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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test (OECD TG 471): negative

in vitro HPRT Test (OECD TG 476): negative

in vitro chromosome aberration assay (OECD TG 473): negative

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:
July 2009
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 the solvent was analytically proved.
Target gene:
mutant histidine gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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 substance formed clear colorless solutions in DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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);
Testings 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.
Statistics:
no statistics perfomed; evaluation based on criteria mentioned above
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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: No indication of a bacteriotoxic effect of the test substance at doses of up to and including 5000 µg per plate.
Executive summary:

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

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb to May 2009
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 the solvent was analytically proved.
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: PAA Ready Mix (PAA, Paching, Austria), consists of MEM, Earle, with supplements.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
To keep the number of spontaneous 6-TG resistant mutants at a low level, cell cultures were subcloned at least every two weeks.
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from the liver of male Aroclor 1254 induced Sprague-Dawley rats.
Test concentrations with justification for top dose:
Both for 1st experiment and independent repeat: 30, 80, 120, 240, 480, 960 µg/mL with and without S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In this solvent the test substance was soluble up to 200 mg/mI.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethyl methanesulphonate, dimethylbenzanthracene
Remarks:
ethyl methanesulfonate used without S9-mix (900 µg/mL); dimethylbenzanthracene used with S9-mix (20 µg/mL).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; In all parts of this study incubation was performed at 37 °C in a humidified atmosphere with about 5 % CO2.
The method is based on the publication of Myhr and DiPaolo (Cancer Res. 38, 2539-2543, 1978).
Treatment Protocol without MetabolicActivation: Exponentially growing V79 cells were plated in culture medium at a final volume of 20 ml in two 75 cm² flasks per concentration (4x10exp6 per flask) including all control groups. After attachment (16-24 hours later), the cells were exposed for 5 hours in 20 ml culture medium with reduced serum content (2 %). The corresponding controls were incubated under the same conditions. Thereafter, cell monolayers were washed with PBS, trypsinized and replated in 20 ml culture medium using 1.5x10exp6 cells per 75 cm² flask and in 5 ml culture medium using 200 cells per Petri dish (diameter 60 mm). Per culture one flask and 3 Petri dishes ware used. The Petri dishes were incubated (normally 6 days) to allow colony development and to determine the cytotoxicity associated with each test substance directly after treatment ("Survival to Treatment").
Cells in 75 cm² flasks were incubated to permit growth and expression of induced mutations. Cells were subcultured (= count 1, normally after 3 days) by reseeding 1.5x10exp6 cells into 20 ml medium in 75 cm² flasks. At the end of the expression period (= count 2, normally a total of 6 days), cultures were reseeded in Petri dishes (diameter 100 mm) at 3x10exp5 cells per dish (8 dishes per culture) in 20 ml culture medium without
hypoxanthine but containing 10 µg/ml 6-TG for selection of mutants. In addition, 200 cells per dish (diameter 60 mm, 3 dishes per culture) were seeded in 5 ml culture medium to determine the absolute cloning efficiency for each concentration. After incubation for 6 to 8 days, the colonies were fixed, stained with Giemsa and counted to determine the number of 6-TG resistant colonies in the 'mutation assay dishes and the number of colonies in the cloning efficiency dishes.
At least two trials will be performed.
Treatment Protocol with Metabolic Activation: The activation assay was performed independently. The procedure was identical to the nonactivation assay except for the addition of 59 mix. In these experiments 19 instead of 20 ml culture medium and additionally 1 ml of 59 mix were added to the
flasks for the treatment period, resulting in a concentration of 5 % S9-mix in the cultures. The number of 6-TG resistant mutants and viability were determined as described above.

DURATION
- Exposure duration: 5 hours.
- Expression time (cells in growth medium): normally 6 days
- Selection time (if incubation with a selection agent): 6 to 8 days, then colonies were fixed.

DETERMINATION OF CYTOTOXICITY: A preliminary cytotoxicity test was conducted without and with metabolic activation using concentrations ranging from 7.8 µg/mL to 2000 µg/ml. Result: No cytotoxic effects above 80 % were induced by the used concentrations in the pretest. However, precipitation of the test item in the culture medium started at 500 µg/ml without S9 mix and at 1000 µg/ml with S9 mix. Due to these findings, the test concentrations for the main testing ranged from 30 µg/ml to 960 µg/ml both without and with metabolic activation
- Method: Cytotoxicity was expressed by comparison of colonies in treated cultures versus negative control cultures (relative cloning efficiency).
Evaluation criteria:
Mutant frequencies will only be used for assessment, if at least 5 dishes per culture were available and relative survival to treatment, relative population growth and absolute cloning efficiency were 10 % or greater.
A trial will be considered positive if a concentration-related and in parallel cultures reproducible increase in mutant frequencies is observed. To be relevant, the increase in mutant frequencies should be at least two to three times that of the highest negative or negative control value observed in the respective trial. If this result can be reproduced in a second trial, the test substance is considered to be mutagenic.
Despite these criteria, a positive result will only be considered relevant, if no significant change in osmolality compared to the negative control can be observed. Otherwise, unphysiological culture conditions may be the reason for the positive result.
A test substance will be judged as equivocal if there is no strictly concentration related increase in mutation frequencies but if one or more concentrations induce a reproducible and biologically relevant increase in mutant frequencies in all trials.
An assay will be considered negative if no reproducible and relevant increases of mutant frequencies were observed.
However, these criteria may be overruled by good scientific judgement.
Statistics:
The statistical analysis relies on the mutant frequencies which are submitted to a weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights. Mutant frequencies based on less than 5 plates and/or on a relative survival to treatment and/or a relative population growth and/or an absolute cloning efficiency below 10 % are not included in the statistical analysis.
The two mutant frequency values obtained per group are, although somewhat related, considered as independent measurements thus increasing the power of the statistical tests applied. Since the protocol of the HPRT assay requires at least two independent trials, the overall analysis without respectively with activation is the most important one for classifying substances into mutagens and non-mutagens. However, separate analyses will be run for each trial in order to examine the consistency of the results.
All acceptable groups are included in the weighted analysis of variance followed by pairwise comparisons to the negative control on a nominal significance level of a = 0.05 using the Dunnett test. The regression analysis part is performed on the basis of the actual concentrations thereby omitting the positive, untreated and negative controls. If there is a significant concentration related increase of the mutant frequency (a = 0.05) in the main analysis the highest concentration will be dropped and the analysis will be repeated. This procedure will be repeated until p > 0.05. In that way eliminated concentrations are flagged correspondingly.
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:
valid
Positive controls validity:
valid
Additional information on results:
Mutation Assay without Metabolic Activation: Relevant test substance induced increases in mutant frequencies could not be found. In addition, the overall statistical analysis reveals no statistically significant increases.
Mutation Assay with Metabolic Activation: The test substance induced no relevant increases in mutant frequencies. In addition, the overall statistical analysis reveals no statistically significant increase.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
For the test substance treated cultures no cytotoxic effects of 80 % to 90 % were induced. However, the substance was tested up to its limits of solubility in the solvent.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Concentrations of up to 2000 µg/ml test substance did not change the pH in the medium of the pre-test.
- Effects of osmolality: The osmolality in the medium of the pre-test was not changed by concentrations of up to 2000 µg/ml.
- Precipitation: Without S9 mix substance precipitation occurred in the medium at the concentration 480 µg/ml and above. With 59 mix precipitation was noted at the concentration 960 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 in concentrations up to and including 960 µg/ml, without and with a metabolising system (S9 mix). The substance induced no decreases in survival to treatment or in relative population growth, without and with S9 mix. However, precipitation of the substance in the culture medium was observed without S9 mix at 480 µg/ml and above and with S9 mix at 960 µg/ml. Without and with S9 mix there was no biologically relevant increase in mutant frequency above that of the negative controls. The positive controls ethyl methanesulfonate and dimethylbenzanthracene induced clear mutagenic effects and demonstrated the sensitivity of the test system and the activity of the S9 mix. Based on these results, the substance is considered to be non-mutagenic in the V79/HPRT.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan - June 2009
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
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: without S9-mix: 300, 600, 900 µg/mL; with S9-mix: 500, 1000, 2000 µg/mL
18 hours treatment: without S9-mix: 50, 100, 200, 500, 800 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Using sonication for 5 minutes the test substance was soluble up to 200 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
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 1-2000 µg/mL and without S9-mix for 18 hours to concentrations of 1-900 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. (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:
cytotoxicity
Remarks:
at 900 µg/mL and above after 4 hours treatment and at 100 µg/mL and above after 18 hours treatment without S9-mix (precipitation at 500 µg/mL); at 2000 µ/mL after 4 hours treatment with S9-mix (precipitation occurred)
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, only the concentrations 100, 200 and 500 µg/mL of the 18 hours treatments were selected for reading. None of the cultures treated with the test substance in the absence or presence of S9 mix showed biologically relevant or statistically significant increased numbers of aberrant metaphases.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In the solubility test concentrations of up to 2000 µg/mL test substance 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 2000 µg/mL.
- Precipitation: Precipitation in the medium occurred at 500 µg/mL and above (without S9 mix) and at 2000 µg/mL (with S9 mix).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Without S9 mix cytotoxic effects were observed at 900 µg/mL after 4 hours treatment and at 100 µg/mL and above after 18 hours treatment. With S9 mix cytotoxic effects were observed at 2000 µg/mL after 4 hours treatment.


Executive summary:

The clastogenic potential of the test substance was evaluated in a chromosome aberration test with Chinese hamster V79 cells according to OECD TG 473. In that for the 4 -hour treatment period test concentrations up to 900 µg/mL and 2000 µg/mL without and with S9-mix were used, respectively, and, for the 18 -hour continuous treatment up to 800 µg/mL without S9 mix. Without S9-mix cytotoxic effects were observed at 900 µg/mL after 4 hours treatment and at 100 µg/ml and above after 18 hours treatment. With S9 mix cytotoxic effects were observed at 2000 µg/mL after 4 hours treatment. Precipitation in the medium occurred at 500 µg/mL and above without S9 mix and at 2000 µg/mL with S9 mix.

None of the chromosome preparations assessed showed biologically relevant or statistically significant increased numbers of aberrant metaphases, therefore the substance is not considered 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 the respective vitro gene mutation assays in bacteria (OECD TG 471) and in mammalian cells (OECD TG 476) and in an in vitro chromosome aberration assay (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.