2,2-dimethyl-3-(3-methylpenta-2,4-dienyl)oxirane

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:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected August 2005; signature: November 2005

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2-dimethyl-3-(3-methylpenta-2,4-dienyl)oxirane

EC Number:

273-870-4

EC Name:

2,2-dimethyl-3-(3-methylpenta-2,4-dienyl)oxirane

Cas Number:

69103-20-4

Molecular formula:

C10H16O

IUPAC Name:

2,2-dimethyl-3-(3-methylpenta-2,4-dienyl)oxirane

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Approximately 4°C, in the dark, under nitrogen.
- Other: Pale yelow

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:

Preliminary test (toxicity test): 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate.
Range-finding test:
Salmonella strains: 15, 50, 150, 500, 1500, 5000 ug/plate.
E.coli strain WP2uvrA·: 50, 150, 500, 1500, 5000 ug/plate.
Main test: 50, 150, 5000, 1500 and 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/ml
but was fully miscible in acetone at the same concentration in solubility checks performed.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

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: in medium; in agar (plate incorporation)

DURATION
- Exposure duration: All of the plates were incubated at 37ºC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one or more bacterial strain with or without metabolic activation.

Statistics:

Statistical methods, 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:

no cytotoxicity

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:

no cytotoxicity

Remarks:

TA100 at 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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. Range finding test

Range-finding test with and without S9
		Number of revertants (mean number of colonies per plate)
S9 Mix	Test substance concentration (ug/plate)	TA100	Mean	TA1535	Mean	WP2uvrA-	Mean	TA98	Mean	TA1537	Mean
-	0	74	89	22	27	24	21	24	20	11	10
		90		32		16		17		7
		102		26		22		19		11
-	50	95	97	29	32	17	17	16	17	8	8
		90		37		19		16		7
		105		30		15		19		9
-	150	94	95	21	24	12	14	15	23	8	10
		C		25		13		15		8
		95		15		17		38		15
-	500	118	105	24	29	20	23	25	23	7	9
		87		34		21		22		15
		110		30		29		22		6
-	1500	89	95	28	26	20	21	18	17	4	7
		106		26		24		12		12
		89		24		18		21		6
-	5000	97	106	33	34	20	20	19	12	5	6
		111		30		18		7		10
		109		39		22		11		4
+	0	85	89	13	21	21	21	24	24	16	11
		93		11		25		25		10
		89		10		16		23		6
+	50	88	81	12	24	20	24	29	24	6	12
		74		12		23		30		13
		82		9		29		12		17
+	150	85	91	11	15	15	15	25	26	15	20
		89		6		17		30		19
		98		7		13		23		26
+	500	72	78	10	21	21	21	22	24	10	14
		79		10		21		23		15
		83		3		20		26		18
+	1500	94	79	1	21	21	21	16	19	19	14
		76		13		22		16		11
		67		10		20		24		13
+	5000	78	79	7	23	22	23	24	15	15	15
		77		11		27		9		17
		81		7		20		13		13
Positive controls		ENNG		ENNG		ENNG		4NQO		9AA
Concentration (ug/plate)		3		5		2		0.2		80
without S9		436	469	193	191	828	858	120	128	1034	869
		487		202		894		146		1160
		484		179		851		118		412
Positive controls		2AA		2AA		2AA		BP		2AA
Concentration (ug/plate)		1		2		10		5		2
With S9		767	834	187	221	314	282	166	214	222	250
		845		245		277		223		273
		891		232		252		254		254

Table 2. Main test

Main test with and without S9
		Number of revertants (mean number of colonies per plate)
S9 Mix	Test substance concentration (ug/plate)	TA100	Mean	TA1535	Mean	WP2uvrA-	Mean	TA98	Mean	TA1537	Mean
-	0	84	94	28	23	20	21	17	18	10	9
		88		17		18		18		9
		111		25		24		19		9
-	50	94	93	29	22	20	22	21	20	8	12
		86		22		20		16		17
		98		29		26		24		10
-	150	109	107	30	21	17	21	17	14	6	8
		116		32		24		15		8
		97		29		33		9		9
-	500	112	109	32	18	16	18	19	20	10	8
		103		30		20		21		4
		113		22		19		21		10
-	1500	96	98	30	18	11	18	18	20	11	11
		102		39		17		30		15
		96		19		25		11		8
-	5000	113	110	31	13	5	13	6	11	5	6
		100		28		23		8		6
		118		32		11		18		6
+	0	103	98	10	10	36	30	21	27	15	19
		96		10		26		29		20
		96		11		27		31		21
+	50	104	103	10	13	24	27	23	24	13	18
		101		19		22		26		24
		105		11		35		24		16
+	150	106	104	11	9	19	21	27	25	11	14
		105		9		24		18		19
		100		7		19		29		13
+	500	101	97	15	13	26	21	23	22	12	13
		102		9		24		22		13
		89		16		12		22		13
+	1500	82	93	12	13	19	23	28	23	11	18
		115		17		24		21		27
		83		11		26		19		15
+	5000	104	93	9	14	25	25	20	20	16	16
		101		18		25		21		12
		73		C		26		19		19
Positive controls		ENNG		ENNG		ENNG		4NQO		9AA
Concentration (ug/plate)		3		5		2		0.2		80
without S9		493	493	307	302	600	600	161	153	1145	1179
		497		323		613		133		1131
		488		277		588		165		1262
Positive controls		2AA		2AA		2AA		BP		2AA
Concentration (ug/plate)		1		2		10		5		2
With S9		482	706	266	249	310	299	264	249	119	189
		807		263		309		237		117
		828		218		278		245		332

BP - Benzo(a)pyrene

2AA - 2-Aminoanthracene

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

4NQO - 4 -Nitroquinoline-1-oxide

9AA - 9 -Aminoacridine

C - Contaminated

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test material 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 Japanese guidelines 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. A preliminary test was performed to determine the toxicity of the test material. A range-finding study was performed to determine the doses used for the main test. In the main test, the plate incorporation method was used and was evaluated at a concentration of up to 5000 µg/plate. Positive controls appropriate for each strain, in the presence and absence of S9 -mix, were included. The test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The vehicle (acetone) 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 test material caused a visible reduction in the growth of the bacterial background lawns at 5000 µg/plate to the majority of the Salmonella strains in the absence of S9 and to TA100 and TA1535 in the presence of S9. No toxicity was noted for Escherichia coli strain WP2uvrA-. 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. It was concluded that, under the conditions of this assay, the test substance 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.