29 22 24	(25) 3.6	7S 8S 12S	(9) 2.6
	500 µg	117 125 129	(124) 6.1	20 32 25	(26) 6.0	22 25 12	(20) 6.8	20 33 22	(25) 7.0	15V 9V 9V	(11) 3.5
	1500 µg	75 77 115	(89) 22.5	5 20 13	(13) 7.5	18 14 14	(15) 2.3	15 20 27	(21) 6.0	10V 9V 10V	(10) 0.6
	5000 µg	23S 12S 17S	(17) 5.5	10S 15S 10S	(11) 2.9	14 12 17	(14) 2.5	18S 28S 18S	(21) 5.8	N/T
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		2AA		2AA
		1 µg		2 µg		0.5 µg		0.5 µg		2 µg
		479 495 705	(560) 126.1	253 271 241	(255) 15.1	169 199 200	(189) 17.6	175 200 204	(193) 15.7	65 66 65	(65) 0.6

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

4NQO  4-Nitroquinoline-1-oxide

9AA      9-Aminoacridine

4NOPD4-nitro-o-phenylenediamine

2AA      2-Aminoanthracene

N/T      Not tested at this dose level

S            Sparse bacterial background lawn

V           Very weak bacterial background lawn

T             Toxic, no bacterial lawn

#           Standard deviation

C            Contaminated

X            Plate unscorable

 

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		TA1538		TA98		TA1537
	Solvent Control (acetone)	106 90 97	(98) 8.0#	14 10 13	(12) 2.1	23 15 17	(18) 4.2	21 23 23	(22) 1.2	8 13 8	(10) 2.9
	0.15 µg	N/T		N/T		N/T		19 18 17	(18) 1.0	N/T
	0.50 µg	N/T		N/T		N/T		15 29 18	(21) 7.4	9 8 12	(10) 2.1
	1.50 µg	N/T		17 17 13	(16) 2.3	N/T		20 25 10	(18) 7.6	11 11 8	(10) 1.7
	5 µg	N/T		15 17 19	(17) 2.0	N/T		17 20 20	(19) 1.7	8 8 11	(9) 1.7
	15 µg	N/T		24 15 13	(17) 5.9	14 9 27	(17) 9.3	17 24 19	(20) 3.6	7 11 7	(8) 2.3
	50 µg	107 95 72	(91) 17.8	25 19 10	(18) 7.5	15 18 15	(16) 1.7	18V 19V 17V	(18) 1.0	13S 2S 3S	(6) 6.1
	150 µg	111 86 91	(96) 13.2	14 17 14	(15) 1.7	22 18 9	(16) 6.7	N/T		8V 8V 10V	(9) 1.2
	500 µg	101 101 95	(99) 3.5	7S 14S 14S	(12) 4.0	17 13 13	(14) 2.3	N/T		N/T
	1500 µg	71 65 74	(70) 4.6	N/T		13S 17S 9S	(13) 4.0	N/T		N/T
	5000 µg	48V 59V 63V	(57) 7.8	N/T		9V 14V 6V	(10) 4.0	N/T		N/T
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		4NOPD		4NQO		9AA
		3 µg		5 µg		5 µg		0.2 µg		80 µg
		391 368 380	(380) 11.5	266 216 275	(252) 31.2	343 344 324	(337) 11.3	144 129 133	(135) 7.8	687 810 709	(735) 65.6
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		TA1538		TA98		TA1537
	Solvent Control (acetone)	80 76 75	(77) 2.6#	18 23 19	(20) 2.5	30 27 34	(30) 3.5	25 17 34	(25) 8.5	14 13 18	(15) 2.6
	0.50 µg	N/T		N/T		N/T		N/T		15 11 12	(13) 2.1
	1.50 µg	N/T		N/T		N/T		N/T		13 9 13	(12) 2.3
	5 µg	N/T		N/T		N/T		N/T		15 17 12	(15) 2.5
	15 µg	N/T		N/T		N/T		N/T		12 12 7	(10) 2.9
	50 µg	75 125 107	(102) 25.3	15 19 24	(19) 4.5	30 34 23	(29) 5.6	34 22 32	(29) 6.4	13S 9S 10S	(11) 2.1
	150 µg	111 86 89	(96) 13.7	14 23 22	(20) 4.9	32 24 24	(27) 4.6	33 35 27	(32) 4.2	6S 6S 11S	(8) 2.9
	500 µg	82 101 90	(91) 9.5	22 17 28	(22) 5.5	18 19 29	(22) 6.1	35 17 24	(25) 9.1	N/T
	1500 µg	90 91 61	(81) 17.0	9 12 7	(9) 2.5	25 17 22	(21) 4.0	28 20 17	(22) 5.7	N/T
	5000 µg	53V 59V 10V	(41) 26.7	4S 3S 13S	(7) 5.5	20 24 24	(23) 2.3	17S 10S 12S	(13) 3.6	N/T
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		2AA		2AA
		1 µg		2 µg		0.5 µg		0.5 µg		2 µg
		635 663 725	(675) 46.6	160 172 165	(165) 6.0	325 328 399	(351) 41.9	302 335 299	(312) 20.0	151 185 193	(178) 18.9

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
Negative
Under the conditions of this study the test item 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 and EU Method B.14 for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 in both the presence and absence of S-9 mix. The test strains were treated with the test item the Ames plate incorporation method 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 range finding test was predetermined based on the results of a preliminary toxicity assay. The dose levels were 0.05 to 5000 µg/plate depending on the specific strain of S.typhimurium. The experiment was repeated on a separate day using a dose range based on Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations. 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. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or based on presence of cytotoxicity (reduction in growth of the bacterial background law) in all tester strains. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 in the presence and absence of S-9 mix.