2-(3,5-dimethylhex-3-en-2-yloxy)-2-methylpropyl cyclopropanecarboxylate

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:

From June 25 to August 05, 2008.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other:

Remarks:

GLP study conducted according to OECD test Guideline No. 473 with the following deviations: tested too high with metabolic activation, excessive variability between duplicate cultures, no dose response and some individual cultures within HCD.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted July 21, 1997.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Dated May 30, 2008.

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

German Statement of GLP compliance. Inspected on 02 September 2006 /Signed on 19 January 2007.

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-{[(2R)-3,5-dimethylhex-3-en-2-yl]oxy}-2-methylpropyl cyclopropanecarboxylate; 2-{[(2S)-3,5-dimethylhex-3-en-2-yl]oxy}-2-methylpropyl cyclopropanecarboxylate

EC Number:

700-118-9

Cas Number:

676532-44-8

Molecular formula:

C16H28O3

IUPAC Name:

2-{[(2R)-3,5-dimethylhex-3-en-2-yl]oxy}-2-methylpropyl cyclopropanecarboxylate; 2-{[(2S)-3,5-dimethylhex-3-en-2-yl]oxy}-2-methylpropyl cyclopropanecarboxylate

Test material form:

liquid

Details on test material:

- Description: Colourless liquid
- Formula: C16H28O3
- Molecular weight: 268,4 g/mol

Specific details on test material used for the study:

- Storage conditions: In the refrigerator at +2 to +8°C, protected from light

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cell line supplied by Laboratory for Mutagenicity Testing, LMP,
Technical University Darmstadt, D-64287 Darmstadt, Germany were stored in liquid nitrogen in the cell bank of RCC Cytotest Cell Research GmbH allowing the repeated use of the same cell culture batch in experiments.
- Suitability of cells: Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability.
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37° C in 80 cm² plastic flasks (GREINER, D-72632 Frickenhausen). About 5 x 10e5 cells per flask were seeded into 15 mL of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe). The cells were subcultured twice weekly. The cell cultures were incubated at 37° C in a humidified atmosphere with 1.5 %
carbon dioxide (98.5 % air).
- Modal number of chromosomes: The cells have a modal chromosome number of 22.
- Periodically checked for karyotype stability: YES; The cells have a stable karyotype.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: in-house preparation by RCC Cytotest Cell Research
- method of preparation of S9 mix: 8-12 weeks old male Wistar rats (from Harlan) were induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and β-Naphthoflavone p.o. (Aldrich, 82024Taufkirhcen) each on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1:3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -80° C. Small numbers of the ampoules were kept at -20°C for up to one week. The protein concentration was 33.6 mg/mL (Lot. no. 080508).
- concentration or volume of S9 mix and S9 in the final culture medium: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl2; 33 mM KCl; 5 mM glucose-6-phosphate; 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath.

Test concentrations with justification for top dose:

- Main Experiment (18-hour/4-hour without S9-mix): 10.8, 21.6, 43.3, 86.6, 173.1, 346.3, 692.5, 1385.0 (p) and 2770.0 (p) µg/mL
- Main Experiment, repeated due to strong cytotoxicity (18-hour/4-hour* without S9-mix): 10.8, 21.6***, 43.3***, 86.6***, 173.1, 346.3, 692.5, 1385.0 (p) and 2770.0 (p) µg/mL
- Main Experiment, repeated due to missing cytotoxicity (18-hour/4-hour** without S9-mix): 0.3, 0.7, 1.4, 2.7, 5.4, 10.8, 21.6, 43.3, 86.6 µg/mL.

- Main Experiment (18-hour/4-hour with S9-mix): 10.8, 21.6, 43.3, 86.6, 173.1, 346.3***, 692.5***, 1385.0*** (p) and 2770.0*** (p) µg/mL.

*: was repeated due to strong cytotoxicity
** was repeated due to missing cytotoxicity
*** Evaluated experimental points
P: phase separation

Detailed explanation for choice of top dose are explained on the Table 7.6.1/1: Dose selection.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (E. Merck, 64293 Darmstadt, Germany, purity: 99.8 %, Lot no.: K38541383811 and K37361983723). The final concentration ofethanol in the culture medium was 0.5 % (v/v).
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

900 µg/mL (7.2 mM). Dissolved in nutrient medium.

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

1.4 µg/mL (5.0 µM). Dissolved in saline (0.9 % NaCl w/v)

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): The cells were seeded into Quadriperm dishes (Heraeus, 63450 Hanau, Germany) that contained microscopic slides (at least 2 chambers per dish and test group). In each chamber x 10000 - 60000 cells were seeded with regard to the preparation time
- Test substance added in medium (serum-free medium with S9-mix and MEM with 10 % FCS without S9-mix)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: 4 hours with S9-mix and without S9-mix.
- Expression time (cells in growth medium): 14 hours with and without S9-mix.
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Colcemid was added (0.2 Og/mL culture medium) to the cultures 15.5 hours after the start of the treatment. The cells on the slides were treated 2.5 hours later, in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). Per experiment two slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, 64293 Darmstadt, Germany).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): at least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. In each experimental group two parallel cultures were set up.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Evaluation of the cultures was performed using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI)
- Any supplementary information relevant to cytotoxicity: for evaluation of cytotoxicity indicated by reduced cell numbers two additional cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hrs in order to determine microscopically the cell number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.

Rationale for test conditions:

This in vitro test was performed to assess the potential of test item to induce structural chromosome aberrations. Evaluation of cytogenetic damage induced in V79 cells (cell line from the lung of the Chinese Hamster) in the absence and the presence of metabolic activation was performed in one experiment at 18 hours preparation interval.

Evaluation criteria:

Acceptability of the assay:
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) The number of aberrations found in the solvent controls falls within the range of historical laboratory control data range: 0.0 - 4.0 % aberrant cells, exclusive gaps.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory’s historical control data:
EMS (400-900 µg/mL) : 7.0-51.0 %
CPA (1.0-2.0 µg/mL): 5.0-44.0 %

Evaluation of results:
A test item is classified as non-clastogenic if:
-the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
- and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 – 5.3% polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria for the test item mentioned above are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant influence of the test item on the pH value was observed. In the main experiment pH solvent = 7.5 versus pH 2770 µg/mL = 7.4.
- Effects of osmolality: No relevant influence of the test item on the osmolality was observed. In main experiment solvent = 417 mOsm versus 2770 µg/mL = 360 mOsm.
- Precipitation: was observed after 4 hours treatment with 1385 Og/mL and above in the presence of S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: In the absence of S9 mix, no statistically significant or biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 2.5 % aberrant cells, excluding gaps) were close to the solvent control value (1.5 % aberrant cells,
excluding gaps) and within the range of the laboratory’s historical control data range (0.0 - 4.0 % aberrant cells excluding gaps). In contrast, in the presence of S9 mix statistically significant and biologically relevant increases in the number of cells carrying structural chromosome aberrations were observed at the three highest evaluated concentrations 692.5, 1385.0, and 2770 µg/mL . The aberration rates of the cells after treatment with the test item (5.5, 6.0, and 4.5 % aberrant cells excluding gaps, respectively) clearly exceeded the laboratory’s historical
control data (0.0 - 4.0 % aberrant cells excluding gaps).
ADDITIONAL INFORMATION ON CYTOTOXICITY: In the absence of S9 mix reduced cell numbers (23.1 % of control) and reduced mitotic indices (37.2 % of control) were observed after treatment with 86.6 µg/mL. In the presence of S9 mix neither reduced mitotic indices nor reduced cell numbers could be observed up to the highest required test item concentration

Applicant's summary and conclusion

Conclusions:

Under the test conditions of this study, the test item is considered as negative for inducing chromosomal mutations in Chinese hamster cell line V79 under  non-activation conditions and weakly clastogenic (ambiguous response) under the activation conditions used in this assay.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 and in compliance with GLP, V79 cells (Chinese hamster cell line) were exposed to the test material diluted in Ethanol in one experiment.

According to the OECD Guideline only one experiment was performed, since the test item was considered to be mutagenic after 4 hours treatment. The chromosomes were prepared 18 hours after start of treatment with the test item. The exposure period was 4 hours with and without metabolic activation.

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (main study) (2770 μg/mL; approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473.
Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation as following:

- Main Experiment (18-hour/4-hour without S9-mix): 10.8, 21.6, 43.3, 86.6, 173.1, 346.3, 692.5, 1385.0 (p) and 2770.0 (p) µg/mL
- Main Experiment, repeated due to strong cytotoxicity (18-hour/4-hour* without S9-mix): 10.8, 21.6***, 43.3***, 86.6***, 173.1, 346.3, 692.5, 1385.0 (p) and 2770.0 (p) µg/mL
- Main Experiment, repeated due to missing cytotoxicity (18-hour/4-hour** without S9-mix): 0.3, 0.7, 1.4, 2.7, 5.4, 10.8, 21.6, 43.3, 86.6 µg/mL.

- Main Experiment (18-hour/4-hour with S9-mix): 10.8, 21.6, 43.3, 86.6, 173.1, 346.3***, 692.5***, 1385.0*** (p) and 2770.0*** (p) µg/mL.

*: was repeated due to strong cytotoxicity
** was repeated due to missing cytotoxicity
*** Evaluated experimental points
P: phase separation

 

In the main experiment phase separation of the test item in culture medium was observed after 4 hours treatment with 1385 µg/mL and above in the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 417 mOsm, pH 7.5 versus 360 mOsm and pH 7.4 at 2770 µg/mL).

 

None of the dose levels up to the cytotoxicity limit of 86.6 µg/mL in the absence of metabolic activation induced significant increases in the frequency of cells with aberrations in a single experiment. In the presence of metabolic activation the substance was not toxic at up to the maximum dose level of 2770 µg/mL equivalent to 10mM, but showed phase separation (precipitation) at the upper two dose levels. Four dose levels were evaluated for aberration frequency and the upper three dose levels exhbited frequencies of cells with aberrations that exceeded the threshold for a positive response. However, there was no dose-response relationship and marked inter-culture variation, rendering the results ambiguous or weakly clastogenic. The substance does not induce structural aberrations in the chromosomes of Chinese hamster cells line V79 under non-activation conditions, but was considered ambiguous or weakly clastogenic in the presence of activation. Both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells.The substance is therefore considered as negative for inducing chromosomal mutations in Chinese hamster cell line V79 under  non-activation conditions and weakly clastogenic (ambiguous response) under the activation conditions used in this assay.

This study is considered as acceptable and satisfies the requirement for chromosome aberration endpoint.