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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 29 October 1998 and 13 november 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: Commission Directive 92/69/EEC, B14 (Ames test).
Principles of method if other than guideline:
The study was conducted according to Safepharm Standard Method 700.04 and was designed to assess the mutagenic potential of the test material using a bacterial / microsome test system. The study was based on the in vitro technique described by Ames and his co-workers and Garner et al, in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli to various concentrations of the test material. The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and US EPA (TSCA) guidelines.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine synthesising gene
Species / strain / cell type:
bacteria, other: Salmonella typhimurium: TA1535, TA1537, TA98 and TA100. Escherichia coli WP2uvrA-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced, rat-liver S9.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 50 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50 ... 5000 µg/plate
Vehicle / solvent:
Solvent: Dimethyl sulphoxide
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
All of the strains were stored at -196C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limitedl lot number 201417 1/03) and incubated at 37 C for approximately 10 hours.

Rationale for test conditions:
Not discussed
Evaluation criteria:
The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible dose-related and statistically (Dunnett's method of linear regression(5)) significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments then this is taken as evidence of a positive response.
Statistics:
Dunnett's method of linear regression(5) is used to determine statistical significance in the increase in the revertant count.
Species / strain:
other: TA100, WP2uvrA-
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(> 5000 µg/plate)
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
(>5000 μg/plate)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(> 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
(> 5000 µg/plate)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(> 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(> 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Observations:
Solvent control plates gave counts of revertant colonies within the normal range.

All positive control chemicals gave increases in revertants, either with or without the metabolising system as appropriate, within expected ranges. No statistically significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of the substance, either with or without metabolic activation. The substance was found to be non-mutagenic under the conditions of this test.
Remarks on result:
other: other: preliminary test
Remarks:
To test cytotoxicity

Please refer to attached pdf document for detailed results.

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Eschericia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and USA EPA (TSCA) guidelines. The dose range was determined in a preliminary toxcity assay and was 50 to 5000 μg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Validity of test

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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Results and conclusions

The test material caused no visible reduction in the growth of the bacterial lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose of 5000 μg/plate.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Commission Directive 92/69/EEC
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian cytogenicity (B10)
Target gene:
Not relevant. Test examines damage to chromosomes.
Species / strain / cell type:
lymphocytes: Human lymphocytes
Details on mammalian cell type (if applicable):
Sufficient whole blood was deawn from 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 16.7 hours under normal experimental conditions
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/á-naphthoflavone induced, rat-liver S9.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 473 ... 1892 µg/ml
Concentration range in the main test (without metabolic activation): 473 ... 1892 µg/ml

Maximum dose level was 1892.1 μg/l was calculated to be equivalent to 10 mM of the test material (MW = 189.21).
Vehicle / solvent:
Dimethyl sulphoxide
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
mitomycin C
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 4 hours
Exposure period (without metabolic activation): 24 hours

Expression time: Not applicable
Selection time: Not applicable

Fixation time: 24 Hours
Statistics:
The frequency of cells with aberratoins (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
> 1892 µg/ml
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: human lyphocytes
Metabolic activation:
without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
946 µg/ml
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: as specified above
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
> 1892 µg/ml
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 1892 µg/ml
Species / strain:
other: as specified above
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
> 1892 μg/ml
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
946 µg/ml
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Observations:
All the negative control cultures gave values of chromosome
aberrations with the expected range for normal human
lymphocytes.

All the positive control cultures gave highly-significant
increases in the frequency of aberrations, indicating the
satisfactory performance of the test and of the activity of
the metabolising system.

The substance was seen to induce no significant,
dose-related increases in the frequency of aberrations in
any of the treatment groups, either with or without S9.

The substance did not induce a significant increase in the numbers of polydiploid cells at any dose level in any of the
treatment cases.

The substance is therefore considered to be non-clastogenic to human lymphocytes in vitro.
Remarks on result:
other: preliminary test - 4-hour pulse exposure
Remarks:
Migrated from field 'Test system'.

Preliminary toxicity test

In the 4-hour pulse exposure cases the test material showed no evidence of toxicity. In the 24-hour continuous exposure case, there was an approximate of 50% mitotic inhibition at 946 μg/ml and 1892 μg/ml. A precipitate of the test material was not observed at any dose level tested. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to the 10 mM concentration (1892 μg/ml) in all the treatment groups.

Experiment 1

The test material was not toxic and there was no dose-related mitotic inhibition. In the with-metabolic activation group there appears to be mitotic synchronisation but this was considered to be a probable artefact, due to a low mitotic index for the A culture in the vehicle control treatment.

Both of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore satisfactory and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the presence or absence of metabolic activation.

The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in ehtier of the treatment cases.

Experiment 2

A dose-related inhibition was observed in the absence of S9 and greater than 50% mitotic inhibition was achieved at 1892 μg/ml. In the presence of S9 there was no mitotic inhibition observed.

All the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore satisfactory and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the presence or absence of metabolic activation.

The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the treatment cases.

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the presence or absence of a liver enzyme metabolising system in either of two separate experiments. BMS 214702-01 was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Methods

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system (S9) with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of activation with a 20-hour expression period; this was Experiment 1. In Experiment 2 the 4-hour exposure with addition of S9 was repeated (using a 2% final concentration of S9), whilst in the absence of activation the exposure time was increased to 24 hours.

The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicilogy: Chromosome Aberration Test" and Method B10 of Commission Directive 92/69/EEC.

Results

All vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control treatments gave 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 material, BMS 214702-01, did not induce any statistically significant increases in the frequency of cells with aberrations in either of two experiments. The test material was shown to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH S2(R1)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
comet assay
Target gene:
Mouse lymphoma thymidine kinase (TK) assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
TK +/-
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of a liver homogenate from rats treated previously with phenobarbital / 5,6-benzoflavone
Test concentrations with justification for top dose:
Nine concentrations of the BMS-214702-01 (ranging from 0.800 to 200 μg/mL, using a 2-fold dose interval) were tested.

Typically, the test item is dosed at a range of concentrations but is only assessed at the highest levels below the toxic level or, if nontoxic (taking into account solubility of the test item in culture medium), at levels up to the standard limit of 0.5 mg/mL or 1 mM, whichever is lower. For pharmaceuticals with unusually low molecular weight (e.g., less than 200), higher test concentrations should be considered as per the ICH S2(R1) guideline. Compounds with limited aqueous solubility are tested up to a level showing visible precipitation in the culture medium. BMS-214702-01 was previously tested in a human lymphocyte chromosome aberration study at concentrations up to 1892 μg/mL (10 mM), with indications of cytotoxicity noted at >= 946 μg/mL in the long exposure regimen only. Therefore, the highest concentration tested was the ICH limit dose of ~1 mM (200 μg/mL).
Vehicle / solvent:
Identity: Potassium phosphate, monobasic, 20 mM, pH 3.2 in sterile water
The solubility of BMS-214702-01 in vehicles compatible with the test system was not assessed prior to the study; information supplied by the Sponsor was taken into account. The Sponsor found BMS-214702-01 to be soluble and stable in potassium phosphate, monobasic, 20 mM, pH 3.2 at 3 mg/mL for up to 12 days of storage at ambient temperature in a non-GLP solubility and stability assessment.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
3 hour treatment in the presence of S9 activation
Details on test system and experimental conditions:
Mouse lymphoma L5178Y TK+/- cells were incubated with the vehicle, in replicates of 4 cultures, or with the test item or positive control for 3 hours (with and without exogenous
metabolic activation) or 24 hours (without exogenous metabolic activation), in duplicate cultures. The metabolic activation system was the S9 fraction of a liver homogenate from rats treated previously with phenobarbital/5,6-benzoflavone. Nine concentrations of the BMS-214702-01 (ranging from 0.800 to 200 μg/mL, using a 2-fold dose interval) were tested. Following a subsequent 48 hour growth recovery period, the cells were transferred to selective medium (in microwell plates) which allowed only the growth of mutant cells into colonies. After a suitable growth period in selective medium, the number of wells containing colonies was evaluated. The mutation frequency for each treatment was calculated, taking into account the viability of the treated cells.

Cell line
Mouse lymphoma L5178Y TK+/- (clone 3.7.2C) cells were originally obtained from the American Type Culture Collection (ATCC) and subsequently maintained in-house. The cell line
was cloned, cleansed, characterized and confirmed to be free of mycoplasma. Frozen aliquots of cells (passage number 13 after receipt from the ATCC) were thawed and used
to inoculate R10p culture medium. The cells were transferred to fresh medium when necessary in order to maintain exponential growth and to obtain sufficient cells for the experiment.

Culture medium
R0p consisted of RPMI 1640 medium supplemented with 0.1% v/v Pluronic F68, 1 mM sodium pyruvate and 50 μg gentamycin per mL. R2p and R10p consisted of R0p supplemented with 2% and 10% v/v heat-inactivated donor herd horse serum respectively. R2p was used for washing steps and intermediate dilutions. Selective medium consisted of R10p containing 5 μg trifluorothymidine (TFT) per mL.

Cell culture
Cells were grown in R10p in an incubator set at 37C with 5% CO2 in a humidified atmosphere.

S9 Mix
The S9 mixture, used as a model of intact mammalian metabolism, was prepared on the day of use and contained 10% v/v S9 fraction (phenobarbital/5,6-benzoflavone induced male rat liver fraction supplied by Moltox) and the following sterile cofactors: 8 mM MgCl2, 33 mM KCl, 100 mM sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 4 mM NADP. The S9 mixture was stored in a refrigerator set to maintain 4°C until required, then held on ice during utilization. A copy of the manufacturer’s quality control certificate for the S9 fraction was retained as raw data.
Rationale for test conditions:
Not mentioned
Evaluation criteria:
Calculation of toxicity:
Relative Total Growth (RTG) is a measure of the survival of the cells over the treatment and expression periods compared with the concurrent vehicle control. It is calculated by multiplying RG0 by the Relative Growth over Days 1 and 2 by relative Day 2 (viability) plating efficiency, i.e. Relative Viability (RV).

Calculation of mutation frequency
The mutation frequency (MF) is calculated in terms of mutant cells per 10^6 viable cells. It is based on the plating efficiencies of the mutation plates and the viability plates. Colonies are characterized as small (<= 1/4 well diameter) or large (> 1/4 well diameter).

Criteria for a valid test
For a test to be considered valid, absolute plating efficiency for the vehicle control cultures should be within an approximate range of 65-120% for the viability plates. Lower plating efficiencies may be acceptable as outlined in ASTM Guideline E 1280-97. Growth over the 2-day expression period for the vehicle controls should be in the range of 8- to 32-fold for the 3-hour treatments and 32- to 180-fold for the 24-hour treatment. Mean spontaneous mutant frequency should exceed 50 mutants per 10^6 viable cells, but remain close to or within the current historical control range of the laboratory. Positive control compounds should cause an increase of at least 300 mutants per 10 viable cells compared to the vehicle control in each treatment regime with approximately 40% or more of the increase reflected in the small colony mutant frequency. Alternatively, the positive controls should increase the small colony mutant frequency by approximately 150 or more small colony mutants per 10^6 viable cells compared to vehicle controls. For toxic substances, dose levels for the main test should cover an appropriate range of toxicity (from approximately 10-20% to 80-90% in terms of RTG). In addition, there should be at least four analyzable (relatively non-toxic) dose levels per treatment regime.
Statistics:
Not mentioned
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
TK +/-
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

The item was not toxic, so the 4 highest dose levels were selected for evaluation in each treatment regime.

All plating efficiencies for vehicle control cultures were within acceptable limits based on the ASTM Guideline E 1280-97. Growth over the 2-day expression period for the vehicle controls (G1 × G2) was in the range of 8- to 32-fold for the 3-hour treatments and 32- to 180-fold for the 24 -hour treatment. The mean spontaneous mutant frequency met the acceptance criteria of exceeding 50 mutants per 10^6 viable cells, but remaining close to or within the current historical control range of the laboratory (see Section 14 for historical negative control results). The

positive control compounds caused increases of at least 300 mutants per 10^6 viable cells with at least 40% of the increase reflected in the small colony mutant frequency, confirming the sensitivity of the test and the effectiveness of the metabolic activation system (see Section 14 for

historical positive control results). The procedures used were therefore shown to be effective, and the test was considered valid.

No substantial increases in mutation frequency were obtained after treatment of cells with BMS-214702-01 in any treatment regime, at dose levels up to 200 μg/mL.

No change in culture medium color compare to the vehicle control or precipitate was observed at the end of treatment.

Conclusions:
No toxicity or precipitate was observed in any of the regimes, in either the absence or presence of metabolic activation. No substantial increases in mutation frequency were obtained after treatment of cells with BMS-214702-01 in any treatment regime, at dose levels up to 200 μg/mL.

It is concluded that BMS-214702-01 did not show any evidence of genotoxicity in this in vitro test when tested in accordance with regulatory guidelines.
Executive summary:

The objective of this study was to determine the potential genotoxicity of BMS-214702-01, a chemical intermediate used in drug synthesis, using a mammalian L5178 TK+/- mouse lymphoma cell mutation test.

Mouse lymphoma L5178Y TK+/- cells were incubated with the vehicle, in replicates of 4 cultures, or with the test item or positive control for 3 hours (with and without exogenous metabolic activation) or 24 hours (without exogenous metabolic activation), in duplicate cultures. The metabolic activation system was the S9 fraction of a liver homogenate from rats treated previously with phenobarbital/5,6-benzoflavone. Nine concentrations of the BMS-214702-01 (ranging from 0.800 to 200 μg/mL, using a 2-fold dose interval) were tested. Following a subsequent 48 hour growth recovery period, the cells were transferred to selective medium (in microwell plates) which allowed only the growth of mutant cells into colonies. After a suitable growth period in selective medium, the number of wells containing colonies was evaluated. The mutation frequency for each treatment was calculated, taking into account the viability of the treated cells.

Responses to the positive controls confirmed the sensitivity of the assay and the effectiveness of the metabolic activation system.

No toxicity or precipitate was observed in any of the regimes, in either the absence or presence of metabolic activation. No substantial increases in mutation frequency were obtained after treatment of cells with BMS-214702-01 in any treatment regime, at dose levels up to 200 μg/mL.

It is concluded that BMS-214702-01 did not show any evidence of genotoxicity in this in vitro test when tested in accordance with regulatory guidelines.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No tests were necessary. In vitro tests showed no evidence of genetic toxicity.

Additional information

Justification for classification or non-classification

Three in vitro genetic toxicity tests (Ames, Chromosome Aberration and mammalian cell gene mutation) were negative. The substance is not considered mutagenic, hence no classification is justified.