Methyl 3,3-dimethylpent-4-enoate

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:

1988-01-26 to 1988-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 3,3-dimethylpent-4-enoate

EC Number:

264-431-8

EC Name:

Methyl 3,3-dimethylpent-4-enoate

Cas Number:

63721-05-1

Molecular formula:

C8H14O2

IUPAC Name:

methyl 3,3-dimethylpent-4-enoate

Details on test material:

- Substance type: ester
- Physical state: colourless liquid
- Purity test date: not reported
- Expiration date of the lot/batch: not reported
- Stability under test conditions: not determined
- Storage condition of test material: at 4°C

Method

Target gene:

his G46 (S. typhimurium TA1535 and TA100)
his C3076 (TA1537)
his D3052 (TA1538 and TA98)
trp (E. coli WP2 uvrA)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium, other: TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced CD (Sprague-Dawley derived) rat liver S9 fraction, diluted 1:10, co-factors glucose-6-phosphate (5mM), NADH (4 mM) and NADP (4 mM)

Test concentrations with justification for top dose:

Dose range finding preliminary toxicity test: 5000, 500, 50, 5 microgram/plate
Test 1 and 2: 2000, 1000, 500, 250, 125 microgram/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO (0.1 mL/plate)
- Justification for choice of solvent/vehicle: not reported

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

3 plates per strain

True negative controls:

no

Positive controls:

yes

Remarks:

3 plates per strain

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation

(TA 1535, TA 100, WP2), 2-nitrofluorene (TA 98, TA 1538), 9-aminoacridine (TA 1537)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

3 plates per strain

True negative controls:

no

Positive controls:

yes

Remarks:

3 plates per strain

Positive control substance:

other: 2-aminoanthracene (all strains)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Pre-culture: 10 hours
- Preincubation period: 20 min at 37°C
- Exposure duration: 72 hours at 37°C

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: 1 plate per concentration, with and without S-9 mix
- Tests 1 and 2: 3 plates per concentration, with and without S-9 mix
- Two identical replicate experiments with the same concentrations: Test 1 and 2

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn for completeness, reduction of revertant colony counts

COUNTING
- Revertant colonies were counted using an automated colony counter

Evaluation criteria:

Positive response:
- statistically significant dose-related increase in number of revertants in two separate experiments

Statistics:

- Mean colony count / plate and standard deviation
- Assessment of statistical significance (not further specified)

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See tables 2 and 3

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Pretest: no or incomplete bacterial lawn at 5000 microgram/plate; mutation test: no cytotoxicity observed at 2000 microgram/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium, other: TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See tables 2 and 3

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Pretest: no or incomplete bacterial lawn at 5000 microgram/plate; mutation test: no cytotoxicity observed at 2000 microgram/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not reported
- Effects of osmolality: Not reported
- Evaporation from medium: Not to be expected, due to low vapor pressure (8.5 hPa at 25°C)
- Water solubility: sufficient (240 mg/L at 25°C)
- Precipitation: none observed
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
- Cytotoxicity to all tester strains detected at 5000 microgram/plate
- Top concentration in main test consequently reduced to 2000 microgram/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- Not reported

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- 5000 microgram/plate, without S-9 mix: No bacterial lawn with TA 100, TA1535, TA 98, TA 1537, and TA 1538; incomplete lawn with WP2 uvra
- 5000 microgram/plate, with S-9 mix: No bacterial lawn with WP2 uvra and TA 1537; incomplete lawn with TA 100, TA1535, TA 98, and TA1538

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 1: Dose range finding test: Revertant colony counts obtained

Material	Test conc.	With /		Reverse	mutation:	Colonies	/ plate
	microgram	without	Base pair	exchange	type	Frame shift	exchange	type
	/ plate	S-9 mix	TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537	TA 1538
Solvent control		-	107	15	68	21	7	18
Test item	5000	-	NL	NL	IL	NL	NL	NL
	500	-	114	9	74	21	4	14
	50	-	108	8	73	17	5	13
	5	-	125	10	62	17	6	14
Solvent control		+	120	12	59	27	7	14
Test item	5000	+	IL	IL	NL	IL	NL	IL
	500	+	96	9	62	16	9	15
	50	+	120	12	64	20	9	11
	5	+	91	14	66	23	12	15

NL No bacterial lawn

IL Incomplete bacterial lawn

Table 2: Test 1 (Mutation test): Mean revertant colonies obtained (mean of three plates)

Material	Test conc.	With /		Reverse	mutation:	Colonies	/ plate
	microgram/plate	without	Basepair	exchange	type	Frameshift	exchange	type
		S-9 mix	TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537	TA 1538
Solvent control		-	115	16	61	22	12	11
Test item	2000	-	74	11	62	17	13	9
	1000	-	96	10	60	20	13	11
	500	-	103	11	66	23	10	7
	250	-	107	11	48	19	16	9
	125	-	110	11	51	19	9	10
Solvent control		+	128	14	61	24	14	12
Test item	2000	+	88	8	51	20	12	13
	1000	+	102	10	56	20	12	6
	500	+	109	11	58	20	9	12
	250	+	121	11	64	23	9	12
	125	+	124	9	52	17	10	13
Positive controls	name		ENNG	ENNG	ENNG	NF	9 AC	NF
	microgram/plate	-	3	5	2	1	80	2
	colonies/plate		357	423	714	86	1586	63
	name		AA	AA	AA	AA	AA	AA
	microgram/plate	+	1	2	20	0.5	2	0.5
	colonies/plate		627	118	388	149	103	131

Table 3: Test 2 (Mutation test): Mean revertant colonies obtained (mean of three plates)

Material	Test conc.	With /		Reverse	mutation:	Colonies	/ plate
	microgram/plate	without	Basepair	exchange	type	Frameshift	exchange	type
		S-9 mix	TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537	TA 1538
Solvent control		-	136	15	66	36	13	16
Test item	2000	-	107	11	69	23	11	17
	1000	-	116	12	64	20	11	14
	500	-	127	11	69	24	10	12
	250	-	112	11	65	26	10	11
	125	-	116	14	64	23	10	12
Solvent control		+	132	12	75	23	15	17
Test item	2000	+	95	9	74	20	13	14
	1000	+	120	16	63	21	12	12
	500	+	127	15	65	20	12	16
	250	+	104	12	65	22	15	12
	125	+	144	10	66	20	10	14
Positive controls	name		ENNG	ENNG	ENNG	NF	9 AC	NF
	microgram/plate	-	3	5	2	1	80	2
	colonies/plate		353	172	941	88	2310	53
	name		AA	AA	AA	AA	AA	AA
	microgram/plate	+	1	2	20	0.5	2	0.5
	colonies/plate		484	112	431	140	79	138

Explanations to tables 2 and 3: Positive control substances

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

9 AC: 9-aminoacridine

NF: 2-nitrofluorene

AA: 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

The test material was found to be non-mutagenic to five Salmonella typhimurium strains and one E. coli strain under the conditions of the assay. The study report is relevant, adequate and reliable for risk assessment, classification and labeling.

Executive summary:

The ability of the test material to induce reverse mutations was assessed in an in vitro bacterial system, Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538, as well as E. coli strainWP2 uvrA (Ames test).

Bacteria were treated with the test compound in a plate incorporation test. A dose range finding test showed cytotoxicity in all tested strains at 5000 microgram/plate. In the mutagenicity tests, five dose levels (between 125 and 2000 microgram/plate) were assayed in triplicate, both with and without metabolic activation (Aroclor 1254 induced rat liver S-9 mix). Two independent experiments were carried out to establish reproducibility. No precipitation and no cytotoxicity were observed in the main tests. All six bacterial strains exhibited mutagenic responses to the appropriate positive control substances. Solvent controls were also tested with each strain, and the mean numbers of spontaneous revertants were found to be acceptable.

No substantial increases in revertant colony numbers of any of the six tester strains were observed following treatment with the test substance at any dose level, with and without metabolic activation (S-9 mix).

The authors hence conclude that no evidence of mutagenic potential of the test item was obtained in the bacterial test system at the dose levels used.