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

- L5178Y/MLA Mammalian Cell Gene Mutation Assay (OECD 476, GLP, K, rel. 1): non mutagenic up to cytotoxic concentration.

- Human lymphocytes chromosome aberration test (OECD 473, GLP, K, rel. 1): non clastogenic up to cytotoxic concentration.

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:
From July 16 to August 03, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD 471 Guideline 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:
inspected on May 13, 1998/ signed on November 05, 1998
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium strains: Histidine gene
Escherichia coli strain WP2uvrA: Tryptophan gene
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 (v/v); S9 from liver of rats injected with Aroclor 1254
Test concentrations with justification for top dose:
Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 µg/plate in TA 98, TA 100 and WP2 uvrA strains, with and without S9 mix, using direct plate incorporation method

Mutagenicity tests without S9 mix (direct plate incorporation method):
- 93.75, 187.5, 375, 750 and 1500 µg/plate: for any tester strains in the first experiment as well as for the WP2 uvrA strain in the second experiment,
- 46.875, 93.75, 187.5, 375 and 750 µg/plate: for the TA 1535 and TA 100 strains in the second experiment,
- 23.438, 46.875, 93.75, 187.5 and 375 µg/plate: for the TA 1537 and TA 98 strains in the second experiment.

Mutagenicity tests with S9 mix:
- 93.75, 187.5, 375, 750 and 1500 µg/plate: for all tester strains in the first experiment (direct plate incorporation method) as well as for the WP2 uvrA strain in the second experiment (pre-incubation method),
- 46.875, 93.75, 187.5, 375 and 750 µg/plate: for Salmonella typhimurium strains in the second experiment (pre-incubation method).
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Test substance was freely soluble in DMSO at 50 mg/mL.

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
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:
other: 2-Anthramine
Remarks:
With S9-mix
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Salmonella typhimurium strains were obtained from B.N. Ames Laboratory (University of California, Berkeley, U.S.A.) whilst
Escherichia coli strain WP2uvrA- was obtained from S. Venitt's Laboratory (lCR, Sutton, England).

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

DURATION
- Preincubation period: 60 minutes, 37 °C

- Exposure duration: 48-72 hours at 37 °C

NUMBER OF REPLICATIONS:
- Preliminary toxicity study: One plate/dose
- Mutation study: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

OTHER:
- After 48 to 72 hours of incubation at 37 °C, revertants were scored with an automatic counter (Artek counter, model 880, O.S.l., 75015 Paris, France).
- The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.
Evaluation criteria:
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
not applicable
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see additional information on results
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: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: Not expected
- Water solubility: None
- Precipitation: None
- Other confounding effects: None

P^$^$^$^$^=RELIMINARY TOXICITY TEST
No precipitate was observed in the Petri plates when scoring the revertants at any dose level.
The test substance was highly toxic at dose levels ≥ 2500 µg/plate, in the three tester strains, both with and without S9 mix.
At dose-levels of 500 and 1000 µg/plate, the test substance was slightly to moderately toxic in the TA 100 strain, both with and without S9 mix.

MUTAGENICITY EXPERIMENTS
1) Experiment without S9 mix:
A slight to strong toxicity was induced in Salmonella typhimurium strains mainly at dose-levels ≥ 750 µg/plate. In the WP2 uvrA strain, a slight to moderate toxicity was noted at 1500 µg/plate.
The test substance did not induce any noteworthy increase in the number of revertants in any of the five strains.

2) Experiment with S9 mix:
In the first experiment, a slight to marked toxicity was generally noted at 1500 µg/plate (direct plate incorporation method).
In the second experiment (preincubation method), a slight to marked toxicity was generally induced at dose-levels ≥ 375 µg/plate.
The test substance did not induce any noteworthy increase in the number of revertants in any of the five strains.

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the untreated controls.

See the attached document for information on tables of results

Conclusions:
The test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471/EU Method B.13/14 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2 uvr A were exposed to the test material diluted in DMSO at the following concentrations:

Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 µg/plate in TA 98, TA 100 and WP2 uvrA strains, with and without S9 mix, using direct plate incorporation method

Mutagenicity tests without S9 mix (direct plate incorporation method):

- 93.75, 187.5, 375, 750 and 1500 µg/plate: for any tester strains in the first experiment as well as for the WP2 uvrA strain in the second experiment,

- 46.875, 93.75, 187.5, 375 and 750 µg/plate: for the TA 1535 and TA 100 strains in the second experiment,

- 23.438, 46.875, 93.75, 187.5 and 375 µg/plate: for the TA 1537 and TA 98 strains in the second experiment.

Mutagenicity tests with S9 mix:

- 93.75, 187.5, 375, 750 and 1500 µg/plate: for all tester strains in the first experiment (direct plate incorporation method) as well as for the WP2 uvrA strain in the second experiment (pre-incubation method),

- 46.875, 93.75, 187.5, 375 and 750 µg/plate: for Salmonella typhimurium strains in the second experiment (pre-incubation method).

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

 

 

No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. Experiment without S9 mix: A slight to strong toxicity was induced in Salmonella typhimurium strains mainly at dose-levels ≥ 750 µg/plate. In the WP2 uvrA strain, a slight to moderate toxicity was noted at 1500 µg/plate. The test substance did not induce any noteworthy increase in the number of revertants in any of the five strains.

Experiment with S9 mix: In the first experiment, a slight to marked toxicity was generally noted at 1500 µg/plate (direct plate incorporation method). In the second experiment (preincubation method), a slight to marked toxicity was generally induced at dose-levels ≥ 375 µg/plate. The test substance did not induce any noteworthy increase in the number of revertants in any of the five strains.

 

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.

 

 

Under the test condition, the test material is not mutagenic with and without metabolic activation to S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.

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

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 14 to September 12, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD 476 Guideline without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on April 12, 2005/ signed on June 09, 2005)
Type of assay:
mammalian cell gene mutation assay
Target gene:
The test involves detection of mutation of mouse lymphoma L5178Y cells. The cells are heterozygous at the thymidine kinase (tk) locus (TK+/-), and forward mutation (TK-/-) is detected by the ability of these cells to divide and form colonies in the presence of trifluorothymidine (TFT), a toxic analogue of thymidine.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source: MRC Cell Mutation Unit, University of Sussex, Brighton
- Type and identity of media: RPMI 1640 medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24 hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196 °C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (5% v/v); S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Preliminary toxicity test: 3.44, 6.89, 13.77, 27.54, 55.08, 110.16, 220.33, 440.65, 881.3, 1762.6 µg/mL

Mutation tests:
- Without S9 mix, Test 1 (3 hours): 25, 50, 100, 200, 210, 220, 230 240, 250 µg/mL
- With S9 mix, Test 1 (3 hours): 25, 100, 200, 250, 300, 350, 400, 450 µg/mL
- Without S9 mix, Test 2 (24 hours): 5, 10, 15, 20, 30, 50, 100, 200, 250 µg/mL
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Test material is soluble in DMSO. No precipitation is observed at doses of 1% in medium.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation; 10 μg/mL (3 hour exposure) 5 μg/mL (24 hour exposure)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with metabolic activation; 2.5 μg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: RPMI 1640 medium
R0: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin.
R10p: R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R30p: R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.
R10p medium was used for cell culture unless otherwise specified.
R20p medium was used for the cloning efficiency plating. This was prepared by mixing equal volumes of R10p and R30p.

DURATION
- Exposure duration:
Preliminary toxicity test: 3 hours in the absence and presence of S9 mix and 24 hours in the absence of S9 mix
Main mutation assay: 3 hours in the absence and presence of S9 mix and 24 hours in the absence of S9 mix
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): 7 days for viability plates and approximately 10 to 14 days for mutant plates
- All incubations were performed at 37 °C in a humidified atmosphere of 5 % CO2 in air.

SELECTION AGENT (mutation assays): Selective medium consisted of R10p containing 4 μg/mL trifluorothymidine (TFT).

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture/dose for test item and 2 cultures for vehicle control
- Main test: 4 cultures for vehicle control, 2 cultures/dose for test item and positive controls

NUMBER OF CELLS EVALUATED: 1.6 and 2000 cells per well plated for assessing cloning efficiency (CE) and mutant frequency (MF), respectively.

DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growth (RSG), Cloning efficiency (CE), Relative cloning efficiency (RCE) and Relative total growth (RTG)
RSG = (Individual SG x 100) / Mean solvent control SG
CE = - InP(0) / No. of cells per well
RCE = (Individual CE x 100) / Mean vehicle control CE
RTG = (RSG x Day2 RCE) / 100

OTHER:
Mutant frequency per 10^6 survivors (MF) was calculated as:: CE selective medium / CE non-selective medium
Evaluation criteria:
The following criteria were applied for assessment of individual assay results using data for Mutant Frequency (MF) where the Relative total growth (RTG) normally exceeded 10%.

The assay was considered valid in accordance with the assay acceptance criteria.

The test agent was regarded as negative if:
- The Induced Mutation Frequency (IMF) for any test concentration was less than the GEF.

If the Induced Mutation Frequency (IMF) of any test concentration exceeded the Global Evaluation Factor (GEF), a linear trend test was applied:
- If the linear trend test was negative, the result was regarded as negative.
- If the linear trend test was positive, this indicated a positive, biologically relevant response.

Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies and the nature of any concentration-related effects.

Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.
Statistics:
The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al., (1989).
Key result
Species / strain:
mouse lymphoma L5178Y cells
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 change in the pH of the medium of more than 1.0 unit compared with the vehicle control was observed at 13.57 μg/mL.
- Precipitation: solution of 173.7 mg/mL, dosed at 1% in medium (final concentration of 1737 μg/mL), showed no precipitate in the culture medium.

PRELIMINARY TOXICITY TEST:
A 3 hour exposure to test item at concentrations from 3.44 to 1762.3 μg/mL in the absence and presence of S9 mix resulted in relative suspension growth (RSG) values from 117% to 0% and from 101% to 0% respectively. A continuous exposure for 24 hours to test item at concentrations 3.44 to 1762.3 μg/mL in the absence of S9 mix resulted in RSG values 87% to 0%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10% to 100% relative total growth (RTG).

MAIN TESTS
In the first and second main tests in the absence and presence of S9 mix, the maximum concentration of the tested material plated for determination of mutant frequency was 250 μg/mL, where RTG was reduced to 20% relative to the concurrent solvent control. There were no increases in induced mutation frequency that exceeded the Global Evaluation at any of the concentrations tested within acceptable levels of toxicity. In all tests the concurrent solvent and positive control were within acceptable ranges.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical control data (24 March, 2004 - September 05, 2005)

See the attached document for information on tables of results

Conclusions:
Under the test conditions, test item is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.
Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk+/-(3.7.2C) mouse lymphoma cells were exposed to test item at the following concentrations:

 

Preliminary toxicity test: 3.44, 6.89, 13.77, 27.54, 55.08, 110.16, 220.33, 440.65, 881.3, 1762.6 µg/mL

 

Mutation tests:

- Without S9 mix, Test 1 (3 hours): 25, 50, 100, 200, 210, 220, 230 240, 250 µg/mL

- With S9 mix, Test 1 (3 hours): 25, 100, 200, 250, 300, 350, 400, 450 µg/mL

- Without S9 mix, Test 2 (24 hours): 5, 10, 15, 20, 30, 50, 100, 200, 250 µg/mL

 

Vehicle and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 5 % (v/v) S9 mix; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with Aroclor 1254.

 

In the preliminary toxicity test, toxicity was observed following a 3 hour exposure to test item in both the absence and presence of S9 mix, and following a 24 hour exposure in the absence of S9 mix. At concentrations from 3.44 to 1762.6 μg/mL, relative suspension growth was reduced from 117% to 0%, from 101% to 0% and from 87% to 0% respectively. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10% to 100% relative total growth (RTG). In the first and second main tests in the absence and presence of S9 mix, the maximum concentration test item plated for determination of mutant frequency was 250 μg/mL, where RTG was reduced to 20% relative to the concurrent solvent control. There were no increases in induced mutation frequency (i.e. the mean mutant frequency of test item minus the mean concurrent solvent control mutant frequency) that exceeded the Global Evaluation at any of the concentrations tested within acceptable levels of toxicity. In all tests the concurrent solvent and positive control were within acceptable ranges. 

 

Under the test conditions, test item is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 08, 2011 to January 24, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD 473 Guideline 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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Acceptable to the Japanese New Chemical Substance Law (METI)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on July 10, 2012/ signed on November 30, 2012)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
mammalian cell line, other: human lymphocytes
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
2 % S9 (final concentration); S9 fraction was obtained from the liver homogenates of male Sprague Dawley rats treated with phenobarbitone and β-naphthoflavone
Test concentrations with justification for top dose:
Preliminary Toxicity Test (Cell Growth Inhibition Test):
0, 6.88, 13.75, 27.5, 55, 110, 220, 440, 880 and 1760 μg/mL; 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation

EXPERIMENT 1: 4 hour treatment followed by 24 hour harvest after the start of treatment
without S9: 0, 25, 50, 100, 200, 300 and 400 μg/mL
with S9 (2%): 0, 25, 50, 100, 200, 300 and 400 μg/mL

EXPERIMENT 2:
without S9: 0, 6.25, 12.5, 25, 50, 100 and 150 μg/mL; 24 hour treatment followed by harvest at the end of treatment
with S9 (1%): 0, 50, 100, 200, 240, 300 and 360 μg/mL; 4 hour treatment followed by 24 hour harvest after the start of treatment
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Formulation preparation:The test item was accurately weighed, dissolved in dimethyl sulphoxide (DMSO) and serial dilutions prepared. Formulated concentrations were adjusted to allow for the stated water/impurity content (4.1%) of the test item. With a molecular weight of 176 the maximum dose level was 1760 μg/mL which was calculated to be equivalent to 10 mM (the maximum recommended dose level). 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:
DSMO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9 mix; 0.4 and 0.2 μg/mL for 4(20)-hour and 24-hour cultures respectively
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Without S9 mix; 5 μg/mL
Details on test system and experimental conditions:
TEST SYSTEM: Cultures prepared from the blood of a human volunteer who had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.

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 37ºC 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: Duplicate cultures/dose

NUMBER OF CELLS EVALUATED:
- Total of 2000 lymphocyte cell nuclei was 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 approximately 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.

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:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Key result
Species / strain:
mammalian cell line, other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Effects of pH: No significant change in pH when the test material was added into media.
Effects of osmolality: Osmolality did not increase by more than 50 mOsm.

RANGE-FINDING/SCREENING STUDIES:
The test item exhibited clear dose-related toxicity with no observed metaphases suitable for scoring at and above 440 µg/mL in both of the 4(20)-hour exposure groups, and at and above 220 µg/mL in the 24-hour continuous exposure group.
Test item-induced precipitate was observed in the parallel blood-free cultures at the end of exposure at 1760 µg/mL in the absence of S9, and at and above 880 µg/mL in the presence of S9.
Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 220 µg/mL in the 4(20)-hour both with and without S9 and 110 µg/mL in the 24-hour continuous exposure groups.

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: No precipitate was observed in the blood cultures, either with or without S9. The results of the mitotic indices (MI) from the cultures after their respective treatments confirm 15% and 23% growth inhibition was achieved with and without S9 at 300 μg/mL, respectively. However, there were no metaphases suitable for scoring present at 400 μg/mL in both exposure groups, indicating a very steep toxicity curve. Thus, the selection of the maximum dose level for metaphase analysis was based on toxicity and was limited to 300 μg/mL in both exposure groups. The test item did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation (S9).
EXPERIMENT 2: No precipitate was observed in the blood cultures, either with or without S9. The results of the mitotic indices (MI) from the cultures after their respective treatments confirm 56% growth inhibition was achieved at 150 μg/mL in the absence of S9 and 65% growth inhibition was observed at 300 μg/mL in the presence of S9. It should be noted that there was an increase in toxicity from Experiment 1 in the presence of S9. The selection of the maximum dose level for metaphase analysis was based primarily on the presence of an approximate 50% growth inhibition of the Mitotic Index. The frequency of chromosome aberrations observed at 300 µg/mL, in the presence of S9 just exceeded the historical maximum of the vehicle controls. However, this dose level was not statistically significant compared to the vehicle control when analysed using Fishers Exact Test.

The test item did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

See the attached document for information on tables of results

Conclusions:
The test substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of 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 (Cell Growth Inhibition Test):

0, 6.88, 13.75, 27.5, 55, 110, 220, 440, 880 and 1760 μg/mL; 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation

EXPERIMENT 1: 4 hour treatment followed by 24 hour harvest after the start of treatment

without S9: 0, 25, 50, 100, 200, 300 and 400 μg/mL

with S9 (2%): 0, 25, 50, 100, 200, 300 and 400 μg/mL

EXPERIMENT 2:

without S9: 0, 6.25, 12.5, 25, 50, 100 and 150 μg/mL; 24 hour treatment followed by harvest at the end of treatment

with S9 (1%): 0, 50, 100, 200, 240, 300 and 360 μg/mL; 4 hour treatment followed by 24 hour 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.

In Experiment 1, the highest evaluable concentration was 300 µg/mL based on toxicity and a lack of metaphases suitable for scoring at the highest test concentration of 400 µg/mL. The results of the mitotic indices (MI) from the cultures after their respective treatments show that 15% and 23% growth inhibition was achieved with and without S9 at 300 µg/mL, respectively. In Experiment 2, there was an increase in toxicity in the presence of S9 compared to Experiment 1. The MI from the cultures after their respective treatments confirmed 56% growth inhibition was achieved at 150 µg/mL in the absence of S9 and 65% growth inhibition was observed at 300 µg/mL in the presence of S9 and there were metaphases suitable for scoring at these dose levels. 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 exceeded or was approximately 50% mitotic inhibition, depending on the exposure group. All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. The positive control substances, mitomycin C and cyclophosphamide gave the expected statistically significant increases in the number of cells with structural chromosome aberrations, which demonstrates the sensitivity of the test system.

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

Test concentration

Statement

1

 

CIT, 1999

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100

E.coli WP2 uvrA

Up to cytotoxic concentration

-S9 : non mutagenic

+S9 : non mutagenic

2

 

HLS, 2005

L5178YTK+/-/MLA test (OECD 476)

K, rel. 1

Gene mutation

L5178Y tk+/-(3.7.2C) mouse lymphoma cells

Up to cytotoxic concentration

-S9 : non mutagenic

+S9 : non mutagenic

3

 

Harlan, 2013

HL/CAT

(OECD 473)

K, rel.1

Chromosomal aberration

Human lymphocyte

Up to cytotoxic concentration

-S9 : non clastogenic

+S9 : non clastogenic

Gene mutation Assays (Tests n° 1 -2):

- A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 with the substance (See Table 1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria under the test condition 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.

- Inability to produce gene mutation was confirmed in mammal cells using an in vitro gene mutation assay in L5178Y tk+/-(3.7.2C) mouse lymphoma cells (L5178Y TK+/- /MLA test) (Test n°2). None of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat experiments whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Therefore, the substance is considered as negative for inducing gene mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of the substance to mammalian cells.

 

Chromosomal aberration (Test n°3)

The clastogenic potential of the test material was determined using an in vitro chromosome aberration test in Human lymphocytes, 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, either with or without metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. The substance does not induce structural aberrations in the chromosomes of Human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells. Therefore, the substance is considered as negative for inducing chromosomal mutations in Human lymphocytes in vitro under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonized classification:

The test material 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 germ cell mutagenicity according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.