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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was not mutagenic in the in-vitro bacterial reversed mutation assay (Ames test).

The chromosome aberration test was valid and the substance is not clastogenic in human lymphocytes under the experimental conditions described in this report.

The substance is mutagenic in the TK mutation test system (OECD 476) under the experimental conditions (with and without S9 mix, up to 2000 µg/ml, where cytotoxicity was observed).

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
At the time the study was performed, no Guideline was compulsory.The study was performed according to the method as described by Ames, B.N. (1975), Mutation Res.31, 347-364.
This method used is widely comparable to OECD 471/EC B. 13/14
Deviations:
yes
Remarks:
No E. coli strain or, alternatively, S. typhymurium TA 102 strain was tested, however, the type and the nature of the 5 strains tested is considered to be sufficient to detect a mutagenic potential of the test item.
GLP compliance:
no
Remarks:
The study was performed under the control of a quality assurance unit similar to GLP.
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0.8, 4, 20, 100 and 500 µg/plate
Selected based on results from preliminary toxicity test with 5µg to 10 mg.
Positive controls:
yes
Positive control substance:
9-aminoacridine
benzo(a)pyrene
other: N-methyl-N’-nitro-N-nitrosoguanidine, 2-aminoanthracene, 2-Nitroflurane
Details on test system and experimental conditions:
Way of application: An aliquot (0.1 mL) of each test substance concentration was added to a sterile tube containing molten, histidine-deficient agar and bacterial suspension and maintained at 45°C. 0.5 mL rat liver microsomal preparation (S-9 mix) was added where appropriate. The contents of the tubes were poured onto plates containing solidified minimal medium (20 mL). All plates were prepared in duplicate and incubated at 37°C for 48 hours.
Pre-incubation time: 48 hours
Other modifications: None
Evaluation criteria:
Total revertant colonies on nutrient plates were counted
Key result
Species / strain:
S. typhimurium, other: all strains tested
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
The test substance was not mutagenic in the in-vitro bacterial reversed mutation assay.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human peripheral blood
Details on mammalian cell type (if applicable):
- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine
(Gibco), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Gibco),
L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively), sodium bicarbonate (1.2 g/I) and 30 U/ml heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 100, 333, 1000, 3330 and 5000 µg/ml

First cytogenetic test (1):
Without and with S9-mix, 3 h exposure time, 24 h fixation time: 1000, 1500, 1750, 2000, 2250 and 2500 µg/ml

Repeat of first cytogenetic test (1A):
Without and withS9-mix, 3 h exposure; 24 h fixation: 1000, 1150, 1250, 1400, 1500 and 1650 µg/ml

Second cytogenetic test (2):
Without S9-mix 24 h exposure time, 24 h fixation time: 28, 33, 56, 75, 100 and 133 μg/ml
Without S9-mix 24 h exposure time, 48 h fixation time: 75, 100, 133, 156, 200 and 250 μg/ml
With S9-mix, 3 h exposure time, 48 h fixation time: 1000, 1150, 1250, 1350, 1400 and 1450 μg/ml

Repeat of second cytogenetic test (2A):
Without S9-mix 24 h exposure time, 24 h fixation time: 56, 75, 100, 150, 175, 200, 220, 250 and 300 μg/ml
With S9-mix, 3 h exposure time, 48 h fixation time: 1400, 1500, 1525, 1550, 1575, 1600, 1625 and 1650 μg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was soluble in culture medium and this has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.
Key result
Species / strain:
lymphocytes: lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

First cytogenetic assay

In the absence of S9-mix, the substance induced a statistically significant increase in the number of cells with chromosome aberrations at the two highest concentrations tested, both when gaps were included and excluded. However, the increase in the number of cells with chromosome aberrations was not dose dependent and the number of aberrant cells was still well within our historical control data range. In addition, the pH at the concentrations tested (1000, 1150 and 1400μg/ml) was 6.85, 6.74 and 6.67 respectively, indicating that the aberrations are observed at concentrations where non-physiological conditions might occur. Therefore, the increase was considered not biologically relevant.

In the presence of S9-mix, the substance induced a statistically significant increase in the number of cells with chromosome aberrations, when gaps were excluded only, at the highest concentration tested (i.e. 1400μg/ml). Since this increase was not dose related, the number of cells with chromosome aberrations was still well within our historical control data range and the increase was observed at a concentration with non-physiological test conditions (i.e. pH was 6.67), it was considered not biologically relevant.

 

Second cytogenetic assay

In the absence of S9-mix, at the 24 h continuous exposure time, the substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

In the absence of S9-mix, at the 48 h continuous exposure time, the substance induced a statistically significant increase in the number of cells with chromosome aberrations both when gaps were included and excluded at all concentrations tested. However, the type of aberrations observed was only breaks, the number of aberrant cells was still well within our historical control data range and did not increase at higher concentrations (i.e.is not dose-dependent).

In addition, the number of aberrant cells reached statistically significance only because of the low number of aberrant cells in the controls (0 out of 200, both when gaps were included and excluded). Therefore, this increase was considered to be not biologically relevant.

Conclusions:
The test was valid and the substance is not clastogenic in human lymphocytes under the experimental conditions described in this report.
Executive summary:

The effects of the substance on the number of chromosome aberrations in cultured peripheral human lymphocytes was observed in a study according to OECD 473 with and without metabolic activation system (Aroclor-1254 induced rat liver S9-mix).

In the first cytogenetic assay, the substance was tested up to 1400 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. Appropriate toxicity was reached at this dose level.

In the second cytogenetic assay, the substance was tested up to 200 μg/ml for a 24 and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. In the presence of 1.8% (v/v) S9-fraction the substance was tested up to 1650 μg/ml for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at these dose levels.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The test was valid and the substance is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Dose range finding test
100 to 5000 μg/ml; No survival at 3330 and 5000 μg/ml

First mutagenicity test

Without S9-mix : 10, 50, 100, 250, 500, 750, 1000, 1250, 1500, 2000 and 2500 μg/ml exposure
medium.
With 59-mix : 10, 50, 100, 250, 500, 750, 1000, 1250, 1500, 2000 and 2500 μg/ml exposure medium.

Second mutagenicity test
Without S9-mix: 100, 250, 500, 750, 1000, 1175, 1350, 1500, 1750, 2000, 2250 and 2500 μg/ml exposure medium.
With 59-mix : 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250 and 2500 μg/ml exposure medium
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Solvent (ethanol) treatment groups were used as the vehicle controls.
- Justification for choice of solvent/vehicle: Suitable for dosing at the required concentration.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
ethylmethanesulphonate
Evaluation criteria:
A test substance was considered positive (mutagenic) in the mutation assay if:
a) lt induced at least a three-fold increase in the mutant frequency compared to the solvent control in a dose-dependent manner; or
b) In case a positive result was repeated, the positive response was reproducible in at least one repeated experiment.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations showed a mutant frequency of at least three-fold compared to the solvent control.
b) The results was confirmed in an independently repeated test.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
The experimental results were not subjected to statistical analysis.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Mutant frequency at the TK-locus

In the absence of S9-mix, the substance showed a 4.2-fold, dose-related, increase in the mutantfrequencyat the TK-locus. However, this increasewasjustwithout our historical controldatarange [0.5- and 7.0 x 10E5 (mean2.9 x 10E5)]. Verification of this result was performed in the second experiment, in which the substance showed a more than five-fold, dose-related, increase in the mutant frequency at the TK-locus.

In the presence of S9-mix, the substance showed 2.9- and 2.8-fold, dose-related, increases in the mutant frequency at the TK-locus.

 

Mutant frequency of the small and large mutant colonies

In the absence of S9-mix,the substanceshowed 50- and 18-fold increases in the mutant frequency of the small colonies in the first and second experiment, respectively and 3.7- and 4.2-fold increases in the mutant frequency of the large colonies.

In the presence of S9-mix,the substanceshowed 5.8- and 4.5-fold increases in the mutant frequency of the small colonies inthefirst and second experiment, respectively and 2.6- and 2.5-fold increases in the mutant frequency of the large colonies.

Conclusions:
In conclusion, the substance is mutagenic in the TK mutation test system (OECD 476) under the experimental conditions (with and without S9 mix, up to 2000 µg/ml, where cytotoxicity was observed).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the data available the substance is not classified according to Regulation 1272/2008/EEC (CLP).