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.

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

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:
21 March to 15 May 2012
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 19-21 July 2011 / signed on 31 August 2011)
Type of assay:
bacterial reverse mutation assay
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 rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/ kg bw/day by oral route).
Test concentrations with justification for top dose:
Preliminary toxicity test (Plate incorporation method): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate in TA100 or WP2uvrA strains with and without S9 mix

Mutation Test – Experiment 1 – Range-finding test (Plate incorporation method):
All Salmonella strains (without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate.
All Salmonella strains (with S9-mix) and E.coli strain WP2uvrA (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate.
Mutation Test – Experiment 2 - Main Test (Pre-Incubation Method):
All Salmonella strains (without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate.
All Salmonella strains (with S9-mix) and E.coli strain WP2uvrA (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 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 50 mg/ml 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 dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated impurity content (1.9%) 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 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, from Syngenta CTL, Alderley Edge, as frozen vials, on 20 March 2007 or from the British Industrial Biological Research Association, on nutrient agar plates, 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 °C for 20 minutes (with shaking) at approximately 130 rpm
- Exposure duration: Plates were incubated at 37 °C for approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: Plates were assessed for visible reduction in the growth of the bacterial background lawns indicating toxicity.

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.
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).
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 judgement 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: A test item precipitate (light and oily in appearance) was observed at 5000 μg/plate.

PRELIMINARY TOXICITY TEST:
The test item was toxic to TA100 from 150 μg/plate and to WP2uvrA from 500 μg/plate.

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 2010 and 2011 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1 and 2: The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 μg/plate after employing plate incorporation methodology (range-finding test) and from 15 μg/plate following the pre-incubation method (main test). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. The test item was tested up to the toxic limit. A test item precipitate (light and oily in appearance) was observed at 5000 μg/plate. The test item was tested up to the toxic limit in the range-finding and main tests, therefore, this observation was only noted in the preliminary toxicity test.

MUTAGENICITY:
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

The test item formulation and S9-mix used in this experiment were both shown to be sterile.

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.

Tables: cf Attached background material.

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-.
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, ST 01 C 11, using both the Ames plate incorporation and pre-incubation methods at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 0.15 and 500 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. The experiment was repeated on a separate day (pre-incubation method) using the same dose range, fresh cultures of the bacterial strains and test item formulations. Additional dose levels and an expanded dose range were selected in each experiment 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 test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 μg/plate after employing plate incorporation methodology (range-finding test) and from 15 μg/plate following the pre-incubation method (main test). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. The test item was tested up to the toxic limit. A test item precipitate (light and oily in appearance) was observed at 5000 μg/plate. The test item was tested up to the toxic limit in the range-finding and main tests, therefore, this observation was only noted in the preliminary toxicity test.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

 

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-.

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:
01 October 2012 to 21 March 2013
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
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:
other: in vitro mammalian chromosome aberration test
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 in the Preliminary Toxicity Test and Experiment 1 and 1% S9 in Experiment 2; 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
0, 7.34, 14.69, 29.38, 58.75, 117.5, 235, 470, 940 and 1880 μg/mL; 4 h exposure time with and without metabolic activation followed by a 20 h recovery period, and a continuous exposure of 24 h without metabolic activation
Justification: The maximum dose was the maximum recommended dose level, the 10 mM concentration.

Main experiments
Experiment 1: 4-hour exposure to the test item followed by 20-hour culture in treatment-free media prior to cell harvest
without S9: 0, 3.75, 7.5, 15, 30, 60 and 120 μg/mL;
with S9 (2%): 0, 15, 30, 60, 80, 100 and 120 μg/mL
Experiment 2:
24-hour continuous exposure to the test item without S9-mix prior to cell harvest: 0, 3.75, 7.5, 15, 30, 40 and 60 μg/mL;
4-hour exposure to the test item with S9-mix (1%) followed by 20-hour culture in treatment-free media prior to cell harvest: 0, 7.5, 15, 30, 60, 80 and 100 μg/mL

Justification: The maximum dose level selected for the main experiments was based on the toxicity seen in the Preliminary Toxicity test and was 120 μg/mL for the 4(20)-hour exposure groups of Experiment 1. In Experiment 2, the maximum dose was 60 μg/mL in the 24-hour exposure group and 100 μg/ml in the 4(20)-hour exposure in the absence of S9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Formulation preparation: The test item was accurately weighed, dissolved in Dimethyl sulphoxide (DMSO) and serial dilutions prepared. The test item formulations in DMSO were dosed at a concentration of 1%. The molecular weight of the test item was supplied as 188.27, therefore the maximum recommended dose level was the 10 mM concentration of 1880 μg/mL. The purity of the test item was 98.1% and an adjustment was made when the test item was formulated for an impurity of 1.9%. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was 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 volunteer who had been previously screened for suitability. The volunteer had not 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 average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

CELL CULTURE: Cells 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 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinised 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 was 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. In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: If greater than 44 chromosomes are scored and the number is a multiple of the haploid count then the cell is classified as a polyploid cell.
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated (E).
Evaluation criteria:
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. The level of spontaneous background aberrations may be slightly elevated above the normal range and the experiment still considered to be valid. In such cases the Study Director will justify the reason for acceptance of the data in the report.
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.
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:
- A cloudy precipitate of the test item was observed in the parallel blood-free cultures at the end of exposure at and above 117.5 μg/mL and a greasy/oily precipitate was seen at and above 940 μg/mL in the 4(20)-hour exposure groups in the absence and presence of S9. In the 24-hour exposure group greasy/oily precipitate and cloudy precipitate were seen at and above 235 μg/mL at the end of the exposure period. Haemolysis was also noted at the end of exposure in the blood cultures of the 4(20)-hour exposure group in the presence of S9 and in the 24-hour exposure group at and above 117.5 μg/mL. In the 4(20)-hour exposure in the absence of S9, haemolysis was seen at and above 58.75 μg/mL at the end of the exposure period.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells suitable for scoring were present up to 117.5 μg/mL in the 4(20)-hour exposure group in the absence of S9 and up to 58.75 μg/mL in the 4(20)-hour exposure group in the presence of S9 and in the 24-hour exposure group.

The maximum dose level selected for the main experiments was based on the toxicity seen in the Preliminary Toxicity test and was 120 μg/mL for the 4(20)-hour exposure groups of Experiment 1. In Experiment 2, the maximum dose was 60 μg/mL in the 24-hour exposure group and 100 μg/mL in the 4(20)-hour exposure in the absence of S9.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1:
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at 60 μg/mL in both exposure groups.
No precipitate was observed at the end of exposure in either exposure group. Haemolysis was noted at the end of exposure at and above 60 and 80 μg/mL in the absence and presence of S9, respectively.
There was a reduction in mitotic index in the 4(20)-hour exposure group in the absence of S9 of 64% at 30 μg/mL and 55% at 60 μg/mL. Although the mitotic index value at 60 μg/mL was based on the scoring of the ‘B’ replicate only, as the ‘A’ replicate was lost due to a technical error, it was considered to give an adequate indication of toxicity for this dose level. The toxicity observed between 30 μg/mL and 60 μg/mL was seen to plateau with greater than optimum toxicity being achieved at both these dose levels. Therefore, the maximum dose level selected for metaphase analysis was the lowest dose level (30 μg/mL) where 64% mitotic inhibition was achieved. Although, the toxicity at 60 μg/mL was marginally less than that observed at 30 μg/mL it was considered that it was unnecessary to score this dose level for chromosome aberrations as greater than optimum toxicity was already achieved at 30 μg/mL. In the presence of S9 there was a steep toxicity curve between 60 μg/mL and 80 μg/mL with no marked reduction in mitotic index at 60 μg/mL and insufficient cells and metaphases for scoring at 80 μg/mL.
The maximum dose level selected for metaphase analysis was 30 and 60 μg/mL in the absence and presence of S9, respectively.

Experiment 2:
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at 40 μg/mL in the absence of S9. In the presence of S9 there were metaphase suitable for scoring at 60 μg/mL.
No precipitate was observed at the end of exposure in either exposure group. Haemolysis was observed at the end of the exposure period at and above 60 μg/mL in the 4(20)-hour exposure group in the presence of S9.
The mitotic index data confirm that the qualitative observations in that dose-related toxicity was observed in the presence of S9 with 29% and 65% mitotic inhibition at 30 and 60 μg/mL respectively. In the absence of S9 the toxicity was slightly inconsistent throughout the dose range but greater than optimum toxicity (50%) was achieved at 40 μg/mL with 67% mitotic inhibition.
The maximum dose level selected for metaphase analysis was 40 and 60 μg/mL in the absence and presence of S9, respectively.

MAIN STUDY RESULTS
Experiment 1 and 2
- 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 at any dose level in either of the exposure groups.

None

Conclusions:
The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either the absence or presence of S9, in two separate experiments. 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

0, 7.34, 14.69, 29.38, 58.75, 117.5, 235, 470, 940 and 1880 μg/mL; 4 h exposure time with and without metabolic activation followed by a 20 h recovery period, and a continuous exposure of 24 h without metabolic activation

 

Main experiments

Experiment 1:

4(20)-hour without S9: 0, 3.75, 7.5, 15, 30, 60 and 120 μg/mL;

4(20)-hour with S9 (2%): 0, 15, 30, 60, 80, 100 and 120 μg/mL

Experiment 2:

24-hour without S9: 0, 3.75, 7.5, 15, 30, 40 and 60 μg/mL;

4(20)-hour with S9 (1%): 0, 7.5, 15, 30, 60, 80, 100 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 (solvent) 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 the satisfactory performance of the test and of the activity of the metabolising system.

 

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either the absence or presence of S9, in two separate experiments.

 

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, 2013

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, 2014

HL/CAT (OECD 473)

K, rel. 1

Chromosome aberration

Human Lymphocytes

-S9

+S9

Up to cytotoxic concentration

-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). A test item precipitate (light and oily in appearance) was observed at 5000 μg/plate in the preliminary test. The test substance was tested up to 150 µg/plate in the main test for all Salmonella strains -S9 and up to 500 µg/plate for E.coli +S9 and -S9, and for all Salmonella strains +S9. 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. 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 (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 I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.