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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a reliable in vitro genotoxicity study (Ames test) the substance was tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and in Escherichia coli strain WP2 uvrA. Concentrations of up to 5000 μg/plate were tested. No evidence of mutagenic activity was seen at any concentration of the substance in either theplate incorporation method or the pre-incubation method. It was concluded that the substance showed no evidence of mutagenic activity in these bacterial strains, with and without metabolic activation.

In a reliable reverse gene mutation assay in bacteria, strains TA 100, TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium and WP2 uvr A pkM 101 of E. coli were exposed to a structurally similar substance (a.i 76.9 %) diluted in water at concentrations of 125 to 2500 µg per plate in the presence and absence of mammalian metabolic activation (co-incubation). There was no evidence of induced mutant colonies over background.

In a mammalian chromosome aberration test performed according to OECD Guideline 473 (1997), human lymphocyte cultures were exposed to a structurally similar substance (no solvent), (100% a.i.), at concentrations between 11.9 - 3200µg/mL with metabolic activation and 20.8 - 3200 µg/mL without metabolic activation. The substance(no solvent)was tested up to cytotoxic or precipitating concentrations. In Experiment II, in the absence of S9 mix, a single increase in chromosomal aberrations was observed, slightly exceeding the laboratory’s historical control data range, but since the value was not statistically significantly increased these findings were considered as biologically irrelevant. A single statistically significant increase was observed in Experiment II, in the presence of S9 mix, but the value was clearly within the range of the laboratory’s historical control data and thus considered as being without biological relevance. It can be concluded that under the experimental conditions reported, the substance did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to cytotoxic and/or precipitating concentrations in the absence and presence of metabolic activation.

In a mammalian gene mutation assay, L5178 Y (mouse lymphoma thymidine kinase locus) cells cultured in vitro were exposed to a structurally similar substance (98 % a.i.). The substance was tested up to cytotoxic concentrations (>/= 16.0 µg/ml without metabolic activation and >/= 40.0 µg/ ml with metabolic activation). There was no evidence of increased number of induced mutant colonies over background.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 April 2017 - 02 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Physical state/Appearance: Yellow to brown paste
Batch: RL55/17
Purity: 100%
Expiry Date: 15 March 2018
Storage Conditions: Room temperature in the dark
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:
S9 Microsomal fractions from rats pre-treated with phenobarbitone and beta-naphthaflavone.
Test concentrations with justification for top dose:
Experiment 1 - The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the substance (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed.
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
Salmonella strains (with and without S9): 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate. E.coli strain WP2uvrA (with and without S9): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Vehicle / solvent:
Dimethyl sulphoxide.
Untreated negative controls:
yes
Remarks:
Untreated.
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Evaluation criteria:
Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
Species / strain:
E. coli WP2 uvr A
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
Conclusions:
The substance is not mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA using the Ames plate incorporation and pre-incubation methods, with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).
Executive summary:

In the in vitro genotoxicity study (Ames test) the substance was tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and in Escherichia coli strain WP2 uvrA. Concentrations of up to 5000 μg/plate were tested. No evidence of mutagenic activity was seen at any concentration of the substance in either theplate incorporation method or the pre-incubation method. It was concluded that the substance showed no evidence of mutagenic activity in these bacterial strains under the test conditions employed.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read-across to K1 study therefore K2 is the maximum Klimisch value.
Justification for type of information:
Read-across approach - see read-across justification in section 13.
Reason / purpose for cross-reference:
read-across source
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
This in vitro experiment assesses the potential of the substance to induce gene mutations by means of a Thymidine Kinase assay using the mouse lymphoma cell line L5178Y. The Thymidine Kinase (TK) system detects base pair mutations, frameshift mutations, small deletions as well as large, non lethal deletions and rearrangements of the relevant chromosomes.
Cells deficient in the heterozygous TK-Iocus due to the forward mutation TK+/- to TK-/- are resistant to the cytotoxic effects of pyrimidine analogues such as trifluorothymidine (TFT).
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 fractions obtained from livers of male Wistar rats which were induced with Phenobarbital (80 mg/kg b.w.) and ß-Naphthoflavone (100 mg/kg b.w.). Final protein concentration of 0.75 mg/mI in the cultures.
Test concentrations with justification for top dose:
The selection of the concentrations was based on data from a pre-experiment.

Experiment I
- with metabolic activation: 2.50, 5.00, 7.50, 10.0, 12.5, 15.0, 20.0, 40.0 and 60.0 µg/ml
- without metabolic activation: 1.00, 3.00, 5.00, 10.0, 12.0, 16.0, 18.0, 20.0, 25.0 and 30.0 µg/ml
Experiment II
- with metabolic activation: 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0 and 55.0 µg/ml
- without metabolic activation: 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, 10.0, 12.0 and 16.0 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: 80 and 400 µg/ml without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: 1.50 µg/ml with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: experiment I short time incubation 4 hours and experiment II long-term incubation 24 hours
- Expression time (cells in growth medium): 72 hours in experiment I and 48 h in experiment II
- Selection time (if incubation with a selection agent): 11 to 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 to 17 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: two

NUMBER OF CELLS EVALUATED: 300000/plate seeded

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, cloning efficiency
Evaluation criteria:
There are several criteria for determining a positive result:
- clear and dose-related increase in the mutant frequency,
- biologically relevant response (at least 2-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) for at least one of the dose groups.
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low large/small colonies ratio (< 4), is an indication for potential clastogenic effects and/or chromosomal aberrations.

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

A test item is considered to be negative if there is no biologically relevant increase in the induction of mutant cells above concurrent control levels, at
any dose level.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations >/= 16 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The selection of the concentrations was based on data from the pre-experiment. Precipitation of the test item in cell culture medium was observed at concentrations of 50.0 µg/ml and higher. The pH-value detected at a test item concentration of 200 µg/ml was 7.2.

TOXICITY:

Growth inhibition was observed in experiment I and II with and without metabolic activation.

In experiment I with metabolic activation the relative total growth (RTG) was 6.85 % for the highest concentration (60.0 µg/ml) evaluated. The highest biologically relevant dose group evaluated with metabolic activation was 40.0 µg/ml with a RTG of 16.19 %. The highest concentration investigated without metabolic activation was 30.0 µg/ml with a RTG of 7.62 %. The highest biologically relevant concentration evaluated without metabolic activation was 20.0 µg/ml with a RTG of 10.19 %.

In experiment II with metabolic activation the relative total growth (RTG) was 13.04 % for the highest concentration (55.0 µg/ml) evaluated. The highest biologically relevant concentration evaluated without metabolic activation was 16.0 g/ml with a RTG of 17.14 %.

MUTAGENICITY:
In experiment I with metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 20-150 mutants per 10exp6 cells). No dose effect relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the controls.

Mutation frequencies with the negative controls were 66.39 - 100.61 mutants/10exp6 cells. The values of the solvent controls were found to be 64.18 - 90.07 mutants/10exp6 cells and 46.56 - 133.57 mutants/10exp6 cells with the test item, respectively. The highest mutation factor (compared to the solvent control values) of 1.73 was found at a concentration of 60.0 µg/ml with a RTG of 6.85 %. The mutation factor found at this dose group was considered as not biologically relevant due to the high toxicity found. The highest biologically relevant concentration evaluated was 40.0 µg/ml and showed a mutation factor of 1.07.

In experiment I without metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 20-150 mutants per 10exp6 cells) except the value (172.70 mutants/10exp6 cells) found at a dose of 25.0 µg/ml. No dose dependency could be observed. Due to the high cytotoxicity of 8.93 % RTG at 25.0 µg/ml the increased mutation factor (above the historical control data) was considered as not biologically relevant. Moreover, if the spontaneous mutant frequency (negative control) is > 100 mutants/10exp6 cell, a doubling of the spontaneous frequency should be required for a definitive positive response. The mutation factor found at a dose of 25.0 µg/ml was 1.45, thus below the threshold value of 2.

Mutation frequencies with the negative controls were 100.99 - 136.72 mutants/10exp6 cells. The values of the solvent controls were found to be 102.49 - 135.07 mutant/10exp6 cells and 65.22 - 172.70 mutants/10exp 6 cells with the test item, respectively. The highest mutation factor (compared to the solvent control values) of 1.45 was found at a concentration of 25.0 µg/ml with a RTG of 8.93 %. The highest biologically relevant dose group
evaluated (20.0 µg/ml) showed a mutation factor of 1.02.

In experiment II with metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 20-150 mutants per 10exp6 cells). No dose effect relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the controls.

Mutation frequencies with the negative controls were 64.80 - 68.94 mutants/10exp6 cells. The values of the solvent controls were found to be 62.71 - 103.15 mutants/10exp6 cells and 40.30 - 88.61 mutants/10exp6 cells with the test item, respectively. The highest mutation factor (compared to the solvent control values) of 1.07 was found at a concentration of 15.0 µg/ml with a RTG of 91.69 %.

In experiment II without metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 20-150 mutants per 10exp6 cells). No dose effect relationship could be observed. The mutation factor of 2, which was found at a dose of 0.50 µg/ml, was considered as not relevant due to the mutant frequency of 136.84 mutants/10exp6 cells, which was within the historical control data.

Mutation frequencies with the negative controls were 76.86 - 111.59 mutants/10exp6 cells. The values of the solvent controls were found to be 64.67 - 72.05 mutants/l0exp6 cells and 49.86 - 136.84 mutants/l0exp6 cells with the test item, respectively. The highest mutation factor (compared to the solvent control values) of 2.00 was found at a concentration of 0.50 µg/ml with a RTG of 83.16 %.

EMS (80 µg/ml and 400 µg/ml) and B[a]P (1.50 µg/ml) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

RELATIONSHIP TO LARGE AND SMALL COLONIES:

Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. A mutation frequency above 2 in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone), indicated by a low large/small colonies ratio (< 4), is an indication for potential clastogenic effects and/or chromosomal aberrations.

In experiment I, with metabolic activation the quotients of large/small colonies of the negative controls were found to be 14.00 and 73.00. For the
solvent controls the quotients noticed were 7.43 and 10.33. The quotient of the highest dose groups were found to be 17.00 (15.0 µg/ml), 14.50
(20.0 µg/ml), 15.00 (40.0 µg/ml) and 10.38 (60.0 µg/ml). No indication of potential clastogenic effects and/or chromosomal aberrations was found in all dose groups evaluated.

In experiment I without metabolic activation the quotients of large/small colonies of the negative controls were found to be 12.60 and 26.00. For the
solvent controls the quotients noticed were 5.91 and 10.43. The quotients of the highest dose groups were found to be 14.50 (18.0 µg/ml), 7.89
(20.0 µg/ml), 4.89 (25.0 µg/mI) and 7.45 (30.0 µg/ml). No indication of potential clastogenic effects and/or chromosomal aberrations was found in
all dose groups evaluated.

In experiment II with metabolic activation the quotients of large/small colonies of the negative controls were found to be 7.38 and 11.60. For the
solvent controls the quotients noticed were 7.67 and 9.40. The quotients of the highest dose groups were found to be 18.00 (40.0 µg/ml), 6.43
(45.0 µg/ml), 17.00 (50.0 µg/ml) and 10.60 (55.0 µg/ml). No indication of potential clastogenic effects and/or chromosomal aberrations was found in all dose groups evaluated.

In experiment II without metabolic activation the quotients of large/small colonies of the negative controls were found to be 7.33 and 8.89. For the
solvent controls the quotients noticed were 4.44 and 6.70. The quotients of the highest dose groups were found to be 9.67 (8.00 µg/ml), 7.63 (10.0 µg/ml), 12.25 (12.0 µg/ml) and 5.25 (16.0 µg/ml). No indication of potential clastogenic effects and/or chromosomal aberrations was found in
all dose groups evaluated.

CONCLUSION

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Rewoquat V 2815 is considered to be
non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
In conclusion, in the described mutagenicity test under the experimental conditions reported, the substance is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
Executive summary:

In a mammalian gene mutation assay, L5178 Y (mouse lympoma thymidine kinase locus) cells cultured in vitro were exposed to the substance (98 % a.i.) at the following concentrations:

Experiment I

- with metabolic activation: 2.50, 5.00, 7.50, 10.0, 12.5, 15.0, 20.0, 40.0 and 60.0 µg/ml

- without metabolic activation: 1.00, 3.00, 5.00, 10.0, 12.0, 16.0, 18.0, 20.0, 25.0 and 30.0 µg/ml

Experiment II

- with metabolic activation: 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0 and 55.0 µg/ml

- without metabolic activation: 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, 10.0, 12.0 and 16.0 µg/ml

The substance was tested up to cytotoxic concentrations (>/= 16.0 µg/ml without metabolic activation and >/= 40.0 µg/ ml with metabolic activation). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of increased number of induced mutant colonies over background.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read-across to K2 study therefore K2 is the maximum Klimisch value.
Justification for type of information:
Read-across approach - see read-across justification in section 13.
Reason / purpose for cross-reference:
read-across source
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: Histidin auxotroph
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
other: Tryptophan auxotroph
Metabolic activation:
with and without
Metabolic activation system:
The 9000 g supernatant of rat-liver homogenate (phenobarbital/ß-naphtoflavone incuded) purchased by CCR GmbH & Co. KG, D-64380 Roßdorf , was used in assays with metabolic activation. The lot number was 30797, microsomal protein-content was 24.7 mg/ml.
Test concentrations with justification for top dose:
Range finding test: Concentration µg/plate: 19.53, 39.06, 78.13, 156.25, 312.50, 625, 1250, 2500, 5000
Main test: In two independent mutation experiments, cells were exposed to concentrations of 125, 250, 500, 1000, 2000 µg/plate (1st assay) and 156.25, 312,50, 625, 1250, 2500 µg/plate (2nd assay) in presence and absence of S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Aqua dest.
- Justification for choice of solvent/vehicle: 50 mg of test substance could be dissolved in 1 ml Aqua dest.
Untreated negative controls:
yes
Remarks:
for all strains with Aqua dest., with and without metabolic activation system
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: at 0.25 µg/plate for TA 100, TA 1535, without metabolic activation
Positive control substance:
2-nitrofluorene
Remarks:
Migrated to IUCLID6: at 0.2 µg/plate for TA 98, without metabolic activation
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: 50 µg/plate for TA 1535, TA 1537 without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: at 1.0 µg/plate for E.coli without metabolic activation
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: at 0.5 µg/plate for TA 100, TA 98, at 2.0 µg/plate for TA 1535, TA 1537, 1.0 µg/plate for E. coli with metabolic activation by S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 h


SELECTION AGENT (mutation assays): histidine


NUMBER OF REPLICATIONS: 2


NUMBER OF PLATES EVALUATED: three per dose


DETERMINATION OF CYTOTOXICITY
- Method: The criterion for a biologically significant bacteriotoxic effect is a either a reduction of the survival of the cells to at least 50% or a reduction of the background growth of auxotrophic cells and the number of revertants.



Evaluation criteria:
Selection of the Test Concentrations
The highest concentration used in the test must either reduce the survival of the cells to at least 50% or reduce the background growth of auxotrophic cells and the number of revertants.
If no toxicity is observed, the survey is carried out with the highest soluble concentration of the test article, however, not exceeding 5 mg/plate for solids and 10 mM of liquids or 200 µl of extracts/plate. The concentration intervals between the test doses have a factor of 5.

Analysis of Results
In the Ames assay a test compound is considered as mutagenic if there is a dose dependent increase in the number of revertant colonies of one or more strains. For the highest non-toxic concentration an increase should be by a factor of about 2 and deemed to be biologically relevant. Any evidence of mutagenic activity must be reproducible in an independent experiment. In addition statistical evaluation may aid data interpretation.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
<= 2500 µg/plate in preliminary tests on TA 100
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
37% at 2500 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test compound reduced the survival of the strain TA 100 at a concentration of 2500 µg per plate to 0.6 % of the control value.
The number of the his--cells of the strain TA 100 at the concentration of 5000 µg/plate was reduced to 15.9 % of the control value. In addition the background growth is reduced at concentrations from 1250 to 5000 µg/plate.
Precipitation of the test compound was observed from concentrations for 1250 to 5000 µg/plate. Therefore the mutagenicity assays were performed in a concentration range from 125 to 2500 µg per plate.


MAIN TEST
Concentrations between 125 and 2500 µg/plate were applied to the bacterial strains TA 1535, TA 1537, TA 98 and TA 100.

No evidence of biologically significant mutagenic activity of test substance was found. The results of the 1st and the 2nd assay indicate, that in the tested concentration range with and without metabolic activation no significant increase in the numbers of his+- or trp+-revertants over the spontaneous values could be detected with the Salmonella tester strains TA 100, TA 1535, TA 98, TA 1537 and E. coli WP2 uvrA pkm (101).

Slight bacteriotoxicity was observed for E. coli WP2 uvr A pKM 101 at the highest concentration tested (2500 µg/plate in the second experiment.

STERILITY CONTROLS
The plates for the sterility control of top agar, S9-mix and test compound showed no growth.

NEGATIE CONTROLS
In all experiments the control plates without mutagen showed a normal number of spontaneous revertants

POSITIVE CONTROLS
The positive controls demonstrated the sensitivity of the indicator strains and the activity of the metabolizing syste.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
In summary, the results indicate that the substance caused no mutagenic effects at concentrations ranging from 125 to 2500 µg/plate in thefive bacterial strains.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 100, TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium and WP2 uvr A pkM 101 of E. coli were exposed to the substance (a.i 76.9 %) diluted in water at concentrations of 125 to 2500 µg per plate in the presence and absence of mammalian metabolic activation (co-incubation).

Significant bacteriotoxic effects were not observed in the main study up to and including the highest concentration tested of 2500 µl/plate. In the range finding study the substance reduced the survival at a concentration of 2500 µg per plate to 0.6 % of the control value. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 and 472, as well as EU Method B.13/14, 29th December 1992 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read-across to K1 study therefore K2 is the maximum Klimisch value.
Justification for type of information:
Read-across approach - see read-across justification in section 13.
Reason / purpose for cross-reference:
read-across source
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Chromosome aberration assays detect the induction of chromosome breakage (clastogenesis). Although mutagenic substances produce structural
chromosome aberrations by a variety of mechanisms, the endpoint is a discontinuity in the chromosomal DNA which is left unrejoined, or rejoined
inaccurately, thus producing a mutated chromosome. Chromosome aberrations are generally evaluated in first post treatment mitoses. The majority of chemical mutagens induce aberration of the chromatid type, but chromosome type aberrations also occur.
Species / strain / cell type:
other: human lymphocytes
Details on mammalian cell type (if applicable):
Blood samples were obtained from healthy donors not receiving medication.
Blood cultures were set up in bulk within 24 hrs after collection in 75 cm² cell culture flasks.
- Type and identity of media: DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1; Life Technologies GmbH, 76339 Eggenstein, Germany) containing 10 % FCS (fetal calf serum) provided by PAA Laboratories GmbH (35091 Cölbe, Germany), the antibiotic solution containing 10,000 U/mL penicillin and 10,000 µg/mL streptomycin (SEROMED, D-12247 Berlin). Additionally, the medium was supplemented with Phytohemagglutinin (PHA, final concentration 3 µg/mL, SEROMED), the anticoagulant heparin (25,000 U.S.P.-U/mL, NATTERMANN, 50829 Köln, Germany), and
HEPES (final concentration 10 mM, Serva, 69115 Heidelberg, Germany).
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar HanIbm rats.
Test concentrations with justification for top dose:
Experiment I
with metabolic activation: 11.9, 20.8, 36.4, 63.7, 111.4, 195.0, 341.2, 597.1, 1044.9, 1828.6 and 3200 µg/mL
without metabolic activation: 20.8, 36.4, 63.7, 111.4, 195.0, 341.2, 597.1, 1044.9, 1828.6 and 3200 µg/mL

Experiment II
with metabolic activation: 10.0, 20.0, 50.0, 125.0, 250.0, 500.0, 750.0, 1000.0, 1250.0 and 1500.0 µg/mL
without metabolic activation: 8.1, 14.2, 25.9, 43.5, 76.2, 133.3, 233.2, 408.2, 714.3 and 1250 µg/mL
Results from the rangefinding assay were used to determine the dose range to be used in the chromosomal aberrations assay.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol 0.5%
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: 880 (ExperimentI) and 770 (Experiment II) µg/mL, positive control without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: 37.5 µg/mL, positive control with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:

Range-finder
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by
the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the mutagenicity assay.
The pre-test phase was performed with 10 concentrations without S9 mix and 11 concentrations with S9 mix of the test item and a solvent and
positive control. All cell cultures were set up in duplicate. Exposure times were 4 hrs (with and without S9 mix). The preparation interval was 22 hrs
after start of the exposure. Additional solvent control cultures (with and without S9 mix) were used in the presence of BrdU (5-bromodeoxyuridine; 6 µg/mL) to reassure the replication time of the cultured lymphocytes.

Dose Selection
The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests
requesting for the top concentration clear toxicity with reduced mitotic indices below 50 % of control, and/or the occurrence of precipitation.
In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate
solvent is possible.
With respect to the ability to formulate a homogeneous suspension of the test item, 3200 µg/mL of test substance
(approx. 4.8 mM) were applied as top concentration for treatment of the cultures in the pre-test. Doses over 3200 µg/mL led to an inhomogeneous
suspension in ethanol that was not applicable. Test item concentrations between 11.9 and 3200 µg/mL, and between 20.8 and 3200 µg/mL (with and without S9 mix, respectively) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test item was observed
before start of treatment at 36.4 µg/mL and above in the absence of S9 mix, and at 20.8 µg/mL and above in the presence of S9 mix. Since the
cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Using reduced mitotic indices as an indicator for toxicity in Experiment I, toxic effects of about 50 % of control were observed after 4 hrs treatment
with 597.1 µg/mL and above in the absence and presence of S9 mix. Considering these toxicity data, 1250 µg/mL (without S9 mix) and 1500 µg/mL
(with S9 mix) were chosen as top concentrations in Experiment II.
The cytogenetic evaluation of higher concentrations in the respective preparation interval (with and without S9 mix) was impossible due to strong test item-induced toxic effects (low metaphase numbers, partially paralleled by poor metaphase quality and precipitation on the slides).

Exposure time 4 hours
The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix), containing the test item.
For the treatment with metabolic activation 50 µL S9 mix per mL medium were used.
Concurrent solvent and positive controls were performed. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The
supernatant with the dissolved test item was discarded and the cells were re-suspended in "saline G" solution (NaCl -8000 mg, KCl -400 mg, glucose x H2O -1100 mg, Na2HPO47H2O - 290 mg, KH2PO4 --150 mg). The washing procedure was repeated once as described.
After washing the cells were re-suspended in complete culture medium and cultured until preparation.

Exposure time 22 hours (without S9 mix)
The culture medium was replaced with complete medium (with 10 % FCS) containing the test item without S9 mix. The culture medium at continuous
treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.
All cultures were incubated at 37° C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).

Preparation of the Cultures
Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL). The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were re-suspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37° C for 20 to 25 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by
dropping the cell suspension onto a clean microscope slide. The cells for evaluation of cytogenetic damage were stained with Giemsa (MERCK, 64293 Darmstadt, Germany) or according to the Fluorescent plus Giemsa technique, respectively.

Analysis of Metaphase Cells
The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" [4]) using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. At least 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment II without metabolic activation, where only 50 metaphase plates were scored. Only metaphases with 46 +/-1 centromer regions were included in the analysis. To describe a
cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 250 metaphase cells (% polyploid
metaphases) was scored.
Evaluation criteria:
A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 %
aberrant cells, excluding gaps).
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, excluding
gaps) and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 – 0.8 % polyploid cells).
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be
considered together. If the criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Species / strain:
lymphocytes: human lymphocytes
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:
With respect to the ability to formulate a homogeneous suspension of the test item, 3200 µg/mL of the test substance (approx. 4.8 mM) were applied as top concentration for treatment of the cultures in the pre-test. Doses over 3200 µg/mL led to an inhomogeneous suspension in ethanol that was not applicable.
Test item concentrations between 11.9 and 3200 µg/mL, and between 20.8 and 3200 µg/mL (with and without S9 mix, respectively) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test item was observed before start of treatment at 36.4 µg/mL and above in the absence of S9 mix, and at 20.8 µg/mL and above in the presence of S9 mix.

Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

In Experiment II, in the absence of S9 mix, a single increase in chromosomal aberrations was observed, slightly exceeding the laboratory’s historical control data range, but since the value was not statistically significantly increased these findings were considered as biologically irrelevant. A single statistically significant increase was observed in Experiment II, in the presence of S9 mix, but the value was clearly within the range of the laboratory’s historical control data and thus considered as being without biological relevance.
 

Polyploid metaphases:
Polyploid metaphases occured, but in both experiments, no biologically relevant increase in the rate of polyploid metaphases was found
after treatment with the test item (0.0 – 0.6 %) as compared to the rates of the solvent controls (0.0 – 0.8 %).


POSITIVE CONTROLS
In both experiments, EMS (880 and 770 µg/mL, respectively) and CPA (37.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
Remarks on result:
other: strain/cell type: human lymphocytes
Remarks:
Migrated from field 'Test system'.

 

Summary of results of the chromosomal aberration study with PC-2007-140 (W 575 no solvent)

Exp.

Preparation interval

Test item concentration in µg/mL

Polyploid cells in %

Mitotic indices in % of control

Incl. gaps

Aberrant cells in % excl. gaps

With exchanges

Exposure period 4 hrs without S9 mix

I

22 hrs

Solvent control

0.8

100.0

2.0

2.0

1.0

 

Positive control

0.2

64.2

10.0

9.0S

0.0

 

20.8

0.2

73.4

3.0

2.0

0.0

 

36.4 P

0.4

82.6

2.5

1.5

0.5

 

341.2 P

0.2

61.3

2.0

1.5

0.0

 

597.1 P

0.4

50.5

0.5

0.5

0.0

Exposure period 22 hrs without S9 mix

II

22 hrs

Solvent control

0.4

100.0

2.0

2.0

0.0

 

Positive# control

0.0

25.3

50.0

49.0S

11.0

 

43.5##

0.2

72.3

5.0

4.5

0.0

 

76.2 P

0.2

67.9

0.0

0.0

0.0

 

133.3 P

0.0

53.6

3.0

3.0

0.0

 

233.2 P

0.0

29.2

2.5

2.0

0.0

Exposure period 4 hrs with S9 mix

I

22 hrs

Solvent control

0.2

100.0

2.0

1.5

0.0

 

Positive control

0.4

49.7

8.5

 8.5S

2.0

 

11.9

0.0

84.7

1.0

1.0

0.0

 

20.8 P

0.4

88.9

1.5

1.0

0.5

 

341.2 P

0.6

65.3

0.0

0.0

0.0

 

597.1 P

0.4

73.4

2.5

2.0

0.0

II

22 hrs

Solvent control

 

0.0

 

100.0

 

2.0

 

1.0

 

0.0

 

Positive control

 

0.0

 

34.6

 

22.0

   20.0S

 

1.0

 

20.0

0.0

120.1

1.5

1.5

0.0

 

50.0 P##

0.0

111.0

3.5

  3.3S

0.0

 

125.0 P

0.0

110.2

1.5

1.5

0.0

 

P -Test item precipitation was observed

S - Aberration frequency statistically significant higher than corresponding control values

# - Evaluation of 50 metaphases per culture

## - Evaluation of 200 metaphases per culture

Conclusions:
It can be concluded that under the experimental conditions reported, the substance did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to cytotoxic and/or precipitating concentrations in the absence and presence of metabolic activation.
Executive summary:

In a mammalian chromosome aberration test performed according to OECD Guideline 473 (1997), human lymphocyte cultures were exposed to the substance (no solvent), (100% a.i.), suspended in ethanol at concentrations between 11.9 - 3200 µg/mL with metabolic activation and 20.8 - 3200 µg/mL without metabolic activation. The substance (no solvent) was tested up to cytotoxic or precipitating concentrations. The following experimental points were microscopically evaluated:11.9, 20.0, 20.8, 50.0, 125.0, 341.2, 597.1 µg/mL with metabolic activation and 20.8, 36.4, 43.5, 76.2, 133.3, 233.2, 341.2, 597.1 µg/mL without metabolic activation. In the absence of S9 mix, reduced mitotic indices of about or below 50 % of control were observed at the highest evaluated concentrations. In the presence of S9 mix, concentrations showing clear cytotoxic effects were excluded from scoring for the endpoint cytogenicity.

In Experiment II, in the absence of S9 mix, a single increase in chromosomal aberrations was observed, slightly exceeding the laboratory’s historical control data range, but since the value was not statistically significantly increased these findings were considered as biologically irrelevant. A single statistically significant increase was observed in Experiment II, in the presence of S9 mix, but the value was clearly within the range of the laboratory’s historical control data and thus considered as being without biological relevance.

Positive controls induced the appropriate response. There was no evidence of Chromosome Aberration induced over background.

This study is classified as acceptable. It satisfies the requirement for Test Guideline In vitro mammalian Chromosome Aberration test OECD 473 for in vitro cytogenetic mutagenicity data.

It can be concluded that under the experimental conditions reported, the substance did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to cytotoxic and/or precipitating concentrations in the absence and presence of metabolic activation.

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 findings of reliable in vitro genotoxicity studies conducted on the substance and on structurally similar substances, classification of the substance is not justified.