3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 April 2016 to 28 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

Refer to main study report

GLP compliance:

yes

Remarks:

Refer to main study report

Type of assay:

other: In Vitro Mammalian Chromosomal Aberration Assay in Chinese Hamster Lung (CHL) or V79 Cells

Test material

Test material information

1

Reference substance name:

3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

EC Number:

814-345-7

Cas Number:

2003244-43-5

Molecular formula:

C14H21N

IUPAC Name:

3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

Details on test material:

- Name of test material (as cited in study report): ES421 Pinyl Nitrile- Molecular weight (if other than submission substance): 203 g/mol- Physical state: Crystallized white powder- Analytical purity: 98.08% (per Certificate of Analysis)- Composition of test material, percentage of components: 2-Norpinene-2-propionitrile, alpha, alpha,6,6-tetramethyl- Purity test date: 08 February 2016- Lot/batch No.: SM15077102- Storage condition of test material: 2-8°C, protected from light

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: International Flavors & Fragrances Inc. Lot/Batch No.SM15077102- Expiration date of the lot/batch: 22 July 2016STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: 2 to 8° C, protected from light- Solubility of the test substance in the solvent/vehicle: Soluble in DMSO at a concentration of approximately 500 mg/mLTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

Method

Target gene:

chromosomes

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED- Source of cells: Sigma Aldrich- Suitability of cells: Stability of karyotype and morphology has made them suitable for genetic toxicity assays with low background aberrations- Cell cycle length, doubling time or proliferation index: Population doubling time of approximately 15 hours- Modal number of chromosomes: 22MEDIA USED- Type and identity of media including CO2 concentration if applicable: complete medium [Dulbecco's Modified Eagle Medium with GlutaMAX™ –I, 1 g/L D-Glucose, 110 mg/L Sodium Pyruvate containing 10% heat inactivated fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin and 2.5 µg/mL Amphotericin B]. The cultures were incubated under standard conditions (37 ± 1C in a humidified atmosphere of 5 ± 1% CO2 in air).- Properly maintained: yes- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was purchased commercially from MolTox (Boone, NC)..

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the doses tested were 0.2, 0.6, 2, 6, 20, 60, 200, 600, and 2000 µg/mL. The top dose tested, 2000 µg/mL, was the limit dose for this assay, as per OECD 473 (adopted 2014). Dose levels for the chromosomal aberration assay were based upon post-treatment toxicity (cell growth inhibition relative to the vehicle control). Doses tested were:Non-activation 4 hour: 5.0, 10, 20, 30, 35, 40, 45, 50, 55, and 60 µg/mLS9-activation 4 hour: 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 µg/mLNon-activation 20 hour: 0.5, 1.0, 2.5, 5.0, 10, 20, 30, 35, 40, 45, and 50 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO - Justification for choice of solvent/vehicle: DMSO was used as the vehicle based on the information provided by the Sponsor, the solubility of the test substance, and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in mediumDURATION- Exposure duration: CHO cells were exposed to the test and control articles for 4 and 20 hours without S9 and for 4 hours with S9, and rinsed. Cells were harvested 20 hours (±30 minutes) after initiation of treatment, which corresponds to 1.5 normal cell cycles.STAIN (for cytogenetic assays): GiemsaNUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 2 in the chromosome aberration assayNUMBER OF CELLS EVALUATED: a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture), whenever possibleDETERMINATION OF CYTOTOXICITY- Method: Cell growth indexOTHER EXAMINATIONS:- Determination of polyploidy: yes- Determination of endoreplication: yes

Evaluation criteria:

Toxicity induced by treatment was based upon inhibition of cell growth and was reported for the cytotoxicity and chromosome aberration portions of the study. The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but were not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.A test article was considered positive if it induced a statistically significant and dose dependent increase in the frequency of aberrant metaphases (p

Statistics:

Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose-responsiveness.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Definitive assay: Cytotoxicity (55 ± 5% cell growth inhibition relative to vehicle control), was observed at doses >/= 35 µg/mL in the non activated 4 and 20-hour exposure groups, and at doses >/= 100 µg/mL in the S9 activated 4-hour exposure group.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: The pH of the highest dose of test substance in treatment medium was 7.5.- Effects of osmolality: The osmolality of the test substance doses in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 120%.- Definition of acceptable cells for analysis: Metaphase cells with 22 ± 2 centromeres were examined under oil immersion RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was conducted in order to determine cytotoxicity and to select doses for the chromosomal aberration assay.HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)- Positive historical control data: Historical positive control chromosomal aberration data for CHL or V79 cells is not available due to limited number of studies conducted.- Negative (solvent/vehicle) historical control data: Historical vehicle control chromosomal aberration data for CHL or V79 cells is not available due to limited number of studies conducted.ADDITIONAL INFORMATION ON CYTOTOXICITY:- Measurement of cytotoxicity used: reduction in cell growth index relative to the vehicle control

Applicant's summary and conclusion

Conclusions:

Under the conditions of the assay described in this report, ES421 Pinyl Nitrile was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using Chinese Hamster Lung (CHL) or V79 cells.

Executive summary:

The test substance,ES421 Pinyl Nitrile, was tested to evaluate the potential to induce structural chromosomal aberrations using Chinese hamster lung (CHL) or V79 cells in both the absence and presence of an of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO)was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 µg/mL, which was the limit dose for this assay, as per OECD 473 (adopted 2014). Cytotoxicity (greater than 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥60 µg/mL in the non‑activated 4 and 20-hour exposure groups, and at doses ≥200 µg/mL in the S9‑activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥600 µg/mL in the non‑activated and S9-acitvated 4-hour exposure groups, and at 2000 µg/mL in the non‑activated 20-hour exposure group. Based upon these results, the doses chosen for the chromosome aberration assay ranged from 5 to 60 µg/mL for the non‑activated 4-hour exposure group, from 20 to 200 µg/mL for the S9-activated 4-hour exposure group, and from 0.5 to 50 µg/mL for the non-activated 20-hour exposure group.

In the chromosome aberration assay, cytotoxicity (55 ± 5% reduction in cell growth index relative to the vehicle control), was observed at doses ≥35 µg/mL in the non‑activated 4 and 20-hour exposure groups, and at doses ≥100 µg/mL in the S9‑activated 4-hour exposure group. The doses selected for evaluation of chromosome aberrations were 10, 20, and 35 µg/mL for the non‑activated 4-hour exposure group; 20, 40, and 100 µg/mL for the S9‑activated 4-hour exposure group; and 5, 20, and 35 µg/mL for the non‑activated 20-hour exposure group.

No significant or dose‑dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

These results indicateES421 Pinyl Nitrilewas negative for the induction of structural and numerical chromosome aberrations in the presence and absence of the exogenous metabolic activation systemin thein vitroMammalian Chromosomal Aberration Assay inChinese Hamster Lung (CHL) or V79 Cells.