Cyclopropanecarboxylic acid, 2-[1-(3,3-dimethylcyclohexyl)ethoxy]-2-methylpropyl ester

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:

06-05-2004 to 21-06-2004

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:

2004

Report date:

2004

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:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: June 2001; signature: January 2002

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

460-490-0

EC Name:

-

Cas Number:

477218-42-1

Molecular formula:

C18H32O3

IUPAC Name:

2-[(1S)-1-[(1R)-3,3-dimethylcyclohexyl]ethoxy]-2-methylpropyl cyclopropanecarboxylate; 2-[(1S)-1-[(1S)-3,3-dimethylcyclohexyl]ethoxy]-2-methylpropyl cyclopropanecarboxylate

Test material form:

liquid

Details on test material:

Physical state: Liquid
- Storage condition of test material: at room temperature
- Other: colourless liquid

Method

Target gene:

histidine or tryptophan locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment 2 (pre-incubation method): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Salmonella strain TA1537 (with and without S9-mix): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Salmonella strain TA98 and E.coli strain WP2uvrA (with and without S9-mix): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Salmonella strains TA100 and TA1535 (with and without S9-mix): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol (purity > 99%)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and relative nontoxicity to the bacteria.
- Other: No precipitation of the test item occurred up to the highest investigated dose.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

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 (pre-incubation)

DURATION
- Exposure duration:
Experiment 1. The appropriate bacterial strain culture, with or without S9-activation mix, as applicable, the test item formulation, solvent or appropriate positive control were incubated at 37°C for 4 hours (with shaking) prior to plating. All testing for this experiment was performed in triplicate. Concurrent negative controls were dosed using the standard plate incorporation method. All of the plates were incubated at 37°C for approximately 48 hours in the dark, and scored for the presence of revertant colonies using an automated colony counting system. Due to reduced background growth, some of the plates were counted manually.

Experiment 2. The procedure for incubation and duration was the same as in Experiment 1, with the exception: In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45°C) was added to each tube. The mixture was poured on minimal agar plates.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

In accordance with the relevant guidelines.

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
4. Statistical analysis of data (as required or applicable).
5. Fold increase greater than two or three times the concurrent solvent control for respective tester strain

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:

Not performed.

Results and discussion

Test results

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

Remarks on result:

other: all strains/cell types tested

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	TA102	TA1535	TA98	TA1537
	Solvent Control	150 (151) 139 13.1 165	111 (129) 122 21.8 153	18 (16) 21 6.2 9	14 (16) 19 2.6 15	6 (8) 7 3.2 12
	33 µg	137 (136) 140 4.6 131	99 (104) 96 10.8 116	18 (18) 14 4.0 22	15 (14) 16 3.2 10	6 (5) 3 1.5 5
	100 µg	141 (137) 132 4.7 139	71 (94) 111 20.5 99	15 (14) 12 1.7 15	17 (17) 15 1.5 18	8 (8) 7 0.6 8
	333 µg	122 (132) 136 9.1 139	112 (111) 108 2.3 112	19 (17) 16 2.1 15	16 (16) 18 2.5 13	7 (8) 6 2.1 10
	1000 µg	143 (134) 137 11.4 121	72 (86) 96 12.5 90	12 (14) 11 3.8 18	17 (16) 15 1.2 17	6 (6) 7 0.6 6
	2500 µg	135 (136) 143 7.0 129	117 (108) 114 13.7 92	13 (15) 19 3.8 12	14 (14) 12 2.0 16	5 (4) 2 2.1 6
	5000 µg	140 (144) 149 4.5 144	98 (103) 112 7.8 99	21 (19) 16 2.5 19	14 (12) 11 2.1 10	3 (4) 5 1.2 3
Positive controls S9-Mix (-)	Name	Sodium Azide	methyl methane sulfonate	Sodium Azide	4-nitro-o-phenylene-idamine	4-nitro-o-phenylene-idamine
	Dose Level	10.0 µg/plate	4.0 µg/plate	10.0 µg/plate	10.0 µg/plate	50.0 µg/plate
	No. of Revertants	632 (574) 537 50.7 554	530 (530) 486 44.0 574	296 (318) 312 26.1 347	124 (135) 145 10.5 136	68 (67) 58 8.1 74
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains			Frameshift strains
		TA100	TA102	TA1535	TA98	TA1537
	Solvent Control	166 (167) 170 3.1 164	120 (136) 141 13.8 146	20 (18) 18 2.0 16	17 (17) 16 1.5 19	16 14 13 1.7 13
	33 µg	140 (154) 163 12.3 159	104 (122) 118 19.8 143	24 (20) 15 4.6 21	12 (13) 13 1.5 15	21 (15) 8 6.5 15
	100 µg	148 (149) 152 2.6 147	111 (121) 128 8.9 124	22 (22) 24 2.5 19	14 (16) 18 2.1 17	10 (13) 21 6.7 9
	333 µg	138 (132) 121 9.8 138	115 (120) 132 10.8 112	19 (21) 23 2.0 21	17 (17) 14 3.0 20	10 (10) 10 0.6 9
	1000 µg	131 (118) 114 11.2 110	67 (78) 79 10.1 87	14 (16) 17 1.7 17	20 (17) 14 3.0 17	15 (12) 11 2.3 11
	2500 µg	102 (106) 116 9.1 99	61 (53) 63 16.2 34	5 (7) 5 2.9 10	21 (20) 20 0.6 20	16 (9) 6 6.1 5
	5000 µg	80 (90) 93 8.5 96	20 (24) 23 4.0 28	4 (2) 1 1.7 1	14 (16) 19 2.5 16	3 (3) 2 0.6 3
Positive controls S9-Mix (+)	Name	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene
	Dose Level	2.5 µg/plate	10.0 µg/plate	2.5 µg/plate	2.5 µg/plate	2.5 µg/plate
	No. of Revertants	1224 (1179) 1206 63.0 1107	757 (821) 840 56.9 866	320 (297) 302 25.9 269	147 (14) 162 15.0 132	201 (179) 177 21.5 158

 

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	TA102	TA1535	TA98	TA1537
	Solvent Control	81 (83) 89 5.3 79	142 (139) 141 4.9 133	23 (19) 16 3.6 18	27 (31) 38 5.9 29	6 (6) 7 1.0 5
	33 µg	85 (78) 74 5.9 76	94 (90) 81 7.8 95	15 (13) 14 2.6 10	18 (21) 25 3.5 21	4 (4) 3 1.0 5
	100 µg	84 (69) 60 5.9 63	88 (93) 99 5.7 91	12 (14) 16 2.1 15	23 (32) 20 1.5 21	3 (4) 6 1.5 4
	333 µg	73 (74) 79 5.0 69	38 (35) 39 6.1 28	9 (10) 11 1.2 9	25 (24) 20 3.6 27	5 (6) 4 2.1 8
	1000 µg	54 (57) 67 8.5 51	27 (23) 17 5.3 25	13 (12) 3 2.3 13	21 (22) 24 1.5 22	8 (8) 8 0.6 7
	2500 µg	48 (49) 54 7.0 46	18 (18) 20 2.5 15	8 (9) 9 1.5 11	25 (24) 28 5.1 18	8 (5) 4 2.3 4
	5000 µg	38 (29) 26 7.6 24	17 (15) 9 4.9 18	7 (9) 10 1.7 10	23 (25) 26 2.1 27	2 (3) 3 1.0 4
Positive controls S9-Mix (-)	Name	Sodium Azide	methyl methane sulfonate	Sodium Azide	4-nitro-o-phenylene-idamine	4-nitro-o-phenylene-idamine
	Dose Level	10.0 µg/plate	4.0 µg/plate	10.0 µg/plate	10.0 µg/plate	50.0 µg/plate
	No. of Revertants	559 (580) 595 18.7 586	801 (794) 798 10.2 782	1266 (1244) 1201 37.0 1264	297 (300) 306 5.5 296	103 (102) 106 4.6 97
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains			Frameshift strains
		TA100	TA102	TA1535	TA98	TA1537
	Solvent Control	101 (167) 108 5.1 98	161 (162) 159 3.6 166	24 (21) 19 2.6 20	29 (28) 26 1.5 28	11 (9) 7 2.1 10
	33 µg	114 (107) 116 13.9 91	101 (105) 119 12.5 95	13 (13) 12 1.5 15	28 (29) 30 1.0 29	11 (9) 6 2.5 9
	100 µg	86 (78) 76 7.6 71	99 (97) 104 7.6 89	12 (14) 16 2.1 13	22 (21) 23 3.2 17	11 (9) 6 2.9 11
	333 µg	57 (46) 41 9.9 39	29 (30) 34 4.0 26	15 (14) 16 2.6 11	25 (28) 31 3.1 27	7 (5) 2 3.5 n.a.
	1000 µg	65 (57) 55 7.6 50	21 (19) 17 2.1 18	6 (10) 9 4.6 15	33 (31) 30 1.5 31	2 (5) 7 2.9 7
	2500 µg	51 (45) 45 6.5 38	10 (14) 18 5.7 n.a.	8 (8) 5 3.0 11	7 (9) 11 2.1 10	4 (2) 0 2.8 n.a.
	5000 µg	28 (35) 37 6.7 41	9 (9) n.a. 0.7 8	5 (5) 6 0.6 5	14 (11) 7 4.9 n.a.	1 (1) 1 0.0 1
Positive controls S9-Mix (+)	Name	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene
	Dose Level	2.5 µg/plate	10.0 µg/plate	2.5 µg/plate	2.5 µg/plate	2.5 µg/plate
	No. of Revertants	599 (684) 768 84.5 685	864 (786) 795 83.4 698	182 (181) 170 10.5 191	655 (585) 548 60.9 551	129 (124) 118 5.7 126

n.a. = not analysable (no distinction possible between colonies and reduced background growth).

 

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 under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 in both the presence and absence of S-9 mix. The test strains were treated with the test substance using the Ames plate incorporation method (Experiment 1) and pre incubation method (Experiment 2) at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (15% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 33 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Six test item dose levels were again selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item the maximum recommended dose level of 5000 μg/plate. The vehicle (ethanol) 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 (plate-incorporation) was selected as the maximum recommended dose level of 5000 μg/plate. For the second mutation test (pre-incubation) the toxic limit was employed as the maximum dose concentration. In the second experiment, reduced background growth was observed in strains TA1535, TA1537, and TA100 at 333 μg/plate and above with metabolic activation, in strains TA98 (with metabolic activation) and TA100 (without metabolic activation) at 1000 ug/plate and above, and in strain TA102 at 33 μg/plate and above with and without metabolic activation. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix. 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) or were within the expected ranges. 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, TA102, TA1535, and TA1537 in the presence and absence of S-9 mix.