1-(5-propyl-1,3-benzodioxol-2-yl)-1-ethanone

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 cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

- Human lymphocytes 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:

29 November 2013 to 28 January 2014

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 10 July 2012 / signed on 30 November 2012)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Purity test date: 30 October 2013
- Storage Conditions: ca. 4 °C in the dark under nitrogen

Target gene:

Histidine and tryptophan for S. typhimurium and E. coli, respectively.

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:

10% S9: S9-mix from the livers of male rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/ kg bw/day by oral route).

Test concentrations with justification for top dose:

Experiment 1 – Range-finding test (Pre-incubation method): 0.015, 0.05, 0.15, 0.5, 1.5, 5 and 15 μg/plate in TA 100 and TA 1535 without and with S9-mix; 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 98, TA 1537 and WP2uvrA strains without and with S9-mix
Justification: Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 - Main Test (Pre-Incubation Method):
Salmonella strain TA98 (with S9-mix) and E.coli strain WP2uvrA (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate.
Salmonella strains TA98 (without S9-mix) and TA1537 (with and without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate.
Salmonella strains TA100 and TA1535 (with and without S9-mix): 0.015, 0.05, 0.15, 0.5, 1.5, 5 and15 μg/plate.
Justification: Up to eight test item dose levels were selected in Experiment 2 (main test) in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
- Preparation of test substance formulation: The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (1.1%) of the test item. All formulations were used within four hours of preparation and were assumed to be stable for this period. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 μm.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

TEST SYSTEM: The bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, and the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 37 ± 3 °C for 20 minutes (with shaking)
- Exposure duration: Plates were incubated at 37 ± 3 °C for approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: Plates were assessed microscopically for evidence of thinning (toxicity).

OTHERS:
After incubation, the plates were scored for the presence of revertant colonies using an automated colony counting system.
*concurrent negative controls were dosed using the standard plate incorporation method

Evaluation criteria:

There are several criteria for determining a positive result. Any one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98 and 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:

E. coli WP2 uvr A

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test item was immiscible in sterile distilled water at 50 mg/mL.
- Precipitation: None

RANGE-FINDING/SCREENING STUDIES:
- Experiment 1 (Range finding test): The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in the absence of S9-mix, initially from 5 μg/plate (TA100 and TA1535), 150 μg/plate (TA98 and TA1537) and 500 μg/plate (WP2uvrA). In the presence of S9-mix, weakened lawns were noted from 5 μg/plate (TA100 and TA1535), 150 μg/plate (TA1537) and 500 μg/plate (TA98 and WP2uvrA). Consequently, the toxic limit was employed in the second mutation test. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

HISTORICAL CONTROL DATA
- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2011 and 2012 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 2 (main test): Main test results once again showed a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the absence and presence of S9-mix, initially from 5 μg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

MUTAGENICITY:
- There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in both experiments. A small, statistically significant increase in TA98 revertant colony frequency was observed in the presence of S9-mix at 50 μg/plate in the main test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 50 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control.

OTHERS:
- 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 the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

None

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 WP2 uvrA-.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test substance using the Ames pre-incubation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for the range-finding test was predetermined and was 0.015 to 5000 μg/plate, depending on bacterial strain type and presence or absence of metabolic activation (S9-mix). The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of the range-finding test and ranged between 0.015 and 1500 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. Negative, vehicle (DMSO) and positive control groups were also included in mutagenicity tests.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in the absence of S9-mix, initially from 5 μg/plate (TA100 and TA1535), 150 μg/plate (TA98 and TA1537) and 500 μg/plate (WP2uvrA). In the presence of S9-mix, weakened lawns were noted from 5 μg/plate (TA100 and TA1535), 150 μg/plate (TA1537) and 500 μg/plate (TA98 and WP2uvrA). Consequently, the toxic limit was employed in the second mutation test. Main test results once again showed a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the absence and presence of S9-mix, initially from 5 μg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in both experiments. A small, statistically significant increase in TA98 revertant colony frequency was observed in the presence of S9-mix at 50 μg/plate in the main test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 50 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control.

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 WP2 uvrA- according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

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:

14 January to 10 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 473 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997 version

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Qualifier:

according to guideline

Guideline:

other: 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test (2011).

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 12-14 March 2014/ signed on 12 May 2014)

Type of assay:

other: in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Purity test date: 30 October 2013
- Storage Conditions: ca. 4 °C in the dark under nitrogen

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Remarks:

primary culture

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

2 % S9 (final concentration); S9 fraction was obtained from the liver homogenates of male rats treated with phenobarbitone and β-naphthoflavone

Test concentrations with justification for top dose:

Preliminary Toxicity Test (Cell Growth Inhibition Test):
0, 8.0, 16.10, 32.19, 64.38, 128.75, 257.5, 515, 1030 and 2060 μg/mL; 4 h exposure time with and without metabolic activation followed by a 20 h expression period, and a continuous exposure of 24 h without metabolic activation
Experiment 1: 4 h treatment followed by 20 h expression period
without S9: 0, 30, 60, 120, 160, 200 and 240 μg/mL
with S9 (2%): 0, 30, 60, 120, 160, 200, and 240 μg/mL
Experiment 2: 4 h treatment followed by 20 h expression period
Without S9: 0, 30, 60, 120, 160, 200 and 240 μg/mL
with S9 (2%): 0, 30, 60, 120, 160, 200 and 240 μg/mL
Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

Vehicle / solvent:

Vehicle
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was miscible in both dimethyl sulphoxide and acetone at 206 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was chosen as the preferred solvent vehicle.
- Formulation preparation: Prior to the start of each experiment, the test item was accurately weighed, formulated in dimethyl sulphoxide 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.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with 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 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. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

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 °C 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) in Experiment 1 and 2; 4 hours (± S9) and 4 hours (-S9) in Preliminary toxicity test
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours (± S9) in Preliminary toxicity test, Experiment 1 and 2

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: Single culture/dose
Main experiments: Duplicate cultures/dose

NUMBER OF CELLS EVALUATED:
- A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
- Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were at least 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. 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). In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Endoreduplicated cells were recorded separately and are included in the polyploid cell total number.

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:

Negative control: The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range.
Positive control: All the positive control chemicals must induce positive responses (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.
Cytotoxicity: There must be at least three analyzable dose levels present for each experiment.

A test item can be classified as non-genotoxic if:
1. The number of induced chromosome aberrations in all evaluated dose groups is within the range of historical control data.
2. No toxicologically or statistically significant increase of the number of structural chromosome aberrat ions is observed following statistical analysis.
A test item can be classified as genotoxic if:
1. The number of induced structural chromosome aberrations is not in the range of historical control data.
2. Either a concentration-related or a statistically significant increase of the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis.
Biological relevance of the results will be considered first. Statistical methods will be used to analyze the increases in aberration data as recommended in the OECD 473 guidelines. However, statistical significance will not be the only determining factor for a positive response.
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.

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.

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

Positive controls validity:

valid

Additional information on results:

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

PRELIMINARY TOXICITY TEST:
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at 2060 μg/mL in the 4(20)-hour exposure group in the absence of metabolic activation (S9-mix), at and above 515 μg/mL in the 4(20)-hour exposure group in the presence of S9-mix and at and above 1030 μg/mL in the continuous exposure group.
- Haemolysis was observed following exposure to the test item at and above 257.5 μg/mL in the 4(20)-hour exposure groups (with and without S9-mix) and at and above 515 μg/mL in the 24-hour continuous exposure group. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
- The test item induced evidence of toxicity in all of the exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 128.75 μg/mL in all three exposure groups.
The selection of the maximum dose level was based on toxicity for both Experiment 1 and Experiment 2.

COMPARISON WITH HISTORICAL CONTROL DATA: All vehicle and solvent controls were in the range of historical laboratory control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1 (Short term exposure both with and without activation):
- No precipitate or haemolysis was observed at the end of exposure in either exposure group.
- In the absence of S9-mix, the mitotic index data (MI) confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and 38% and 50% mitotic inhibition was achieved at 200 and 240 μg/mL, respectively. Similarly, in the presence of S9-mix, 17%, 41% and 69% mitotic inhibition was observed at 160, 200 and 240 μg/mL, respectively.
- The maximum dose level selected for metaphase analysis was, therefore, the maximum concentration of the test item tested (240 μg/mL) for both exposure groups.

Experiment 2 (Short term exposure both with and without activation):
- No precipitate or haemolysis was observed at the end of exposure in either exposure group.
- A moderate dose-related inhibition of mitotic index was observed in both exposure groups. In the absence of S9-mix, 21%, 42% and 60% mitotic inhibition was achieved at 160, 200 and 240 μg/mL, respectively. In the presence of S9-mix, 17% and 80% mitotic inhibition was achieved at 200 and 240 μg/mL, respectively. The excessive toxicity at 240 μg/mL precluded this dose from metaphase analysis.
- The maximum dose level selected for metaphase analysis was, therefore, 200 and 240 μg/mL, in the presence and absence of S9-mix, respectively.

MAIN STUDY RESULTS:
Experiment 1 (Short term exposure both with and without activation):
- The test item induced a statistically significant increase in the frequency of cells with structural chromosome aberrations in the absence of metabolic activation at 240 μg/mL, which was accompanied by optimum toxicity. The test item also induced an increase in the frequency of cells with structural chromosome aberrations in the presence of metabolic activation, at 240 μg/mL, although this was not statistically significant and was accompanied by excessive toxicity (MI value of 69%).
- The test item did not induce a statistically significant increase in the numbers of polyploid cells (which were mostly endoreduplicated) at any dose level in either of the exposure groups.

Experiment 2 (Short term exposure both with and without activation):
- The test item did not induce any statistically significant increases in the frequency of cells with chromosome 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 (with one incidence of endoreduplication noted) at any dose level in either of the exposure groups.

None

Conclusions:

Although the test item induced a statistically significant increase in the frequency of cells with chromosome aberrations in the absence of a liver enzyme metabolizing system, it was not reproduced in a second experiment or in the presence of S9-mix and considered to have no toxicological significance. 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, 8.0, 16.10, 32.19, 64.38, 128.75, 257.5, 515, 1030 and 2060 μg/mL; 4 h exposure time with and without metabolic activation followed by a 20 h expression period, and a continuous exposure of 24 h without metabolic activation

Experiment 1: 4 h treatment followed by 20 h expression period

without S9: 0, 30, 60, 120, 160, 200 and 240 μg/mL

with S9 (2%): 0, 30, 60, 120, 160, 200, and 240 μg/mL

Experiment 2: 4 h treatment followed by 20 h expression period

without S9: 0, 30, 60, 120, 160, 200 and 240 μg/mL

with S9 (2%): 0, 30, 60, 120, 160, 200 and 240 μg/mL

Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

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 (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials 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.

In Experiment 1, the test item induced a statistically significant increase in the frequency of cells with structural chromosome aberrations in the absence of metabolic activation at 240 μg/mL, which was accompanied by optimum toxicity. The test item also induced an increase in the frequency of cells with structural chromosome aberrations in the presence of metabolic activation, at 240 μg/mL, although this was not statistically significant and was accompanied by excessive toxicity (MI value of 69%). However, it was not reproduced in a second experiment or in the presence of S9-mix and considered to have no toxicological significance. 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.

 

Therefore, the variable toxicity and non-reproducible response observed between two experiments is often indicative of toxicity driven mechanisms and not true genotoxic mechanisms. This is further supported by the lack of exchange-type aberrations and can be considered to have little or no biological relevance.

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

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   Harlan, 2014	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, E. coli WP2	-S9 +S9	Up to cytotoxic concentration	-S9 : non mutagenic +S9 : non mutagenic
2   Harlan, 2015	HL/CAT (OECD 473) K, rel. 1	Chromosome aberration	Human Lymphocytes	-S9 +S9	Up to cytotoxic concentrations	-S9 : non clastogenic +S9 : non clastogenic

 

Gene mutation Assays (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). There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in both experiments. A small, statistically significant increase in revertant colony frequency was observed in the presence of S9-mix at 50 μg/plate in the main test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. 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 guideline No. 473), which measures the potential of a substance to increase the incidence 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 two separate experiments. The substance does not induce structural aberrations in the chromosomes of human lymphocytes under activation and non-activation conditionsusing a dose range that included a dose level that induced approximately 50% mitotic inhibition, whereas all the positive control items induced significant increases in the frequency of aberrant cells indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. 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.

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.