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:
01-28 October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test 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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
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:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Purity test date: 04 November 2014
- Storage condition of test material: Refrigerated at 4°C in the dark under nitrogen
Target gene:
Histidine and tryptophan
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 rats treated with phenobarbitone/β-naphthoflavone
Test concentrations with justification for top dose:
Mutation Test – Experiment 1 – Range-finding test (Pre-incubation method):
All Salmonella strains (without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Justification: The maximum concentration was 5000 μg/plate (the maximum recommended dose level).

Mutation Test – Experiment 2 - Main Test (Pre-Incubation Method):
Salmonella strain TA98 and E.coli strain WP2uvrA (without S9-mix) and all tester strains (with S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate.
Salmonella strains TA100, TA1535 and TA1537 (without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate.
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 item 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. Formulated concentrations were adjusted to allow for the stated water/impurity content (2.1%) 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 aluminosilicate pellets with a nominal pore diameter of 4 x 10^-4 microns.
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 strains of bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 and from 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: in agar (plate incorporation); 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: The plates were viewed microscopically for evidence of thinning (toxicity).

OTHERS:
- The concurrent negative controls were dosed using the standard plate incorporation method
- After incubation, the plates were scored for the presence of revertant colonies using an automated colony counting system.
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: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES
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 (TA100, TA1535 and TA1537) and 500 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix, weakened lawns were noted to all of the tester strains from 500 μg/plate. Consequently, the toxic limit of the test item was employed in the second mutation test.e appropriate:

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 2012 and 2013 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY
Main test results once again showed 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 (TA100, TA1535, TA98 and TA1537) and 500 μg/plate (WP2uvrA). In the presence of S9-mix, weakened lawns were noted to all of the tester strains from 500 μg/plate.

MUTAGENICITY RESULTS
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. Small, statistically significant increases in TA100 revertant colony frequency were observed (presence of S9-mix) at 15 and 50 μg/plate in the main test. These increases were 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 15 and 50 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.2 times the concurrent vehicle control.

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.

Conclusions:
Under the test condition, test item 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
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 TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames pre-incubation method (under anaerobic conditions) 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 (Experiment 1) was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) 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 0.15 and 500 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. Seven test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item. Negative, vehicle (dimethyl sulphoxide) 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. 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 (TA100, TA1535 and TA1537) and 500 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix, weakened lawns were noted to all of the tester strains from 500 μg/plate. Consequently, the toxic limit of the test item 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 the absence of S9-mix, initially from 150 μg/plate (TA100, TA1535, TA98 and TA1537) and 500 μg/plate (WP2uvrA). In the presence of S9-mix, weakened lawns were noted to all of the tester strains from 500 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. Small, statistically significant increases in TA100 revertant colony frequency were observed (presence of S9-mix) at 15 and 50 μg/plate in the main test. These increases were 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 15 and 50 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.2 times the concurrent vehicle control.

 

Under the test condition, test item 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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 October 2014 to 29 January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 473 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
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)
Qualifier:
according to
Guideline:
other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 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: 04 November 2014
- Storage condition of test material: Refrigerated at 4°C in the dark under nitrogen
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:
2% S9; S9 fraction was obtained from the liver homogenates of rats treated with phenobarbitone and β-naphthoflavone
Test concentrations with justification for top dose:
Preliminary Toxicity Test (Cell Growth Inhibition Test)
0, 6.57, 13.15, 26.30, 52.59, 105.19, 210.38, 420.75, 841.5 and 1683 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)
Justification: The maximum dose was the maximum recommended dose level, the 10 mM concentration.

Main experiments
Experiment 1:
4(20)-hour without S9-mix: 0, 13.13, 26.25, 52.5, 105, 157.5 and 210 μg/mL;
4(20)-hour with S9 (2%) 0, 26.25, 52.5, 105, 131.25, 157.5, 210 and 420 μg/mL
Experiment 2:
24-hour without S9-mix: 0, 6.56, 13.13, 26.25, 52.5, 78.75, 105 and 210 μg/mL
Justification: The selection of the maximum dose level was based on toxicity for the 4(20)-hour exposure groups and the continuous exposure group.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was insoluble in culture medium (MEM) at 16.83 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 168.3 mg/mL in solubility checks performed in-house.
- Formulation preparation: 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 the main experiment a sample of fresh blood, drawn from the peripheral circulation of a volunteer, will be used within 30 hours of collection. The volunteer was a non-smoker, not knowingly having been exposed to high levels of radiation, hazardous chemicals or recent viral infection, and his/her lymphocytes had previously been shown to respond well to positive control chemicals. The cell cycle time for lymphocytes from each donor will have been determined by using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT) for the laboratory donor pool. Any specific cell cycle time delay induced by the test item will not be accounted for directly. However, the cell harvest time-point will be at approximately 1.5 x AGT and therefore any test item-induced cell cycle delay should be accommodated. Currently the average in-house time for approximately 1.5 x AGT is 24 hours.
After the Cell Growth Inhibition Test the sponsor requested that the study be updated to comply with the new OECD guidelines, therefore the donor for the main experiment was in the age range of 18 to 35 years old.
Preliminary Toxicity Test: male, aged 45 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: 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 150 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. 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:
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.
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 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):
- Cloudy precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 420.75 μg/mL in the 4(20)-hour exposure group in the presence of S9-mix and at 1683 μg/mL in 4(20)-hour exposure group in the absence of S9-mix. None was observed 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 841.5 μg/mL in the 4(20)-hour exposure group in the absence of S9-mix and in the continuous exposure group. None was observed in the 4(20)-hour exposure group in the presence of S9-mix.
- Haemolysis was observed following exposure to the test item at harvesting at and above 210.38 μg/mL in the 4(20)-hour exposure group in the absence of S9-mix, at and above 420.75 μg/mL in the 4(20)-hour exposure group in the presence of S9-mix and at and above 52.59 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.
- Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 210.38 μg/mL in the 4(20)-hour exposure in the presence of metabolic activation and at 105.19 μg/mL in the absence of metabolic activation (S9-mix). The maximum dose with metaphases present in the 24-hour continuous exposure was also 105.19 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.

HISTORICAL CONTROL DATA (mean ± standard deviation)
- Positive historical control data:
cells with aberrations (-gaps):
4(20)-hour exposure without S9%: 39.86 ± 14.04
4(20)-hour exposure with S9 (1%): 30.66 ± 9.65
4(20)-hour exposure with S9 (2%): 25.32 ± 10.09
24-hour exposure without S9: 39.09 ± 14.57
% cells with polyploids:
4(20)-hour exposure without S9%: 0.02 ± 0.13
4(20)-hour exposure with S9 (1%): 0.02 ± 0.12
4(20)-hour exposure with S9 (2%): 0.06 ± 0.21
24-hour exposure without S9: 0.02 ± 0.013

- Negative (solvent/vehicle) historical control data:
cells with aberrations (-gaps):
4(20)-hour exposure without S9%: 0.48 ± 0.56
4(20)-hour exposure with S9 (1%): 0.40 ± 0.48
4(20)-hour exposure with S9 (2%): 0.53 ± 0.52
% cells with polyploids:
4(20)-hour exposure without S9%: 0.05 ± 0.17
4(20)-hour exposure with S9 (1%): 0.02 ± 0.11
4(20)-hour exposure with S9 (2%): 0.09 ± 0.24
24-hour exposure without S9: 0.07 ± 0.22

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No precipitate was observed at the end of exposure, in any of the three exposure groups.
- Haemolysis was observed following exposure to the test item at and above 52.5 μg/mL in the 4(20)-hour exposure group in the absence of S9-mix, and at and above 105 μg/mL in the 4(20)- hour exposure group in the presence of S9-mix. Haemolysis was also observed following exposure to the test item at and above 26.25 μg/mL in the 24 hour continuous exposure group.
- The mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 35% and 63% mitotic inhibition was achieved at 157.5 μg/mL and 210 μg/mL respectively in the absence of S9-mix. In the presence of S9-mix a dose-related inhibition of mitotic index of 45% was observed at 210 μg/mL. In the continuous exposure group in the absence of S9-mix a dose-related inhibition of mitotic index of 47% was observed at 78.75 μg/mL.
- Dose selection was based on toxicity and although the toxicity was marginally outside the requirements stated in the OECD 473 Guidelines it is considered that the test item had been adequately tested.

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 the main experiment. 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, 6.57, 13.15, 26.30, 52.59, 105.19, 210.38, 420.75, 841.5 and 1683 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 experiments

Experiment 1:

4(20)-hour without S9-mix: 0, 13.13, 26.25, 52.5, 105, 157.5 and 210 μg/mL;

4(20)-hour with S9 (2%) 0, 26.25, 52.5, 105, 131.25, 157.5, 210 and 420 μg/mL

Experiment 2:

24-hour without S9-mix: 0, 6.56, 13.13, 26.25, 52.5, 78.75, 105 and 210 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 (DMSO) 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, in the main experiment using a dose range that included a dose level that achieved near optimum toxicity.

 

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, 2015a

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, 2015b

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 assay:

A bacterial reverse mutation Assay (Ames test) was performed according to the OECD guideline 471 and in compliance with GLP (Harlan, 2015a, rel.1). Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames pre-incubation method (under anaerobic conditions) 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).

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. Small, statistically significant increases in TA100 revertant colony frequency were observed (presence of S9-mix) at 15 and 50 μg/plate in the main test. These increases were 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 15 and 50 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.2 times the concurrent vehicle control.

The substance is therefore considered as non-mutagenic according to the Ames test.

Chromosomal aberration:

The clastogenic potential of the substance was determined using the in vitro chromosome aberration test in human lymphocytes (Harlan, 2015b, rel.1) which measures the potential of a substance to increase the incidence of structural chromosome aberrations in cultured human lymphocytes. The study was performed according to the OECD guideline 473 and in compliance with GLP.

In the main experiment, the test item did not induce any statistically significant increases in the frequency of cells with aberrations, either in the presence or absence of metabolic activation in either of two separate experiments, using a dose range that included a dose level that achieved near optimum toxicity whereas all the positive control items induced significant inreases in the frequency of aberrant cells indicating that the sensitivity of the assay ad the efficacy of the S9 -mix is were validated. The substance is therefore considered as negative for inducing chromosomal aberrations in human lymphocyte cells uder activation and non-activation condictions 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.