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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

These endpoints were fulfilled using read across from 3,7-dimethyloct-6-enenitrile (EC 257-288-8 / CAS 51566-62-2), for which the following results were obtained.

The mutagenic potential of the test item was assessed according to the method of Ames et al. (1975). Incorporation of the test liquid with the bacteria at levels up to 0.06 mg per plate did not increase the numbers of his+ revertants with any of the five tester strains, either in the presence or in the absence of the S-9 mix. From the present results it can be concluded that the test item did not reveal any mutagenic activity in the plate incorporation assay with S. typhimurim TA 1535, TA 1537, TA 1538, TA 98 or TA 100 in the presence or absence of the liver microsome activation system under the test conditions employed in this evaluation.

The mutagenic potential of the substance was also assessed in mammalian cells according to OECD 476. It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, citronellylnitril is considered to be non-mutagenic in this HPRT assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 1980

Reliability:

2 (reliable with restrictions)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Ames et al. (1975)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

In the preliminary test conducted to assess the toxicity of the test liquid for the bacteria 0.1mg test liquid per plate appeared slightly toxic as revealed by a less dense background lawn of bacterial growth.
Top dose: 0.06mg test liquid per 0.1mL methanol per plate

Vehicle / solvent:

- Vehicle/solvent used: methanol

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

0mg test liquid per 0.1mL methanol per plate

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: hycanthone methanesulphonate (1), 2-aminoanthracene (2)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: 1 - 4 x 10 9 cells / ml

DURATION
- Exposure duration:three days

NUMBER OF REPLICATIONS: triplicate

Rationale for test conditions:

Based on the test method Ames et al. (1975).

Evaluation criteria:

The colonies (revertants which are histidine independent) are counted and the background lawn of bacterial growth is examined microscopically.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

0.06mg test liquid per 0.1 mL methanol per plate, with S9 mix: background lawn of bacterial growth less dense than in concomitant control plates

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not valid

Positive controls validity:

not specified

Additional information on results:

See attachment.

Conclusions:

Incorporation of the test liquid with the bacteria at levels up to 0.06 mg per plate did not increase the numbers of his+ revertants with any of the five tester strains, either in the presence or in the absence of the S-9 mix.
From the present results it can be concluded that the test item did not reveal any mutagenic activity in the plate incorporation assay with S. typhimurim TA 1535, TA 1537, TA 1538, TA 98 or TA 100 in the presence or absence of the liver microsome activation system under the test conditions employed in this evaluation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Study period:

November 1980

Reliability:

2 (reliable with restrictions)

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:

other: Ames et al. (1975)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

In the preliminary test conducted to assess the toxicity of the test liquid for the bacteria 0.1mg test liquid per plate appeared slightly toxic as revealed by a less dense background lawn of bacterial growth.
Top dose: 0.06mg test liquid per 0.1mL methanol per plate

Vehicle / solvent:

- Vehicle/solvent used: methanol

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

0mg test liquid per 0.1mL methanol per plate

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: hycanthone methanesulphonate (1), 2-aminoanthracene (2)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: 1 - 4 x 10 9 cells / ml

DURATION
- Exposure duration:three days

NUMBER OF REPLICATIONS: triplicate

Rationale for test conditions:

Based on the test method Ames et al. (1975).

Evaluation criteria:

The colonies (revertants which are histidine independent) are counted and the background lawn of bacterial growth is examined microscopically.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

0.06mg test liquid per 0.1 mL methanol per plate, with S9 mix: background lawn of bacterial growth less dense than in concomitant control plates

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not valid

Positive controls validity:

not specified

Additional information on results:

See attachment.

Conclusions:

Incorporation of the test liquid with the bacteria at levels up to 0.06 mg per plate did not increase the numbers of his+ revertants with any of the five tester strains, either in the presence or in the absence of the S-9 mix.
From the present results it can be concluded that the test item did not reveal any mutagenic activity in the plate incorporation assay with S. typhimurim TA 1535, TA 1537, TA 1538, TA 98 or TA 100 in the presence or absence of the liver microsome activation system under the test conditions employed in this evaluation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan, Cytotest Cell research GmbH, In den Leppsteinwiesen 19, 64380 Rossdorf, Germany

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

See any other information on material and methods.

Vehicle / solvent:

- Solvent: DMSO
- Justification for choice of solvent: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures. The final concentration of DMSO in the culture medium was 0.5% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
- Expression/fixation time: Three or four days after treatment 1.5×10^6 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5×10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days.

NUMBER OF REPLICATIONS:
- The study was performed in two independent experiments, using identical experimental procedures.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Acceptability of the assay:
The gene mutation assay is considered acceptable if it meets the following criteria: The numbers of mutant colonies per 10e6 cells found in the solvent controls falls within the laboratory historical control data. The positive control substances should produce a significant increase in mutant colony frequencies. The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.
Evaluation of results: A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS GENOTOXICITY:
- No relevant and reproducible increase in mutant colony numbers/10e6 cells was observed in the main experiments up to the maximum concentration. An isolated increase of the mutation frequency exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the second culture of experiment I without metabolic activation at 67.5 µg/mL. However, the increase was based on a rather low mutation frequency of the solvent control of just 4.5 colonies per 10e6 cells. Furthermore, the effect was not reproduced in the parallel culture under identical experimental conditions and the absolute value of the mutation frequency remained within the historical range of solvent controls. Therefore, the increase of the induction factor was judged as biologically irrelevant fluctuation.

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH/osmolality: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: No precipitation was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

RANGE-FINDING/SCREENING STUDIES:
- In the range finding pre-experiment test item concentrations between 12.2 and 1560 µg/mL (≈10 mM) were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 48.8 µg/mL and above without metabolic activation and at 780 µg/mL and above with metabolic activation following 4 hours treatment. After 24 hours treatment a strong cytotoxic effect occurred even at the lowest concentration of 12.2 µg/mL. From the next higher to the maximum concentration the cell growth was completely inhibited.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the first experiment relevant cytotoxic effects indicated by a relative cloning efficiency or cell density below 50% in both parallel cultures occurred in 45.0 µg/mL and above, without metabolic activation, and at 380 µg/mL with metabolic activation. In the second experiment cytotoxic effects as described above were noted at 18.0 µg/mL and above without metabolic activation and at 384 µg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, citronellylnitril is considered to be non-mutagenic in this HPRT assay.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan, Cytotest Cell research GmbH, In den Leppsteinwiesen 19, 64380 Rossdorf, Germany

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

See any other information on material and methods.

Vehicle / solvent:

- Solvent: DMSO
- Justification for choice of solvent: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures. The final concentration of DMSO in the culture medium was 0.5% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
- Expression/fixation time: Three or four days after treatment 1.5×10^6 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5×10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days.

NUMBER OF REPLICATIONS:
- The study was performed in two independent experiments, using identical experimental procedures.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Acceptability of the assay:
The gene mutation assay is considered acceptable if it meets the following criteria: The numbers of mutant colonies per 10e6 cells found in the solvent controls falls within the laboratory historical control data. The positive control substances should produce a significant increase in mutant colony frequencies. The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.
Evaluation of results: A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS GENOTOXICITY:
- No relevant and reproducible increase in mutant colony numbers/10e6 cells was observed in the main experiments up to the maximum concentration. An isolated increase of the mutation frequency exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the second culture of experiment I without metabolic activation at 67.5 µg/mL. However, the increase was based on a rather low mutation frequency of the solvent control of just 4.5 colonies per 10e6 cells. Furthermore, the effect was not reproduced in the parallel culture under identical experimental conditions and the absolute value of the mutation frequency remained within the historical range of solvent controls. Therefore, the increase of the induction factor was judged as biologically irrelevant fluctuation.

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH/osmolality: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: No precipitation was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

RANGE-FINDING/SCREENING STUDIES:
- In the range finding pre-experiment test item concentrations between 12.2 and 1560 µg/mL (≈10 mM) were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 48.8 µg/mL and above without metabolic activation and at 780 µg/mL and above with metabolic activation following 4 hours treatment. After 24 hours treatment a strong cytotoxic effect occurred even at the lowest concentration of 12.2 µg/mL. From the next higher to the maximum concentration the cell growth was completely inhibited.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the first experiment relevant cytotoxic effects indicated by a relative cloning efficiency or cell density below 50% in both parallel cultures occurred in 45.0 µg/mL and above, without metabolic activation, and at 380 µg/mL with metabolic activation. In the second experiment cytotoxic effects as described above were noted at 18.0 µg/mL and above without metabolic activation and at 384 µg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, citronellylnitril is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

This endpoint was fulfilled using read across from 3,7-dimethyloct-6-enenitrile (EC 257-288-8 / CAS 51566-62-2), for which the following results were obtained.

The cytogenicity of the test substance was assessed according to OECD Guideline 474. 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.

Link to relevant study records

Reference

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.

Reason / purpose for cross-reference:

reference to same study

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on negative results in in-vitro and in-vivo studies conducted, the substance does not need to be classified for germ cell mutagenicity.