3,7-dimethylocta-2,6-dienenitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test compound Geranyl nitrile is likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Data ia from BAuA report

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

In vitro chromosome aberration study was performed on 3,7-dimethylocta-2,6-dienenitrile using V79 cells.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

other: V79 cells

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

No data

Test concentrations with justification for top dose:

0 – 1,000 μg/ml

Vehicle / solvent:

No data available

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: EMS

Details on test system and experimental conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

mammalian cell line, other: V79 cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

-The negative controls (vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line
-Both positive control chemicals, i.e. EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations
-Other confounding effects: The test substance caused a clear, statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl.gaps after adding a metabolizing system.
No increase in the frequency of cells containing numerical aberrations was demonstrated.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
positive with and without

Mutagenic effects were known in chromosome aberration assay with and without metabolic activation study when exposed to V79 cells by 3,7-dimethylocta-2,6-dienenitrile.

Executive summary:

Chromosomal aberration test was performed to determine the mutagenic nature of the test compound 3,7-dimethylocta-2,6-dienenitrile. V79 cells were used for the study and the test chemical was used at dose levels of 0 – 1,000 μg/ml. The test system was used with and without metabolic activation.The test substance caused a clear, statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps after adding a metabolizing system. No increase in the frequency of cells containing numerical aberrations was demonstrated. The negative controls (vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line. Both positive control chemicals, i.e. EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. 3,7-Dimethylocta-2,6-dienenitrile was considered to be a chromosome damaging (clastogenic) agent under in vitro conditions in V79 cells.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Data ia from BAuA report

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Mammalian erthocyte micronucleus assay was performed to determine the mutagenic nature of the test compound 3,7-dimethylocta-2,6-dienenitrile in vivo.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

No details available

Route of administration:

oral: gavage

Vehicle:

Vehicle: olive oil

Details on exposure:

No data

Duration of treatment / exposure:

For 1st experiment:24-h and 48-h
For 2nd experiment:24-h

Frequency of treatment:

single

Post exposure period:

No data

Remarks:

Doses / Concentrations:
312.5, 625.0, 1250.0 mg/kg bw
Basis:
no data

No. of animals per sex per dose:

5 animals per group

Control animals:

not specified

Positive control(s):

cyclophosphamide and vincrist

Tissues and cell types examined:

No data

Details of tissue and slide preparation:

No data

Evaluation criteria:

number of polychromatic erythrocytes (PCEs) containing small micronuclei (MN).

Statistics:

No data

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range:No data
- Solubility: No data
- Clinical signs of toxicity in test animals: squatting posture, poor general state, eyelid closure, hyperacitivity
- Evidence of cytotoxicity in tissue analyzed:
- Rationale for exposure:
- Harvest times:
- High dose with and without activation:
- Other: Both of the positive control chemicals (cyclophosphamide and vincristine),
led to the expected increase in the rate of polychromatic erythrocytes
containing small or large micronuclei.

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay): The administration of the test substance led to evident signs of toxicity
to a statistically significant and dose-dependent increase in the number of polychromatic erythrocytes (PCEs) containing small micronuclei (MN). The positive response was observed in two experiments carried out independently of each other.

- Ratio of PCE/NCE (for Micronucleus assay):An inhibition of erythropoiesis determined from the ratio of PCEs to normochromatic erythrocytes (NCE) was detected at 1,250 mg/kg after a sacrifice interval of 48h.
- Appropriateness of dose levels and route:
- Statistical evaluation:
For 1st experiment(24-h): probability p<0.01
For 1st experiment(48-h):Probability p<0.05
For 2nd experiment(24-h):Probability p<0.01

1^stexperiment:24-h interval

Concentration(mg/kg)	Micronuclei count in 1000 PCEs
0	1.8
312.5	1.2
625	4.5
1250	6.0

1^stexperiment:48-h interval

Concentration	Micronuclei count per 1000 PCEs
0 mg/kg	1.6
1250 mg/kg	6.3

2^ndexperiment:24-h interval

Concentration	Micronuclei count per 1000 PCEs
0 mg/kg	1.3
1250 mg/kg	6.9

Conclusions:

Interpretation of results (migrated information): positive
Mutagenic effects were known when the test substance 2,6-octadienenitrile, 3,7-dimethyl was exposed to NMRI male mouse in the in vivo micronucleus test.

Executive summary:

In vivo micronucleus test was performed to study the mutagenic effects of the test substance 2,6 -octadienenitrile, 3,7-dimethyl. The dose concentration of test chemical was 312.5, 625.0, 1250.0 mg/kg bw administered to animals by gavage route in olive oil. Two experiments were carried out, 1^stat 24 and 48 h interval and 2^ndexperiment at 24-h interval,The positive response was observed in two experiments carried out independently of each other.There was no increase in cells with large micronuclei. An inhibition of erythropoiesis determined from the ratio of PCEs to normochromatic erythrocytes (NCE) was detected at 1,250 mg/kg after a sacrifice interval of 48h. Both of the positive control chemicals (cyclophosphamide and vincristine), led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. The test chemical causes evident signs of toxicity and statistically significant and dose-dependent increase in the number of polychromatic erythrocytes (PCEs) containing small micronuclei (MN). Hence, the chemical 2,6 -octadienenitrile, 3,7-dimethyl is mutagenic in micronucleus test when exposed to NMRI mouse.

Additional information

Additional information from genetic toxicity in vitro:

Gene toxicity in vitro:

Data from various articles were reviewed to determine the mutagenic nature of the test compound Geranyl nitrile. The summary is as mentioned below:

Chromosomal aberration test was performed (BAuA report, 2003) to determine the mutagenic nature of the test compound 3,7-dimethylocta-2,6-dienenitrile (CAS no 5146 -66 -7). V79 cells were used for the study and the test chemical was used at dose levels of 0 – 1,000 μg/ml. The test system was used with and without metabolic activation.The test substance caused a clear, statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps after adding a metabolizing system. No increase in the frequency of cells containing numerical aberrations was demonstrated. The negative controls (vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line. Both positive control chemicals, i.e. EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. 3,7-Dimethylocta-2,6-dienenitrile was considered to be a chromosome damaging (clastogenic) agent under in vitro conditions in V79 cells.

 

Another study was performed by Kerfoot et al (2003). In vitro chromosome aberration test was performed to evaluate the genetic effects of 2,6 -octadienenitrile, 3,7-dimethyl (CAS no 5146 -66 -7). The test substance was tested for its clastogenic potential in the in vitro chromosome aberration test in V79 cells. Exposure duration was 4 hours and cells were sampled after 18 hours, both with and without metabolic activation. The test substance caused a clear, statistically significant and dose-dependent increase in the number of structurally aberrant metaphases after metabolic activation over a dose range of 250-1000 µg/ml. Mutagenic effects were known when the test substance 2,6-Octadienenitrile, 3,7-dimethyl was exposed to V79 cells with metabolic activation in chromosome aberration test.

In Bacterial genotoxicity study (BAuA report, 2013) to Salmonella typhimurium strain TA1535, TA100, TA1537, TA98, Escherichia coli treated with 3,7-dimethylocta-2,6-dienenitrile at dose levels of 20 – 5,000 μg/plate (SPT) and 20 – 2,500 μg/plate (PIT) with and without metabolic activation system. The test was performed according to Standard plate test (SPT) and preincubation test (PIT). No precipitation of the test substance was found. A bacteriotoxic effect was observed under all test conditions (SPT: 500 - 1,000 μg/plate; PIT: 100 – 500 μg/plate)). An increase in the number of revertants was not observed in the SPT or in the PIT with or without S-9 mix. Hence, the chemical 3,7-dimethylocta-2,6-dienenitrile was considered to be non-mutagenic to Salmonella typhimurium by bacterial genetoxicity test.

Gene toxicity in vivo:

The various articles reviewed have been summarized to determine the mutagenic nature of the test compound Geranyl nitrile in vivo:

In vivo micronucleus test was performed (BAuA report, 2013) to study the mutagenic effects of the test substance 2,6 -octadienenitrile, 3,7-dimethyl (CAS no 5146-66-7). The dose concentration of test chemical was 312.5, 625.0, 1250.0 mg/kg bw administered to animals by gavage route in olive oil. Two experiments were carried out, 1^stat 24 and 48 h interval and 2^ndexperiment at 24-h interval,The positive response was observed in two experiments carried out independently of each other.There was no increase in cells with large micronuclei. An inhibition of erythropoiesis determined from the ratio of PCEs to normochromatic erythrocytes (NCE) was detected at 1,250 mg/kg after a sacrifice interval of 48h. Both of the positive control chemicals (cyclophosphamide and vincristine), led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. The test chemical causes evident signs of toxicity and statistically significant and dose-dependent increase in the number of polychromatic erythrocytes (PCEs) containing small micronuclei (MN). Hence, the chemical 2,6 -octadienenitrile, 3,7-dimethyl is mutagenic in micronucleus test when exposed to NMRI mouse.

 

In another study (BAuA report, 2013), in vivo micronucleus test was performed to study the mutagenic effects of the test substance 2,6 -octadienenitrile, 3,7-dimethyl (CAS no 5146-66-7) by OECD guideline 474 under GLP condition. The dose concentration of test chemical was 0,625,1250 mg/kg bw administered to animals by gavage route. The administration of the test substance led to evident signs of toxicity and to a statistically significant and dose-dependent increase in the number of polychromatic erythrocytes (PCEs) containing small micronuclei (only 24 h) interval investigated. There was no increase in cells with large micronuclei. No inhibition of erythropoiesis determined from the ratio of PCEs to normochromatic erythrocytes (NCE) was detected. The positive control (cyclophosphamide) led to the expected increase in the rate of polychromatic erythrocytes containing small micronuclei. Hence, the chemical 2,6 -octadienenitrile, 3,7-dimethyl is considered to be mutagenic in micronucleus test and is likely to classify for gene mutation in vivo.

 

In yet another study (BAuA report, 2013), in vivo micronucleus test was performed to study the mutagenic effects of the test substance 2,6 -octadienenitrile, 3,7-dimethyl (CAS no 5146-66-7) by OECD guideline 474 under GLP condition. Two experiments were carried out with 3 applications and single application, for 3 applications the dose concentration used were 0, 200 and 400 mg/kg (6-h and 24-h interval) and in single application doses included were 0 and 1200 mg/kg. There was no increase in cells with large micronuclei. The positive controls (cyclophosphamide, vincristine) led to the expected increase in the rate of PCEs containing small and large micronuclei. Inhibition of erythropoiesis determined from the ratio of PCEs to normochromatic erythrocytes (NCE) was detected at 200 and 400 mg/kg bw. Hence,the chemical 2,6 -octadienenitrile, 3,7-dimethyl is considered to be mutagenic in micronucleus test when tested in mouse and hence is likely to classify for gene mutation in vivo.

 

In vivo micronucleus test carried out (BASF, 2003) to evaluate the mutagenic potency of chemical 2, 6 -octadienenitrile, 3,7-dimethyl (CAS no 5146-66-7) in NMRI male mouse with the dose concentration of 625 and 1250mg/kg . The animals were sacrificed 24 hours (all doses) or 48 hours (top dose only) after a single oral administration. A dose-dependent and statistically significant increase in polychromatic erythrocytes containing small micronuclei (clastogenic activity) was observed at 625 mg/kg (24 hours) and 1,250 mg/kg body weight (24 and 48 hours). Hence, the test chemical 2, 6 -octadienenitrile, 3,7-dimethyl is considered to be mutagenic in micronucleus test.

 

In vivo chromosomal aberration assay was performed to study the mutagenic effects of the test substance 2, 6 -octadienenitrile, 3,7-dimethyl (CAS no 5146-66-7, BAuA, 2013). The dose concentration of test chemical was 375, 750, 1,250 and 1,500 administered to animals by gavage route in corn oil. 2 experiments carried out 24 h and at 48-h sampling time. The mean aberration frequencies (excl. gaps) for the test concentration was calculated. The administration of the test substance led to evident signs of toxicity and to a statistically significant and biologically relevant enhancement of the aberration frequencies as compared to the vehicle control value. Only the observed increase at 24h / 1500 mg/kg bw was statistically significant. The positive control (Adriblastin, 5 mg/kg bw) showed the expected statistically significant response (aberration rate excl. gaps 5.2%). No reduction of the mitotic indices could be observed after treatment with the test item, indicating that the test item was not cytotoxic for spermatogonial cells. Hence, the test chemical 2, 6 -octadienenitrile, 3,7-dimethyl was considered to be mutagenic in Chromosome aberration assay of spermatogonial cells and hence is likely to classify for gene mutation in vivo.

 

Based on the data summarized, the test chemical Geranyl nitrile is likely to classify as a gene mutant in vitro.

Justification for selection of genetic toxicity endpoint

Data is from K4 article

Justification for classification or non-classification

Based on the data summarized, the test chemical Geranyl nitrile is likely to classify as a gene mutant in vitro.