2-methyl-3-butenenitrile

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:

From 1991-02-19 to 1991-05-31

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD 471, only 4 strains were tested (TA100, TA1535, TA97 and TA98).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four strains were tested (TA102 or E.Coli were not tested)

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-3-butenenitrile

EC Number:

240-596-1

EC Name:

2-methyl-3-butenenitrile

Cas Number:

16529-56-9

Molecular formula:

C5H7N

IUPAC Name:

2-methylbut-3-enenitrile

Details on test material:

- Name of test material (as cited in study report): 2-methyl-3-butenenitrile
- Substance type: no data
- Physical state: colorless liquid
- Isomers composition: no data
- Purity test date: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: assumed to be stable in this assay, no evidence of instability was observed.
- Storage condition of test material: no data
- Other: no data

Method

Target gene:

Histidine revertant.

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 100, TA 1535, TA 97 and TA 98

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from male Crl:CD rat injected i.p. with Aroclor 1254

Test concentrations with justification for top dose:

0, 10, 50, 100, 500, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based on information supplied with the test material.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (Sigma Lot 33F-0816); 2-nitrofluorene (Aldrich Lot JPO3222JJ); sodium azide (Sigma Lot 26F-0434); ICR-191 Acridine (Raylo Chemicals Lot 178). See details in table 7.6.1/1

Remarks:

Deionized water was the solvent used for NAAZ and ICR-191 whereas for other positive indicators the solvent was DMSO.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION:
- Exposure duration: 48 hours at 37C°

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: three

DETERMINATION OF GENOTOXICITY
- Method: revertant colony growth

Evaluation criteria:

A two-fold increase in the number of revertant colonies and a dose response relationship were considered as positive result.

Statistics:

Doses with and without activation were ranked and results from a strain were analyzed individually by multiple linear regression. Comparisons were made between each dose/concentration and the solvent control (0 rank) using the mean square error estimate. All comparisons were at the 95% level of confidence (alpha = 0.05).

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 100, TA 1535, TA 97 and TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See details in Tables 7.6.1/2 and 7.6.1/3

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No data


RANGE-FINDING/SCREENING STUDIES:
No data

COMPARISON WITH HISTORICAL CONTROL DATA:
No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Not applicable

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: TA 100, TA 1535, TA 97 and TA 98

Any other information on results incl. tables

Table 7.6.1/2: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation

TFE Concentration (µg/plate)	TA 100		TA1535		TA 97		TA 98
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	102	10	14	3	92	3	19	5
10	104	4	-	-	-	-	-	-
50	108	13	20	2	101	11	12	2
100	99	7	24	4	108	8	14	2
500	98	7	13	6	113	18	13	3
1000	106	10	18	2	106	12	12	2
2500	96	9	14	5	111	6	13	2
500	107	6	15	5	141	22	15	5
Positive control**	515	14	481	29	1211	43	1164	98

*Solvent control = negative control: 100 µL DMSO

**Mutagens positive controls:

- NAAZ-2 (2 µg/plate) for TA 100 and TA 1535 strains

- ICR-191-2 (2 µg/plate) for TA 97 strain

- 2NF-25 (25 µg/plate) for TA 98

 

 

Table 7.6.1/3: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (S9 mix)

 

 

TFE Concentration (µg/plate)	TA 100		TA1535		TA 97		TA 98
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	116	16	16	1	133	8	28	6
10	117	9	-	-	-	-	-	-
50	120	6	12	5	128	7	24	10
100	107	2	16	4	136	12	31	4
500	105	5	13	2	130	13	24	5
1000	119	8	12	3	135	17	24	5
2500	108	20	12	5	145	20	23	3
500	100	14	18	2	135	36	26	4
Positive control**	1440	168	242	27	1235	86	1530	88

*Solvent control = negative control: 100 µL DMSO

**Mutagens positive controls:

- 2AA-1 (2 µg/plate) for TA 100 and TA 97 strains

- 2AA-2 (2 µg/plate) for TA 1535 and TA 98 strains

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

There was no evidence of induced mutant colonies over background in this study.

Executive summary:

In an in vitro genetic toxicity study, 2 -Methyl-3 -butenenitrile was tested for mutagenic activity in Salmonella typhirium strains TA100, TA1535, TA 97 and TA98, with and without an activation system (S9 -Mix), at concentrations of 0, 10, 50, 100, 500, 1000, 2500 and 5000 µg/plate. This study was performed according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) and GLP.

No signs of toxicity were observed up to the higest dose level investigated with either strain both in the presence or absence of S9 metabolism.

The test substance did not induce a 2 -fold increase in the number of revertant colonies of either strain investigated, with or without the presence of S9, at any concentration.

Marked increases in the number of revertant colonies were obtained following treatment with the positive control reference substances, this demonstrating the validity of the test system. Under the test conditions of this study, there were no indication that the test substance has mutagenic potential.