Reaction mass of 3,5-dimethylcyclohex-3-ene-1-carbaldehyde and 2,4-dimethylcyclohex-3-ene-1-carbaldehyde

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05/11/2021-02/09/2022

Reliability:

1 (reliable without restriction)

Data source

Reference

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 3,5-dimethylcyclohex-3-ene-1-carbaldehyde and 2,4-dimethylcyclohex-3-ene-1-carbaldehyde

EC Number:

943-728-2

Molecular formula:

C9H14O

IUPAC Name:

Reaction mass of 3,5-dimethylcyclohex-3-ene-1-carbaldehyde and 2,4-dimethylcyclohex-3-ene-1-carbaldehyde

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Sponsor; SHT036
- Purity, including information on contaminants, isomers, etc.: 99.48% (sum of isomers)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store at laboratory conditions, protected from heat and light

Method

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: Male chinese hamster fibroblast cell line; European Collection of Animal Cell Cultures (ECACC), Great Britain
- Normal cell cycle time (negative control): 14 hours

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Number of passages if applicable: Genotoxicity evaluations - 6th experiment: passage 5 and 7th experiment: passage 6

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: DMEM with high glucose (with L-Glutamine and Sodium Pyruvate L-glutamin), Foetal Bovine Serum, Penicillin-Streptomycin; 37 °C ± 1 °C; 5% CO2

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Delor 106-induced rat liver S9 metabolic activation; Each culture in experiments with metabolic activation (+MAS) contained 37 uL of S9 and 37 uL of cofactors solution.

Test concentrations with justification for top dose:

Preliminary toxicity tests: 25 - 5000 µg/mL
3 hrs with metabolic activation: 150, 175, 200 µg/mL
3 hrs without metabolic activation: 50, 75, 125 µg/mL
24 hrs without metabolic activation: 11.25, 22.5, 45 µg/mL

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicate
- Number of independent experiments: 2 (3 hr with and without S9; 24 hr without S9)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hr and 24 hour treatments
- Harvest time after the end of treatment (sampling/recovery times):
3 hrs – cells washed and sampled after 24 ± 1 hours since the beginning of treatment.
24 hrs – cells sampled at end of treatment.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: 3 hrs treatment:
21 µL of cytochalasin B per culture was added (the final concentration of cytochalasin B in cultures was 4.2 ug/mL). Cultures were then cultivated and sampled after 24 ± 1 hours since the beginning of treatment.
24 hr treatment: 12 uL of cytochalasin B (final concentration of cytochalasin B in cultures was 2.4 ug/mL) for 24 hrs

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Cultures were harvested 24 ± 1 hours after the beginning of treatment (after about 1.5 to 2 cell cycles). At first cultures were treated by trypsin (0.5 ml of trypsine per culture flask) for 3 minutes. Then 2.5 ml of DMEM were added to flasks and cell suspensions were centrifuged (1600 rpm, 5 minutes, 4 °C). Supernatants were aspirated and cell suspensions were treated by hypotonic solution (RT, about 5 minutes) and then they were centrifuged (1600 rpm, 5 minutes, 4 °C). After removing of hypotonic solution, fixation solution was added to cultures and cultures were centrifuged again (1600 rpm, 5 minutes, 4 °C). The addition of fixation solution and centrifugation were repeated once. Suspensions were then dropped on clear microscopic slides. Preparations were let to dry at laboratory temperature and then slides were stained by Giemsa Romanowski staining solution.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 1000 cells were evaluated at each concentration for cytotoxicity evaluation and at least 2000 binucleated cells were evaluated at each concentration for genotoxicity evaluation.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The micronuclei were scored in the binucleated cells only. The micronuclei in the binucleated cells with irregular shapes, in the binucleated cells where the two nuclei differ greatly in size, the binucleated cells without cytoplasm or binucleated cells with poorly spread multi-nucleate cells was not scored.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Cell proliferation is characterized by Cytokinesis-Block Proliferation Index (CBPI).

Evaluation criteria:

Cytotoxicity is indicated by increasing of % cytotoxicity. % cytotoxicity is determined by ratio of CBPIT at tested concentration to CBPIC at the solvent control. The upper limit of % cytotoxicity for the highest test concentration for determination of genotoxicity effect is 45 +/- 5 %. If this limit is exceeded the lower concentrations are need to use.

Genotoxicity is indicated by increasing of number of cells with micronuclei in comparison to the negative control (two-fold increase rule) and/or by dependence of increasing number of cells with micronuclei on dose (dose-response relationship).

Test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the following three conditions are met concurrently:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control (Fisher´s exact test, Two-fold increase rule)
• the dependence of increasing number of cells with micronuclei on concentration (Cochran Armitage trend test, dose response relationship) is evident
• any of the results are outside the distribution of the historical negative control data

When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and/or gain or loss in this test system.

Test chemical is considered clearly negative if, in all experimental conditions examined, the following three conditions apply concurrently:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are inside the distribution of the historical negative control data.

In case of equivocal results, further testing with modification of experiment conditions could be used for clarification.

Statistics:

In evaluation of results the Two-fold increase rule and statistical method Cochran Armitage trend test were used. For increasing of number of cells with micronuclei comparison to the negative control is used Fisher´s exact test.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

3 hrs

Metabolic activation:

with

Genotoxicity:

other: ambiguous; p=0.008 at 200 μg/mL

Remarks:

p=0.008 at 200 μg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

40.5% at 200 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

3 hr

Metabolic activation:

without

Genotoxicity:

other:

Remarks:

ambiguous; p<0.0001 at 125 μg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

38.8% at 125 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

24 hrs

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

51.3% at 45 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable):
Overall, seven experiments were performed, five experiments with short exposure and two experiments with extended exposure. The results from the 2nd, 3rd, 4th and 5th experiments were used for evaluation of cytotoxicity and the results from the 6th and 7th experiments were used for evaluation of genotoxicity.
In more detail, the 1st experiment was stopped before addition of the test item, because the contamination in cultures was observed. In the 2nd experiment all concentrations were cytotoxic and in the 3rd experiment, the concentration near the threshold of cytotoxicity 45 ± 5% was not found so another experiment was performed. In the 4th experiment with and without metabolic activation (short treatment) the concentrations in the range 45 ± 5% of cytotoxicity were found, however the genotoxicity of the negative controls were outside the laboratory historical range, so that the results from the 4th experiment was not used for genotoxicity evaluation. Optimal concentrations found in the 4th experiment were used in the 6th experiment with 3-hour treatment and genotoxicity evaluation was done. From the 5th experiment´s preparations only cytotoxicity evaluation was done. In the 7th experiment (long treatment), cytotoxicity as well as genotoxicity evaluation were done.

STUDY RESULTS

In this study in the 6th and 7th experiments:

Criterion 1
In the negative control normal cell cycle time or proliferation index should be determined.
Cell cycle length of V79 cells is evaluated for every new batch of cells. The V79 cell cycle length used in this study is 14 hours, which is in accordance with the cell line information. The criterion 1 was fulfilled.

Criterion 2
The cytotoxicity of the highest three concentrations selected for genotoxicity evaluation should not be higher than 45 ± 5 %. Concurrently, at least 2000 binucleated cells should be scored in each concentration of genotoxicity evaluation.
In the 6th experiment (short treatment) without metabolic activation the cytotoxicity in the highest evaluated concentration was 38.8% without metabolic activation and 40.5% with metabolic activation. See Tables No. 5 and 6 in Annex 1. In the 7th experiment the cytotoxicity at the highest evaluated concentration was 51.3%, but as the result was negative, this is acceptable (Deviations). See Tables No. 5 and 6 in Annex 1. The criterion 2 was fulfilled.

Criterion 3
Number of micronuclei in negative and positive controls should be in the 95% control limits of historical laboratory data distribution.
The number of cells with micronuclei in negative controls is in the 95% control limits of historical laboratory data distribution. The number of micronuclei in positive control Cyclophosphamide is higher than the 95% control limits of historical laboratory data distribution. The criterion 3 was fulfilled.

Criterion 4
Negative control value of average number of micronuclei per 1000 binucleated cells will be compared with appropriate positive control value. The result of positive control should be ≥ 2x negative control value. Concurrently, CBPI of negative control should be in the range 1.3 2.2.

CBPI of negative control (untreated culture) was 2.13 in the 6th experiment and 2.12 in the 7th experiment. The result of positive control was ≥ 2x negative control value and according to Fisher´s exact test the increase was statistically significant. The criterion 4 was fulfilled.

Micronucleus test in mammalian cells:

- Results from cytotoxicity measurements:
In the 6th experiment with short exposure the cytotoxicity ≥ 45 ± 5 % was not observed in any concentration (with as well as without metabolic activation) so all concentrations were used for genotoxicity evaluation.

In the 7th experiment with extended exposure without metabolic activation the cytotoxicity slightly above 45 ± 5 % was observed in concentration 45 µg/mL. However, the concentration of 45 µg/mL was nevertheless used to evaluate genotoxicity (see Deviations).

- Genotoxicity results

An equivocal effect was found in evaluated non-cytotoxic concentrations from short treatment experiments with and without metabolic activation.

On the basis of the evaluation criteria, the test item in the 6th experiment (short exposure with and without metabolic activation):
• show a statistically significant increase at the top concentration only (confirmed by Fisher´s exact test) compared with concurrently solvent negative control (Table 7)
• did not show evidently the dependence of increasing number of cells with micronuclei on concentration (dose-response relationship, confirmed by Cochran Armitage trend test) (Table 7)
• four results are outside the distribution of the historical negative control data (27.2 – 39.0 cells with micronuclei per 2000 binucleated cells without metabolic activation and 30.0 – 42.3 cells with micronuclei per 2000 binucleated cells with metabolic activation according to 95% laboratory range) (Table 7).

On the basis of the evaluation criteria, the test item in the 7th experiment (extended exposure without metabolic activation):
• did not show a statistically significant increase (confirmed by Fisher´s exact test) compared with concurrently solvent negative control (Table 8)
• did not show evidently the dependence of increasing number of cells with micronuclei on concentration (dose-response relationship, confirmed by Cochran Armitage trend test) (Table 8)
• all result from the test concentrations is outside the distribution of the historical negative control data (27.2 – 39.0 cells with micronuclei per 2000 binucleated cells without metabolic activation according to 95% laboratory range) (Table 8).

On the basis of informations above, the test item is not considered as clearly positive or clearly negative. The result of the study is equivocal. Further experiments of the in vitro micronucleus test were unlikely to yield new results and were therefore not performed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Refer to Annex 3, Table 10, 11
- Negative (solvent/vehicle) historical control data: Refer to Annex 3, Table 9

Remarks on result:

other: To be updated with QSAR prediction for negative result

Any other information on results incl. tables

Deviations:

No deviation from method described in OECD Test Guideline 487.

There were two deviations against Study Plan.

1) In the 6th experiment, cyclophosphamide anhydrous recommended in OECD 487 was used instead of cyclophosphamide monohydrate, which was previously used in our laboratory and for which a database of historical controls has been created. A different form of cyclophosphamide was used because the cyclophosphamide monohydrate used in the previous experiments had already been used up and in order to follow OECD guidelines, cyclophosphamide anhydrous was ordered for further testing. Cyclophosphamide anhydrous meets the criteria for a positive control: a statistically significant increase in binucleated cells with micronuclei relative to the negative control and cytotoxicity less than 45 ± 5%. The control range for cyclophosphamide anhydrous was taken from the literature (Ref. 3 and 4) and the result of cyclophosphamide anhydrous in the 6th experiment falls within this range.

2) In the 7th experiment the highest evaluated concentration caused cytotoxicity > 45 ± 5%. Although the concentration was slightly cytotoxic (51.3%), no genotoxic effect was observed at this concentration during 24-hour exposure.

 

These deviations had no impact on the study.

Applicant's summary and conclusion

Conclusions:

Under the experimental design described above, it is not clear whether the test item, Reaction mass of 3,5-dimethylcyclohex-3-ene-1-carbaldehyde and 2,4-dimethylcyclohex-3-ene-1-carbaldehyde, had a genotoxic effect in the V79 cell line cells under any experimental conditions. The result of the micronucleus test was therefore considered equivocal.

Executive summary:

In an in vitro cytogenicity study (mammalian cell micronucleus test) (OECD 487/GLP), Chinese hamster lung fibroblast V79 cells were exposed to the test item (99.48% (sum of isomers)) in DMSO. The concentrations were 150, 175, 200 µg/mL (3 hrs) in the presence of Delor 106-induced rat liver S9 metabolic activation, 50, 75, 125 µg/mL (3 hrs) and 11.25, 22.5, 45 µg/mL (24 hrs) without metabolic activation.

In the 3hr experiment without metabolic activation, the cytotoxicity in the highest test concentration 125 µg/mL was 38.8%. In the 3hr experiment with metabolic activation, the cytotoxicity in the highest test concentration 200 µg/mL was 40.5%. In the 24hr experiment, the cytotoxicity in the highest test concentration 45 µg /mL was 51.3%.

In the 3 hr experiment with and without metabolic activation, a statistically significant increase in binucleated cells with micronuclei was observed at the top concentrations only (p=0.008 at 200 μg/mL with metabolic activation; p<0.0001 at 125 μg/mL without metabolic activation), however dose-response was not observed. As all the criteria for a clear positive or negative response were not met, the conclusion was equivocal. In the 24 hr experiment, a statistically significant increase in binucleated cells with micronuclei was not observed at any concentration, though results from all concentrations were outside the distribution of the historical negative control data. As all the criteria for a clear positive or negative response were not met, the conclusion was equivocal.

Under the experimental design described above, it is not clear whether the test item, Reaction mass of 3,5-dimethylcyclohex-3-ene-1-carbaldehyde and 2,4-dimethylcyclohex-3-ene-1-carbaldehyde, had a genotoxic effect in the V79 cell line cells under any experimental conditions. The result of the micronucleus test was therefore considered equivocal.