[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-6-methoxy-3,6,8,8-tetramethyl-1H-3a,7-methanoazulene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-11-02 until 2016-01-21

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 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:

[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-6-methoxy-3,6,8,8-tetramethyl-1H-3a,7-methanoazulene

EC Number:

267-510-5

EC Name:

[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-6-methoxy-3,6,8,8-tetramethyl-1H-3a,7-methanoazulene

Cas Number:

67874-81-1

Molecular formula:

C16H28O

IUPAC Name:

[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-6-methoxy-3,6,8,8-tetramethyl-1H-3a,7-methanoazulene

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate fraction (S9) and cofactors

Test concentrations with justification for top dose:

First experiment:
In the pulse treatment group without S9-mix: 120, 110, 100, 90, 80, 70, 60, 50, 25 and 12.5 μg/mL
In the pulse treatment group with S9-mix: 180, 170, 160, 150, 140, 130, 120, 110, 50, and 12.5 μg/mL

Second experiment:
In the pulse treatment group without S9-mix: 50, 45, 40, 35, 30, 25, 20, 15, 10 and 5.0 μg/mL
In the continuous group without S9-mix: 30, 25, 20, 15, 10, 7.5 μg/mL
In the continuous group without S9-mix (repeat): 60, 55, 50, 45, 40, 35, 30, 25, 20, 15 and 10 μg/mL

Vehicle / solvent:

dimethylsulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Vinblastine sulphate,

Remarks:

In the presence of S9-mix: Cyclophosphamide; In the absence of S9-mix: Mitomycin C and Vinblastine sulphate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
First experiment pulse treatment:
- Preincubation period (cell growth): 48 hours with phytohaemagglutinin
- Exposure duration: 4 hours pulse treatment method
- Recovery time: 20 hours with cytochalasin B

Second experiment continuous treatment method:
- Preincubation period (cell growth): 48 hours with phytohaemagglutinin
- Exposure duration: 24 hours with cytochalasin B

NUMBER OF REPLICATIONS: 2

SPINDLE INHIBITOR: cytochalasin B
STAIN: DNA-specific dye (acridin-orange)

NUMBER OF CELLS EVALUATED:
- Cytotoxicity: At least 500 cells per slide (in total 1000 cells per dose level)
- Micronucleus formation: Two thousand binucleated cells per concentration (1000 per culture)

DETERMINATION OF CYTOTOXICITY
- Cytokinesis-Block Proliferation Index

OTHER:
Solubility, pH, and osmolality were tested prior to the experiment to determine the dose range.

Evaluation criteria:

The study was considered valid if the clastogenic and aneugenic positive controls gave a statistically significant increase in the number of binucleated cells containing micronuclei and if the solvent controls (DMSO) were within the historical data of the test facility.
The response was considered positive if all of the following criteria are met:
- at least one of the test concentrations exhibits a statistically significant increase compared to the concurrent negative control.
- the increase is dose-related in at least in one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical solvent control data.

A response was considered negative if all of the following criteria are met:
- none of the test concentrations exhibits a statistically significant increase compared to the concurrent negative control.
- there is no dose-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical negative control data.

A test substance was considered equivocal if the response was neither positive or negative even after further investigation.

Statistical methods were used as an aid in evaluating the test results. Both biological relevance and statistical analysis were considered in evaluation of the response. Biological relevance was evaluated by comparison of the test results with the test facility’s historical range of the solvent control.

Statistics:

The frequencies micronuclei found in the cultures treated with the test substance and positive control cultures were compared with those of the concurrent solvent control using the Chi-square test (one-sided). The results were considered statistically significant when the p-value of the Chisquare test was less than 0.05. In addition, a trend test was performed on the frequencies of micronuclei found in the cultures treated with the different test substance concentrations using the statistical program GenStat (version 17), Poisson regression with logarithmic link function. The results of the trend test were considered statistically significant when the p-value was less than 0.05.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

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:

- Solubility of the test substance and measurements: In the solubility test, it was observed that DSMO was a suitable vehicle for the test substance. The stock concentration appeared as a clear colorless solution. After preparation of the final concentrations in the culture medium without serum, the concentrations of 50, 100 and 200 μg/mL appeared to be slightly turbid. The next lower concentration (25 μg/mL) was very slightly turbid when compared to the solvent control. At the lowest concentration (12.5 μg/mL) no aberrant findings were observed. In addition, visual observations were recorded after ca. 24 hours incubation at ca. 37°C in humidified air containing ca. 5% CO2. Moreover, at the highest test substance concentration, a slightly turbid solution with a few minuscule oil droplets were observed and at the lower concentrations no differences were observed when compared to the cultures shortly after preparation. The osmolality and pH values were determined shortly after preparation at ambient temperature.

- Micronuclei induction as a result of treatment with the solvent control and positive controls: In the performed experiments, the solvent control (1% DMSO) was within the range of historical data of the test facility. Treatment with the positive control substances Cyclophosphamide, Mitomycin C and Vinblastine Sulphate resulted in statistically significant increases in the number of binucleated cells containing micronuclei, when compared to the numbers found in the concurrent solvent control cultures. This demonstrated the validity of the in vitro micronucleus test.

- Cytotoxicity observed in the first and second experiment: In the first experiment, in the pulse treatment group with S9-mix, the test substance showed a dose-dependent cytotoxicity (i.e. 180, 140 and 110 μg/mL showed 52%, 26% and 9% cytotoxicity, respectively). In addition, no visually aberrant findings were observed during the performance of this pulse treatment group. The positive control substance cyclophosphamide (20 μg/ml) showed 57% cytotoxicity. In the pulse treatment group without S9 mix, the test substance concentrations (120 - 50 μg/mL) were severely cytotoxic to the cells demonstrated by the absence of cells on the slides. The lowest two test substance concentrations (25 and 12.5 μg/mL) showed 42% and 15% cytotoxicity, respectively. Due to the steep toxicity-response curve observed in this pulse treatment group, the criteria for selecting a maximum concentration based on cytotoxicity (55 ± 5 %) as stated in the OECD test guideline 487 were not met. Therefore, the pulse treatment group without S9-mix was repeated using a concentration range of 50 - 5 μg/mL. In this repeat, the test substance concentrations (50 - 35 μg/mL) were severely cytotoxic to the cells demonstrated by the absence of cells on the slides. The next lower concentrations (30, 25, 20 and 15 μg/mL) showed 73%, 57%, 45% and 13% cytotoxicity, respectively. At the lowest concentrations (10 and 5 μg/mL) no cytotoxicity was observed when compared to the concurrent control cultures. The positive control substance Mitomycin C showed 60% cytotoxicity. In the second experiment, in the continuous treatment group without S9-mix, the highest two test substance concentrations 30 and 25 μg/mL showed 38% and 14% cytotoxicity respectively. The next lower concentrations (20 - 7.5 μg/mL) did not show any cytotoxicity. The positive control substance Vinblastine sulphate (0.025μg/mL showed 64% cytotoxicity. At the highest test substance concentration, the required percentage of cytotoxicity (55 ± 5 %) as stated in the OECD test guideline 487 was not obtained and therefore this group was repeated in the third experiment. In this repeat, the test substance concentrations (60, 55, 50, 45, 40, 35 and 30 μg/mL) showed 78%, 67%, 56%, 50%, 48%, 25% and 18%, respectively. At the lowest concentrations (30 - 10 μg/mL) no cytotoxicity was observed when compared to the concurrent control cultures. The positive control substance Vinblastine sulphate (0.0125μg/mL) showed 77% cytotoxicity.

- Micronuclei induction as a result of treatment with the test substance: In the first experiment, four dose levels of the treatment group with S9-mix (12.5, 50, 140 and 180 μg/mL) of the test substance, together with the solvent control (DMSO) and positive control were analysed for micronucleus induction in binucleated lymphocytes. In the pulse treatment group with S9-mix, the test substance did not show a statistically significant, dose-dependent increase in the number of binucleated cells containing micronuclei at any of the concentrations analysed when compared to the concurrent solvent cultures. In addition, the number of binucleated cells containing micronuclei were within the historical data range of the test facility. In the second experiment, in the repeat pulse treatment group without S9-mix, four dose levels of the treatment group with S9-mix (10, 15, 20 and 25 μg/mL) of the test substance, together with the solvent control (DMSO) and positive control were analysed for micronucleus induction in binucleated lymphocytes. In the pulse treatment group without S9-mix, the found number of binucleated cells with micronuclei was only marginally above the historical data range of the test facility. According to the OECD test guideline 487, additional scoring was performed. As a consequence, 3000 instead of 2000 binucleated cells per selected concentration were examined for the presence of micronuclei. After additional scoring, the number of cells with micronuclei was within the historical data range and no statistically significant trend in the micronuclei frequency was observed. In the second experiment, in the continuous treatment group the required percentage of cytotoxicity was not obtained and therefore this group was repeated without performing micronuclei induction analysis in binucleated cells. In the repeated continuous treatment group (third experiment), three dose levels (25, 35 and 50 μg/mL) of the test substance, together with the solvent controls (DMSO 1% and 2%) and positive control were analysed for micronucleus induction in binucleated lymphocytes. In this group, the test substance did not show a statistically significant, dose-dependent increase in the number of binucleated cells containing micronuclei at any of the concentrations analysed when compared to the concurrent solvent cultures. In addition, at the test facility an historical solvent control distribution for final concentrations of 1% and 2%. DMSO was not established for the continuous treatment group when co-exposed with cytochalasin B according to the updated OECD test guideline 487. Therefore, the numbers of binucleated cells containing a micronucleus were compared with the limited historical data at the test facility and were also compared with data presented in the literature (M. Fenech et al, 2003 and E. Lorge et al, 2006). As a result, the number of binucleated cells containing micronuclei were within the historical data range of the test facility.

Applicant's summary and conclusion

Conclusions:

Under the test conditions OECD 487 and GLP the test substance was not clastogenic and/or aneugenic to cultured human lymphocytes.

Executive summary:

In accordance with OECD 487 and GLP, the test substance was examined for its potential to induce micronuclei in cultured inucleated human lymphocytes, in both the absence and presence of a metabolic activation system (S9- mix). Duplicate cultures were used in all experiments. Cytotoxicity was determined from the Cytokinesis-Block Proliferation Index (CBPI). In the first experiment, in the presence and absence of S9-mix the treatment/recovery time was 4/20 hours (pulse treatment). In the second experiment, in the continuous treatment group the treatment/recovery time was 24 hours. Solvent control (DMSO) and positive controls were run in parallel. In the first experiment, in the pulse treatment group with S9-mix a dose-dependent cytotoxicity was observed. In the pulse treatment group without S9- mix the test substance concentrations (50-120 μg/mL) were severely cytotoxic to the cells as demonstrated by the absence of cells on the slides. At the lowest concentrations (25 and 12.5 μg/mL), the required cytotoxicity of 55 ± 5% according to the OECD test guideline 487 was not met and therefore the pulse treatment group without S9-mix was repeated in a second experiment. In this repeated experiment, at the highest concentrations (35 - 50 μg/mL), the test substance was severely cytotoxic to the cells as demonstrated by the absence of cells on the slides. At the lower concentrations (5 - 30 μg/mL) a dose-dependent cytotoxicity was observed. In the pulse treatment groups both with (12.5, 50, 140 and 180 μg/mL) and without S9-mix (10, 15, 20 and 25 μg/mL), concentrations of the test substance, together with the solvent control (DMSO) and positive controls were analysed for micronucleus induction in binucleated lymphocytes. In both pulse treatment groups with and without S9-mix, the test substance did not show a statistically significant, dose-dependent increase in the number of binucleated cells containing micronuclei at any of the concentrations analysed when compared to the concurrent solvent cultures. In addition, the number of binucleated cells containing micronuclei were within the historical data range of the test facility. In the second experiment, in the continuous treatment group, the cells were treated with test substance concentrations ranging from 30 to 0.313 μg/mL. The highest concentration (30 μg/mL) induced a cytotoxicity up to 38% when compared to the concurrent solvent control. As a consequence, the required cytotoxicity of 55 ± 5% accordingly to the OECD test guideline 487 was not met and therefore the continuous treatment group was repeated in a third experiment. In the repeated continuous treatment group a dose-dependent cytotoxicity was observed. Three dose levels (25, 35 and 50 μg/mL) of the test substance, together with the solvent control (DMSO) and positive control were analysed for micronucleus induction in binucleated lymphocytes. In this group, the test substance did not show a statistically significant, dose-dependent increase in the number of binucleated cells containing micronuclei at any of the concentrations analysed when compared to the concurrent solvent cultures. In addition, the number of binucleated cells containing micronuclei were within the historical data range of the test facility and the obtained results were comparable to the data presented in the literature. From the results obtained in the in vitro micronucleus test it is concluded that, under the conditions used in this study, the test substance was not clastogenic and/or aneugenic to cultured human lymphocytes.