2-(3,3-dimethylcyclohex-1-enyl)-2,5,5-trimethyl-1,3-dioxane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to the maximum concentrations in S. typhimurium TA97a, TA98, TA100, TA102 and TA1535.

- Chromosome aberration test (OECD 473, GLP, 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:

From 28 July to 13 August 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

08 April 2015.

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

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 GieBen (Batch nos. 3424, 3366, 3393)
- method of preparation of S9 mix : produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
- concentration of S9 in the final culture medium: S9-fraction 10% v/v

Test concentrations with justification for top dose:

Mutagenicity test:
Main test: 50, 150, 500, 1500 and 5000 μg/plate in TA97a, TA98, TA100, TA102 and TA1535 with and without S9 under the direct plate incorporation method.
Confirmation test: 156, 313, 625, 1250, 2500 and 5000 μg/plate in TA97a, TA98, TA100, TA102 and TA1535 with and without S9 under the pre-incubation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: the solubility of the test item was determined in demineralised water, dimethyl sulfoxide and ethanol. The test item is only soluble in ethanol in a concentration of 50 g/L. Therefore, a test item solution containing 50 g/L in ethanol was prepared for both experiments.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-Nitro-1,2-phenylene Diamine

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-amino-anthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min at 37 +/-1°C
- Exposure duration: 37 +/- 1°C for 48 h

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: A reduction in the number of colonies in a dose-dependent manner compared to negative control for any strain and condition might indicate cytotoxicity.

OTHER:
- After an incubation of about 48 hours at about 37 ºC, the number of colonies per plate was counted.
Data are presented as the number of colonies present per plate (mean ± standard deviation). The R ratio is calculated as follows:
R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item
- Sterility test: The sterility of the test item was assayed by adding of 5 mg/plate to a minimal agar plate and incubating at 37°C for 48h. No growth was observed in the minimal agar plate after incubation with the test item.
- Solubility test: Solubility was assessed as precipitation in the final mixture under the actual test conditions. Observation of precipitation by naked eye indicates insolubility.

Evaluation criteria:

Several criteria are used for determining a positive result: a dose-response 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.
Positive results from the bacterial reverse mutation test indicate that a test item induces point mutations or frame-shifts in the genome of the tested bacterial strains.
Negative results from the test indicate that under the test conditions, the test item neither mutagenic nor-pro-mutagenic in the tested experimental system.

Statistics:

None

Key result

Species / strain:

bacteria, other: S. typhimurium TA97a, TA98, TA100, TA102 and TA1535.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 5000 µg/plate in TA98, TA100 and TA1535 (second experiment).

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: None
- Precipitation: None
- Other confounding effects: None

CYTOTOXICITY TEST:
A decrease in the number of revertant colonies >50% compared to solvent reference was observed, indicating cytotoxicity of the test item. The lowest cytotoxic concentration was 20.6 µg/plate and it was used as the highest formulation for Ames test.

MUTAGENICITY TEST:
- No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation.
- No dose response 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.

OTHERS:
- Sterility test showed no contamination during the study.

None

Conclusions:

Under the test condition, the test mateiral is not mutagenic in presence and absence of metabolic activation in S. typhimurium strains S. typhimurium TA97a, TA98, TA100, TA102 and TA1535.

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 (TA97a, TA98, TA100, TA102 and TA1535) were exposed to the test item diluted in ethanol at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).

 

Main test: 50, 150, 500, 1500 and 5000 μg/plate in TA97a, TA98, TA100, TA102 and TA153 with and without S9 under the direct plate incorporation method

Confirmation test: 156, 313, 625, 1250, 2500 and 5000 μg/plate in TA97a, TA98, TA100, TA102 and TA1535 with and without S9 under the pre-incubation method.

 

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

 

Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data. All positive controls showed valid ratios (R) above 2.5.

 

Cytotoxic effect was observed at 5000 µg/plate. No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation. No dose response was observed in any of the tested bacterial strains.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA97a, TA98, TA100, TA102 and TA1535.

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

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 21 March to 17 June 2019.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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:

- Molecular Weight: 238.4
- Storage condition of test material: Room temperature in the dark.

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: female, aged 28 years (preliminary toxicity test). Female, aged 28 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): 0, 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.

- Main Experiment:
0, 4, 8, 16, 24, 32, 40, 48 and 64 µg/mL, 4(20)-hour without S9-mix
0, 8, 16, 32, 48, 64 and 80 µg/mL with (2%) S9-mix
0, 4, 8, 16, 24, 32, 40, 48 and 64 µg/mL 24-hour without S9-mix.
Justification: The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level for the 4(20)-hour exposure groups and toxicity for the 24-hour exposure group (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 DMSO at 200 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 achievable 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:

TEST SYSTEM: for each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
- The details of the donors used are:
* Preliminary Toxicity Test: female, aged 28 years
* Main Experiment: female, aged 28 years
CELL CULTURE: 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 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

DURATION
- Exposure duration: 4 hours (± S9) and 24 hours (-S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours (± S9)

SPINDLE INHIBITOR (cytogenetic assays): Mitotic activity was arrested by addition of demecolcine (Colcemid 0.1 μg/mL), two hours before the harvest time.

STAIN (for cytogenetic assays): 5 % Giemsa

NUMBER OF REPLICATIONS:
Preliminary toxicity test: quadruplicate cultures for the vehicle control/dose
Main experiments: Duplicate cultures/dose

NUMBER OF CELLS EVALUATED:
- 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.
- 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.
In addition, 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.


DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: If greater than 44 chromosomes are scored and the number is a multiple of the haploid count then the cell is classified as a polyploid cell.
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated (E).

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:

no cytotoxicity, but tested up to precipitating concentrations

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
- Effects of pH: No significant change in pH when the test item was added into media.
- Effects of osmolality: Osmolality did not increase by more than 50 mOsm.
- Precipitation: Yes

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 at and above 250 μg/mL in the continuous exposure group.
- Hemolysis was observed following exposure to the test item at and above 31.25 μg/mL in the 4(20)-hour exposure group in the absence of S9, at and above 15.63 μg/mL in the 4(20)-hour exposure group in the presence of S9 and at and above 62.5 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 and 62.5 μg/mL in the 4(20)-hour exposures in the absence and presence of metabolic activation (S9), respectively. The maximum dose with metaphases present in the 24-hour continuous exposure was 62.5 μg/mL. The results of the mitotic index of the Cell Growth Inhibition. The test item demonstrated marked toxicity in all three exposure groups.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level for the 4(20)-hour exposure groups and toxicity for the 24-hour exposure group.

MAIN STUDY RESULTS
- The qualitative assessment of the slides determined that there was a shift in toxicity in the 4(20)-hour exposure group in the absence of S9 and there were metaphases present at 64 μg/mL in this exposure group. The toxicity observed in the 4(20)-hour exposure in the presence of S9 and in the 24-hour exposure group was similar to that observed in the Preliminary Toxicity Test and there were metaphases suitable for scoring at the maximum dose level tested in both these exposure groups.
- Precipitate observations were made at the end of exposure in blood-free cultures and precipitate was noted at and above 64 μg/mL in the 4(20)-hour exposure groups in the presence and absence of S9. This level of precipitate was similar to that seen in the Preliminary Toxicity Test in these exposure groups. In the 24-hour exposure group precipitate was observed at and above 48 μg/mL at the end of exposure. This was a lower dose level than that where precipitate was observed in the Preliminary Toxicity Test and the reason for this is unclear but may be due to the subjective nature of performing precipitate observations and the test item being formulated at a lower concentration for the Main Experiment. Hemolysis was observed at and above 16 μg/mL in the 4(20)-hour exposures in the absence and presence of S9.
- The results of the mitotic indices (MI) from the cultures after their respective exposures are presented as cell growth indices in Form 1, Form 2, and Form 3 of Appendix 2. They confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in either of the 4(20)-hour exposure groups up to the lowest precipitating dose level, 64 μg/mL. In the 24-hour exposure group a dose-related inhibition of mitotic index was observed and 62% mitotic inhibition was demonstrated at 48 μg/mL which was also the lowest precipitating dose level.
The maximum dose level selected for metaphase analysis was the lowest precipitating dose level (64 μg/mL) for the 4(20)-hour exposure groups and for the 24-hour exposure group it was 48 μg/mL where near optimum toxicity was achieved and it was also the lowest precipitating dose level.
- 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 and 64 μg/mL;

4(20)-hour with S9 (2%): 0, 8, 16, 32, 48, 64 and 80 μg/mL;

24-hour without S9-mix: 0,4, 8, 16, 24, 32, 40, 48 and 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 the lowest precipitating dose level. The 24-hour exposure group also demonstrated near optimum toxicity (62% mitotic inhibition) at the lowest precipitating dose level.

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
LAUS, 2015	Ames Test (OECD 471) K, rel. 1)	Gene mutation	TA 1535, TA97a, TA 98, TA 100 and TA 102	-S9 +S9	Up to limit concentration (in DMSO)	-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) :

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 conditions, 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°3)

The clastogenic potential of the substance was determined using anin vitrochromosome 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.