4,8-Cyclododecadien-1-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19-11-2014 to 14-01-2015

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:

other: US EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

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:

(3E,7E)-cyclododeca-3,7-dien-1-one; (4E,8Z)-cyclododeca-4,8-dien-1-one; (4Z,8E)-cyclododeca-4,8-dien-1-one

EC Number:

941-893-5

Cas Number:

15229-79-5

Molecular formula:

C12H18O

IUPAC Name:

(3E,7E)-cyclododeca-3,7-dien-1-one; (4E,8Z)-cyclododeca-4,8-dien-1-one; (4Z,8E)-cyclododeca-4,8-dien-1-one

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Keep in the dark at 0 - 10 °C, under nitrogen
- Other: Colourless

Method

Target gene:

histidine operon (Salmonella strains)
tryptophan operon (E.coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/β-naphthoflavone activated rat liver S9

Test concentrations with justification for top dose:

Experiment 1 (pre-incubation method): range-finding test: 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Experiment 2 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 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 following the change in test methodology. The dose levels were selected based on the results of Experiment 1.
TA 100 (- MA); TA 98, TA 1537 (+ MA); WP2 uvrA (+/- MA): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate
TA 98, TA 1535, TA 1537 (- MA); TA 1535, TA 100 (+ MA): 0.15, 0.5, 1.5, 5, 15, 50, 150 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, Dimethyl sulphoxide was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

WP2 uvrA: 2 µg/plate, TA 100: 3 µg/plate, TA 1535: 5 µg/plate (without activation)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537: 80 µg/plate (without activation)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

TA 98: 0.2 µg/plate (without activation)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 100: 1 µg/plate, TA 1535 & TA 1537: 2 µg/plate, WP2 uvrA: 10 µg/plate (with activation)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA 98: 5 µg/plate (with activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (pre-incubation)

DURATION
- Exposure duration: 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media. 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). A number of manual counts were performed, predominantly due to colonies spreading, thus distorting the actual plate count.

SELECTION AGENT (mutation assays): histidine or tryptophan deficient agar

NUMBER OF REPLICATIONS: triplicate plates, experiment repeated

NUMBER OF CELLS EVALUATED: the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were assessed microscopically for evidence of thinning (toxicity). Several manual counts were required, predominantly due to the revertant colonies spreading slightly, thus distorting the actual plate count.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth and condition of background lawn.

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:

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:

Reduction in background lawn from 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction in background lawn at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls (mean, n=3 plates)

Concentration (µg/plate)	TA 100		TA 1535		WP2 uvrA		TA 98		TA 1537
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
Solvent control	77	92	16	9	22	30	22	18	6	7
1.5	73	78	14	12	20	23	17	21	6	6
5	72	87	18	12	16	26	26	15	11	8
15	68	81	9	9	19	28	23	19	12	11
50	79	86	12	11	24	27	17	20	13	8
150	61	78*	11*	12*	22	22	19*	19	5*	10
500	48*	76*	11*	0**	14*	23*	9*	23*	7*	4*
1500	0**	42***	0**	0***	0**	21*	0***	8**	0***	5*
5000	0***	0***	0***	0***	0***	14**	0***	0***	0***	0***
Positive control	519	496	2242	128	679	131	195	115	639	272

* Sparse bacterial background lawn

** Very weak bacterial background lawn

*** Toxic, no bacterial background lawn

Table 2 : Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls (mean, n=3 plates)

Concentration (µg/plate)	TA 100		TA 1535		WP2 uvrA		TA 98		TA 1537
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
Solvent control	72	69	20	11	13	23	20	16	12	9
0.15	NT	71	9	11	NT	NT	16	NT	6	NT
0.5	67	75	17	11	10	20	18	20	5	9
1.5	78	75	15	12	10	18	19	12	7	9
5	70	67	15	9	13	20	19	18	9	9
15	74	67	11	9	15	22	15	20	10	7
50	57*	78*	7*	8*	12	19	14*	17	9*	7
150	58*	56*	7**	7*	11	12	16*	13*	8*	8*
500	44**	NT	NT	NT	9*	15*	NT	14*	NT	5*
Positive control	794	649	120	167	431	232	159	226	150	216

NT not tested

* Sparse bacterial background lawn

** Very weak bacterial background lawn

Table 3. Spontaneous Mutation Rates (Concurrent Negative Controls): Experiment 1 and 2, respectively

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2urvA		TA98		TA1537
82		12		20		27		13
86	(88)	8	(9)	19	(20)	13	(12)	11	(12)
95		8		21		27		13
Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2urvA		TA98		TA1537
88		13		20		16		11
74	(78)	9	(11)	16	(18)	18	(15)	33	(8)
73		12		19		10		11

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
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 and US EPA OCSPP harmonized guideline for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance 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 substance using the pre incubation methods 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 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of the range-finding test and ranged between 0.15 and 500 μg/plate, depending on bacterial strain type and presence or absence of metabolic activation (S9-mix). 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 following the change in test methodology. The dose range was amended following the results of Experiment 1 and ranged between 0.15 and 500 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. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in the absence of S9-mix, initially from 150 μg/plate (TA1535, TA98 and TA1537) and 500 μg/plate (TA100 and WP2uvrA). In the presence of S9-mix, weakened lawns were noted from 150 μg/plate (TA100 and TA1535) and 500 μg/plate (TA98, TA1537 and WP2uvrA). Consequently, the toxic limit was employed in the second mutation test. Visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains, initially from 50 μg/plate in the absence of S9-mix and from 50 μg/plate (TA100 and TA1535) and 150 μg/plate (TA98 and TA1537) in the presence of S9-mix. Toxicity was also noted to E.coli strain WP2uvrA at 500 μg/plate in both the absence and presence of 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). A small, statistically significant increase in TA1537 revertant colony frequency was observed in the absence of S9-mix at 50 μg/plate in the range-finding test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. The individual revertant counts at the statistically significant level were within the historical vehicle control range for the strain and the maximum increase was only two-fold the concurrent vehicle control. 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.