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Genetic toxicity in vitro

Description of key information

Ames test, OECD TG 471, negative, Hertner 1995

Mammalian cell gene mutation test, OECD TG 476, negative, Ogorek 1996

Chromosome aberration test, OECD TG 473, negative, Zeugin 1996

All available data were assessed according to the current standard of the genotoxicity testing strategy. Studies falling within this strategy and representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The available data are considered sufficient to demonstrate that the test substance is not genotoxic.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Sep 1995 to 26 Sep 1995
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:
1983
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Version / remarks:
1987
Qualifier:
according to
Guideline:
other: 84/449/EEC B.14
Version / remarks:
1992
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
MHW Japan, 1986
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his- (S. typhimurium), trp- (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 mix
Test concentrations with justification for top dose:
Preliminary Cytotoxicity Assay (with and without metabolic activation): 20.58, 61.73, 185.19, 555.56, 1666.67, 5000.00 µg/plate
Bacterial Assay (with and without metabolic activation): 5000, 2500, 1250, 625 and 312.5 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
cyclophosphamide
mitomycin C
other: 2-Aminoanthracene (2-AA); 4-Nitroquinoline (4-NQ)
Details on test system and experimental conditions:
PRELIMINARY CYTOTOXICITY ASSAY: A range finding test was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA with and without metabolic activation at six concentrations (20.58 to 5000.0 µg/plate) of the test substance and one negative control. The plates were inverted and incubated for about 48 hours at 37±1.5°C in darkness. After incubation they were evaluated by counting the colonies and determining the background lawn. One plate per concentration and negative control was used.

METHOD OF APPLICATION: in agar (plate incorporation);

PROTOCOL:
- Bacterial cultures were prepared from frozen stocks and grown in liquid nutrient broth medium by incubating overnight for 8 hours. 0.1 mL of the overnight cultures were mixed with 2 mL of top agar, either 0.5 ML of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 mL of the activation mixture (experiments with activation) and 0.1 mL of a solution of the test substance, the positive control or the solvent and poured on minimal agar in Petri dishes. Each Petri dish contained about 20.0 mL of minimal agar. The top agar was composed of 0.6% agar and 0.6% NaCl. In the experiment with Salmonella the top agar was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM (+)biotin dissolved in water. In the experiment with E. coli it was supplemented with 10% of 0.5 mM L-tryptophan dissolved in water.
- The test substance (purity 98.6%) in DMSO was tested on five histidine-auxotrophic strains of Salmonella typhimurium and one tryptophan-auxotrophic strain of Escherichia coli, by plate incorporation at five concentrations (312.5 to 5000.0 µg/plate). Two independent assays were performed, both experiments with and without S9 metabolic activation. After preparation, the plates were inverted and incubated for about 48 hours at 37±1.5°C and evaluated by colony counting and determining the background lawn. Concurrent strain-specific mutagens were applied to all strains as positive controls in both experiments.
- For all strains triplicate plates were used for all test substance and positive control treatments. For solvent controls 5 plates were used.

Evaluation criteria:
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. A positive response in a (valid) individual experiment is achieved when one or both of the following criteria are met:
- At least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: TA98, TA1535, TA1537, E. coli WP2 uvrA.
- A reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least factor of 1.5 for strains TA100 or TA102
- Generally a concentration-related effect should be demonstrable.
Statistics:
None.
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA102, TA1535 and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY CYTOGENICITY ASSAY: Normal background growth was observed with both strains tested. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000.0 µg/plate with and 5000.0 µg/plate without metabolic activation.

MUTAGENICITY ASSAY: In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced with increasing concentration. Therefore, the test substance exerted no toxic effect on the growth of the bacteria. The positive controls produced a marked increase of the number of revertant colonies.
Conclusions:
In this GLP compliant study, performed according to OECD TG 471, the test substance and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli
Executive summary:

In a reverse gene mutation assay in bacteria, the test substance (purity 98.6%) in dimethylsulphoxide (DMSO) was tested on five histidine-auxotrophic strains (TA98, TA100, TA102, TA1535 and TA1537) of Salmonella typhimurium and on the tryptophan-auxotrophic strain WP2uvrA of Escherichia coli, by plate incorporation, at concentrations of 0 (solvent control), 312.5, 625, 1250, 2500 and 5000 µg/plate. Two independent assays were performed, both experiments with and without S9 metabolic activation (S9 fraction from Arochlor induced rat liver). After preparation, the plates were inverted and incubated for about 48 hours at 37±1.5°C and evaluated by colony counting and determining the background lawn. Concurrent strain-specific mutagens were applied to all strains as positive controls in both experiments. In the range finding test normal background growth occurred with both strains, with or without metabolic activation and no appreciable cytotoxicity was observed at 5000 µg/plate, the highest concentration evaluated. The test substance did not increase the number of revertants in any of the strains used, with or without metabolic activation, in either experiment when compared to the vehicle controls. There was no evidence of toxicity to the bacteria at any concentration, in either experiment. The positive controls produced a marked increase of the number of revertant colonies. The test substance and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used in the study.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Feb 1996 to 03 Jun 1996
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:
1983
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1992
Qualifier:
according to
Guideline:
EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)
Version / remarks:
1987
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
MAFF Japan 1985
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
MEDIA USED
- Type and identity of media: Nutrient Nature F-12 supplemented with 10% foetal calf serum + Penicillin/Streptomycin 100 units/mL/100 µg/mL
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S-9 mix
Test concentrations with justification for top dose:
Absence of S-9 mix
- Original study, Experiment 1 (21 h treatment): 35.47, 70.94, 141.88, 283.75, 567.5, 1135, 2270, 4545 µg/mL
- Confirmatory study, Experiment 1 (21 h treatment): 212.81, 283.75, 425.63, 567.5, 851.25, 1135, 1702.5, 2270 µg/mL
- Confirmatory study, Experiment 3 (45 h treatment): 212.81, 283.75, 425.63, 567.5, 851.25, 1135, 1702.5, 2270 µg/mL

Presence of S-9 mix
- Original study, Experiment 2 (3 h treatment / 18 h recovery): 35.47, 70.94, 141.88, 283.75, 567.5, 1135, 2270, 4545 µg/mL
- Confirmatory study, Experiment 2 (3 h treatment / 18 h recovery): 425.63, 567.5, 851.25, 1135, 1702.5, 2270, 3405, 4540 µg/mL
- Confirmatory study, Experiment 4 (3 h treatment / 42 h recovery): 425.63, 567.5, 851.25, 1135, 1702.5, 2270, 3405, 4540 µg/mL

JUSTIFICATION FOR HIGHEST CONCENTRATION: Final concentrations higher than 4540.00 µg/mL of culture medium could not be achieved due to solubility limitations. Slight cytotoxicity was observed at the highest concentration.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO 10 µL/mL, 1%
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration without S-9 mix: 21 and 45 h treatment time
- Exposure duration with S9-mix: 3 h treatment time

NUMBER OF REPLICATIONS: quadruplicates

SPINDLE INHIBITION
- INHIBITOR (cytogenetic assays): Colcemid 0.4 µg/mL
- ADMINISTRATION TIME: 2 h (before cell harvest)

CELL HARVEST TIME AFTER TERMINATION OF TREATMENT:
- without S-9 mix (21 hour treatment): 0h
- with S-9 mix (3 hour treatment): 18 h
- without S-9 mix (21 hour treatment): 0 h
- with S-9 mix (3 hour treatment): 18 h
- without S-9 mix (45 hour treatment): 0 h
- with S-9 mix (3 hour treatment): 42 h

SELECTION OF CONCENTRATIONS FOR CHROMOSOME ANALYSIS: The highest concentration used or the lowest concentration which suppresses mitotic activity by approximately 50-80% compared to the control group was selected as the highest for the analysis of chromosome aberrations together with two lower concentrations. For the determination of the mitotic index (M.I.) the preparations from the various cultures were examined first, uncoded. The percentages of mitotic suppression in comparison with the controls were evaluated by counting at least 2000 cells from one slide each of the treatment groups and the negative control group. The determination of the mitotic coefficient was performed for each experiment separately. From the results of corresponding original run, eight suitable concentrations were determined for the experiments of the confirmatory study.

SCORING OF THE SLIDES: Prior to analysis the selected slides were coded, likewise the cultures treated with the vehicle alone as well as the positive control. Whenever possible 200 well spread metaphase figures with 17 to 21 centromeres from two cultures (100 metaphases per replicate culture) in the vehicle control and in the treated groups were scored. At least fifty metaphases were scored in the positive controls (25 per replicate culture). The slides were examined for the following structural aberrations:
- specific aberrations (chromatid and chromosome deletions (including breaks, deletions and fragments), chromatid exchanges (including triradials, quadriradials, endfusions, acentric rings), chromosome exchanges (including dicentrics, polycentrics, centric and acentric rings).
- multiple aberrations: metaphases containing more than 10 aberrations of different types or more than 5 aberrations of one particular type (excluding gaps).
- unspecific aberrations: gaps (chromatid- and chromosome-)
In addition the frequency of polyploid metaphases (multiples of ‘2n’, >30 centromers, including endoreduplication figures) was recorded.

The coordinates of all metaphases were recorded using a computerised coordinate reading system.
Evaluation criteria:
CRITERIA FOR A POSITIVE RESPONSE
The test substance is generally considered to be active in the Chinese Hamster cells if the following conditions are met:
- The percentage of metaphases containing specific aberrations in a treatment group is higher than 6.0 (based on historical negative control range) and differs statistically significantly from the respective value of the negative control.
- A concentration-related response should be demonstrable.

CRITERIA FOR A NEGATIVE RESPONSE
The test substance is generally considered to be inactive in the Chinese Hamster cells if the following conditions are met:
- The percentage of metaphases containing specific aberrations in all treatment groups is less than or equal to 6.0 (based on historical negative control range) and does not differ statistically significantly from the respective value of the negative control.
Statistics:
In the preliminary tests the data were assessed for flask effects (dependence of cells within each culture) using a chi-squared test. The nonsignificant result of this test means there is no substantial evidence to conclude a flask effect (although a flask effect still might exist). Accordingly, a chi-squared test for trend was performed modelling all cells in a given experiment as independent. That is, the individual cell is taken as the experimental unit. Consequently the power of the test is substantially increased.
Key result
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:
valid
Positive controls validity:
valid
Additional information on results:
- The incidence of specific chromosomal aberrations in the test substance -treated groups, at all concentrations at the 21 hour sampling interval, both with and without metabolic activation, were not significantly different from negative control values. At the 45 hour sampling interval, with metabolic activation, 0% metaphases with specific chromosomal aberrations was scored in the negative control cultures. Therefore, a statistically significant increase in the incidence of cells with specific chromosomal aberrations (1.5%) occurred at a concentration of 3405.0 µg/mL. This value of 1.5% is within the historical control range and does not fulfil the criteria for a positive response.
- Non-specific chromosomal aberrations (chromatid gaps) were encountered in all experiments within the generally observed frequency.
- Treatment of CHO cells with the positive control materials produced a high incidence of specific chromosomal aberrations in both experiments, demonstrating the sensitivity of the test system.

Table: Mitotic index (% of control) of CHO cells

Original study

Confirmatory study

Experiment

1

2

Experiment

1

3

Experiment

2

4

S-9

-

+

-

+

Treatment (h)

21

3

Treatment (h)

21

45

Treatment (h)

3

3

Recovery (h)

0

18

Recovery (h)

0

0

Recovery (h)

18

42

test substance tech. (µg/mL)

 

 

test substance tech. (µg/mL)

 

 

test substance tech. (µg/mL)

 

 

4540.0

0.00

9.20

2270.0

4.95

 

4540.0

10.40

11.50

2270.0

1.50

11.90

1702.5

7.00

 

3405.0

11.15

14.15

1135.0

9.10

13.05

1135.0

8.40

2.40

2270.05

11.90

17.15

567.5

10.80

14.40

851.25

9.90

9.30

1702.5

b

b

283.75

11.30

a

567.5

10.25

10.35

1135.0

b

b

141.88

a

a

425.63

a

11.25

851.25

b

b

70.94

a

a

283.75

a

11.90

567.5

b

b

35.47

a

a

212.81

a

a

425.63

14.10

18.40

solvent control

11.8

14.8

solvent control

11.25

13.35

 

13.0

16.05

Concentrations with underlined mitotic index were used for chromosome analysis

a         When 3 subsequent concentrations with a frequency of ≥70% mitosis in relation to the solvent control are found, the evaluation of the lower concentration is omitted.

b         When the 3 highest concentrations show a frequency of ≥70% mitosis in relation to the solvent control, the evaluation of the lower concentrations, with exception of the lowest, is omitted.

ANALYSIS OF TEST SUBSTANCE:

The concentration values found were 125.2% and 114.0% of the calculated concentrations, thus indicating a sufficient stability of the test substance in the vehicle. Since the value found at the lowest concentrated stock solution used in the original mutagenicity test was slightly higher than the calculated concentration, the respective reserve samples and highest concentrated solution were analysed in addition. The values found were 120.4% and 112.3% respectively. The slight increased value at the lowest concentration has no influence on the test results.

Conclusions:
In this GLP compliant study, performed according to OECD TG 473, there was no evidence of a clastogenic effect of the test substance in in vitro cultures of Chinese hamster ovary cells.
Executive summary:

The test substance (purity 98.6%) in dimethylsulphoxide (DMSO) was tested on Chinese hamster ovary cells in vitro, by incorporation in the culture medium, in quadruplicate. In the experiments without metabolic activation, the test substances was treated for 21 hours (concentrations: original experiment: 283.75, 567.50 and 1135.00 µg/mL and confirmatory experiment: 1135.00, 1702.50 and 2270.00 µg/mL) and for 45 hours (concentration: 851.25, 1135.00 and 1702.50 µg/mL). Final concentrations higher than 2270.00 µg/mL of culture medium could not be scored due to cytotoxicity. Cytotoxicity was observed at concentrations of 2270.00 µg/mL and higher. Mitomycin C (0.2 µg/mL) was used as a positive control in the 21 hours experiments. In the experiments with metabolic activation the test substance was treated for 3 hours followed by 18 hours recovery period (concentration: original experiment: 1135.00, 2270.00 and 4540.00 µg/mL; confirmatory experiment: 2270.00, 3405.00 and 4540.00 µg/mL), and for 3 hours followed by 42 hours recovery period (concentration: 2270.00, 3405.00 and 4540.00 µg/mL). Final concentrations higher than 4540.00 µg/mL of culture medium could not be achieved due tosolubility limitations. Slight cytotoxicity was observed at the highest concentration. Cyclophosphamide (20.0 µg/mL) was used as a positive control in the 3 hours/18 hours experiments, for 21 hours without metabolic activation and for 3 hours (with 18 hours recovery) with metabolic activation. Two hours prior to harvesting, the cultures were treated with 0.4 µg/mL colcemid to arrest cells in metaphase. The cells were spread on glass slides, air dried and stained. A cytotoxicity test was performed as an integral part of the study by determination of the percentage mitotic suppression in at least 2000 cells from one slide/group. Whenever possible two hundred well spread metaphase figures with 19 to 21 centromeres from two cultures (100 metaphases per replicate culture) in each group were scored. At least fifty metaphases were scored in the positive controls (25 per replicate culture). The slides were examined blind for specific and non-specific structural aberrations. In both the experiments performed without and with metabolic activation no biologically relevant increase in the number of metaphases containing specific chromosomal aberrations was observed. The incidence of aberrant cells was within the historical control range at all doses assessed. Non-specific chromosomal aberrations (chromatid gaps) were encountered in all experiments within the generally observed frequency. Treatment of CHO cells with the positive control materials produced a high incidence of specific chromosomal aberrations in both experiments, demonstrating the sensitivity of the test system. There was no evidenceof a clastogenic effect of the test substance in in-vitro cultures of Chinese hamster ovary cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Oct 1995 to 13 Dec 1995
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:
1984
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1987/88
Qualifier:
according to
Guideline:
EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)
Version / remarks:
1987
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
MEDIA USED
- Type and identity of media: Ham’s F10 medium supplemented with 10% foetal calf serum and antibiotics. During treatment the foetal calf serum in the medium was reduced to 3%
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S-9 mix
Test concentrations with justification for top dose:
- 1st EXPERIMENT, with S-9 mix: 123.3, 370.0, 1110 and 3333 µg/mL
- 1st EXPERIMENT, without S-9 mix: 61.7, 185.0, 555.0 and 1665 µg/mL
- 2nd EXPERIMENT, with S-9 mix: 416.25, 832.5, 1665 and 3330 µg/mL
- 2nd EXPERIMENT, without S-9 mix: 277.5, 555.0, 1110 and 2220 µg/mL

JUSTIFICATION OF TOP DOSE: The highest concentrations used in the experiments were determined in an initial cytotoxicity range-finding test in which 12 concentrations of the test substance, from 1.63 to 3333.3 µg/mL (limit of solubility), were tested with and without S9 metabolic activation.
Vehicle / solvent:
- Dimethylsulfoxide (DMSO, maximum 1% final concentration in culture medium)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: N-Nitrosodimethylamine
Details on test system and experimental conditions:
PRELIMINARY CYTOTOXICITY ASSAY:
The test substance was tested at twelve concentrations up to 3333.30 µg/mL. Higher concentrations could not be tested due to solubility limitations in the vehicle. The cultures were exposed to the test substance for 5 hours in the presence of S9 mix and for 21 hours in the absence of S9 mix and two vehicle controls were also tested. The treatment was terminated by washing the cultures with phosphate buffered saline (PBS). Compound-induced cytotoxicity was estimated by cloning efficiency immediately after treatment. The cultures were counted and diluted so that 100 cells were seeded per 9.6 cm2 in 3 mL of growth medium. After seven to eight days of growth the cultures were fixed and stained with Giemsa and the surviving colonies determined with the aid of an electronic colony counter or by the naked eye.

MUTAGENICITY ASSAY:
- 2.5-5.0x10^6 cells of passage P 29 (original experiment) and passage P 24 (confirmatory experiment) were plated in 30 mL growth medium and incubated overnight. The growth medium was replaced for 5 hours by treatment medium and S9 activation mixture, or for 21 hours by treatment medium alone. In each assay, cultures were treated in duplicate with four test chemical concentrations, a positive and a negative control.
- The treatment was terminated by washing the cell layer extensively with PBS. After washing, the cells were suspended by trypsinisation, pelleted, resuspended in fresh growth medium and counted with a haemocytometer or electronic coulter counter, diluted with fresh growth medium and re-plated at 2x10^6 cells. The cultures were incubated at 37°C for 7-8 days during which the cells could recover and divide to express the mutant phenotype. The cultures were sub-cultured after the second or third day transferring 2x10^6 cells to a fresh flask to maintain exponential growth during the expression phase.
- In parallel cytotoxicity of the compound was estimated from the cloning efficiency immediately after treatment. The counted cell suspension of each concentration level was further diluted so that 100 cells were seeded per 9.6 cm2 in 2.5 mL of growth medium and incubated at 37°C. The number of colonies which developed within 7-8 days in these cultures reflected the viability at the end of the treatment (survival values).
- At the end of the expression period the cultures were trypsinised, pelleted, resuspended in fresh growth medium and counted with a haemocytometer or electronic coulter counter. The cell suspension of each culture was diluted with fresh growth medium and an aliquot replated into four flasks (75 cm2 growth area) each containing 2x10^6 cells for the mutant selection. The high-density cultures were subjected to the mutant selection procedure by supplementing the growth medium with 8 µg/mL 6-thioguanine (6-TG). Only cells mutated at the hprt locus could survive the 6-TG treatment. The number of colonies formed in these flasks during the following days reflected the overall number of mutations induced by the treatment with the test substance or the mutagen (positive control). After 7-8 days incubation at 37°C, the cultures were fixed and stained with Giemsa. The mutant clones were counted with the naked eye.
- In parallel the viability at the end of the expression period was estimated from the cloning efficiency. The remaining cell suspensions from the various expression cultures were further diluted such that 100 cells were seeded per 9.6 cm2 in 2.5 mL of growth medium and were incubated at 37°C. The number of colonies which developed within these low-density cultures reflected the viability at the end of the expression period (viability values).
Evaluation criteria:
The test substance will be considered to be mutagenic if:
- The assay is valid according to the acceptance criteria
- The mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalised mutant clones in the treated and untreated cultures differs by more than 20.
- There is a significant dose-relationship as indicated by the linear trend analysis.
- The effects described above are reproducible.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines described in the following reference:
Arlett, CF., Smith DM, Green MHL, McGregor DB, Clarke GM, Cole J and Asquith JC (1990). Mammalian cell gene mutation assays based upon colony formation. In: Statistical Evaluation of Mutagenicity Test Data (ed Kirkland, D.J.) Cambridge University press, pp 66-101
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Tested to limit of solubility
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY CYTOTOXICITY ASSAY: In the preliminary cytotoxicity test, the highest concentration with metabolic activation produced 49% growth inhibition whereas complete growth inhibition occurred without metabolic activation. At the next lower concentration 67% growth inhibition occurred.

MUTAGENICITY ASSAY: In both experiments, with and without metabolic activation, no significant increase in the mutation frequencies was observed following exposure to the test substance. In contrast, both DMN and EMS induced clear increases in mutation frequencies. Cytotoxicity is not observed during main mutation test. Not dose limiting.
Conclusions:
In this GLP compliant study, performed according to OECD TG 476, the test substance or its metabolites did not induce gene mutations in cultured V79 Chinese hamster cells with or without metabolic activation.
Executive summary:

In a mammalian cell cytogenetics assay, CH V79 cell cultures/primary lymphocyte cultures were exposed to the test substancewith or without metabolic activation. The highest concentrations used in the experiments were determined in an initial cytotoxicity range-finding test in which 12 concentrations of the test substance, from 1.63 to 3333.3 mg/mL (limit of solubility), were tested withand without S9 metabolic activation. In the gene mutation assay, the test substance (purity 98.6%) in DMSO was tested on Chinese hamster V79 cells by incorporation in the growth medium, at concentrations of 0 (solvent control), 123.3, 370.0, 1110 and 3333 mg/mL with S9 metabolic activation, and 0 (solvent control), 61.7, 185.0, 555.0 and 1665mg/mL without metabolic activation. Two independent assays were performed. In the second experiment, concentrations of 0 (solvent control), 416.25, 832.5, 1665 and 3330 mg/mL with S9 metabolic activation, and 0 (solvent control), 277.5, 555.0, 1110 and 2220 mg/mL without metabolic activation were used. Duplicate cultures were exposed to the test substance, solvent alone or positive control mutagens for 5 hours with S9 and for 21 hours without S9. After exposure, the cultures were incubated at 37°C for 7-8 days for expression during exponential growth. At the end of the expression period the cultures were counted with a haemocytometer or Coulter counter. Mutant selection was by exposure to 6-thio-guanine followed by incubation at 37°C for 7-8 days, after which the cultures were fixed and stained and the mutated clones counted by eye. Cytotoxicity was estimated from the cloning efficiency immediately after exposure for determination of survival values. Viability at the end of expression was estimated from the cloning efficiency.In the preliminary cytotoxicity test, the highest concentration with metabolic activation produced 49% growth inhibition whereas complete growth inhibition occurred without metabolic activation. At the next lower concentration 67% growth inhibition occurred. In both experiments, with and without metabolic activation, no significant increase in the mutation frequencies was observed following exposure to the test substance. In contrast, the positive control substances DMN and EMS produced highly statistically significant increases in mutation frequencies. The test substance or its metabolites did not induce gene mutations in cultured V79 Chinese hamster cells either in the presence or absence of metabolic activation.

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

Genetic toxicity in vivo

Description of key information

Micronucleus test, OECD 474, not clastogenic nor aneugenic, Hertner 1995

A reliable and valid in vivo micronucleus assay conducted under GLP is available demonstrating that the substance did not have clastogenic or aneugenic effects in mice receiving a single oral dose of the test substance at various dose levels.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Sep 1995 to 22 Nov 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
1987
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
1987
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
Tif:MAGf
Remarks:
(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 24 - 38 g
- Housing: Housed individually or 2 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.5-21.0
- Humidity (%): : 44-76
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
26 Sep 1995 to 22 Nov 1995
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Bistilled water
- Amount of vehicle: 10 mL/kg
Frequency of treatment:
Single dose
Post exposure period:
16, 24 and 48h. An overview of the terminations times for the different treatment and control groups can be found in Table 1 in 'Any other information on materials and methods incl. tables'.
Dose / conc.:
312.5 mg/kg bw/day (actual dose received)
Dose / conc.:
625 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
1 250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Vehicle control: 15 animals/sex
Positive control: 5 animals/sex
312.5 and 625 mg/kg bw: 5 animals/sex
1000 mg/kg bw: 15 males and 5 females (+3 reserve males)
1250 mg/kg bw: 10 females (+3 reserve females)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide
- Route of administration: Oral (gavage)
- Doses / concentrations: 64 mg/kg
Tissues and cell types examined:
Femoral bone marrow / polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A preliminary MTD study was performed in mice by the fixed dose procedure at dose levels up to 2000 mg/kg, to determine the highest dose level producing significant toxicity but not death. A solubility test was conducted to determine the highest applicable dose level.

DETAILS OF SLIDE PREPARATION: Bone marrows were harvested from the femurs with foetal calf serum. The bone marrow suspension was centrifuged and the cells re-suspended in foetal calf serum and smears made. The slides were air-dried and stained with May-Grűnwald/ Giemsa solution.

METHOD OF ANALYSIS: Slides were coded and scored blind. Slides of 5 animals/ sex/ dose, showing good differentiation between mature and polychromatic erythrocytes were scored. For each animal the ratio of polychromatic to normochromatic erythrocytes was determined and 2000 polychromatic erythrocytes were scored for micronuclei.
Evaluation criteria:
- Negative effect – if there was no statistically significant difference (Chi-Square ≤ 3.84; p ≥ 0.05) between the mean number of micronucleated PCEs in the groups treated with the test substance and that of the respective negative control and the former did not exceed the range accepted for the negative control (≤ 0.20%).
- Positive Effect - if, in any group with the test substance, the mean number of micronucleated PCEs exceeds the value of 0.20% and if there was a statistically significant difference (Chi-Square > 3.84; p < 0.05) of the number of micronucleated PCEs in comparison with the negative control.
Statistics:
The significance of differences was assessed by the Chi-Square-Test (p<0.5).
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 0 - 2000 mg/kg bw
- Solubility: Bidistilled water was found to be the most appropriate vehicle giving a suspension at the highest dose of 3750 mg/kg.
- Clinical signs of toxicity in test animals: Both animals treated with 2000 mg/kg died within 1-3 hours of treatment. Animals dosed with 1250 mg/kg showed clinical signs. Clinical signs were also recorded at the lower doses. In the confirmatory experiment both animals survived but showed clinical signs. 1250 mg/ was selected as the high dose for the micronucleus test.


RESULTS OF DEFINITIVE STUDY
- Toxicity: The dose level of 1250mg/kg bw caused death in 7/13 females and this dose was reduced to 1000 mg/kg. Treatment at 1000mg/kg bw produced signs of toxicity including lethargy, hunched posture and ataxia. Some animals treated at 625mg/kg bw also showed reduced locomotor activity.
- Ratio of PCE/NCE: The ratios of polychromatic to normochromatic erythrocytes after treatment with the test substance indicated no effects on erythropoiesis at any dose level (Table 1 in 'Any other information on results, incl. tables').

Table 1. Percentage of MNPCE (PCE/NCE ratio) at different preparation times

Treatment

Dose

 

% MNPCE (PCE/NCE ratio)

 

16 hours

24 hours

48 hours

Vehicle Control

10 mL/kg

males

females

combined

0.03 (0.68)

0.03 (1.27)

0.03 (0.98)

0.07 (0.63)

0.02 (1.20)

0.05 (0.92)

0.04 (0.63)

0.08 (1.02)

0.06 (0.83)

test substance1

1000 mg/kg

males

females

combined

0.04 (0.74)

0.06 (0.91)

0.05 (0.83)

0.09 (0.87)

0.05 (0.75)

test substance

2

1250 mg/kg

female

-

0.04 (0.81)

0.02 (1.16)

test substance

625 mg/kg

males

females

combined

-

0.01 (0.68)

0.01 (0.88)

0.01 (0.78)

-

 

test substance

312.5 mg/kg

males

females

combined

-

0.02 (0.84)

0.00 (0.86)

0.01 (0.85)

-

Positive control

64 mg/kg

males

females

combined

 

3.28* (0.91)

1.10* (0.95)

2.19* (0.93)

 

* p < 0.05 (Chi-Square Test)

10000 PCEs counted per group

1Due to symptoms of toxicity the dose level was reduced to 1000 mg/kg.

2Females for 24 and 48 hours sampling were treated with 1250 mg/kg, which proved highly toxic.

Conclusions:
Under the conditions of the test, performed accoring to OECD 474 under GLP, the test substance was not clastogenic or aneugenic in the in vivo mouse bone marrow micronucleus test.
Executive summary:

In an in vivo bone marrow micronucleus assay, performed accoring to OECD 474 under GLP, five male and five female Tif:MAGf young adult mice per group were given a single oral dose of 1000 (or 1250 females 24 and 48 hours), 625.0 or 312.5 mg/kg bw test substance. In addition, five male and five female mice were dosed with10 mL/kg bw bidistilled water (vehicle control) and five male and five female mice with 64 mg/kg bw cyclophosphamide (positive control). From the high dose and negative control groups the animals were killed 16, 24 or 48 hours after dosing. From the intermediate and low dose and positive control group animals were sacrificed 24 hours after application. Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei.

The highest dose of 1250 mg/kg applied to females (24 and 48 hours) caused significant toxicity, manifested as premature death of several animals 4 to 5 hours after treatment. The high dose of 1000 mg/kg applied to males (all sampling times) and to females (16 hour sampling time) caused clinical signs in several animals. In all dosage groups assessed at the different periods post treatment, no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes was observed when compared to the respective negative control group. The test system positive control, induced statistically and biologically significant increases in the frequency of micronucleated polychromatic erythrocytes at the 24 hour sampling time.

In conclusion, under the conditions of the test, the test substance was neither clastogenic nor aneugenic in the in vivo mouse bone marrow micronucleus test.

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

Additional information

In vitro gene mutation in bacteria

In a reverse gene mutation assay in bacteria (Hertner, 1995), the test substance (purity 98.6%) in dimethylsulphoxide (DMSO) was tested on five histidine-auxotrophic strains (TA98, TA100, TA102, TA1535 and TA1537) of Salmonella typhimurium and on the tryptophan-auxotrophic strain WP2uvrA of Escherichia coli, by plate incorporation, at concentrations of 0 (solvent control), 312.5, 625, 1250, 2500 and 5000 µg/plate. Two independent assays were performed, both experiments with and without S9 metabolic activation (S9 fraction from Arochlor induced rat liver). After preparation, the plates were inverted and incubated for about 48 hours at 37±1.5°C and evaluated by colony counting and determining the background lawn. Concurrent strain-specific mutagens were applied to all strains as positive controls in both experiments. In the range finding test normal background growth occurred with both strains, with or without metabolic activation and no appreciable cytotoxicity was observed at 5000 µg/plate, the highest concentration evaluated. The test substance did not increase the number of revertants in any of the strains used, with or without metabolic activation, in either experiment when compared to the vehicle controls. There was no evidence of toxicity to the bacteria at any concentration, in either experiment. The positive controls produced a marked increase of the number of revertant colonies. The test substance and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used in the study.

An additional reliable reverse gene mutation assay in bacteria according to GLP and OECD TG 471 is available as a supporting study (Desperade 1999), covering Salmonella typhimurium strains TA100, TA102, TA1535, TA98 or TA1537. The test substance did not induce gene mutations in these strains under the various experimental conditions.

In vitro gene mutation in mammalian cells

In a mammalian cell cytogenetics assay (Ogorek, 1996), CH V79 cell cultures/primary lymphocyte cultures were exposed to the test substance with or without metabolic activation. The highest concentrations used in the experiments were determined in an initial cytotoxicity range-finding test in which 12 concentrations of the test substance, from 1.63 to 3333.3 mg/mL (limit of solubility), were tested with and without S9 metabolic activation. In the gene mutation assay, the test substance (purity 98.6%) in DMSO was tested on Chinese hamster V79 cells by incorporation in the growth medium, at concentrations of 0 (solvent control), 123.3, 370.0, 1110 and 3333 mg/mL with S9 metabolic activation, and 0 (solvent control), 61.67, 185.0, 555.0 and 1665 mg/mL without metabolic activation. Two independent assays were performed. In the second experiment, concentrations of 0 (solvent control), 416.25, 832.5, 1665 and 3330 mg/mL with S9 metabolic activation, and 0 (solvent control), 277.5, 555.0, 1110 and 2220 mg/mL without metabolic activation were used. Duplicate cultures were exposed to the test substance, solvent alone or positive control mutagens for 5 hours with S9 and for 21 hours without S9. After exposure, the cultures were incubated at 37°C for 7-8 days for expression during exponential growth. At the end of the expression period the cultures were counted with a haemocytometer or Coulter counter. Mutant selection was by exposure to 6-thio-guanine followed by incubation at 37°C for 7-8 days, after which the cultures were fixed and stained and the mutated clones counted by eye. Cytotoxicity was estimated from the cloning efficiency immediately after exposure for determination of survival values. Viability at the end of expression was estimated from the cloning efficiency. In the preliminary cytotoxicity test, the highest concentration with metabolic activation produced 49% growth inhibition whereas complete growth inhibition occurred without metabolic activation. At the next lower concentration 67% growth inhibition occurred. In both experiments, with and without metabolic activation, no significant increase in the mutation frequencies was observed following exposure to the test substance. In contrast, the positive control substances DMN and EMS produced highly statistically significant increases in mutation frequencies. The test substance or its metabolites did not induce gene mutations in cultured V79 Chinese hamster cells either in the presence or absence of metabolic activation.

In vitro cytogenicity

The test substance (purity 98.6%) in dimethylsulphoxide (DMSO) was tested on Chinese hamster ovary cells in vitro, by incorporation in the culture medium, in quadruplicate (Zeugin, 1996). In the experiments without metabolic activation, the test substances was treated for 21 hours (concentrations: original experiment: 283.75, 567.50 and 1135.00 µg/mL and confirmatory experiment: 1135.00, 1702.50 and 2270.00 µg/mL) and for 45 hours (concentration: 851.25, 1135.00 and 1702.50 µg/mL). Final concentrations higher than 2270.00 µg/mL of culture medium could not be scored due to cytotoxicity. Cytotoxicity was observed at concentrations of 2270.00 µg/mL and higher. Mitomycin C (0.2 µg/mL) was used as a positive control in the 21 hours experiments. In the experiments with metabolic activation the test substance was treated for 3 hours followed by 18 hours recovery period (concentration: original experiment: 1135.00, 2270.00 and 4540.00 µg/mL; confirmatory experiment: 2270.00, 3405.00 and 4540.00 µg/mL), and for 3 hours followed by 42 hours recovery period (concentration: 2270.00, 3405.00 and 4540.00 µg/mL). Final concentrations higher than 4540.00 µg/mL of culture medium could not be achieved due to solubility limitations. Slight cytotoxicity was observed at the highest concentration. Cyclophosphamide (20.0 µg/mL) was used as a positive control in the 3 hours/18 hours experiments, for 21 hours without metabolic activation and for 3 hours (with 18 hours recovery) with metabolic activation. Two hours prior to harvesting, the cultures were treated with 0.4 µg/mL colcemid to arrest cells in metaphase. The cells were spread on glass slides, air dried and stained. A cytotoxicity test was performed as an integral part of the study by determination of the percentage mitotic suppression in at least 2000 cells from one slide/group. Whenever possible two hundred well spread metaphase figures with 19 to 21 centromeres from two cultures (100 metaphases per replicate culture) in each group were scored. At least fifty metaphases were scored in the positive controls (25 per replicate culture). The slides were examined blind for specific and non-specific structural aberrations. In both the experiments performed without and with metabolic activation no biologically relevant increase in the number of metaphases containing specific chromosomal aberrations was observed. The incidence of aberrant cells was within the historical control range at all doses assessed. Non-specific chromosomal aberrations (chromatid gaps) were encountered in all experiments within the generally observed frequency. Treatment of CHO cells with the positive control materials produced a high incidence of specific chromosomal aberrations in both experiments, demonstrating the sensitivity of the test system. There was no evidence of a clastogenic effect of the test substance in in-vitro cultures of Chinese hamster ovary cells.

Other in vitro studies

The substance was investigated for DNA-damaging effects on rat and mouse hepatocytes in vitro under GLP and according to OECD TG 482. The compound was dissolved in DMSO and tested at various concentrations up to the highest soluble concentration or to concentrations resulting in considerable cell toxicity in original and confirmatory experiments. In all experiments, none of the concentrations induced a significant increase in the mean gross or net nuclear grain counts when compared to the solvent control group, despite a reduction of 80% in hepatocyte viability in some experiments. The percentage distribution of gross and net nuclear grain counts was not substantially shifted to higher values. In contrast, the positive controls used in the tests produced a marked increase both in the nuclear and in the net nuclear grain counts.

Under the given experimental conditions, no evidence of induction of DNA damage by the substance or by its metabolites was obtained that could be interpreted as suggestive of a genotoxic property of the test substance in vitro.

In vivo micronucleus tests

In an in vivo bone marrow micronucleus assay, performed according to OECD 474 under GLP, five male and five female Tif:MAGf young adult mice per group were given a single oral dose of 1000 (or 1250 females 24 and 48 hours), 625.0 or 312.5 mg/kg bw test substance. In addition, five male and five female mice were dosed with10 mL/kg bw bidistilled water (vehicle control) and five male and five female mice with 64 mg/kg bw cyclophosphamide (positive control). From the high dose and negative control groups the animals were killed 16, 24 or 48 hours after dosing. From the intermediate and low dose and positive control group animals were sacrificed 24 hours after application. Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei.

The highest dose of 1250 mg/kg applied to females (24 and 48 hours) caused significant toxicity, manifested as premature death of several animals 4 to 5 hours after treatment. The high dose of 1000 mg/kg applied to males (all sampling times) and to females (16 hour sampling time) caused clinical signs in several animals. In all dosage groups assessed at the different periods post treatment, no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes was observed when compared to the respective negative control group. The test system positive control, induced statistically and biologically significant increases in the frequency of micronucleated polychromatic erythrocytes at the 24 hour sampling time.

In conclusion, under the conditions of the test, the test substance was neither clastogenic nor aneugenic in the in vivo mouse bone marrow micronucleus test.

Justification for classification or non-classification

Based on the available data classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.