[No public or meaningful name is available]

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:

17-04-2000 to 02-06-2000

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:

2000

Report date:

2000

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: February 2000 ; signature: April 2000

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

451-230-7

EC Name:

-

Cas Number:

55739-89-4

Molecular formula:

C10H18O

IUPAC Name:

2-ethyl-4,4-dimethylcyclohexan-1-one

Test material form:

liquid

Details on test material:

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

Method

Target gene:

histidine or tryptophan locus

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:
TA100 and WP2urvA : 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 1 (plate incorporation method):
All Salmonella strains: 0, 15, 50, 150, 500, 1500, 5000 µg/plate
WP2urvA: 0, 50, 150, 500, 1500, 5000 µg/plate

Experiment 2 (plate incorporation method):
TA98, TA1537 and WP2urvA: 0, 50, 150, 500, 1500, 5000 µg/plate
TA100, TA1535: 0, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Not reported, however DMSO is a standard vehicle for the OECD TG 471 guideline with available HCD information.

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 (plate incorporation)

DURATION
- Exposure duration:
Experiment 1. All of the plates were incubated at 37ºC 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. All of the plates were incubated at 37ºC 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).

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. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
4. Biological relevance against in-house historical control ranges.

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

Additional information on results:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The current Positive HCD dataset is presented in the full study report.
- Negative (solvent/vehicle) historical control data: The current background spontaneous revertant counts in concurrent untreated controls and/or or vehicle controls ; historic negative controls are presented in the full study report.

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		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	119 105 105	(110) 8.1#	23 22 24	(23) 1.0	18 16 18	(26) 7.5	11 15 12	(13) 2.1	4 6 14	(8) 5.3
	15 µg	114 109 113	(112) 2.6	24 26 28	(26) 2.0	N/T		13 15 12	(13) 1.5	3 8 13	(8) 5.0
	50 µg	105 123 102	(110) 11.4	20 22 31	(26) 2.0	20 12 17	(26) 7.5	18 28 13	(20) 7.6	11 10 14	(12) 2.1
	150 µg	124 114 112	(117) 6.4	20 20 29	(24) 5.9	7 18 11	(26) 7.5	20 21 10	(17) 6.1	7 10 14	(9) 1.5
	500 µg	85 102 103	(97) 10.1	20 19 25	(23) 5.2	21 12 18	(26) 7.5	14 C 25	(20) 7.8	9 9 10	(9) 0.6
	1500 µg	116S 84S 109S	(103) 16.8	23S 29S 23S	(25) 3.5	14 6 11	(31) 7.0	17 23 18	(19) 3.2	13 12 6	(10) 3.8
	5000 µg	0V 0V 0V	(0) 0.0	0T 0T 0T	(0) 0.0	17S 20S 19S	(19) 1.5	21S 24S 21S	(22) 1.7	0V 0V 0V	(0) 0.0
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
		507 524 544	(525) 18.5	366 273 265	(301) 56.1	680 704 694	(693) 12.1	120 99 120	(113) 12.1	1789 1561 1500	(1617) 152.3
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)	119 114 118	(117) 2.6#	13 18 10	(14) 4.0	13 9 19	(14) 5.0	40 34 33	(36) 3.8	10 9 10	(10) 0.6
	15 µg	133 133 129	(132) 2.3	12 13 15	(13) 1.5	N/T		30 39 30	(33) 5.2	9 16 15	(13) 3.8
	50 µg	114 113 130	(119) 9.5	14 10 23	(16) 6.7	21 15 16	(17) 3.2	39 28 34	(34) 5.5	15 7 12	(11) 4.0
	150 µg	107 110 113	(110) 3.0	23 9 15	(16) 7.0	14 17 15	(15) 1.5	25 42 30	(32) 8.7	9 14 17	(13) 4.0
	500 µg	124 119 125	(123) 3.2	12 12 19	(14) 4.0	17 17 8	(14) 5.2	40 17 27	(28) 11.5	13 18 12	(14) 3.2
	1500 µg	122 93 115	(110) 15.1	11S 17S 9S	(12) 4.2	17 13 14	(15) 2.1	25 21 23	(23) 2.0	12 4 12	(9) 4.6
	5000 µg	0V 0V 0V	(0) 0.0	0T 0T 0T	(0) 0.0	19 19 15	(18) 2.3	30 34 48	(37) 9.5	14S 10S 8S	(11) 3.1
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
		2005 1967 2141	(2038) 91.5	239 229 217	(228) 11.0	426 420 414	(420) 6.0	358 284 296	(313) 39.7	502 578 566	(549) 40.9

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

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		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	118 132 99	(116) 16.6#	33 26 26	(28) 4.0	20 25 21	(22) 2.6	27 27 20	(25) 4.0	12 8 10	(10) 2.0
	15 µg	106 87 121	(105) 17.0	28 26 34	(29) 4.2	N/T		N/T		N/T
	50 µg	115 111 114	(113) 2.1	35 18 28	(27) 8.5	18 24 18	(20) 3.5	14 21 24	(20) 5.1	6 5 7	(6) 1.0
	150 µg	122 111 109	(114) 7.0	28 22 22	(24) 3.5	27 25 18	(23) 4.7	20 18 32	(23) 7.6	8 6 11	(8) 2.5
	500 µg	108 133 120	(120) 12.5	30 24 27	(27) 3.0	24 27 24	(25) 1.7	13 21 21	(18) 4.6	12 3 11	(8) 2.5
	1500 µg	127 118 122	(122) 4.5	20 25 30	(25) 5.0	21 27 16	(21) 5.5	20 20 23	(21) 1.7	4 11 10	(9) 4.9
	5000 µg	0V 0V 0V	(0) 0.0	14S 19S 12S	(15) 3.6	19S 22S 22S	(21) 1.7	20S 21S 18S	(20) 1.5	0V 0V 0V	(8) 3.8
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
		323 299 247	(290) 38.9	157 161 173	(164) 8.3	585 554 545	(561) 21.0	105 143 151	(133) 24.6	736 961 859	(852) 112.7
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)	103 109 94	(102) 7.5#	20 11 14	(15) 4.6	13 18 19	(17) 3.2	33 38 38	(36) 2.9	23 10 22	(18) 7.2
	15 µg	107 109 106	(107) 1.5	14 12 16	(14) 2.0	N/T		N/T		N/T
	50 µg	100 114 109	(108) 7.1	15 15 14	(15) 0.6	30 17 14	(20) 8.5	30 33 33	(32) 1.7	9 19 13	(14) 5.0
	150 µg	95 106 111	(104) 8.20	11 16 16	(14) 2.9	18 18 17	(18) 0.6	32 30 26	(29) 3.1	16 12 19	(16) 3.5
	500 µg	117 106 112	(112) 5.5	13 11 14	(13) 1.5	24 25 26	(25) 1.0	37 39 34	(37) 2.5	16 12 16	(15) 2.3
	1500 µg	108 112 79	(100) 18.0	5 12 13	(10) 4.4	27 15 22	(21) 6.0	26 23 36	(28) 6.8	17 18 17	(17) 0.6
	5000 µg	75S 111S 51S	(79) 30.2	12S 13S 11S	(12) 1.0	20 12 17	(16) 4.0	38 22 55	(38) 16.5	14S 13S 8S	(12) 3.2
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
		1834 1949 1953	(1912) 67.6	585 643 X	(614) 41.0	1052 1009 986	(1016) 33.5	379 355 408	(381) 26.5	491 541 603	(545) 56.1

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 B.13/14, 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 the Ames plate incorporation method 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 Experiment 1 range finding test was predetermined based on the results of a preliminary toxicity assay. Experiment 1 (plate incorporation method): All Salmonella strains: 0, 15, 50, 150, 500, 1500, 5000 µg/plate and WP2urvA- : 0, 50, 150, 500, 1500, 5000 µg/plate. The experiment was repeated on a separate day using a slightly amended dose-range TA98, TA1537 and WP2urvA: 0, 50, 150, 500, 1500, 5000 µg/plate and TA100, TA1535: 0, 50, 150, 500, 1500, 5000 µg/plate. Additional dose levels (in excess of the five stated in the protocol) were included to ensure a minimum of four non-toxic dose levels. The vehicle (DMSO) 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 absence of cytotoxicity (reduction in growth of the bacterial background law) in all tester strains. No test item precipitate was observed on the plates at up to 5000 μg/plate. 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.