Cyclohexane-1,3-dione

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:

The experimental phase of this study was performed between 23 January 2008 and 19 February 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2010

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 15-09-2009 Date of Signature: 26-11-2009

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,3-Cyclohexanedione

IUPAC Name:

1,3-Cyclohexanedione

Details on test material:

Sponsor's identification: 1,3-Cyclohexanedione
Description: Pale yellow powder
Batch number: 1073312
Date received: 14 January 2008
Storage conditions: room temperature in the dark

Method

Target gene:

Histidine for Salmonella.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced 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 one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
Sterile distilled water.

- Justification for choice of solvent/vehicle:
The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in - house

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA102

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 1,8-Dihydroxyanthraquinone: 10 µg/plate

Remarks:

With S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix

Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA102

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9 mix

Migrated to IUCLID6: mitomycin C: 0.5 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation
Dunnetts Linear Regression Analysis

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Small decreases in revertant colony frequency were noted at 5000 µg/plate for several of the tester strains; however there was no visible reduction in the growth of the bacterial background lawn at any dose level.

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:

Small decreases in revertant colony frequency were noted at 5000 µg/plate for several of the tester strains; however there was no visible reduction in the growth of the bacterial background lawn at any dose level.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house.
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- Formulated concentrations were adjusted to allow for the stated water/impurity content (3%) of the test material.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Small decreases in revertant colony frequency were noted at 5000 µg/plate for several of the tester strains; however there was no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

RESULTS

Preliminary ToxicityTest

The test material was non-toxic to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) Metabolic Activation	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	110	73	84	68	95	93	74	88	85	77	70
+	TA100	85	75	76	77	80	93	64	72	82	65	62

Mutation Test

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented inTable1and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5.

Small decreases in revertant colony frequency were noted at 5000 µg/plate for several of the tester strains; however there was no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table1               Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		TA102		TA98		TA1537
93		21		221		22		25
89	(91)	20	(21)	207	(208)	27	(23)	7	(14)
92		22		196		20		10

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		TA102		TA98		TA1537
88		29		260		22		19
74	(88)	22	(27)	254	(268)	20	(22)	10	(14)
102		30		291		25		13

Table 2               Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 08 February 2008						To: 11 February 2008
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type								Frameshift type
		TA100		TA1535		TA102				TA98		TA1537
-	0	73 67 92	(77) 13.1#	17 17 14	(16) 1.7	300 257 258			(272) 24.5	25 21 12	(19) 6.7	15 12 11	(13) 2.1
-	50	75 93 76	(81) 10.1	20 14 14	(16) 3.5	274 265 285			(275) 10.0	14 11 19	(15) 4.0	14 11 10	(12) 2.1
-	150	66 81 68	(72) 8.1	18 19 14	(17) 2.6	289 274 291			(285) 9.3	17 13 10	(13) 3.5	9 15 12	(12) 3.0
-	500	65 69 77	(70) 6.1	20 20 14	(18) 3.5	271 294 257			(274) 18.7	11 10 9	(10) 1.0	5 10 14	(10) 4.5
-	1500	65 62 63	(63) 1.5	12 16 17	(15) 2.6	255 249 278			(261) 15.3	14 9 13	(12) 2.6	10 7 11	(9) 2.1
-	5000	63 57 57	(59) 3.5	11 9 9	(10) 1.2	240 266 236			(247) 16.3	14 11 9	(11) 2.5	7 4 11	(7) 3.5
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		MMC				4NQO		9AA
		3		5		0.5				0.2		80
		390 392 387	(390) 2.5	77 96 101	(91) 12.7	1974 948 917	(1280) 601.5			289 272 288	(283) 9.5	373 414 309	(365) 52.9

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

4NQO   4-Nitroquinoline-1-oxide

9AA     9-Aminoacridine

MMC   Mitomycin C

#            Standard deviation

 

Table 3               Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 08 February 2008						To: 11 February 2008
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type								Frameshift type
		TA100		TA1535		TA102				TA98		TA1537
+	0	91 73 66	(77) 12.9#	13 10 11	(11) 1.5	258 254 242			(251) 8.3	25 22 22	(23) 1.7	6 6 7	(6) 0.6
+	50	77 67 78	(74) 6.1	9 10 10	(10) 0.6	238 260 234			(244) 14.0	21 19 21	(20) 1.2	7 5 6	(6) 1.0
+	150	60 78 67	(68) 9.1	12 14 12	(13) 1.2	250 209 236			(232) 20.8	20 18 19	(19) 1.0	4 7 7	(6) 1.7
+	500	67 76 64	(69) 6.2	11 10 9	(10) 1.0	269 241 242			(251) 15.9	14 10 17	(14) 3.5	7 4 4	(5) 1.7
+	1500	54 47 62	(54) 7.5	11 10 11	(11) 0.6	264 258 244			(255) 10.3	20 17 17	(18) 1.7	3 6 6	(5) 1.7
+	5000	43 54 43	(47) 6.4	10 7 7	(8) 1.7	225 228 228			(227) 1.7	11 10 7	(9) 2.1	3 7 6	(5) 2.1
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		DAN				BP		2AA
		1		2		10				5		2
		1269 1054 756	(1026) 257.6	201 177 162	(180) 19.7	1257 1280 1296	(1278) 19.6			576 803 717	(699) 114.6	243 196 243	(227) 27.1

2AA     2-Aminoanthracene

BP         Benzo(a)pyrene

DAN     1,8-Dihydroxyanthraquinone

#            Standard deviation

PLEASE SEE OVERALL REMARKS for Tables 4 & 5 (Experiment 2)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction.

The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using the plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended slightly, following the results of Experiment 1, and was 15 to 5000 µg/plate

An additional dose level was included in Experiment 2 to allow for slight decreases in revertant colony frequency noted in the first experiment.

Results.

The vehicle (sterile distilled water) 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.

Small decreases in revertant colony frequency were noted at 5000 µg/plate for several of the tester strains; however there was no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.