(3R)-3,7-dimethyloct-6-enenitrile

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: micronucleus test

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, no restrictions, fully adequate for assessment.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The structures of the target and source substances are identical and differ only with respect to the ratio of enantiomers where the target substance is a single pure L-isomer and the source substance is an equimolar mixture of L and D isomers.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance, L-Citronellyl nitrile, is a mono-constituent substance (EC No. 695-909-8, CAS no. 35931-93-2).
The source substance, DL-Citronellyl nitrile, is a mono-constituent substance (EC No. 257-288-8, CAS no. 51566-62-2).
The source and target substances are both of high purity with a low concentration of impurities.

3. ANALOGUE APPROACH JUSTIFICATION
The read across hypothesis is based on structural similarity where the only difference between target and source molecules is the enantiomeric ratio. In a non-chiral environment the target and source chemicals will have identical properties but in the chiral environment of living organisms the enantiomers may possess different carcinogenicity and teratogenicity (in a chiral environment, stereoisomers might experience selective absorption, protein binding, transport, enzyme interactions and metabolism, receptor interactions, and DNA binding). Therefore, as a precaution for the developmental toxicity endpoint it is suggested that the NOAEL 250 mg/kg bw/day for L-Citronellyl nitrile is used instead of 500 mg/kg bw/day, as it is not known which form is more potent in vivo. All other endpoints are considered to be acceptable for this substance assuming that 50% of the target compound is available in the test material.

4. DATA MATRIX
Please refer to the data matrix included in the read-across justification document attached in Section 13.2.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

Experimental Toxicology and Ecology, BASF Aktiengesellschaft, 67056 Ludwigshafen, Germany

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH.
- Age at study initiation: 5-8 weeks
- Weight at study initiation: mean weight 27 g
- Housing: During the acclimation period, animals were housed in Makrolon cages, separately according to sex. Before the start of the treatment the animals were transferred to Makrolon cages, type Ml, and housed individually.
- Diet: Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: Drinking water from bottles, ad libitum.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours).

Route of administration:

oral: gavage

Vehicle:

- Vehicle: olive oil
- Justification for choice of solvent/vehicle: Due to the insufficient solubility of the test substance in water, olive oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

All test substance formulations were prepared immediatly before administration.

Frequency of treatment:

single administration

Post exposure period:

24 and 48 hours after administration in the highest dose group of 2000 mg/kg bw and in the vehicle controls.
24 hours in the test groups of 500 and 1000 mg/kg bw and in the positive control groups.

Remarks:

Doses / Concentrations:
500, 1000, and 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide for clastogenicity and vincristine for spindle poison effects.

Tissues and cell types examined:

polychromatic and normochromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- In a pretest for the determination of the acute oral toxicity, 2000 mg/kg body weight recommended as the highest dose according to the OECD Guideline were survived by all animals (male and female). The clinical signs observed were squatting posture and eyelid closure (male and female) and the general state of the animals was poor (only males). Therefore, a dose of 2000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
- Preparation of the bone marrow: The bone marrow was prepared according to the method described by SCHMID, W. and SALAMONE, M. et al. The two femora of the animals sacrificed by cervical dislocation were prepared by dissection and removing all soft tissues. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 mL/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µI fresh FCS. 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
- Staining of the slides: The slides were stained in eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes. After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes. Subsequently, the slides were stained in Giemsa solution (15 mL Giemsa, 185 mL purified water) for about 15 minutes. After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2000 polychromatic erythrocytes (PCEs) from each of the male and female animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded: - Number of polychromatic + normochromatic erythrocytes; - Number of polychromatic + normochromatic erythrocytes containing micronuclei., - Number of small/large micronuclei.

Evaluation criteria:

Acceptance criteria: The mouse micronucleus test is considered valid if the following criteria are met: The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable cells, i.e. ≥ 2 000 PCEs and a clear differentiation between PCEs and NCEs. The ratio of PCEs/NCEs in the untreated animals (negative control) has to be within the normal range for the animal strain selected. The number of cells containing micronuclei in negative control animais has to be within the range of the historical control data both for PCEs and for NCEs. The two positive control substances have to induce a significant increase in the number of PCEs containing small and large micronuclei within the range of the historical control data or above.
Assessment criteria: A finding is considered positive if the following criteria are met: Significant and dose-related increase in the number of PCEs containing micronuclei. The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range. A test substance is considered negative if the following criteria are met: The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.

Statistics:

The statistical evaluation of the data was carried out using the program System MUKERN (BASF Aktiengesellschaft). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determination for the U test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

male at 2000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- The single oral administration of olive oil in a volume of 10 mL/kg bw led to 2.0‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or after the 48-hour sacrifice interval. After the single administration of the highest dose of 2000 mg/kg bw, 2.6 ‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1.9 ‰ after 48 hours. In the two lower dose groups, rates of micronuclei of about 2.5 ‰ (1000 mg/kg bw group) and 2.0 ‰ (500 mg/kg bw group) were detected after a sacrifice interval of 24 hours in each case. The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals. Thus, the test substance Citroneltylnitril did not Iead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not deviate from the vehicle control value at any of the sacrifice intervals and was within the historical control range. A slight inhibition of erythropoiesis induced by the treatment of mice with Citronellylnitril was detected from about 500 mg/kg body weight onward.
-The single oral administration of the vehicle in a volume of 10 mL/kg body weight was tolerated by all animals without any signs or symptoms. The female animals tolerated the single oral treatment without any clinical signs. The male animals showed signs of toxicity but only at the highest dose of 2000 mg/kg bw (squatting posture and eyelid closure).

Conclusions:

Under the experimental conditions chosen here, the test substance Citronellylnitril has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

Experimental Toxicology and Ecology, BASF Aktiengesellschaft, 67056 Ludwigshafen, Germany

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

3,7-dimethyloct-6-enenitrile

EC Number:

257-288-8

EC Name:

3,7-dimethyloct-6-enenitrile

Cas Number:

51566-62-2

Molecular formula:

C10H17N

IUPAC Name:

3,7-dimethyloct-6-enenitrile

Details on test material:

- Name of test material: Citronellylnitril
- Test substance No.: 03/0333-1
- Batch No.: PBG-Ch. 00000277LO Betr.-Ch. RCN-02.003
- Date of manufacture: November 27, 2002
- Purity: 97.4 area - %
- Physical state, appearance: Colorless liquid
- Storage: Room temperature (N2 conditions)
- Stability: At least 4 hours (analytically verified)

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH.
- Age at study initiation: 5-8 weeks
- Weight at study initiation: mean weight 27 g
- Housing: During the acclimation period, animals were housed in Makrolon cages, separately according to sex. Before the start of the treatment the animals were transferred to Makrolon cages, type Ml, and housed individually.
- Diet: Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: Drinking water from bottles, ad libitum.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle: olive oil
- Justification for choice of solvent/vehicle: Due to the insufficient solubility of the test substance in water, olive oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

All test substance formulations were prepared immediatly before administration.

Frequency of treatment:

single administration

Post exposure period:

24 and 48 hours after administration in the highest dose group of 2000 mg/kg bw and in the vehicle controls.
24 hours in the test groups of 500 and 1000 mg/kg bw and in the positive control groups.

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, and 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide for clastogenicity and vincristine for spindle poison effects.

Examinations

Tissues and cell types examined:

polychromatic and normochromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- In a pretest for the determination of the acute oral toxicity, 2000 mg/kg body weight recommended as the highest dose according to the OECD Guideline were survived by all animals (male and female). The clinical signs observed were squatting posture and eyelid closure (male and female) and the general state of the animals was poor (only males). Therefore, a dose of 2000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
- Preparation of the bone marrow: The bone marrow was prepared according to the method described by SCHMID, W. and SALAMONE, M. et al. The two femora of the animals sacrificed by cervical dislocation were prepared by dissection and removing all soft tissues. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 mL/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µI fresh FCS. 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
- Staining of the slides: The slides were stained in eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes. After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes. Subsequently, the slides were stained in Giemsa solution (15 mL Giemsa, 185 mL purified water) for about 15 minutes. After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2000 polychromatic erythrocytes (PCEs) from each of the male and female animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded: - Number of polychromatic + normochromatic erythrocytes; - Number of polychromatic + normochromatic erythrocytes containing micronuclei., - Number of small/large micronuclei.

Evaluation criteria:

Acceptance criteria: The mouse micronucleus test is considered valid if the following criteria are met: The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable cells, i.e. ≥ 2 000 PCEs and a clear differentiation between PCEs and NCEs. The ratio of PCEs/NCEs in the untreated animals (negative control) has to be within the normal range for the animal strain selected. The number of cells containing micronuclei in negative control animais has to be within the range of the historical control data both for PCEs and for NCEs. The two positive control substances have to induce a significant increase in the number of PCEs containing small and large micronuclei within the range of the historical control data or above.
Assessment criteria: A finding is considered positive if the following criteria are met: Significant and dose-related increase in the number of PCEs containing micronuclei. The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range. A test substance is considered negative if the following criteria are met: The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.

Statistics:

The statistical evaluation of the data was carried out using the program System MUKERN (BASF Aktiengesellschaft). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determination for the U test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

male at 2000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- The single oral administration of olive oil in a volume of 10 mL/kg bw led to 2.0‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or after the 48-hour sacrifice interval. After the single administration of the highest dose of 2000 mg/kg bw, 2.6 ‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1.9 ‰ after 48 hours. In the two lower dose groups, rates of micronuclei of about 2.5 ‰ (1000 mg/kg bw group) and 2.0 ‰ (500 mg/kg bw group) were detected after a sacrifice interval of 24 hours in each case. The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals. Thus, the test substance Citroneltylnitril did not Iead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not deviate from the vehicle control value at any of the sacrifice intervals and was within the historical control range. A slight inhibition of erythropoiesis induced by the treatment of mice with Citronellylnitril was detected from about 500 mg/kg body weight onward.
-The single oral administration of the vehicle in a volume of 10 mL/kg body weight was tolerated by all animals without any signs or symptoms. The female animals tolerated the single oral treatment without any clinical signs. The male animals showed signs of toxicity but only at the highest dose of 2000 mg/kg bw (squatting posture and eyelid closure).

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, the test substance Citronellylnitril has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.