Reaction mass of stereoisomers of 4-METHYLENE-2-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-TETRAHYDRO-PYRAN and; 4-METHYL-6-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-3,6-DIHYDRO-2H-PYRAN and; 4-METHYL-2-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-3,6-DIHYDRO-2H-PYRAN

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- 1 Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to the maximum concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

- Chromosome aberration test (OECD 473, K, rel. 1): non clastogenic up to cytotoxic concentrations.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-25 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 471 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Directive 2000/32/EC

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2009-07-13 and 2009-07-22 / Signed on 2009-08-3 / Valid until 2011-08-003

Type of assay:

bacterial reverse mutation assay

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

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH
- method of preparation of S9 mix : not detailed
- concentration of S9 in the final culture medium: S9-fraction 10% v/v
- quality controls of S9: enzymatic activity, sterility, metabolic capability (certificate included in the study report)

Test concentrations with justification for top dose:

Cytotoxicity test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 100 without S9 under the direct plate incorporation method.

Mutagenicity test:
Main test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.
Confirmation test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2 (pKM101) with and without S9 under the pre-incubation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol 96%
- Appearance in solvent: Colourless liquid solution
- Basis for dose calculation: The test item was used as provided
- Formulation conditions: Room temperature
- Formulation frequency: Daily
- Formulation preparation: The highest exposure concentration was 5 μL/plate. Further lower concentrations were prepared by 1:3 serial dilutions in the selected solvent from the highest concentration.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol (95%)

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol (95%)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

SOURCE OF TESTER STRAINS: Strains of salmonella typhimurium and E. Coli were obtained from Moltox. Bacterial strains used for the study were grown from controlled Working Banks obtained from Master Banks (generated in Vivotecnia).

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 37 °C for 48 h

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity evaluation of the test item was based on the decrease in the number of revertant colonies, or a clearing or diminution of the background lawn.

OTHER:
- After an incubation of about 48 hours at about 37 °C, the number of colonies per plate was counted.
- The number of revertant colonies per plate was counted and recorded by an automatic colony counter. Average plate counts was presented with the mean and the standard deviation for each set of triplicates per test item concentration and was used to calculate the ratio of colonies per exposed plate compared to the corresponding negative control.
- Test item solubility: The solubility of the test item was evaluated in a standard solvent panel (milliQ water, ethanol 96%, DMSO and corn oil). Observation of precipitation by the unaided eye indicated that the test item is not soluble. The test item was found to be soluble when diluted 1/3 in ethanol (96%).
- Test item sterility assay: The sterility of the test item was assayed by adding of 5 μL/plate to a minimal agar plate and incubating at 37 °C for 48 h. No growth was observed in the minimal agar plate after incubation with the test item.

Evaluation criteria:

The criteria used for determining a positive result take into account a dose-response effect in the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
A result is considered positive whenever the number of revertants of the test item treated plates is increased when compared to the solvent treated plates according to the following criteria: 2 fold for S.typhimurium TA98, TA 100 and E.coli WP2(pKM101); 3 fold for S.typhimurium TA1535 and TA1537.
Biological relevance of the results was also considered.

Statistics:

None

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test item was found to be soluble when diluted 1/3 in ethanol (96%).
- Precipitation: None
- Other confounding effects: None

CYTOTOXICITY TEST:
No cytotoxic activity was observed by the test item in the bacterial system at a concentration of 5 μL/plate.

MUTAGENICITY TEST:
- None of the concentrations assayed for the test item showed an increase in the R value either with or without S9 metabolic activation regardless of the procedure.
- No dose response for the test item was observed in any of the tested bacterial strains.

HISTORICAL CONTROL DATA
- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data of the test facility.

Ames test acceptance criteria:

The bacterial reverse mutation test for the test item was considered valid as the following criteria were met:

The mean solvent control counts complied with Vivotecnia historical data for each strain.

The mean reference item control counts complied with Vivotecnia historical data for each strain.

Conclusions:

Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2(pKM101) were exposed to the test substance diluted in ethanol (95%) at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).

Cytotoxicity test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 100 without S9 under the direct plate incorporation method. 

Mutagenicity test:

Main test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.

Confirmation test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2 (pKM101) with and without S9 under the pre-incubation method.

Vehicle and positive control groups were also included in mutagenicity tests.

 

Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data.

 

No cytotoxic effect was observed. None of the concentrations assayed for the test item showed an increase in the R value either with or without S9 metabolic activation regardless of the procedure. No dose response for the test item was observed in any of the tested bacterial strains.

 

Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 05 March to 28 May 2019.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD TG 473 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE).

Version / remarks:

Guidelines of 31 March 2011.

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (Inspected on 2018-08-21 / Signed on 2018-11-18).

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Physical state: Pale yellow liquid
- Storage condition of test material: Room temperature in the dark until 19 February 2019 and approximately 4°C, in the dark thereafter.

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For lymphocytes:
- Sex, age and number of blood donors: male, aged 29 years (preliminary toxicity test). Male, aged 21 years (Main experiment).
- Whether whole blood or separated lymphocytes were used: for each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability.
- Whether blood from different donors were pooled or not: no, one donor for each experiment.
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA).

MEDIA USED :
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % fetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air.

Additional strain / cell type characteristics:

other: Not applicable.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Covance laboratory (Lot No. PB/βNF S9 31/08/18), stored at approximately -196 °C.
- method of preparation of S9 mix: S9 fraction was obtained from the liver homogenates of male rats treated with Phenobarbitone/Beta-naphthoflavone.
- concentration or volume of S9 mix and S9 in the final culture medium : the final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test concentrations with justification for top dose:

- Preliminary Toxicity Test (Cell Growth Inhibition): 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL, 4h exposure time with and without metabolic activation followed by a 20h recovery period (4(20)-hour with (2%) and without S9-mix), and a continuous exposure of 24h without metabolic activation (24-hour without S9-mix).
Justification: The maximum dose was the maximum achievable dose level using acetone as the solvent.

- Main Experiment: 4, 8, 16, 32, 40, 48, 56 and 64 µg/mL, 4(20)-hour with (2%) and without S9-mix and 24-hour without S9-mix.
Justification: The maximum dose level selected for the main experiment was the lowest precipitating dose level (64 μg/mL) for all three exposure groups with toxicity being taken into account in the selection of dose levels (preliminary toxicity test).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test item was insoluble in Minimal Essential Medium at 20 mg/mL and dimethyl sulphoxide at 200 mg/mL and 100 mg/mL but was soluble in acetone at 200 mg/mL in solubility checks performed in-house.
- Formulation preparation: Due to the sensitivity of human lymphocytes to acetone, the formulations were dosed at 0.5% in 50 μl aliquots. Consequently, the maximum practical concentration was 1000 μg/mL.The test item was accurately weighed, formulated in acetone and serial dilutions prepared. The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation (2% S9-mix).

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: quadruplicate cultures for the control; duplicate culture per dose levels
- Number of independent experiments: 2

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Exp. 1: 4 hours (± S9) / Exp. 2: 24 hours (-S9)
- Harvest time after the end of treatment (sampling/recovery times): 24 hours

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): Mitotic activity was arrested by addition of demecolcine (Colcemid 0.1 μg/mL), two hours before the harvest time.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.
When the slides were dry, they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Where possible 1000 cells per culture were evaluated for the incidence of metaphase cells and expressed as the mitotic index and as a percentage of the vehicle control value.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Where possible, 300 consecutive well-spread metaphases from each concentration were counted, 600 from the vehicle control (150 per replicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides
- Determination of polyploidy / endoreplication: cells with 69 chromosomes or more were scored as polyploid cells (including endoreduplicated cells) and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)

Rationale for test conditions:

Human peripheral blood lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration Test.

Evaluation criteria:

The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• Concurrent positive control chemicals should induce responses that are compatible with those generated in historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data of pH: No significant change in pH when the test item was added into media.
- Data of osmolality: Osmolality did not increase by more than 50 mOsm.
- Possibility of evaporation from medium: not expected (vapour pressure = 0.313 Pa)
- Water solubility: Insoluble at 20 mg/mL.

PRELIMINARY TOXICITY TEST (CELL GROWTH INHIBITION TEST):
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 62.5 μg/mL, in the 4(20)-hour exposure groups and in the 24-hour continuous exposure group.
- Hemolysis was observed following exposure to the test item at and above 3.91 μg/mL in the 4(20)-hour exposure groups and at and above 15.63 μg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
- Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 31.25 μg/mL in the 4(20)-hour exposure group in the absence of S9 and in the 24-hour exposure group. In the 4(20)-hour exposure group in the presence of S9 there were metaphases present up to the maximum dose level tested (1000 μg/mL) but the numbers of metaphases present were much reduced above 62.5 μg/mL. The lowest precipitating dose level in the presence of S9 was 62.5 μg/mL and therefore this was the maximum dose level selected for mitotic index evaluation in this exposure group. The test item demonstrated a sharp toxicity curve in all three exposure groups.
The maximum dose level selected for the main experiment was the lowest precipitating dose level (64 μg/mL) for all three exposure groups with toxicity being taken into account in the selection of dose levels.

MAIN STUDY RESULTS
- Concurrent vehicle negative and positive control data: all of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.
All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
- Results from cytotoxicity measurements:
- The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Cell Growth Inhibition Test and that there were metaphases suitable for scoring present up to 56 μg/mL in the 4(20)-hour exposure in the absence of S9 and up to 64 μg/mL in the presence of S9 and in the 24-hour exposure group.
- Precipitate observations were made at the end of exposure in blood-free cultures and precipitate was noted at and above 56 μg/mL in all three exposure groups.
- Hemolysis was observed at and above 16 μg/mL in the 4(20)-hour exposure groups and at and above 8 μg/mL in the 24-hour exposure group.
- In the 4(20)-hour exposure group in the absence of S9, 44% mitotic inhibition was achieved at 56 μg/mL, the lowest precipitating dose level. In the 24 hour exposure group 45% mitotic inhibition was achieved at 40μg/mL The dose levels of 48 μg/mL and 56μg/mL achieved 66% and 82% mitotic inhibition, respectively and were considered too toxic for metaphases analysis. In the presence of S9, no dose-related inhibition of mitotic index was observed up to the lowest precipitating dose level of 56 μg/mL.
- The maximum dose level selected for metaphase analysis for the 4(20)-hour exposure groups in the absence and presence of S9 was 56 μg/mL, the lowest precipitating dose level and for the 24-hour exposure group was 40 μg/mL where the toxicity was close to optimum.
- Genotoxicity results:
- The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
- The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups. There was no indication of endoreduplication noted.

HISTORICAL CONTROL DATA (mean ± standard deviation)
- Positive historical control data:
cells with aberrations (-gaps):
4(20)-hour exposure without S9%: 25.13 ± 13.14
4(20)-hour exposure with S9 (2%): 16.22 ± 7.00
24-hour exposure without S9: 26.81 ± 12.28
% cells with polyploids:
4(20)-hour exposure without S9%: 0.01 ± 0.06
4(20)-hour exposure with S9 (2%): 0.03 ± 0.12
24-hour exposure without S9: 0.02 ± 0.11

- Negative (solvent/vehicle) historical control data:
cells with aberrations (-gaps):
4(20)-hour exposure without S9%: 0.48 ± 0.40
4(20)-hour exposure with S9 (2%): 0.54 ± 0.53
24-hour exposure without S9: 0.36 ± 0.43

% cells with polyploids:
4(20)-hour exposure without S9%: 0.04 ± 0.13
4(20)-hour exposure with S9 (2%): 0.03 ± 0.10
24-hour exposure without S9: 0.02 ± 0.07

None.

Conclusions:

Under the test conditions, test item did not induce any statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured human lymphocytes were exposed to test item at the following concentrations:

 

Preliminary Toxicity Test (Cell Growth Inhibition Test)

0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL; 4 h exposure time with and without metabolic activation followed by a 20 h recovery period (4(20)-hour with and without S9-mix), and a continuous exposure of 24 h without metabolic activation (24-hour without S9-mix)

 

Main experiment

4(20)-hour without S9-mix: 0, 4, 8, 16, 32, 40, 48, 56, 64 μg/mL;

4(20)-hour with S9 (2%): 0,4, 8, 16, 32, 40, 48, 56, 64 μg/mL;

24-hour without S9-mix: 0,4, 8, 16, 32, 40, 48, 56, 64 μg/mL;

 

Mitotic activity was arrested by addition of colcemid at 0.1 μg/mL for each culture, two hours before the harvest. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Vehicle and positive controls were also included in this test.

 

All vehicle (Acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9- mix were validated.

 

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was either the lowest precipitating dose level as in the 4(20)-hour exposure groups in the absence and presence of S9 or induced near optimum toxicity with 45% mitotic inhibition as in the 24-hour exposure group.

Under the test conditions, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

This study is considered as acceptable and satisfies the requirement for chromosome aberration 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 VIVOTECNIA, 2013a	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, E. coli WP2	-S9 +S9	Up to limit concentration (in ethanol)	-S9 : non mutagenic +S9 : non mutagenic
2 COVANCE, 2019	CAT (OECD 473) K, rel. 1	Chromosomal aberration	Human Lymphocytes	-S9 +S9	Up to cytotoxicity	-S9 : non clastogenic +S9 : non clastogenic

Gene mutation Assay (Test n° 1):

One 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 tests 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 chromosome aberration test in human lymphocytes (OECD 473), which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured human lymphocytes.

None of the dose levels up to the cytotoxicity limit with the substance, either in the presence or absence of metabolic activation, induced significant increases in the frequency of cells with aberrations in either of three experiments. The substance does not induce structural aberrations in the chromosomes of human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells.The substance is therefore considered as negative for inducing chromosomal mutations in human lymphocyte cells under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonized classification:

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

 

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the CLP and to the GHS.