Cinnamonitrile

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Evaluation of genotoxicity of nitrile fragrance ingredients using in vitro and in vivo assays

Author:

S.P. Bhatia, V.T. Politano, A.M. Api

Year:

2013

Bibliographic source:

Food and Chemical Toxicology 59 (2013) 784–792

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

In vivo micronucleus assay was performed to determine the mutagenic nature of cinnamyl nitrile

GLP compliance:

not specified

Type of assay:

other: In vivo micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Cinnamonitrile

EC Number:

217-552-5

EC Name:

Cinnamonitrile

Cas Number:

1885-38-7

Molecular formula:

C9H7N

IUPAC Name:

cinnamonitrile

Details on test material:

- Name of test material (IUPAC name): (E)-3-phenylprop-2-enenitrile
- Common name: Cinnamyl nitrile
- Molecular formula: C9H7N
- Molecular weight: 129.161 g/mol
- Smiles notation: N#C\C=C\c1ccccc1
- InChl: 1S/C9H7N/c10-8-4-7-9-5-2-1-3-6-9/h1-7H/b7-4+
- Substance type: Organic

Specific details on test material used for the study:

- Name of test material: Cinnamyl nitrile
- IUPAC name: (E)-3-phenylprop-2-enenitrile
- Molecular formula: C9H7N
- Molecular weight: 129.161 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: No data
- Impurities (identity and concentrations): No data

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: The test chemical was soluble in corn oil
- Concentration of test material in vehicle:
Main Experiment (males/females):
0, 50, 100, 200 or 200 mg/Kg bw

Repeat experiment (males only):
0, 150 or 150 mg/Kg bw

- Amount of vehicle (if gavage or dermal): The individual volume administered was adjusted to the animal’s body weight.
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data

Details on exposure:

For oral route
PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in corn oil to provide doses of 0, 50, 100, 200 or 200 mg/Kg bw (males and females) in the main experiment and 0, 150 or 150 mg/Kg bw in the repeat experiment for males only

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

Duration of treatment / exposure:

24 hrs and 48 hrs

Frequency of treatment:

Once

Post exposure period:

No data

Doses / concentrations

Remarks:

Main Experiment (males/females):
0, 50, 100, 200 or 200 mg/Kg bw

Repeat experiment (males only):
0, 150 or 150 mg/Kg bw

No. of animals per sex per dose:

Total:
Main experiment:
0 mg/Kg bw: 6 males and 6 females
50 mg/Kg bw: 6 males and 6 females
100 mg/Kg bw: 6 males and 6 females
200 mg/Kg bw: 12 males and 12 females (24 hrs)
200 mg/Kg bw: 12 males and 12 females (48 hrs)

Repeat experiment:
0 mg/Kg bw: 6 males and 6 females
150 mg/Kg bw: 6 males and 6 females (24 hrs)
150 mg/Kg bw: 6 males and 6 females (48 hrs)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: No data
- Doses / concentrations: 40 mg/Kg bw

Examinations

Tissues and cell types examined:

Bone marrow polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary test was carried out for maximal tolerance in mice. The test materials were administered orally once daily for 2 days at doses of up to 500 mg/kg for cinnamyl nitrile. Based on the results of this preliminary testing, doses were determined for the main study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Main study:
24 hrs (0, 50, 100, 200 mg/Kg bw)
48 hrs (200 mgKg bw)

Repeat study:
24 hrs (150 mg/Kg bw)
48 hrs (150 mgKg bw)

DETAILS OF SLIDE PREPARATION: At least one slide was made from each bone marrow sample. A minimum of 2000 polychromatic erythrocytes were analyzed per animal for micronuclei.

METHOD OF ANALYSIS: A test material was classified as mutagenic if it induced either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. A test material that failed to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes was considered non-mutagenic in this system.

OTHER: The animals of all dose groups were examined for acute toxic symptoms at intervals of 1, 2–4, 6, 24 and 48 h after administration of the test material.

The animals were sacrificed using CO2 followed by bleeding.

Evaluation criteria:

A test material was classified as mutagenic if it induced either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. A test material that failed to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes was considered non-mutagenic in this system.

Statistics:

Statistical significance at the 5% level (p < 0.05) was conducted using non-parametric Mann Whitney test

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

not specified

Toxicity:

yes

Remarks:

Cinnamyl nitrile at 200 mg/kg was lethal in seven male mice and in one female mice

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: No mutagenic potential

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: up to 500 mg/kg
- Solubility: No data
- Clinical signs of toxicity in test animals: No data
- Evidence of cytotoxicity in tissue analyzed: No data
- Rationale for exposure: No data
- Harvest times: No data
- High dose with and without activation: No data
- Other: No data

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No data
- Induction of micronuclei (for Micronucleus assay): No
- Ratio of PCE/NCE (for Micronucleus assay):
- Appropriateness of dose levels and route: Oral route
- Statistical evaluation: Statistical significance at the 5% level (p < 0.05) was conducted using non-parametric Mann Whitney test

Applicant's summary and conclusion

Conclusions:

Cinnamyl nitrile did not induce micronuclei formation in bone marrow isolated from male and female NMR1 mice and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

In vivo micronucleus assay was performed to determine the mutagenic nature of cinnamyl nitrile. The study was performed as per OECD 474 using male and female NMR1 mice. The test chemical was dissolved in corn oil and used at dose levels of0, 50, 100, 200 mg/Kg bw for 24 hrs and 200 mg/Kg bw for 48 hrs in the main study and 150 mg/Kg bw for 24 and 48 hrs in the repeat study. Cinnamyl nitrile at 200 mg/kg was lethal in seven male mice and in one female mouse. Due to the low survival rates of the male mice, mutagenicity could not be assessed. Hence, additional males were treated with 150 mg/kg and prepared at 24 and 48 h post treatment.The mice were given a single oral dose of the test chemical and were observed for acute toxic symptoms at intervals of 1, 2–4, 6, 24 and 48 h after administration of the test material. The animals were sacrificed using CO2 followed by bleeding and observed for micronuclei formation in bone marrow cells. In the main stusy, Cinnamyl nitrile did not induce micronuclei formation in bone marrow isolated from male and female NMR1 mice. In the repeat experiment, the number of PCEs was not substantially decreased when compared to the mean value of PCEs of the vehicle control thus indicating that cinnamyl nitrile did not exert any cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval or dose level after administration of the test item. The mean values of micronuclei observed after treatment with cinnamyl nitrile were below or near to the value of the vehicle control group. Based on the observations made, cinnamyl nitrile is non-mutagenic as per the criteria mentioned in CLP regulation.