[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): not mutagenic up to limit or cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 & E.coli WP2uvrA-.

- Micronucleus test in vitro (OECD 487, GLP, K, Rel.1): not clastogenic and not aneugenic up to cytotoxic concentrations in human peripheral blood lymphocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 19, 2016 to February 17, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD TG 487 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2014

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: cultured human peripheral blood lymphocytes
- Suitability of cells: yes
- Normal cell cycle time (negative control): 15 to 17 hours

For lymphocytes:
- Sex, age and number of blood donors: one male adult donor (nonsmoker without a history of radiotherapy, chemotherapy, or drug usage, and lacking current viral infections); 18-35 years
- Whether whole blood or separated lymphocytes were used: whole blood cultures
- Whether blood from different donors were pooled or not: not
- Mitogen used for lymphocytes: phytohemagglutinin M.

MEDIA USED
- Type and composition of media: The medium was HEPES-buffered RPMI 1640 culture medium supplemented with approximately 20% (v/v) heatinactivated fetal bovine serum (FBS), penicillin (100 units/mL), streptomycin (100 μg/mL), L-glutamine (2 mM) and 2% phytohemagglutinin M (PHA).
- CO2 concentration: 2-6 %
- Humidity level: humidified atmosphere
- Temperature: 37°C ± 2°C

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

cytochalasin B (6 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

DRF: from 19.8 to 1000 μg/mL
Main: 24-hour / without S9: 35.0 to 143 μg/mL; 3-hour without S9: 84.4 to 243 μg/mL; 3-hour with S9: 104 to 300 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl formamide (DMF).
- Justification for choice of solvent/vehicle: In a solubility assessment with DMSO, the test article formed a white, opaque, non-viscous suspension at 200 mg/mL. The formulation was prepared by vortex mixing and sonication at 37°C.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: 2

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours (+/- S9), 24 hours (-S9)
- Harvest time after the end of treatment (sampling/recovery times):

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: cytochalasin B (final concentration of 6 μg/mL), 20 hours for the 3-hours treatment groups; 24 hours for the 24-hours treatment group
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): All cultures were harvested approximately 24 hours after initiation of treatment. The cultures were centrifuged, the supernatant discarded, and the cells were swollen with 75 mM KCl, fixed in methanol: glacial acetic acid (3:1, v/v), dropped onto glass slides, and air dried. The slides were appropriately stained with acridine orange.
- Number of cells spread and analysed per concentration: a minimum of 200 cells per culture (when available) for the DRF, and a minimum of 500 cells per culture for the Micronucleus Assay
- Slide analysis:
Scoring was carried out using fluorescence microscopy. Binucleate cells were only included in the analysis if all of the following criteria were met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.
A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.
For each treatment regime, two vehicle control cultures were analyzed for micronuclei. Slides from the positive control treatments were checked to ensure that the system was operating satisfactorily.
All slides for analysis were coded by an individual not connected with the scoring of the slides, such that analysis was conducted under blind conditions. Labels with only the study number, assay type, experiment number, the sex of the donor and the code were used to cover treatment details on the slides.
At least three concentrations were scored for micronucleus induction. Where possible, at least one thousand binucleate cells from each culture (2000 per concentration) were analyzed for micronuclei. The number of cells containing micronuclei and the number of micronuclei per cell on each slide were recorded.
Micronucleus analysis was not conducted on slides generated from the Range-Finder treatments.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method:
CBPI = Number of mononucleate cells + (2 x number binucleate cells) + (3 x number multinucleate cells) / total number of cells in treated cultures
Relative CBPI (%) = (CBPI of treated cultures – 1) / (CBPI of vehicle controls – 1) x100
Cytotoxicity (%) is expressed as (100 – Relative CBPI).

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account, for example consistency of response within and between concentrations, or effects occurring only at very toxic concentrations (Thybaud et al., 2007).

Statistics:

The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989).
The proportion of MNBN cells for each treatment condition was compared with the proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989). A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant. Additionally, the number of micronuclei per binucleate cell were obtained and recorded.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cf. Tables of results in attached background documents

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: A solubility test was performed under Covance Study Number 8350026 and DMF was selected as the vehicle for this assay.
The top dose tested for the test article was based on OECD criteria, 10 mM or 2 mg/mL (2 μL/mL for liquid test materils), which ever was the lowest. The molecular weight of the test material was 250.38 g/mol, so 2 mg/mL, was lower than the 10 mM (2503.8 μg/mL).
Due to the limited solubility of the test article in the culture media, top dose used for this test article in the test system was 1000 μg/mL.
- Precipitation and time of the determination:
Dose Range-finding Assay: At the time of treatment, precipitate was observed at ≥343 μg/mL in the approximate 24 hour treatment without S9, and in the 3 hour treatments with and without S9. At the end of treatment (or at wash), precipitate was observed at ≥700 μg/mL in the 3 hour treatments with and without S9. At the time of harvest, precipitate was observed at ≥490 μg/mL in the approximate 24 hour treatment without S9 and at ≥343 μg/mL in the 3 hour treatments with and without S9.
Main Assay: No precipitate or hemolysis was observed at any tested concentration at the time of treatment or at harvest.
- Definition of acceptable cells for analysis: In the main assay, 51% cytotoxicity was observed at 93.8 μg/mL. This concentration along with two lower concentrations, 35.0 and 68.4 μg/mL, producing 11 and 35% cytotoxicity, respectively, were selected for MN evaluation.

RANGE-FINDING/SCREENING STUDIES:
Hemolysis was observed at ≥490 μg/mL at the end of the treatment in the 3 hour treatment and at the time of harvest in the approximate 24 hour treatment, without S9. Slides were prepared and scored to calculate CBPI for the measurement of cytotoxicity. In the approximate 24 hour treatment without S9, 66% cytotoxicity was observed at 118 μg/mL. In the 3 hour treatment without S9, 44% cytotoxicity was observed at 168 μg/mL. The next higher concentration, 240 μg/mL, was excessively cytotoxic and produced 75% cytotoxicity. In the 3 hour treatment with S9, 65% cytotoxicity was observed at 240 μg/mL. Based on the cytotoxicity data, concentrations were selected for the micronucleus assay (B1).

STUDY RESULTS
In the assay without metabolic activation with a 3 hour treatment, CBPI data are presented in Table 8.7. Dose levels of 129, 143, and 177 μg/mL were analyzed for MNBN (Table 8.8). Statistical Analysis data are presented in Table 8.9.
No precipitate or hemolysis was observed at any tested concentration at the time of treatment, end of treatment, or at harvest. In this test condition, 58% cytotoxicity was observed at 177 μg/mL. This concentration along with two lower concentrations, 129 and 143 μg/mL, producing 9 and 24% cytotoxicity, respectively, were selected for MN evaluation.
In the assay with metabolic activation with a 3 hour treatment, CBPI data are presented in Table 8.10. Dose levels of 177, 219, and 243 μg/mL were analyzed for MNBN (Table 8.11). Statistical Analysis data are presented in Table 8.12.
No precipitate or hemolysis was observed at any tested concentration at the time of treatment, end of treatment, or at harvest. In this test condition, 61% cytotoxicity was observed at 243 μg/mL. This concentration along with two lower concentrations, 177 and 219 μg/mL, producing 19 and 39% cytotoxicity, respectively, were selected for MN evaluation.
A statistically significant increase in the MNBN frequency was observed at 35.0 μg/mL in the approximate 24 hour treatment without S9. However, the MNBN frequency (0.60%) observed at this concentration was within the vehicle historical control range (0.10 to 1.10%) for the approximate 24 hour treatment without S9 in the male donors. Therefore, the statistically significant increase observed at this concentration was considered as biologically non-relevant. No statistically significant increase in the MNBN frequencies was observed in the 3 hour treatments with or without S9.

DATA ACCEPTABILITY AND VALIDITY
All positive and vehicle control MNBN frequencies were within acceptable ranges, except the 3 hour treatments with S9 (1.80%) and without S9 (3.60%), where the positive controls MNBN frequencies which were below the historical control ranges for with S9 (2.07 to 7.55%) and without S9 (8.55 to 16.78%) test conditions in the male donors. However, as the MNBN frequency of the positive control in these two test conditions were statistically significantly higher compared to the concurrent vehicle control, this was accepted as valid. All acceptance criteria were met and the study was accepted as valid

Conclusions:

The test article was considered negative for inducing micronuclei in the binucleated cells of human peripheral blood lymphocytes from a male donor when evaluated up to the limit of cytotoxicity in the approximate 24 hour treatment without S9 and 3 hour treatments with and without S9.

Executive summary:

An in vitro micronucleus test was performed according to OECD Guideline 487 and in compliance with GLP.

The objective of the study was to evaluate the clastogenic and aneugenic potential of the test article by its effects on the frequency of micronuclei in cultured human peripheral blood lymphocytes treated with and without rat liver metabolizing system (S9). The test article was formulated in dimethyl formamide (DMF). Initially a dose range-finding (DRF) assay was performed in single cultures to select doses for the micronucleus (MN) assay where duplicate cultures were used. Different concentrations of the test article were used with a dosing volume of 1% (v/v). Vehicle and positive controls were used concurrently. Due to the limited solubility of the test article in the culture media, top dose used for in the test system was 1000 μg/mL.

In the DRF assay, human peripheral blood lymphocytes (HPBL) were treated with the test item at concentrations ranging from 19.8 to 1000 μg/mL for 3 hours with and without S9 and approximately 24 hours without S9. Precipitate was observed at the end of treatment (or at wash) at ≥700 μg/mL in the 3 hour treatments with and without S9, and at ≥490 μg/mL in the approximate 24 hour treatment without S9. Slides were scored to calculate the cytokinesis block proliferation index (CBPI) for the measurement of cytotoxicity.

Based on the DRF results, Sclareolide was evaluated in the micronucleus assay (MN) in the human peripheral blood lymphocytes (HPBL) from a male donor at concentrations ranging from 35.0 to 143 μg/mL, in the approximate 24 hour treatment without S9, and from 84.4 to 243 μg/mL in the 3 hour treatments without S9. The test article was also evaluated at concentrations ranging from 104 to 300 μg/mL in the 3 hour treatment with S9. No precipitate was observed at the end of the treatment at any tested concentration in any treatment condition with or without S9. In the approximate 24 hour treatment without S9, 51% cytotoxicity was observed at 93.8 μg/mL. This concentration along with two lower concentrations, 35.0 and 68.4 μg/mL, producing 11 and 35% cytotoxicity, respectively, were selected for MN evaluation. In the 3 hour treatment without S9, 58% cytotoxicity was observed at 177 μg/mL. This concentration along with two lower concentrations, 129 and 143 μg/mL, producing 9 and 24% cytotoxicity, respectively, were selected for MN scoring. In the 3 hour treatment with S9, 61% cytotoxicity was observed at 243 μg/mL. This concentration along with two lower concentrations, 177

and 219 μg/mL, producing 19 and 39% cytotoxicity, respectively, were selected for MN evaluation.

A statistically significant increase in the frequency of micronucleated binucleated (MNBN) cells was observed at 35.0 μg/mL in the approximate 24 hour treatment without S9. However, the MNBN frequency (0.60%) observed at this concentration was within the vehicle historical control range (0.10 to 1.10%) for this test condition in the male donors. Therefore, the statistically significant increase was considered as biologically non-relevant. No statistically significant increase in the MNBN frequencies was observed in the 3 hour treatments with or without S9. The MNBN frequencies of the

vehicle and the positive controls fell within the acceptable range. All acceptance criteria were met and the study was accepted as valid.

 

All vehicle (acetone) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that induced approximately 50% reduction in CBPI.

The test article was considered negative for inducing micronuclei in the binucleated cells of human peripheral blood lymphocytes from a male donor when evaluated up to the limit of cytotoxicity in the approximate 24 hour treatment without S9 and 3 hour treatments with and without S9.

This study is considered as acceptable and satisfies the requirement for in vitro micronucleus study.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 15, 2016 to january 27, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 471 and in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene for Salmonella and tryptophan gene for E.coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

Initial: 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 µg/plate.
Confirmatory: 5.00, 16.0, 50.0, 160, 500, and 1600 µg/plate in TA98, TA100, TA1535, TA1537; 16.0, 50.0, 160, 500, 1600, and 5000 µg/plate in WP2uvrA.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl formamide (DMF)
- Justification for choice of solvent/vehicle: In a solubility assessment with DMSO, the test article formed a white, opaque, non-viscous suspension at 200 mg/mL. The formulation was prepared by vortex mixing and sonication at 37°C.
Solubility was further evaluated in DMF. The test article formed non-viscous, transparent, clear, colorless solutions at 6.25, 12.5, 25, 50, 100, and 200 mg/mL. All formulations were prepared by vortex mixing. Upon addition to RPMI 1640 culture media, precipitate was observed at final concentrations of 0.125, 0.25, 0.50, 1.00, and 2.00 mg/mL. The formulation was subsequently diluted to a final concentration of 0.0625 mg/mL, where no precipitate was observed. After 24 hours of incubation at 37±2°C, precipitation was observed at concentrations ≥0.125 mg/mL. DMF was selected as the vehicle for this study.

Untreated negative controls:

yes

Remarks:

Without S9-mix

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: IC-191 [2 µg/plate / TA1537 (Lot No. SLBM9359V)

Untreated negative controls:

yes

Remarks:

With S9-mix

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 52 ± 4 hours at 37 ± 2°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

- OTHER: ACCEPTANCE CRITERIA
The assay was to be considered valid if the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges
2. The positive control chemicals induced increases in revertant numbers of ≥3-fold (in strains TA98, TA100, TA1535, TA1537, or WP2uvrA,) the concurrent vehicle control confirming discrimination between different strains, and an active S9 preparation.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥2.0-fold (in strains TA98, TA100 or WP2uvrA) or ≥3.0-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.
The test article was considered positive in this assay if the above criterion was met.
The test article was considered negative in this assay if the above criterion was not met.
Biological relevance was taken into account on a case-by-case basis, for example consistency of response within and between concentrations and between experiments, where applicable. Other criteria were used in reaching a conclusion about the study results (e.g., comparison to historical control values, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

Statistics:

None

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination:
Initial: In the presence of S9, precipitate was observed at 5000 μg/plate in all tester strains except WP2uvrA, where precipitate was observed at ≥1600 μg/plate. In the absence of S9, precipitate was observed at ≥1600 μg/plate in all tester strains except WP2uvrA, where precipitate was observed at 5000 μg/plate.
Repeat: In the repeat experiment, Precipitate was observed at ≥1600 μg/plate in all tester strains in the absence of S9. In the presence of S9, precipitate was observed at 5000 μg/plate in WP2uvrA.


STUDY RESULTS
- Concurrent vehicle negative and positive control data: The individual mutagenicity plate counts were averaged to give mean values. From the data it can be seen that the mean vehicle control counts fell within the laboratory’s historical ranges. All the positive control chemicals induced increases in revertant numbers of ≥3-fold in all tester strains when compared with the concurrent vehicle control. The study therefore demonstrated assay functionality and was accepted as valid.
- Signs of toxicity:
In the initial experiment, toxicity, as evident by the reduction in the bacterial background lawn and/or reduction in the mean number of revertant colonies, was observed in all Salmonella tester strains at ≥500 μg/plate in the absence of S9, except TA1535, where toxicity was observed at ≥160 μg/plate. In the presence of S9, toxicity was observed at ≥1600 μg/plate in all tester strains.
In the repeat experiment, toxicity, as evident by the reduction in the bacterial background lawn and/or reduction in the mean number of revertant colonies, was observed in all Salmonella tester strains at 1600 μg/plate in the presence of S9. In the absence of S9, toxicity was observed at ≥500 μg/plate in all Salmonella tester strains and at ≥1600 μg/plate in WP2uvrA.
- Individual plate counts: cf attachment
- Mean number of revertant colonies per plate and standard deviation: cf. attachment

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: cf attachment
- Negative (solvent/vehicle) historical control data: cf attachment

Conclusions:

The test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA- were exposed the test material diluted in DMF both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method.

The test material was evaluated in the initial mutagenicity assay in all five tester strains at concentrations of 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate in the presence and absence of S9. Precipitate was observed at 5000 μg/plate in all tester strains in the presence of S9, except WP2uvrA where precipitate was observed at ≥1600 μg/plate. In the absence of S9, precipitate was observed at ≥1600 μg/plate in all tester strains, except WP2uvrA, where precipitate was observed at 5000 μg/plate. Toxicity was observed at ≥1600 μg/plate in all tester strains in the presence of S9 and at ≥500 μg/plate in all Salmonella tester strains in the absence of S9, except TA1535, where toxicity was observed at ≥160 μg/plate.

Based on the results of the initial mutagenicity assay, an independent confirmatory mutagenicity assay was conducted in tester strains TA98, TA100, TA1535, and TA1537 at concentrations of 5.00, 16.0, 50.0, 160, 500, and 1600 μg/plate, and in strain WP2uvrA at concentrations of 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate in the presence and absence of S9. Precipitate was observed at 5000 μg/plate in WP2uvrA in the presence of S9. In the absence of S9, precipitate was observed at ≥1600 μg/plate in all tester strains. Toxicity was observed in all Salmonella tester strains at 1600 μg/plate in the presence of S9. In the absence of S9, toxicity was observed at ≥500 μg/plate in all Salmonella strains and at ≥1600 μg/plate in WP2uvrA.

No increase in the mean number of revertant colonies was observed at any tested concentration in any tester strain in the presence or absence of S9. All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

 

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1  (Covance, 2017)	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 1538, TA 98, TA 100 TA 102	-S9 +S9	Up to limit or cytotoxic concentration	-S9 : weak mutagenic +S9 : weak mutagenic
2 (Covance, 2017)	MNT vitro (OECD 487) K, rel.1	Chromosomal aberration	Human lymphocytes	-S9 +S9	Up to cytotoxic concentration	-S9 and +S9 Not clastogenic -S9 and +S9 Not aneugenic

Gene mutation Assay (Test n° 1):

- A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

 

Chromosomal aberration (Test n°2)

The clastogenic potential of the substance was determined using an in vitro micronucleus test in human lymphocytes (OECD 487), which measures the potential of a substance to increase the incidence of micronuclei in cultured human lymphocytes. The test item was cytotoxic to human lymphocytes but it did not induce any statistically significant increases in the frequency of cells with micronuclei, in any of the exposure groups, using a dose range that included a dose level that induced a sufficient reduction in the cytokinesis block proliferation index (CBPI). Both positive and negative controls validated the sensitivity of the assay. Therefore, the substance is considered as non-clastogenic and non-aneugenic under the conditions used in this assay.

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification for human health according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification:

Based on the available data, no self-classification is proposed regarding germ cell mutagenicity according to the CLP and to the GHS.