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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A test article of low purity was tested in an Ames, Chromosomal Aberration and in a HPRT assay. All studies yielded negative results. New tests will be performed with high purity test material following ECHA Decision Number: CCH-D-2114440074-60-01/F.

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Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hprt
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/b-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
19.5, 39.1, 78.1, 156.3, 1250.0, 2500.0, 5000.0 mg/L (experiment 1; 4h, without S9)
25, 50, 100, 625, 1250, 2500, 5000 mg/L (experiment 2; 24h, without S9)
19.5, 39.1, 78.1, 156.3, 312, 1250, 2500 mg/L (experiment 1; 4h, with S9)
25, 50, 100, 200, 400, 600, 800 mg/L (experiment 2; 4h, with S9)
Vehicle / solvent:
Dimethylsulfoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9:300 μg/mL ethyl methanesulfonate, with S9: 20 μg/mL methylcholanthrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 - 24h
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium): 5 - 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: Population doubling time 12 - 16h

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should fall within our historical negative control data range of 0 – 15.95 mutants per 10exp6 clonable cells
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies (historical positive control data given in Appendix to study report)
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a dos-response relationship.
Statistics:
Due to the negative findings, a statistical evaluation was not carried out.
Species / strain:
Chinese hamster Ovary (CHO)
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: not influenced
- Effects of osmolality: not influenced
- Precipitation: test substance precipitation in culture medium at the end of treatment was observed at about 50 μg/mL and above

RANGE-FINDING/SCREENING STUDIES:
In the pretest for toxicity 5000 μg/mL was used as top concentration both with and without S9 mix at 4-hour exposure time and without S9 mix at 24-hour exposure time. The cloning efficiency 1 (survival) was determined as toxicity indicator for dose selection and various parameters were checked for all or at least for some selected doses. In the pretest the parameters pH value and osmolarity were not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, no test substance precipitation in the vehicle DMSO was observed in the stock solution (Test group: 5000 μg/mL). In culture medium test substance precipitation occurred at 78.1 μg/mL and above at all test conditions. After 4 and 24 hours treatment in the absence of S9 mix cytotoxicity indicated by reduced relative cloning efficiency of about or below 20% relative survival was observed at 2500 μg/mL and above. In addition, in the presence of S9 mix, clearly reduced relative cloning efficiency was observed after 4 hours treatment with 625 μg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutant frequencies at any concentration were close to the range of the concurrent vehicle control values and within the range of the historical negative control data. The mutation frequencies of the vehicle control groups were within the historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of mutant colonies induced by the positive control substances EMS and MCA clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control were observed in the 1st and 2nd Experiment in the presence of S9 mix at least in the highest applied concentrations. In both experimental parts in the absence of metabolic activation no distinct cytotoxicity was obtained. In detail, with S9 mix, there was a decrease in the number of colonies from 625 μg/mL (CE1 relative: 8.3%) onward in the 1st Experiment and from 400 μg/mL (CE1 relative: 17.6%) onward in the 2nd Experiment. The cell densities were distinctly reduced.
Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Executive summary:

The substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without

the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the

following doses were tested and the doses in bold type were evaluated in this study:

1st Experiment

without S9 mix (4-hour exposure period): 0; 19.5; 39.1; 78.1; 156.3; 1250.0; 2500.0; 5000.0 μg/mL

with S9 mix (4-hour exposure period): 0; 19.5; 39.1; 78.1; 156.3; 312.5; 625.0; 1250.0 μg/mL

2nd Experiment

without S9 mix (24-hour exposure period): 0; 25; 50; 100; 625; 1250; 2500; 5000 μg/mL

with S9 mix (4-hour exposure period): 0; 25; 50; 100; 200; 400; 600; 800 μg/mL

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations. Cytotoxicity indicated by reduced survival (CE1) of below 20% of control was observed in both experiments in the presence of metabolic activation only. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence 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:
1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
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:
other: Japanese Guidelines for Screening Toxicity Testings of Chemicals: Testing Methods for New Chemical Substances
Deviations:
no
Qualifier:
according to
Guideline:
other: Code of Federal Regulations, Title 40, Subpart F Genetic Toxicology
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Non-specific.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimal Essential Medium (MEM) supplemented with 10% fetal calf serum.
- Periodically checked for Mycoplasma contamination: Yes.
- Periodically checked for karyotype stability: Yes.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9.
Test concentrations with justification for top dose:
- 10, 30, 50, 100, 300, and 1000 µg/mL for cultures exposed for 4 hours (fixation interval of 18 hours) with metabolic activation and 18 hours without metabolic activation
- 50, 100, 300, and 1000 µg/mL for cultures exposed for 4 hours (fixation interval of 28 hours) with metabolic activation and 28 hours without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO) (1% (v/v) in culture medium)
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity to the cells.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
(Final Concentration: 600 µg/ml = 4.8 mM) Migrated to IUCLID6: (without metabolic activation).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
(Final Concentration: 0.71 µg/ml = 2.5 µM Migrated to IUCLID6: (with metabolic activation).
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium.

DURATION
- Exposure duration: 4 hours with metabolic activation, and 18 and 28 hours without metabolic activation.
- Expression time (cells in growth medium): 15.5 and 25.5 hours after the start of the treatment.
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 hours after the start of the treatment.

SPINDLE INHIBITOR (cytogenetic assays):
- Name: Colcemid
- Concentration: 0.2 µg/ml culture medium
- Time schedule for addition: 2.5 hours before harvest of cells (15.5 and 25.5 hours after start of exposure)

STAIN (for cytogenetic assays): Giemsa (E. Merck, D-64293 Darmstadt).

NUMBER OF REPLICATIONS: Two experiments with duplicate cell cultures/experiment

NUMBER OF CELLS EVALUATED: Per culture 100 metaphase plates were scored for structural chromosome aberrations. The mitotic index was determined in a sample of 1000 cells per culture of each test group.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index and cell count.

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

OTHER: Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates.
Evaluation criteria:
The chromosome aberration assay performed in our laboratory is considered acceptable if it meets the following criteria:

a) The number of structural aberrations found in the negative and/or solvent controls falls within the range of our historical laboratory control data: 0.00 % - 4.00 %.
b) The positive control substances should produce significant increases of the number of cells with structural chromosome aberrations.

A test article is classified mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosome aberrations or a significant and reproducible positive response for at least one of the test points. A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosome aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
Statistics:
Statistical significance was confirmed by means of the Fischer's exact test. However, both biological and statistical significance should be considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 1000 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 300 µg/mL for 18 hours; > 100 µg/mL for 28 hours
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 300 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 1000 µg/mL for 18 hours; >300 µg/mL for 28 hours
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>300 µg/mL for 18 hours; >1000 µg/mL for 28 hours
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant shift in the pH-value of culture medium was observed after adding the test article.
- Effects of osmolality: No relevant shift in the osmolarity of culture medium was observed after adding the test article.
- Precipitation: Precipitation of the test article in culture medium was observed 4 h after start of treatment at 300 µg/mL and above (in the absence and the presence of S9 mix) except in experiment II in the absence of S9 mix where precipitation occurred from 200 µg/ml and above).

RANGE-FINDING/SCREENING STUDIES:
Test article concentrations between 3 and 5000 μg/mL (with and without S9 mix) were chosen for the assessment of the cytotoxic potential. In the absence of S9 mix toxic effects indicated by reduced cell numbers below 50 % of control were observed after treatment with 100 μg/mL and above. In the presence of S9 mix reduced cell numbers below 50 % of control were observed after treatment with 1000 μg/mL and above.
- Precipitation: In the pre-test and in the cytogentic experiments precipitation of the test article was observed 4 hours after start of treatment at concentrations of 300 μg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
In both experiments, in the absence and presence of S9 mix the test article did not increase the frequency of cells carrying structural aberrations. The aberration rates of the cells after treatment with the test article (Experiment 1: 0.0 % - 2.5 %; Experiment 2: 0.0 % - 3.5 %) were in or near to the range of the solvent control values (Experiment I: 0.5 % - 2.5 %; Experiment II: 1.0 % - 3.5 %) and within the range of the historical control data: 0.0 % - 4.0 %. In conclusion, no significant or dose-related increases in the frequency of cells with aberrations or polyploid metaphases were seen.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix, the cell numbers were reduced to ≤ 50 % of control at this experimental point, except in experiment II at interval 28 h (63.5% of control). In the presence of S9 mix, the cell number was distinctly reduced at interval 28 hours after treatment with 1000 μg/mL (48.1 % of control). However, toxicity was observed only in the presence of test article precipitation. The above mentioned concentrations were the highest concentration which could be evaluated for cytogenetic damage. The cytogenetic evaluation of concentrations higher than 1000 µg/mL in the respective intervals (with and without S9 mix) was impossible due to strong test article induced toxic effects (reduced cell numbers and/or low metaphase numbers, partially paralleled by poor metaphase quality).

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test article (Experiment I: 1.5%-4.5%; Experiment II: 1.5 % - 6.0 %) as compared to the rates of the solvent controls (Experiment I: 1.0 % - 3.0 %; Experiment II: 4.0 % - 5.0 %).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: in preliminary test

Table 1. Evaluated experimental points after treatment with the test article

Preparation interval

Test No.

Without S9 mix concentrations in μg/mL

With S9 mix concentrations in μg/mL

18 hours

I

30.0

50.0

100.0P

300.0P

30.0

50.0

100.0P

1000.0P

18 hours

II

30.0

50.0

100.0

300.0P

30.0

50.0

100.0P

300.0P

28 hours

I

 

 

50.0

100.0P

 

 

50.0

300.0P

28 hours

II

 

 

100.0

300.0P

 

 

50.0

1000.0P

P = precipitation occurred. 

Conclusions:
Under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be non-mutagenic in this chromosome aberration test.
Executive summary:

The test article dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The chromosomes were prepared 18 h and 28 h after start of treatment with the test article. The treatment interval was 4 h with metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. In a pre-test, test article concentrations between 3 and 5000 µg/ml (with and without S9 mix) were chosen for the assessment of the cytotoxic potential. Toxic effects indicated by reduced cell numbers below 50 % of control were observed after treatment with 100 µg/ml and above in the absence and 1000 µg/ml and above in the presence of S9 mix. Precipitation of the test article in culture medium was observed 4 h after start of treatment at 300 µg/ml and above (in the absence and the presence of S9 mix). Following concentration ranges were applied in the cytogenetic experiments:

   without S9 mix  with S9 mix
 Experiment I 10 -1000 µg/ml   10 -1000 µg/ml
 Experiment II  10 -300 µg/ml  10 -1000 µg/ml

Test article precipitation occurred 4 h after start of treatment at 100 µg/ml and above (with and without S9 mix) except in experiment II in the absence of S9 mix (200 µg/ml and above). No relevant shift in the osmolarity or pH-value of culture medium was observed

after adding the test article. In the absence of S9 mix, in both experiments the mitotic indices and/or the cell numbers were distinctly reduced after treatment with 300 µg/ml. In the presence of S9 mix reduced mitotic indices and/ or cell numbers were observed after treatment with 300 µg/ml (exp. I, 28 h interval) or 1000 µg/ml (exp. I,18 h interval; exp. II, 28 h interval). Neither significant nor biologically relevant increases in the number of cells carrying structural chromosome aberrations were observed after treatment with the test article. In addition, no biologically relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be non-mutagenic in this chromosome aberration test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. Typhimurium: Histidine operon.
- E. Coli: trpE gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9 mix
Test concentrations with justification for top dose:
Concentration range in the main tests (with metabolic activation): 312.5, 625, 1250, 2500 and 5000 µg/plate
Concentration range in the main tests (without metabolic activation): 312.5, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
Solvent: Dimethylsulfoxide
- Justification for choice of solvent/vehicle: Prior to commencing testing the solubility of the test substance was assessed at 50 mg/ml in water and dimethyl sulphoxide. The test substance was immiscible with water but fully miscible with dimethyl sulphoxide. Therefore dimethyl sulphoxide was chosen as the solvent.
Untreated negative controls:
yes
Remarks:
(sterility controls)
Negative solvent / vehicle controls:
yes
Remarks:
(Dimethyl sulfoxide)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Remarks:
with and without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 3 days at 37°C.

NUMBER OF REPLICATIONS: 2 experiments á 3 plates per experiment.

DETERMINATION OF CYTOTOXICITY
- Method: reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

POSITIVE CONTROLS
without metabolic activation:
TA 1535: N-ethyl-N-nitro-N-nitrosoguanidine, 5 µg/plate
TA 100: N-ethyl-N-nitro-N-nitrosoguanidine, 3µg/plate
WP2 uvrA: N-ethyl-N-nitro-N-nitrosoguanidine, 2µg/plate
TA 1537: 9-aminoacridine, 80µg/plate
TA 98: 2-nitrofluorene,1 µg/plate

with metabolic activation for strains:
TA 1535 and TA 1537: 2-aminoanthracene: 2 µg/plate
TA 98: 2-aminoanthracene, 0.5 µg/plate
TA 100: 2-aminoanthracene, 1 µg/plate
WP2 uvrA: 2-aminoanthracene, 10 µg/plate
Evaluation criteria:
The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test substance is assessed by applying the following criteria:

(a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system.

(b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system.

Statistics:
No statistical analysis was performed.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test article in solution occurred with all strains from concentrations of 1250 µg/plate and above.

RANGE-FINDING/SCREENING STUDIES:
Four concentrations of test substance were assessed for toxicity using the five tester strains. The highest concentration was 50 mg/ml of test substance in the chosen solvent, which provided a final concentration of 5000 µg/plate. Three 10-fold serial dilutions of the highest concentration were also
tested. The chosen solvent, dimethyl sulphoxide, was used as the negative control. The test item was not toxic towards the tester strains. Therefore
5000 µg/plate was chosen as the top dose level in the mutation tests.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The test article shows no evidence of mutagenic activity in this bacterial system.
Executive summary:

In this in vitro assessment of the mutagenic potential of the test item, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and a tryptophan dependent mutant of Escherichia coli (WP2 uvr A) were exposed to the test substance, diluted in dimethyl sulphoxide which was also used as a negative control. Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats. In the preliminary toxicity test with dose levels of up to 5000 µg/plate no toxicity was observed. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 2500, 1250, 625 and 312.5 µg/plate. No evidence of mutagenic activity was seen at any dose in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the

metabolising activity of the liver preparations. It is concluded that, when tested in dimethyl sulphoxide, the test article shows no evidence of mutagenic activity in this bacterial system.

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Test material of low purity was tested in three genotoxicity assays, summarized below.

In vitro Gene Mutation - Bacteria Cells

Using a test procedure that complies with the requirements of OECD TG 471, the test article was tested in a Bacterial Reverse Mutation Test using the plate incorporation method. Following strains were used: Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 as well as Escherichia coli (WP2 uvr A). In each of two runs, the test article was tested in triplicates at concentrations of 0; 312.5; 625; 1,250; 2,500 and 5,000 µg/plate in the presence and absence of Aroclor-induced rat liver S-9 mix. The vehicle was dimethyl sulfoxide. Cytotoxicity was absent up to 5000 µg/plate. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. There was no evidence of induced mutant colonies over background up to the maximum dose of 5000 µg/ plate both in the presence and absence of metabolic activation.

 

In vitro Gene Mutation - Mammalian Cells

The substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro . The study followed OECD testing guideline 476 and the principles of GLP. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested and evaluated in this study:

1st Experiment

without S9 mix (4-h exposure): 0; 19.5; 39.1; 78.1; 156.3; 1,250.0; 2,500.0; 5,000.0 µg/mL

with S9 mix (4-h exposure): 0; 19.5; 39.1; 78.1; 156.3; 312.5; 625.0; 1,250.0 µg/mL

2nd Experiment

without S9 mix (24-h exposure): 0; 25; 50; 100; 625; 1,250; 2,500; 5,000 µg/mL

with S9 mix (4-h exposure): 0; 25; 50; 100; 200 µg/mL

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations. Cytotoxicity indicated by reduced survival of below 20% of control was observed in both experiments in the presence of metabolic activation only. On the basis of the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.

 

In Vitro Cytogenetics

In a mammalian in-vitro cytogenetic assay (chromosome aberration) that complies with the test requirements of OECD TG 473, Chinese hamster lung fibroblasts (V79) were exposed to the test article in dimethyl sulfoxide (1% final concentration) at concentrations of 10; 30; 50; 100; 300; and 1,000 µg/mL for 4 hours (fixation interval of 18 hours) with metabolic activation and 18 hours without metabolic activation. Another set of cultures were exposed to concentrations of 50; 100; 300; and 1,000 µg/mL for 4 hours (fixation interval of 28 hours) with metabolic activation and 28 hours without metabolic activation. Cytotoxic effects were evident at 100 µg/mL and above. However, toxicity was observed only in the presence of test article precipitation (> 100 µg/mL). Positive controls induced the appropriate response. Per culture, 100 metaphases were scored for structural chromosome aberrations. There was no evidence of chromosome aberration induced over background. Both in the absence and presence of S9-mix, the test article did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations or the number of polyploid cells.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation 1272/2008. Based on the criteria laid down in Regulation (EC) No.1272/2008, classification for genetic toxicity is not warranted.