[(3,7-dimethyl-6-octenyl)oxy]acetaldehyde

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 07, 2014 to August 26, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

[(3,7-dimethyl-6-octenyl)oxy]acetaldehyde

EC Number:

231-324-2

EC Name:

[(3,7-dimethyl-6-octenyl)oxy]acetaldehyde

Cas Number:

7492-67-3

Molecular formula:

C12H22O2

IUPAC Name:

2-[(3,7-dimethyloct-6-en-1-yl)oxy]acetaldehyde

Test material form:

liquid

Specific details on test material used for the study:

The test article was supplied by Givaudan as a liquid on 18 November 2013 and identified as follows.:
- Batch: VE00275479
- Purity: 92.3%
- Expiration date: 23 September 2014

Method

Species / strain

Species / strain / cell type:

mammalian cell line, other: Binucleated cells in human peripheral blood lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

The in vitro metabolic activation system (Maron and Ames, 1983) consisted of a rat liver post-mitochondrial fraction (S9) and an energy-producing system (NADP plus isocitric acid). Various hepatic P450 isoenzyme levels are increased by treatment of the rats with Aroclor 1254 (single concentration of 500 mg/kg) which were sacrificed 5 days later (Ames et al., 1975) (Molecular Toxicology, Inc., Lot No. 3234). The S9 fraction, prepared in potassium chloride, was retained frozen at -60 to -80°C until use.

Test concentrations with justification for top dose:

The dose range-finding assay was conducted at concentrations of 13.4, 19.2, 27.4, 39.2, 56.0, 80.0, 114, 163, 233, 333, 476, 680, 972, 1388, and 1983 µg/mL for 3 and ~24 hours without metabolic activation and 3 hours with metabolic activation. All cultures were harvested at approximately 24 hours from the initiation of treatment.

Vehicle / solvent:

The vehicle control article was Dimethylsulfoxide (DMSO), supplied by Sigma-Aldrich, with the following information/characteristics.
1st Batch: SHBC3313V (Purity: >= 99.9%; expiration date: 31 May 2014)
2nd Batch: SHBC0839V (Purity: >= 99.7%; expiration date: 31 August 2015)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Human venous blood from healthy adult donors (nonsmokers without a history of radiotherapy, chemotherapy, or drug usage, and lacking symptoms of current viral infections) was drawn into sterile, heparinized "vacutainers". Whole blood cultures were initiated in 15 mL centrifuge tubes by adding approximately 0.6 mL of fresh heparinized blood into a sufficient volume of culture medium so that the final volume was 10 mL in the assay without metabolic activation after the addition of the test article in its chosen vehicle, or 10 mL in the assay with metabolic activation after the addition of the test article in its chosen vehicle and the S9 mix.

Evaluation criteria:

- Criteria for a Positive Response: A test article was considered to have produced a positive response if it induced a statistically significant, dose-dependent increase in the frequency of BNMN.
- Criteria for a Negative Response: A test article was considered to have produced a negative response if no statistically significant or dose dependent increases in the frequency of BNMN were observed.
- Criteria for an Equivocal Response: Despite extensive testing, a test article may produce results that are neither clearly positive nor clearly negative. In those rare instances (e.g., statistically significant or dose dependent increases), the test article may be considered to have produced equivocal responses.
Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, etc.). In such cases, the Study Director will clearly report and describe such any considerations.

Results and discussion

Test results

Key result

Species / strain:

mammalian cell line, other: Binucleated cells in human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Initially a dose range-finding (DRF) assay was performed in single cultures to select doses for the micronucleus (MN) assay where duplicate cultures were used. Different concentrations of the test article were used with a dosing volume of 1% (v/v). Vehicle and positive controls were tested concurrently. Based on the current OECD 487 guideline, the top concentration of the test article used in the DRF assay was 10 mM. In the DRF assay, human peripheral blood lymphocytes (HPBL) were treated with Citronelloxyacetaldehyde at concentrations ranging from 13.4 to 1983 μg/mL for 3 hours with and without S9 and for approximately 24 hours without S9. Precipitate was observed at the time of treatment at ≥333 µg/mL in the approximate 24 hour treatment and at ≥233 µg/mL in the 3 hour treatment without S9. In the 3 hour treatment with S9, precipitate was observed at ≥680 µg/mL at the time of treatment. All cultures were harvested approximately 24 hours after the initiation of treatment. Slides were scored to calculate the cytokinesis block proliferation index (CBPI) for the measurement of cytotoxicity.
Based on the DRF results, Citronelloxyacetaldehyde was evaluated at concentrations ranging from 2.16 to 100 µg/mL in the approximate 24 hour treatment without S9. In the 3 hour treatment, Citronelloxyacetaldehyde was evaluated at concentrations ranging from 24.5 to 125 µg/mL without S9 and from 37.5 to 250 µg/mL with S9. No precipitate was observed in the 3 hour or the approximate 24 hour treatments. Precipitate was observed at the time of treatment at 250 µg/mL in the 3 hour treatment with S9. In the approximate 24 hour treatment, approximately 66% cytotoxicity was observed at 70.0 µg/mL. This concentration along with three lower concentrations, 35.8, 44.8, and 56.0 µg/mL producing approximately 7, 24 and 45% cytotoxicity, respectively, were selected for MN scoring. In the 3 hour treatment without S9, concentrations of 38.3, 47.8, 65.6, and 72.9 µg/mL producing approximately 15, 31, 50, and 65% cytotoxicity, respectively, were selected for MN scoring. In the 3 hour treatment with S9, concentrations 95.7, 120, and 133 µg/mL producing approximately 14, 43, and 48% cytotoxicity, respectively, were selected for MN scoring. No statistically significant increases in the BNMN frequency were observed at any analyzed concentration in any treatment condition. The BNMN frequencies of the vehicle and the positive controls fell within the acceptable range. All acceptance criteria were met and the study was accepted as valid.

Citronelloxyacetaldehyde was considered negative for inducing micronuclei in the binucleated cells in human peripheral blood lymphocytes when analyzed up to the limit of cytotoxicity in the approximate 24 hour treatment without S9 and in the 3 hour treatment with and without S9.

Executive summary:

The objective of this in vitro study was to evaluate the ability of Citronelloxyacetaldehyde, and/or its metabolites to induce micronuclei in cultured human lymphocytes using cytochalasin-B-induced cytokinesis block methodology in the presence and absence of an exogenous rat liver metabolic activation system (S9). The test article was formulated in dimethyl sulfoxide (DMSO). Initially a dose range-finding (DRF) assay was performed in single cultures to select doses for the micronucleus (MN) assay where duplicate cultures were used. Based on the results of the DRF, the concentrations in the definitive study ranged from 2.16 to 100 µg/mL in the approximate 24 hour treatment without S9. In the 3 hour treatment, Citronelloxyacetaldehyde was evaluated at concentrations ranging from 24.5 to 125 µg/mL without S9 and from 37.5 to 250 µg/mL with S9.

No statistically significant increases in the BNMN frequency were observed at any analyzed concentration in the approximate 24 hour treatment without S9 and in the 3 hour treatment with and without S9. The BNMN frequencies of the vehicle and the positive controls fell within the acceptable range. All acceptance criteria were met and the study was accepted as valid.

Citronelloxyacetaldehyde was considered negative for inducing micronuclei in the binucleated cells in human peripheral blood lymphocytes.