2-methyl-3-butenenitrile

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

other information

Study period:

No data

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Relevant methodological deficiencies: cytotoxicity not studied, administrated volume was higher than recommended, animals were sacrificed 6 hours after the last administration instead of 18-24 hours. Purity and composition not stated.

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:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

: Relevant methodological deficiencies: cytotoxicity not studied, administrated volume was higher than recommended, animals were sacrificed 6 hours after the last administration instead of 18-24 hours. Purity and composition not stated.

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Type of assay:

micronucleus 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): methyl-2 butene-3 nitrile
- Substance type: no data
- Physical state: colorless lquid
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: no data

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Carworth Farm Lane-Petter
- Age at study initiation: no data
- Weight at study initiation: 25 to 30 g
- Assigned to test groups randomly: no data
- Fasting period before study: no
- Housing: no data
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data


IN-LIFE DATES: no data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 0.4 and 2 µg/mL
- Amount of vehicle (if gavage or dermal): 0.25 mL
- Lot/batch no. (if required): no data
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
2-methyl-3-butenenitrile was diluted in arachis oil at concentrations of 0.4 and 2 µg/mL

DIET PREPARATION
no data

Duration of treatment / exposure:

30 hours.

Frequency of treatment:

Twice administration at 24-hr interval

Post exposure period:

6 hours (animals are sacrified six hours after the last administration of 2-methyl-3-butenenitrile)

Doses / concentrations

Remarks:

Doses / Concentrations:
2 x 0.01 and 2 x 0.05 mL/kg
Basis:
nominal conc.

No. of animals per sex per dose:

10 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Benzene (Prolabo, lot n° 79.018)
- Justification for choice of positive control(s): benzene is a recognised clastogenic substance
- Route of administration: oral gavage
- Doses / concentrations: Benzene was diluted in arachis oil at concentrations of 75 µg/mL

Examinations

Tissues and cell types examined:

Femur bone marrow (2000 polychromatic erythrocytes per animal)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
based on a preliminary study

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Treatment was administered twice at 24-hr interval, and animals were sacrified 6 hours after the last administration. Then, samples were collected.

DETAILS OF SLIDE PREPARATION:
Femur bone marrow was rescued in foetal calf serum. After centrifugation, the pellet was homogenized and a drop was spread on slide. The smear was dried and coloured with May Grünwald Giemsa.

METHOD OF ANALYSIS:
Femur bone marrow was harvested and the polynucleus cell count was performed on 2 000 polychromatophyle erythrocytes. Two different people each read 1 000 polychromatophyle erythrocytes. A mean of both results was performed.

Evaluation criteria:

% of polychromatic erythrocytes with micronuclei

Statistics:

Statistical analysis consists in a comparison of both means determined with the student method.

Results and discussion

Test results

Sex:

male

Genotoxicity:

other: nonconclusive

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
> at 0.5 mL/kg, 5/5 animals died some hours after the administration
> at 0.1 ml/kg, 4/5 animals died in the 3 hr after the administration
> at 0.05 ml/kg, animals were prostrated during the hr after administration
- Dose range: 0.05, 0.1 and 0.5 mL/kg
- Solubility: not much soluble in water
- Clinical signs of toxicity in test animals: animals died some hours after the 2-methyl-3-butenenitrile administration at concentration of 0.1 and 0.5 mL/kg
- Evidence of cytotoxicity in tissue analyzed: no data
- Rationale for exposure: no data
- Harvest times: no data


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): see Table 7.6.2/2
- Ratio of PCE/NCE (for Micronucleus assay): no data
- Appropriateness of dose levels and route: no data
- Statistical evaluation: no statistically significant increase in the  number of micronuclei was observed in the treated animals.

Any other information on results incl. tables

7.6.2/1: 2M3BN administration and toxicity

Substance administered (mL/kg)	Number of animals
	Treated	Died	Alife
Vehicle Arachis oil	10	0	10
2M3BN 2 x 0.01 2 x 0.05	10 10	0 0	10 10
Benzene 2 x 1.875	10	0	10

Table 7.6.2/2: Micronucleus assay results

Substance	Doses mL/kg (PO	Number of animals	Percentage of P.E. with micronuclei ±2 Sm
Control vehicle	-	10	0.14± 0.05
2M3BN	2 x 0.01 2 x 0.05	10 10	0.21± 0.05 0.21 ± 0.05
Benzene	2 x 1.875	10	6.59± 1.59

P.E.: polychromatophyle erythrocytes

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: nonconclusive
Under the test conditions, no conclusion can be established.

Executive summary:

In a genetic toxicity in vivo study, a micronucleus assay was performed on male swiss mice. 2 -methyl-3 -butenenitrile was admistered by gavage twice at 24 -hours interval at dose concentration of 0.01 and 0.05 mL/kg. Arachis oil was used as vehicle.

This study was performed equivalent to OECD Guideline 474, but with no GLP compliance. A preliminary study was performed to determine dose.

Animals were sacrified 6 hours after the second administration of 2 -methyl-3 -butenenitrile. Polychromatophyle erythocyte sampling was performed in the femur. 2000 cells were coloured with May Grünwald-Giemsa and analysed for micronuclei.

Positive control (benzene) and negative control (vehicle) were valids.

Only the percentage of micronuclei in polychromatic erythrocytes is determined. There is no information concerning proportion of immature erythrocytes among total erythrocytes and the accessibility of the product to the bone marrow. The cytotoxicity of the product is missing. There are several other deviations. Therefore no conclusion can be established (Kr:3)