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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro genemutations in bacterial cells (OECD TG 471): negative

In vitro cytogenicity, in vitro micronucleus test (OECD TG 487): negative
In vitro gene mutations in mammalian cells, MLA (OECDTG 490): negative

Link to relevant study records

Reference

Reference 1

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

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

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)

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

other: L5178Y tk +/- 3.7.2C line

Metabolic activation:

with and without

Metabolic activation system:

Rat aroclor S9 liver homogenate

Test concentrations with justification for top dose:

Dose range finding: 0, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, 100, and 200 µg/mL
Main experiment: 0, 3, 6, 12, 17, 24, 35, 39, 43, 48, 53, 59, 66, 73, 81, and 90 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Remarks:

methylmethanesulfonate without metabolic activation and 3-methylcholanthrene with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Selection time (if incubation with a selection agent): 13 days

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity of the test substance was determined by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG).

Evaluation criteria:

A response was considered to be positive if the induced MF (MF of the test substance minus that of the vehicle negative control) was more than 126 mutants per 1,000,000 clonable cells (Aaron et al., 1994; Clive et al., 1995). A response was considered to be equivocal if the induced mutant frequency was more than 88 mutants (but not more than 126 mutants) per 1,000,000 clonable cells. Any apparent increase in MF at concentrations of the test substance causing more than 90% cytotoxicity and with no evidence of mutagenicity at RTG > 10%, was considered to be an artefact and not indicative of genotoxicity.

The test substance was considered to be mutagenic in the gene mutation test at the TK-locus if a concentration-related increase in MF was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed.

The test substance was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the MF nor a reproducible positive response at any of the test substance concentrations.

Both numerical significance and biological relevance were considered together in the evaluation. No statistical analysis was performed.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

Dose range finding test
In the dose range finding test, single cultures were exposed for 24h in the absence of S9-mix to 10 concentrations of the test susbatnce ranging from 0.4 to 200 μg/ml. After 4 and 24 hours aliquots were taken to assess the cell yield. After 4h, at the two highest concentrations tested (200 and 100 μg/ml), the cells were heavily affected (flocculated) due to exposure to the test substance. The next lower concentration (50 μg/ml) also showed signs of cytotoxicity. At concentrations of 25 μg/ml and lower, no abnormalities were observed. After incubation for 24h a dose related cytotoxicity was observed from a concentration of 25 μg/ml onwards. At a concentration of 50 μg/ml cell viability was about 30%. Approximately 48h after start of treatment, cytotoxicity was observed at concentrations of 25 μg/ml and higher. Based on the observations during the dose range finding test, it was decided to use 90 μg/ml, as the highest dose level for the main test, in both the absence and presence of a metabolic activation system (S9-mix).

Main test
- Positive and negative controls: MMS and MCA were used as positive control substances in the absence and in the presence of S9-mix, respectively; DMSO served as a negative control. In both exposure groups the negative controls were within historical background ranges and treatment with the positive controls yielded the expected significant increase in MF compared to the negative control. Therefore, the study was considered valid. Concentration levels and visual observations before and after treatment In the main test, the concentrations of the test substance ranged from 3 to 90 μg/ml in both the absence and presence of S9-mix. At the start and end of the treatment in the presence of S9-mix, no visibly aberrant effects (e.g. flocculation/precipitation of the test substance or lysed cells) were observed. In the absence of S9-mix, at the start of the treatment, the culture medium was slightly more turbid at the highest six concentrations (53 – 90 μg/ml) when compared to the negative control. These observations are in line with the solubility data obtained in the in vitro micronucleus test (V20717/07). At the end of treatment, the culture medium was less turbid (suggesting cell lysis) at the highest nine concentrations (39 – 90 μg/ml) when compared to the negative control. Both in the absence and presence of S9-mix, at the end of the treatment period, viability of the cells (as determined with trypan blue dye exclusion) at the highest concentration (90 μg/ml) was 16% and 14 %, respectively.

- Cytotoxicity: In the absence of S9-mix, the test substance was toxic to the cells. Following 4h exposure, the relative initial cell yield and/or relative suspension growth (RSG) and/or relative total growth (RTG) were reduced by more than 10% at and above 3 μg/ml. The concentrations of the test substance evaluated for mutagenicity were 17, 12, 6 and 3 μg/ml; the RTG at these concentrations was 3%, 24%, 78% and 93% respectively. In the presence of S9-mix the test substance was also toxic to the cells. The initial cell yield and/or RSG and/or RTG were reduced by more than 10% at and above 35 μg/ml. The four highest concentrations evaluated for mutagenicity were 90, 81, 73 and 66 μg/ml; the RTG at these concentrations was 0.01%, 41%, 24% and 43%, respectively.

- Mutagenicity: Following 4h exposure, in the absence and presence of S9-mix, no increase in the mutant frequency (MF) by more than 88 or 126 mutants per 1,000,000 clonable cells, i.e. no equivocal or positive response, compared to the negative control was observed at any dose level.

- Colony sizing: Colony sizing was not performed as no positive responses were observed in cultures exposed to the test substance.

Discussion
Although the fact that in the absence of S9-mix only 4 single analysable concentrations were available may be considered limited, the minimal criteria of the OECD 490 are met and these concentrations reflect the complete cytotoxicity range. In addition, the dose-response with respect to cytotoxicity is very steep and the concentrations are already very closely spaced and higher, lower or intermediate concentrations are not expected to give any additional information. In the presence of S9-mix, the cytotoxicity criteria are not met. However, also in this case, the test is considered valid. Although no concentration resulting in an RTG between 10 and 20% is present, the dose-response relationship with respect to cytotoxicity is very steep and concentrations are already very closely spaced. This is reflected by the fact that at a concentration of 81 μg/ml, the RTG is 41%, whereas at the next higher concentration of 90 μg/ml the RTG is 0.01%. The lower concentration of 73 μg/ml resulted in an RTG of 24%, which was very close to the RTG of 10-20%. Furthermore, the OECD test guideline 490 claims that if the maximum concentration is based on cytotoxicity, the highest concentration should aim to achieve between 20 and 10% RTG for the MLA. As it is anticipated that a repeat of the study using possibly even more closely spaced concentrations will not lead to a different result. The OECD guideline 490 states that in cases where the short-term treatment yields negative results, and there is information suggesting the need for longer treatment (e.g., nucleoside analogs, poorly soluble substances), consideration should be given to conducting the test with longer treatment. As there is no information suggesting the need for longer treatment (i.e. the test substance is not a nucleoside analog or a poorly soluble substance), longer treatment (24 hour treatment) was not conducted.

Conclusions:

Under the conditions of the test the substance is not mutagenic.

Executive summary:

The test substance was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, according to OECD 490 and in compliance with GLP. A preliminary dose range finding test was performed to determine the dosage of the main experiment. A single experiment was conducted. In this experiment, 15 single cultures were treated for 4h in the presence and absence of S9-mix. The test substance was dissolved in DMSO. The highest concentration evaluated for mutagenicity was 17.1 μg/ml and 90 μg/ml in the absence and presence of S9-mix, respectively, limited by cytotoxicity. Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively and DMSO served as negative control. In both the absence and presence of S9-mix no increase in mutant frequency (MF) by more than 88 or 126 mutants per 1,000,000 clonable cells, i.e. no equivocal or positive response, compared to the negative control was observed at any of the dose levels tested. It is concluded that under the conditions used in this study, the test substance is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (S9-mix) when exposed for 4h.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli Wp2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: TA100 and WP2 uvrA 0, 50, 150, 500, 1500, 5000 µg/plate with and without S9 mix.
Experiment 1:
- TA 100, TA1535, TA98, TA1537: 0, 1.5, 5, 15, 50, 150, 500, and 1500 µg/plate without S9 mix.
- WP2 uvrA: 0, 15, 50, 150, 500, 1500, and 5000 µg/plate without S9 mix.
- TA100, TA1535, TA98, TA1537: 0, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate with S9 mix.
- WP2 uvrA: 0, 50, 150, 500, 1500, and 5000 µg/plate with S9 mix.
Experiment 2:
- TA 100, TA1535, TA98, TA1537: 0, 0.5, 1.5, 5, 15, 50, 150, and 500 µg/plate without S9 mix.
- WP2 uvrA: 0, 15, 50, 150, 500, 1500, and 5000 µg/plate without S9 mix.
- TA 100, TA1535, TA98, TA1537: 0, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate with S9 mix.
- WP2 uvrA: 0, 15, 50, 150, 500, 1500, and 5000 µg/plate with S9 mix.

Vehicle / solvent:

- Solvent used: Acetone
- Justification for choice of solvent: The test item was immiscible in sterile distilled water and DMSO at 50 mg/mL but was fully miscible in acetone at 100 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

- S9 mix: N-ethyl-N-nitro-N-nitrosoguanidine (WP2 uvrA, TA100, TA1535), 9-aminoacridine (TA1537) 4-nitroquinoline-N-oxide (TA98).+ S9 mix: benzo(a)pyrene (TA98), 2-aminoanthracene (TA100, TA1535, TA1537, WP2 uvrA).

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: after 48 hours of incubation the plates were assessed for numbers of revertant colonies using an automated colony counter and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

EVALUATION CRITERIA
- A dose related increase in mutant frequency over the dose range tested
- A reproducible increase at one or more concentrations
- Biological relevance against in-house historical control ranges
- Statistical analysis of data as determined by UKEMS
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).

ACCEPTANCE CRITERIA
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- All tester strain cultures should be in the range of 0.9 to 9E9 bacteria per mL.
- Diagnostic mutagens (postive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9 mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test item dose levels.
- There should be no evidence of excessive contamination.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli Wp2 uvrA

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:

Preliminary Toxicity Test
- The test item was toxic to TA 100 from 150 and 500 µg/plate in the absence and presence of S9-mix respectively and toxic to WP2uvrA at 5000 µg/plate in the absence of S9-mix and non-toxic in the presence of S9-mix. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation Test
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.
- The test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains initially from 50 µg/plate in the absence of S9-mix and at 500 µg/plate in the presence of S9-mix. Weakened lawns were also noted to Escherichia coli strain WP2uvrA at 5000 µg/plate in both the absence and presence of S9-mix. The test item was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on bacterial strain type and Experiment number. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation.

Conclusions:

Under the conditions of the test the substance is not mutagenic

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 and according to GLP principles. The test was performed in two independent preincubation experiments, both in the absence and presence of S9-mix. The dose levels were selected based on the dose range finding experiment and ranged from 0.5 to 5000 µg/plate depending on the strain tested and the presence or absence of S9 -mix. The strains tested were Salmonella typhimurium strains TA 100, TA1535, TA98, TA1537 and Escherichia coli strain WP2uvrA. The tests were performed in triplicate and adequate negative and positive controls were included. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. Under the condition of the test, the substance was considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 and according to GLP principles. The test was performed in two independent preincubation experiments, both in the absence and presence of S9-mix. The dose levels were selected based on the dose range finding experiment and ranged from 0.5 to 5000 µg/plate depending on the strain tested and the presence or absence of S9 -mix. The strains tested were Salmonella typhimurium strains TA 100, TA1535, TA98, TA1537 and Escherichia coli strain WP2uvrA. The tests were performed in triplicate and adequate negative and positive controls were included. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. Under the condition of the test, the substance was considered to be non-mutagenic.

In vitro micronucleus test:

In accordance with OECD 487 and GLP, the test substance was examined for its potential to induce micronuclei in cultured nucleated 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 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 continuous treatment group, dose-dependent cytotoxicity was observed. 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.

Mouse lymphoma test:

The test substance was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, according to OECD 490 and in compliance with GLP. A preliminary dose range finding test was performed to determine the dosage of the main experiment. A single experiment was conducted. In this experiment, 15 single cultures were treated for 4h in the presence and absence of S9-mix. The test substance was dissolved in DMSO. The highest concentration evaluated for mutagenicity was 17.1 μg/ml and 90 μg/ml in the absence and presence of S9-mix, respectively, limited by cytotoxicity. Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively and DMSO served as negative control. In both the absence and presence of S9-mix no increase in mutant frequency (MF) by more than 88 or 126 mutants per 1,000,000 clonable cells, i.e. no equivocal or positive response, compared to the negative control was observed at any of the dose levels tested. It is concluded that under the conditions used in this study, the test substance is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (S9-mix) when exposed for 4h.

Justification for classification or non-classification

The Ames, micronucleus and mouse lymphoma test were negative and therefore the substance does not have to be classified for genotoxicity in accordance with Regulation (EC) No. 1272/2008.