Reaction mass of (1RS)-1-[(1RS)-3,3-dimethylcyclohexyl]ethyl [(2RS,3RS)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1RS)-3,3-dimethylcyclohexyl]ethyl [(2SR,3SR)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1SR)-3,3-dimethylcyclohexyl]ethyl [(2RS,3RS)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1SR)-3,3-dimethylcyclohexyl]ethyl [(2SR,3SR)-3-ethyloxiran-2-yl]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:

29-01-2014 to 04-03-2014

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:

2014

Report date:

2014

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 (1RS)-1-[(1RS)-3,3-dimethylcyclohexyl]ethyl [(2RS,3RS)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1RS)-3,3-dimethylcyclohexyl]ethyl [(2SR,3SR)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1SR)-3,3-dimethylcyclohexyl]ethyl [(2RS,3RS)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1SR)-3,3-dimethylcyclohexyl]ethyl [(2SR,3SR)-3-ethyloxiran-2-yl]acetate

EC Number:

950-412-8

Molecular formula:

C16H28O3

IUPAC Name:

Reaction mass of (1RS)-1-[(1RS)-3,3-dimethylcyclohexyl]ethyl [(2RS,3RS)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1RS)-3,3-dimethylcyclohexyl]ethyl [(2SR,3SR)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1SR)-3,3-dimethylcyclohexyl]ethyl [(2RS,3RS)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1SR)-3,3-dimethylcyclohexyl]ethyl [(2SR,3SR)-3-ethyloxiran-2-yl]acetate

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: In refrigerator 4°C in the dark, under nitrogen
- Other: clear 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 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method): 0, 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 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/guideline limit of the test item. The dose levels were selected based on the results of Experiment 1.

All Salmonella strains (without S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50 500 μg/plate.
Salmonella strain TA100 and TA1537 (with S9-mix) and E.coli strain WP2uvrA (without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
Salmonella strain TA98 and TA1537 (with S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
E.coli strain WP2uvrA (with S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.

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 in solubility checks performed. Dimethyl sulphoxide was selected as the vehicle.
- Other: Formulated concentrations were (if required) adjusted by an appropriate factor to allow for the stated purity of the test item.

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 (pre-incubation) ; Experiment 2. in medium; in agar (pre-incubation)

DURATION
- Exposure duration:
Experiment 1. 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer (or S9-activation mix, as applicable) and 0.1 mL of the test item formulation, solvent or 0.1 mL of appropriate positive control were incubated at 37 °C± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. After setting, the plates were placed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) during the incubation procedure. 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± 3 °C 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).

Experiment 2. The procedure for incubation and duration was the same as in Experiment 1.

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 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:

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 1535, TA 1537, TA 98 and TA 100

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

Note: The experimental procedure for Experiment 1 tested up to 5000 µg/plate was repeated with limited dose range due to excessive toxicity in the original test. The data for the Salmonella strains given in Experiment 1, refer to the repeat experiment only. Original Experiment 1 data for the Salmonella strains was discarded as there were less than four non-toxic doses.

 

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)	78          (75) 82          8.9# 65	15          (13) 73          4.9 16	25          (21) 17          4.0 20	9             (16) 20          6.4 20	20          (19) 24          5.6 13
	0.015 µg	53         (65) 79          13.1 64	8           (12) 12          2.1 11	N/T	20         (23) 25          2.5 23	13         (14) 17          3.1 11
	0.05 µg	56          (66) 71         9.0 72	11          (11) 12         1.5 9	N/T	23          (15) 11         6.9 11	9             (18) 25         8.2 20
	0.15 µg	83         (82) 82          1.5 80	1           (7) 9             5.3 11	N/T	25         (18) 16          6.2 13	21         (15) 9             6.0 15
	0.5 µg	69         (66) 63          3.1 67	3           (8) 5             6.4 15	N/T	20         (19) 17          1.5 19	15         (15) 17          2.5 12
	1.5 µg	74         (89) 100        13.3 92	11         (12) 12          1.0 13	23         (19) 12          6.4 23	16         (17) 13          4.0 21	11         (16) 15          6.1 23
	5 µg	61         (63) 75          11.6 52	8           (8) 8             0.6 9	23         (16) 16          7.5 8	24         (25) 27          2.1 23	12         (15) 19          3.5 15
	15 µg	45 S      (54) 57 S       7.9 60 S	12 S      (9) 3 S         4.9 11 S	12         (15) 15          2.5 17	20 S      (18) 13 S       4.4 21 S	3 S        (3) 0 S         3.5 7 S
	50 µg	79 S       (72) 88 S       21.0 48 S	11 S       (12) 13 S       1.0 12 S	24          (15) 16          9.5 5	19 S       (13) 12 S       5.1 9 S	3 S         (6) 9 S         3.1 5 S
	150 µg	N/T	N/T	11          (15) 20          4.5 15	N/T	N/T
	500 µg	N/T	N/T	23 S      (24) 31 S       7.0 17 S	N/T	N/T
	1500 µg	N/T	N/T	15 S      (17) 24 S       6.2 12 S	N/T	N/T
	5000 µg	N/T	N/T	12 S       (15) 13 S      4.4 20 S	N/T	N/T
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
		788       (842) 913        64.2 825	1576     (1649) 1613      96.8 1759	520       (388) 414        146.2 231	128       (143) 154        13.5 147	3702      (3014) 2663      595.6 2678
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)	75          (83) 94          10.0# 79	11          (15) 25          8.7 9	32          (35) 36          2.6 37	13          (23) 28          8.7 28	5             (8) 11          3.0 8
	1.5 µg	76         (69) 68          6.1 64	23         (15) 12          7.4 9	19         (25) 23          6.7 32	24         (24) 23          0.6 24	15          (12) 9             3.1 11
	5 µg	63         (77) 68          19.5 99	12         (13) 8             5.6 19	27         (27) 35          8.0 19	19         (19) 19          0.6 20	9             (10) 13          2.6 8
	15 µg	87         (84) 95          13.3 69	11         (10) 9             1.2 11	21         (25) 36          10.0 17	24         (22) 27          5.7 16	5           (9) 12          3.5 9
	50 µg	75         (86) 96          10.5 87	8           (9) 11          1.5 9	24         (23) 21          1.5 23	23         (21) 21          2.0 19	5           (7) 11          3.5 5
	150 µg	80         (77) 67          8.5 83	15 S      (10) 8 S         4.0 8 S	25         (22) 21          2.6 20	24 S      (25) 25 S       1.5 27 S	9           (9) 11          2.0 7
	500 µg	82 S      (79) 102 S    24.6 53 S	9 S        (8) 7 S         1.2 9 S	17         (19) 12          8.2 28	17 S      (19) 19 S       1.5 20 S	5 S         (8) 13 S      4.6 5 S
	1500 µg	68 V     (65) 68 V      5.2 59 V	4 V        (7) 9 V        2.6 8 V	20 S      (21) 20 S       1.7 23 S	15 S      (18) 21 S       3.1 17 S	7 S        (5) 1 S         3.8 8 S
	5000 µg	74 V     (73) 74 V      1.2 72 V	1 V        (3) 5 V        2.0 3 V	24 S      (20) 20 S       4.5 15 S	13 S      (9) 8 S         3.2 7 S	7 S         (5) 4 S         1.7 4 S
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
		548        (588) 547        69.6 668	155        (138) 124        15.7 135	182        (153) 140        25.5 136	122        (117) 127        13.2 102	132        (134) 120        15.6 151

 

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)	68          (77) 87          9.6# 75	15          (17) 19          2.0 17	25          (20) 11          7.8 24	8             (16) 19          6.7 20	12          (13) 17          4.0 9
	0.05 µg	72         (81) 84          7.6 86	12         (10) 9             1.7 9	N/T	25         (18) 17          6.6 12	8           (10) 15          4.0 8
	0.15 µg	87          (81) 72         7.8 83	9             (9) 9           0.6 8	N/T	17          (15) 15         2.5 12	12          (11) 5           5.6 16
	0.5 µg	72         (69) 61          7.4 75	9           (8) 8             0.6 8	15         (18) 19          2.6 20	13         (13) 11          2.0 15	13         (8) 3             5.0 7
	1.5 µg	72          (67) 61          5.7 69	13          (12) 13          1.2 11	12          (12) 16          3.5 9	19          (17) 17          2.0 15	7             (10) 7             5.8 17
	5 µg	60          (64) 69          4.5 64	11          (10) 11          1.2 9	15          (16) 15          1.2 17	12          (14) 15          1.7 15	5             (8) 9             2.3 9
	15 µg	60 S      (69) 71 S       7.8 75 S	7 S        (9) 13 S       3.5 7 S	16         (16) 9             7.5 24	20         (17) 21          5.5 11	11 S      (9) 11 S       3.5 5 S
	50 µg	74 S      (66) 61 S       7.0 63 S	7 S        (6) 5 S         1.2 7 S	21         (15) 12          4.9 13	11 S      (9) 8 S         1.7 8 S	1 S        (3) 4 S         2.1 5 S
	150 µg	N/T	N/T	25          (19) 17         5.3 15	N/T	N/T
	500 µg	N/T	N/T	15 S       (14) 17 S      3.1 11 S	N/T	N/T
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
		746       (802) 859        56.5 802	450       (435) 370        59.4 486	1056     (913) 877        128.4 807	202       (189) 162        23.1 202	568        (559) 492       62.5 616
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          (71) 74          3.1# 68	23          (19) 15          4.0 19	24          (26) 27          1.7 27	29          (23) 19          5.5 20	12          (12) 12          0.0 12
	0.15 µg	N/T	13         (12) 13          1.2 11	N/T	16         (19) 20          2.3 20	N/T
	0.5 µg	83          (80) 80         3.5 76	19          (17) 19         4.0 12	N/T	28          (25) 24         2.3 24	4             (7) 5           4.4 12
	1.5 µg	64         (64) 67          3.0 61	16         (15) 16          1.7 13	25         (27) 24          3.8 31	16         (17) 17          0.6 17	9           (8) 9             1.2 7
	5 µg	67          (75) 84          8.5 74	8             (15) 24          8.2 13	16          (23) 28          6.2 25	21          (23) 27          3.8 20	13          (11) 12          2.1 9
	15 µg	67          (70) 72          2.9 72	15          (16) 16          0.6 16	29          (28) 23          5.0 33	20          (21) 25          3.2 19	5             (11) 7             8.1 20
	50 µg	75         (79) 83          4.0 78	24         (17) 15          6.2 12	23         (25) 24          2.6 28	12         (17) 17          4.5 21	3           (9) 8             6.6 16
	150 µg	78         (78) 90          12.5 65	20 S      (14) 11 S       4.9 12 S	17         (17) 15          2.5 20	21 S      (24) 29 S       4.6 21 S	5           (10) 7             6.4 17
	500 µg	59 S       (52) 48 S      6.4 48 S	N/T	27          (18) 13         8.1 13	N/T	9 S         (5) 3 S        3.2 4 S
	1500 µg	N/T	N/T	16 S       (15) 13 S      1.5 15 S	N/T	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
		746       (802) 859        56.5 802	450       (435) 370        59.4 486	1056     (913) 877        128.4 807	202       (189) 162        23.1 202	568        (559) 492       62.5 616

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

N/T: Not tested at this dose level

S: Sparse bacterial background lawn

T: Toxic, no bacterial background lawn

V: Very weak bacterial background lawn

#: Standard deviation

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, 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 item 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 item using the Ames pre incubation 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). Formulated concentrations were adjusted by an appropriate factor to allow for the stated purity of the test item.The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. However, due to excessive toxicity, a repeat experiment had to be performed (Salmonella strains dosed in the absence of S9-mix only) employing a dose range of 0.015 to 50 μg/plate. The second experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Seven test item dose levels were 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 dose range was amended following the results of Experiment 1 and ranged between 0.05 and 1500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. 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 item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. However the Salmonella strains dosed in the absence of S9-mix had to be repeated and were tested up the toxic limit (i.e. 50 μg/plate based on the first experiment). In the first mutation test, the test item induced a visible reduction in the growth of the bacterial background lawns of all the tester strains in both the presence and absence of S9-mix. Consequently, for the second mutation test the toxic limit was employed as the maximum dose concentration. Results from the second mutation test (pre-incubation method) confirmed the toxicity previously noted with weakened bacterial background lawns observed. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9 mix. 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). 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 E.coli strain WP2uvrA- in the presence and absence of S-9 mix.