Registration Dossier

Administrative data

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

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 November 2013 to 14 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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
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. certificate)
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: 14 January 2014
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): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix
Justification: Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 - Main Test (Pre-Incubation Method):
Salmonella strain TA100 (without S9-mix) and Salmonella strains TA98, TA1535 and TA1537 and E.coli strain WP2uvrA (with S9-mix): 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate.
Salmonella strains TA98, TA1535 and TA1537 and E.coli strain WP2uvrA (without S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate.
Salmonella strain TA100 (with S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Justification: Seven 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 DMSO at the same concentration in solubility checks performed in-house. DMSO was therefore selected as the vehicle.
- Preparation of test substance formulation: The test item was accurately weighed and approximate half-log dilutions prepared in DMSO by mixing on a vortex mixer on the day of each experiment. 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, on 04 August 1995 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. Several manual counts were required, predominantly due to revertant colonies spreading slightly, thus distorting the actual plate count.

*concurrent negative controls were dosed using the standard plate incorporation method
Rationale for test conditions:
Experiment 1 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 - Seven 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.
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:
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): 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 150 μg/plate (TA1537), 500 μg/plate (TA1535, TA98 and WP2uvrA) and 1500 μg/plate (TA100). In the presence of S9-mix, weakened lawns were noted from 1500 μg/plate (TA1535, TA98, WP2uvrA and TA1537) and at 5000 μg/plate (TA100). 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, initially from 150 μg/plate (TA1537) and 500 μg/plate (TA100, TA1535, TA98 and WP2uvrA) in the absence of S9-mix. In the presence of S9-mix, weakened lawns were noted from 500 μg/plate (TA1535) and 1500 μg/plate (TA100, TA98, WP2uvrA and TA1537). 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 TA1537 revertant colony frequency was observed in the absence of S9-mix at 150 μg/plate in the range-finding 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 response was accompanied by weakened bacterial background lawns and was, therefore considered spurious.

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 1.5 to 5000 μg/plate. 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 0.5 and 5000 μg/plate, depending on bacterial strain type and presence or absence of metabolic activation (S9-mix). Seven 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. Negative, vehicle (DMSO) and positive control groups were also included in mutagenicity tests.

 

The vehicle 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. 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 150 μg/plate (TA1537), 500 μg/plate (TA1535, TA98 and WP2uvrA) and 1500 μg/plate (TA100). In the presence of S9-mix, weakened lawns were noted from 1500 μg/plate (TA1535, TA98, WP2uvrAand TA1537) and at 5000 μg/plate (TA100). Consequently, depending on bacterial strain type, the same maximum dose level and 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, initially from 150 μg/plate (TA1537) and 500 μg/plate (TA100, TA1535, TA98 and WP2uvrA) in the absence of S9-mix. In the presence of S9-mix, weakened lawns were noted from 500 μg/plate (TA1535) and 1500 μg/plate (TA100, TA98, WP2uvrA and TA1537). 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 TA1537 revertant colony frequency was observed in the absence of S9-mix at 150 μg/plate in the range-finding 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 response was accompanied by weakened bacterial background lawns and was, therefore considered spurious.

 

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- .

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.
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 11 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 473 (1997) without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Qualifier:
according to
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. certificate)
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: 29 January 2015
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
1 and 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, 8.45, 16.9, 33.8, 67.59, 135.19, 270.38, 540.75, 1081.5 and 2163 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, 67.5, 135, 270, 405, 540 and 1080 μg/mL
with S9 (2%): 0, 135, 270, 405, 540, 810 and 1080 μg/mL
Experiment 2:
without S9: 33.75, 67.5, 135, 202.5, 270, 405 and 540 μg/mL; 24 h treatment followed by harvest at the end of treatment
with S9 (1%): 0, 135, 270, 405, 540, 810 and 1080 μg/mL; 4 h treatment followed by 20 h expression period
The selection of the maximum dose level was based on toxicity for the 4(20) h exposure groups and the continuous exposure group used in Experiment 2.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Formulation preparation: The test item was insoluble in culture medium (MEM) at 21.63 mg/mL but was soluble in DMSO at 216.3 mg/mL in solubility checks performed in-house. The test item was accurately weighed, dissolved in DMSO 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 h 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, 4 hours (+S9) and 24 hours (-S9) in Experiment 2
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours (± S9) in 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. If there was a dose-related increase in endoreduplicated cells then they are reported separately.

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).
Rationale for test conditions:
The selection of the maximum dose level was based on toxicity for the 4(20) h exposure groups and the continuous exposure group used in Experiment 2.
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 aberrations 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:
Cloudy precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 135.19 μg/mL, in the 4(20)-hour exposure groups and at and above 1081.5 μg/mL in the continuous exposure group. Greasy/oily precipitate of the test item was also observed in the parallel blood-free cultures at the end of the exposure, at and above 1081.5 μg/mL, in the 4(20)-hour exposure group in the presence of S9-mix and continuous exposure group. In the 4(20)-hour exposure group in the absence of S9-mix greasy/oily precipitate was observed at and above 540.75 μg/mL.
Haemolysis was observed following exposure to the test item at harvesting at and above 540.75 μg/mL in the 4(20)-hour exposure group in the absence of S9-mix, and at and above 1081.5 μg/mL in the 24-hour continuous exposure group and in the 4(20)-hour exposure group in the presence of S9-mix. Haemolysis 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 1081.5 μg/mL in the 4(20)-hour exposure in the presence of metabolic activation and at 540.75 μg/mL in the absence of metabolic activation (S9-mix). The maximum dose with metaphases present in the 24-hour continuous exposure was 270.38 μg/mL. The test item induced evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level was based on toxicity for the 4(20)-hour exposure groups and the continuous exposure group used in 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)
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 at 810 μg/mL in the presence of metabolic activation (S9-mix). In the absence of metabolic activation (S9-mix) the maximum dose level of the test item with metaphases suitable for scoring was 405 μg/mL.
Precipitate observations were made at the end of exposure, greasy/oily precipitate was noted at 1080 μg/mL levels tested in both exposure groups.
Haemolysis was observed following exposure to the test item at and above 67.5 μg/mL in the 4(20)-hour exposure group in the absence of S9-mix, and at and above 540 μg/mL in the 4(20)-hour exposure group in the presence of S9-mix.
The mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 53% mitotic inhibition was achieved at 405 μg/mL in the absence of S9-mix. In the presence of S9-mix a dose-related inhibition of mitotic index of 35% was observed at 810 μg/mL.

Experiment 2 (Continuous exposure without activation and short term with activation)
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the dose level of 405 μg/mL in the absence of S9-mix. In the presence of S9- mix, the maximum test item dose level with metaphases suitable for scoring was 1080 μg/mL.
A greasy/oily precipitate of the test item was observed at the end of exposure, at and above 810 μg/mL, in the 4(20)-hour exposure group and at and above 405 μg/mL, in the 24-hour continuous exposure group.
Haemolysis was observed following exposure to the test item at and above 270 μg/mL in the 24-hour continuous exposure group, and at and above 810 μg/mL in the 4(20)-hour exposure group in the presence of S9-mix.
A dose-related inhibition of mitotic index was observed, and that 40% mitotic inhibition was achieved at 405 μg/mL in the absence of S9-mix. In the presence of S9-mix a dose-related inhibition of mitotic index of 55% was observed at 1080 μg/mL.

MAIN STUDY 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.

None

Conclusions:
The 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, in either of two separate experiments. The test item was therefore considered to be nonclastogenic 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, 8.45, 16.9, 33.8, 67.59, 135.19, 270.38, 540.75, 1081.5 and 2163 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, 67.5, 135, 270, 405, 540 and 1080 μg/mL

with S9 (2%): 0, 135, 270, 405, 540, 810 and 1080 μg/mL

Experiment 2:

without S9: 33.75, 67.5, 135, 202.5, 270, 405 and 540 μg/mL; 24 h treatment followed by harvest at the end of treatment

with S9 (1%): 0, 135, 270, 405, 540, 810 and 1080 μg/mL; 4 h treatment followed by 24 h harvest after the start of treatment

 

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.

 

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition.

 

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). 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. A small, statistically significant increase in TA1537 revertant colony frequency was observed in the absence of S9-mix at 150 μg/plate in the rangefinding 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 conditions using 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 Regulation (EC) No. 1272/2008 (CLP) and to the GHS.