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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Stability in vehicle analytically confimed.
Target gene:
mutant histidine gene
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 100, TA 1537, TA 98, TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from the liver of Aroclor 1254 induced male Sprague Dawley rats.
Test concentrations with justification for top dose:
plate incorporation assay: 0, 50, 158, 500, 1581, 5000 µg/plate with and without S9 mix
preincubation assay: 0, 50, 158, 500, 1581, 5000 µg/tube with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item formed clear colorless solutions in DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Na-azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), mitomycin C (only TA 102 in plate incorporation assay), cumene hydroperoxide (only TA 102 in preincubation assay), 2-aminoanthracene.
Remarks:
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C and cumene hydroperoxide were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for initial testing; independent repeat as preincubation testing (preincubation for 20 min. at 37 °C);
Testing for each strain and dose with and without S9 mix was performed in triplicate, which is also valid for solvent and positive controls.

DETERMINATION OF CYTOTOXICITY
- background growth
- mutant count: A toxic effect was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls.
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Species / strain:
S. typhimurium, other: TA 1535, TA 100, TA 1537, TA 98, TA 102
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:
None of the five strains concerned showed in the plate incorporation test a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed on the plates.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Doses up to and including 500 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had only a weak, strain-specific bacteriotoxic effect. Due to the weakness of this effect this range could nevertheless be used for assessment purposes.
Executive summary:

The test substance was investigated in a bacterial reverse mutation (Ames) test according to OECD TG 471 on Salmonella typhimurium TA 1535, TA 100, TA, 1537, TA 98, and TA 102. The test substance was considered to be non-mutagenic without and with S9 mix in the initially performed plate incorporation as well as in the preincubation modification, performed as independent repeat.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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)
Version / remarks:
(1997)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
Stability in vehicle analytically confirmed.
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (Seromed, 12247 Berlin, Germany) with supplements
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from the liver of male Phenobarbital/ beta-Naphthoflavone induced Wistar rats.
Test concentrations with justification for top dose:
Experiment I: 12.5, 25, 50, 100, 200, 250 µg/ml with and without S9-mix
Experiment II: 6.3, 12. 5, 25, 50, 100, 200 µg/ml with and without S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethyl methanesulphonate, dimethylbenzanthracene
Remarks:
ethyl methanesulfonate used without S9-mix (150 µg/mL); dimethylbenzanthracene used with S9-mix (1.1 µg/mL).
Details on test system and experimental conditions:
METHOD OF APPLICATION: Two independent experiments I and II were performed.
24 h after cells were seeded in plastic culture flasks in complete medium (MEM with 10 % FCS), the medium was replaced with serum-free medium containing the test item either without or with S9-mix. After 4 h this medium was replaced with complete medium following two washing steps with "saline G". The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment.
Three days after treatment 1.5x10exp6 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3-5x10exp5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH-solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)

DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days

DETERMINATION OF CYTOTOXICITY: A pre-test on toxicity was performed. Toxicity of the test item is indicated by a reduction of the cloning efficiency. The highest concentration used (200 µg/mL) was limited by the solubility of the test item in DMSO and aqueous medium. Test item concentrations between 1.6 µg/mL and 200 µg/mL were used to evaluate toxicity in the presence and absence of metabolic activation (4 hours treatment). No relevant cytotoxic effect was observed up to the maximum concentration tested with and without metabolic activation following.
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A test item is classified as mutagenic
- if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
- there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.6-31.7 mutants per 10exp6 cells) a concentration related increase of the mutations within all experiments of this study was also taken into consideration.
Statistics:
A linear regresson (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using Systat 11 (Systat Software, Inc, 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.5. However, both, biological and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The cell cultures were evaluated at the following concentrations: Exp. I: 25, 50, 100, 200 and 250 µg/mL without and with S9-mix; Exp. II: 12.5, 25, 50, 100 and 200 µg/mL without and with S9-mix. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: The osmolality and pH-value were determined in the solvent control and the maximum concentration of the pre-experiment without metabolic activation. There was no relevant shift of osmolality and pH value of the medium even in the stock solution of the test item.
- Water solubility: The highest concentration used (200 µg/mL) was limited by the solubility of the test item in the solvent and in the medium.
- Precipitation: Precipitation of the test item in the medium was observed starting at 100 µg/mL.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No relevant toxic effects occurred up to the maximal concentration of 200 µg/mL
Executive summary:

The test substance was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476. The highest concentration used (200 µg/mL) was limited by the solubility in the solvent and in the medium. Precipitation in the medium was observed starting at 100 µg/mL. No relevant toxic effects occurred up to the maximal concentration of 200 µg/mL. Without and with S9 mix the test item induced no substantial and reproducible dose dependent increase of the mutant frequency. The positive controls ethyl methanesulfonate and dimethylbenzanthracene induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the S9 mix. Based on these results, the test substance is considered to be non-mutagenic in the V79/HPRT-assay.

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)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Stability in vehicle analytically confirmed.
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: PAA Ready Mix (PAA, Paching, Austria), consists of MEM, Earle, with supplements.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from the liver of Aroclor 1254 induced male Sprague Dawley rats.
Test concentrations with justification for top dose:
4 hours treatment: 0, 14, 28, 56, 112, 224 µg/mL without and with S9-mix
18 hours treatment: 0, 14, 28, 56, 112, 224 µg/mL without S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In this solvent the test item was soluble up to 83.33 mg/ml.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C, cyclophosphamide
Remarks:
mitomycin C used without S9-mix (0.1 µg/ml for 4 hour treatment and 0.03 µg/ml for 18 hour treatment); cyclophosphamide used with S9-mix (2 µg/ml).
Details on test system and experimental conditions:
PRETESTING: Cells were exposed with and without S9-mix for 4 hours to concentrations of 1-800 µg/mL and without S9-mix for 18 hours to concentrations of 14-224 µg/mL. As indicators of cytotoxic effects, numbers of surviving cells (survival index) were used.

TREATMENT RPOTOCOL FOR MAIN TESTING: The general protocol was similar to published procedures (e.g. Dean and Danford, in: Mutagenicity testing - a practical approach, (ed. Venitt and Parry) IRL Press, Oxford, 1984).
Chromosomes were prepared 18 and 30 hours (for 4 hour treatment) or 18 hours (for 18 hour treatment) after start of treatment with test substances. The treatment interval was 4 hours with and without metabolic activation and 18 hours without metabolic activation. In each experimental group cultures were set up in duplicate.

NUMBER OF CELLS EVALUATED: Chromosomes of approximately 200 metaphases per concentration, 100 metaphases from each of two parallel cultures, were examined. The classes of structural chromosome damage were defined and recorded by using essentially the terminology of Rieger and Michaelis (Die Chromosomenmutationen, VEB Gustav-Fischer Verlag, Jena, 1967). Both chromatid and chromosome-type aberrations were assessed.
Polyploid metaphases were recorded.

SPINDLE INHIBITOR (cytogenetic assays): 0.2 ml Colcemid-solution (40 µg/ml water) was added to each flask two hours prior to the end of the incubation period.

STAIN (for cytogenetic assays): 3 % Giemsa solution

DETERMINATION OF CYTOTOXICITY: was assessed in the pre-test as well as in the main-study.
- Method: Cell survival was determined in the presence and absence of S9 mix.

OTHER: Influence on pH and osmolality was assessed.
Evaluation criteria:
- An increased incidence of gaps of both types without concomitant increase of other aberration types was not considered as indication of a clastogenic effect.
- A test was considered positive, if there was a relevant and statistically significant increase in the aberration rate.
- A test was considered negative, if there was no such increase at any time interval.
- A test was also considered negative, if there were statistical significant values, which were, however, within the range of historical negative controls.
- A test was considered equivocal, if there was an increase of aberrant metaphases above the range of historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. A test was also considered equivocal, if its result was implausible.
Statistics:
Pair-wise comparison of treated and positive control groups to the respective solvent control group.
The numbers of metaphases with aberrations excluding gaps were compared. The statistical analysis followed the recommendations outlined by Richardson et al. (1989). The statistical analysis followed the recommendations outlined by Richardson et. al. (Analysis of data from in vitro cytogenetic assays in: Statistical evaluation of mutagenicity test data, (ed. Kirkland) Cambridge University Press, Cambridge, 1989). The one-sided chi2-test was used for the statistical evaluation.
A difference was considered to be significant, if the probability of error was below 5 %.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Based on the results of the survival index and the precipitation in the medium, the following concentrations were selected for reading: 56, 112 and 224 µg/mL, 4 hours treatment, both without and with S9-mix; 56, 112 and 224 µg/mL, 18 hours treatment, without S9-mix. None of the cultures treated with the test item in the absence or presence of S9 mix showed statistically significant or biologically relevant increases of numbers of metaphases with aberrations.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In the solubility test concentrations of up to 800 µg/mL did not change the pH in the medium.
- Effects of osmolality: The osmolality in the medium of the pre-test was not changed by concentrations of up to 800 µg/mL .
- Precipitation: Without and with S9 mix substance precipitation occurred in the medium at 224 µg/mL.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In comparison to the solvent control, the survival indices in the cultures treated with the test substance (up to the limit of solubility) were not relevantly reduced, both without and with S9-mix.
Executive summary:

The clastogenic potential of the test substance was evaluated in a chromosome aberration test on Chinese Hamster V79 cells according to OECD TG 473 with concentrations up to 224 µg/mL without and with S9-mix for the 4 -hour treatment period and without S9-mix for the 18 -hour treatment period. Without and with S9-mix cytotoxic effects were not observed up to 224 µg/mL after 4 hours treatment or after 18-hour treatment. Precipitation in the medium occurred at 224 µg/mL.

None of the cultures treated with the test item in the absence and in the presence of S9-mix showed biologically relevant or statistically significant increased numbers of aberrant metaphases, therefore the test substance is considered not to be clastogenic for mammalian cells in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The substance did not show genetic toxicity in two in vitro gene mutation assays according to OECD TG 471 and OECD TG 476 (Ames and HPRT, respectively) and in an in vitro chromosome aberration assay according to OECD TG 473, all conducted without and with a metabolic activating system.

Justification for classification or non-classification

No classification required for genetic toxicity according to Regulation (EC) No 1272/2008, Annex I.