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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Salmonella typhimurium/ Escherichia coli reverse mutation assay (OECD 471): Positive.

Mutagenicity in mammalian cells (HPRT assay according to OECD 476): Negative.

Micronucleus test (OECD 487): negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Jun 2016 - 07 Jul 2016
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:
21 Jul 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)
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Batch identification: 001-150606
- Date of production: 26 May 2015
- Physical state, appearance: solid, black
- Content: Approx. 98%
- Test substance No.: 16/0074-1
Target gene:
his for S. typhimurium
trp for E. Coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Standard plate test with and without S9 mix

2nd Experiment
Strains: TA 100, TA 1537 and TA 98
Doses: 0; 0.3; 1; 3.3; 10; 33 and 100 μg/plate (TA 100)
0; 0.1; 0.3; 1; 3.3; 10 and 33 μg/plate (TA 1537 and TA 98)
Type of test: Standard plate test with and without S9 mix

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.
Vehicle / solvent:
- Vehicle used: The test substance was dissolved in dimethyl sulfoxide (DMSO).
- Justification for choice of vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Please refer to 'any other information on materials and methods incl. tables'
Details on test system and experimental conditions:
METHOD OF APPLICATION: standard plate test (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours at 37°C in the dark
NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control

NUMBER OF CELLS EVALUATED:
all revertants / colonies counted

DETERMINATION OF CYTOTOXICITY
Following incubation, the bacterial colonies (his+ revertants for Salmonella typhimurium, trp+ revertants for E. coli) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

EVALUATION
- Mutagenicity: Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.
- Toxicity: Toxicity detected by a decrease in the number of revertants (factor ≤ 0.6) and/or clearing or diminution of the background lawn (= reduced his- or trp- background growth) was recorded for all test groups both with and without S9 mix in all experiments. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.
- Solubility: If precipitation of the test material was observed, it would be recorded. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.
Rationale for test conditions:
Mutagenicity was observed in the standard plate test. Therefore, the standard plate test was repeated instead of the prival modification.
Evaluation criteria:
ACCEPTANCE CRITERIA
Generally, the experiment was considered valid if the following criteria were met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- Fresh bacterial culture containing approximately 10^9 cells per mL were used.

ASSESSMENT CRITERIA
The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see 'Additional information on results'
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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see 'Additional information on results'
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Test substance precipitation was found from about 100 μg/plate onward with and without S9 mix.

CYTOTOXICITY
A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was only observed using the tester strains TA 1535 and E. coli WP2 uvrA, depending on the strain and test conditions about 1000 μg/plate onward.

ADDITIONAL INFORMATION ON MUTAGENICITY
TA 100
- without S9 mix: Increase of revertants at concentrations of 100 up to 5000 μg/plate in the 1st Experiment and at a concentration of 100 μg/plate in the 2nd Experiment.
- with S9 mix: Increase of revertants at concentrations of 100 up to 5000 μg/plate in the 1st Experiment and at concentrations of 33 and 100 μg/plate in the 2nd Experiment.
TA 98
- without S9 mix: Increase of revertants at concentrations of 33 up to 5000 μg/plate in the 1st Experiment and at concentrations of 0.3 up to 33 μg/plate in the 2nd Experiment.
- with S9 mix: Increase of revertants at concentrations of 33 up to 5000 μg/plate in the 1st Experiment and at concentrations of 1.0 up to 33 μg/plate in the 2nd Experiment.
TA 1537
- without S9 mix: Increase of revertants at concentrations of 33 up to 5000 μg/plate in the 1st Experiment and at concentrations of 10 and 33 μg/plate in the 2nd Experiment.
- with S9 mix:
Increase of revertants at concentrations of 33 up to 5000 μg/plate in the 1st Experiment and at concentrations of 3.3, 10 and 33 μg/plate in the 2nd Experiment.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Aug 2016 to 27 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
28 Jul 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
other: in vitro gene mutation test in mammalian cells
Specific details on test material used for the study:
- Name as cited in study report: Orasol Black X51
- Test substance No.: 16/0074-1
- Batch: 001-150606
- Purity: ca. 95% can be assumed
- Physical state / color: Solid / black
- Storage conditions: Room temperature
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELL LINE AND STORAGE
The CHO (Chinese hamster ovary) cell line Is a permanent cell line derived from the Chinese hamster and has
- a high proliferation rate (doubling time of about 12 to 16 hours)
- a high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.
Stocks of the CHO cell line (1-mL portions) are maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.

CULTURE MEDIA
All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)

CULTURE MEDIUM
Ham's F12 medium containing stable glutamine and hypoxanthin supplemented with 10% (v/v) fetal calf serum (FCS)

TREATMENT MEDIUM (without S9 mix)
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) FCS.

TREATMENT MEDIUM (with S9 mix)
Ham's F12 medium containing stable glutamine and hypoxanthine.

PRETREATMENT MEDIUM ("HAT" medium)
Ham's F12 medium supplemented with:
- hypoxanthine (13.6 x 10^-3 mg/mL)
- aminopterin (0.18 x 10^-3 mg/mL)
- thymidine (3.88 x 10^-3 mg/mL)
- 10% (v/v) FCS

SELECTION MEDIUM ("TG" medium)
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with:
- 6-thioguanine (10 µg/mL)
- 10% (v/v) FCS
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced S9 fraction from Wistar rats
Test concentrations with justification for top dose:
In the pretest for toxicity based on the purity of the test substance 2100.0 μg/mL the test substance was used as top concentration both with and without S9 mix at 4 hour exposure time. The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses. Based on the data and the observations from the pretest and taking into account the current guidelines, the following doses were selected:

- 1st experiment, 4h exposure without S9 mix: 31.3, 62.5, 125, 250, 500 and 1000 µg/mL
- 1st experiment, 4h exposure with S9 mix: 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL

- 2nd experiment, 4h exposure without S9 mix: 50, 100, 200, 400, 800 and 1000 µg/mL
- 2nd experiment, 4h exposure with S9 mix: 12.5, 25, 50, 100, 200 and 400 µg/mL

Unfortunately, in the 2nd Experiment with S9 mix the positive control substance did not lead to an increase in the mutant frequency within the historical positive control data range. Nevertheless, the increase in this positive control group was statistically significant compared to the respective vehicle control group and it fulfilled the criteria for a positive mutagenic observation. However, to have a clear proof of the validity of this experimental part, it was repeated, designated 3rd Experiment:

- 3rd experiment, 4h exposure with S9 mix: 12.5, 25, 50, 100, 200 and 400 µg/mL
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: Due to insolubility of the test substance in water, DMSO was selected as vehicle, which has been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Test substance preparation: the substance was dissolved in DMSO. The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve a solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance solutions were prepared immediately before administration.
- Cell density at seeding: 20E+06 cells in 40 mL
- Conditions: in this study, all incubations were performed at 37°C with a relative humidity of ≥ 90% in a 5% (v/v) CO2 atmosphere.
- Preparation of test cultures: cell stocks (1.0-mL portions) stored in liquid nitrogen were thawed at 37°C in a water bath. 0.5 mL of stock cultures were pipetted into 25 cm2 plastic flasks containing 5 mL Ham's F12
medium (incl. 10% [v/v] FCS). After 24 hours, the medium was replaced to remove any dead cells. At least 2 passages were performed before cells were taken for the experiment. A further passage was also necessary in order to prepare test cultures.
- Pretreatment of cells with "HAT" medium: during the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium. 0.8 - 1E+06 cells were seeded per flask (175 cm²) and incubated with "HAT" medium for 3 - 4 days. A subsequent passage in Ham's F12 medium incl. 10% (v/v) FCS was incubated for a further 3 - 4 days.
- Attachment period: for each test group, about 20E+06 logarithmically growing cells per flask (300 cm²) were seeded into about 40 mL Ham's F12 medium supplemented with 10% (v/v) FCS and incubated for about 20 to 24 hours.

DURATION
- Attachment period: 20 - 24 hours
- Exposure duration: 4 hours
- Expression time: 7 - 9 days
- Selection time: 6 - 7 days
- Fixation time: from day 16

SELECTION AGENT: 6-thioguanine (10 μg/mL)

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: fixation occurred with methanol. Cells were stained with Giemsa.

DETERMINATION OF CYTOTOXICITY
Method: cloning efficiency (CE1. survival; CE2. viability)

OTHER EXAMINATIONS:
pH, osmolality, solubility and cell morphology
Rationale for test conditions:
In the 2nd Experiment with S9 mix the positive control substance did not lead to an increase in the mutant frequency within the historical positive control data range. Nevertheless, the increase in this positive control group was statistically significant compared to the respective vehicle control group and it fulfilled the criteria for a positive mutagenic observation. However, to have a clear proof of the validity of this experimental part, it was repeated, designated 3rd Experiment.
Evaluation criteria:
A test substance is considered to be clearly positive if all following criteria are met:
- A statistically significant increase in mutant frequencies is obtained.
- A dose-related increase in mutant frequencies is observed.
- The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of the laboratory’s historical negative control data (95% control limit).

Isolated increases of mutant frequencies above the historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity. A test substance is considered to be clearly negative if the following criteria are met:
- Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
- The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of the laboratory’s historical negative control data (95% control limit).
Statistics:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carriedout using one-sided Fisher's exact test with Bonferroni-Holm correction (7, 8). The calculation was performed using R.
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:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH values were not relevantly influenced by test substance treatment. In this study, in the absence of S9 mix, test substance precipitation was observed macroscopically in culture medium at the end of treatment at about 400 μg/mL and above in both experiments. In the presence of S9 mix precipitation was observed at 500 μg/mL in the 1st Experiment, at 400 μg/mL in 2nd Experiment and at 100 μg/mL and above in the 3rd Experiment.

RANGE-FINDING/SCREENING STUDIES:
The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses. In the pretest the pH value was not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, precipitation of the test substance in the vehicle DMSO was not observed in the stock solution (Test group: 2100.0 μg/mL). In culture medium, test substance precipitation occurred by the end of treatment at concentrations of 525.0 μg/mL and above in the absence of S9 mix and at 131.3 μg/mL and above in the presence of S9 mix. After 4 hours treatment in the absence of S9 mix, cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20% relative survival at 525.0 and 1050.0 μg/mL. In the presence of S9 mix, a clearly reduced relative cloning efficiency was observed after treatment with 525.0 μg/mL and above.

CELL MORPHOLOGY
After 4 hours treatment in both experiments in the absence of metabolic activation the cell morphology and attachment of the cells was adversely influenced (grade > 2) from about 500 μg/mL onward. Besides, in the 1st Experiment in the presence of S9 mix cell morphology and attachment of the cells was adversely influenced (grade > 2) at 500 μg/mL.

CYTOTOXICITY
Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were observed in all experiments in the absence and presence of S9 mix at least at the highest applied concentrations. Without S9 mix, in the 1st and 2nd Experiment after an exposure period of 4 hours there was a decrease in the number of colonies at the highest applied concentration of 1000 μg/mL (CE1 relative: 2.5% and 5.2%, respectively). Due to low cell densities these test groups were discontinued. In addition, with S9 mix, after an exposure period of 4 hours there was a decrease in the number of colonies in the 1st Experiment from 250 μg/mL onward (CE1 relative: 6.7%) and in the 2nd Experiment from 200 μg/mL onward (CE1 relative: 6.8%). In the 3rd Experiment at 400 μg/mL cell viability at the end of the experiment were clearly reduced (CE2 relative: 15.1%).

MUTANT FREQUENCY
No biologically relevant increase in the number of mutant colonies was observed with or without S9 mix. Test groups showing strong cytotoxicity and/or clear test substance precipitation in culture medium at the end of exposure period may lead to artifactual findings. Thus, test groups which fulfill these criteria were excluded from assessment of the genotoxicity as recommended by the current OECD Guideline No. 476.
The test groups evaluated for gene mutations:
- 1st Experiment without S9 mix: 0;31.3; 62.5; 125.0; 250.0 and 500.0 μg/mL
- 1st experiment with S9 mix: 0; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0 μg/mL
- 2nd Experiment without S9 mix: 0; 100.0; 200.0; 400.0 and 800.0 μg/mL
- 2nd Experiment with S9 mix: 0; 12.5; 25.0; 50.0; 100.0; 200.0; 400.0 μg/mL
- 3rd Experiment with S9 mix: 0; 25.0; 50.0; 100.0; 200.0; 400.0 μg/mL
Based on this limitations, in all three experiments after 4 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 0.00 – 9.85 per E+6 cells) were close to or within the respective vehicle control values (MFcorr.: 1.09 – 7.34 per E+6 cells) and within the range of our historical negative control data (MFcorr.: 0.00 – 9.93 per E+6 cells).

HISTORICAL CONTROL DATA (corrected mutant frequencies)
- Positive historical control data (EMS): 138.82 (42.47 - 360.93)
- Positive historical control data (DMBA): 98.14 (21.52 - 189.14)
- Negative (vehicle) historical control data without S9: 2.53 (range 0 - 6.48)
- Negative (vehicle) historical control data with S9: 2.25 (range 0 - 9.93)
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 Jul 2016
Qualifier:
according to guideline
Guideline:
other: EU Method B.49 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
06 July 2012
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Details on mammalian cell type (if applicable):
CELLS USED
The TK6 cell line is a permanent human B lymphoblastoid cell line. It shows following characteristics what makes it suitable for this test system:
- high proliferation rate (doubling time of about 12 - 14 hours)
- suspension culture
- p53 proficiency (Cell cycle checkpoint)
- stable karyotype
The TK6 cell line has shown its suitability to detect aneugenic effects in the Micronucleus test in vitro either in the absence and presence of CytB.
Stocks of the TK6 cell line (1-mL portions incl. 7% [v/v] DMSO) have been maintained at -196°C in liquid nitrogen. Before being used, each batch will be checked for
- mycoplasma contamination,
- karyotype stability,
- growth characteristics.

MEDIA USED
- Culture medium: RPMI 1640 medium (containing a L-glutamine source and antibiotics) supplemented with 10% (v/v) fetal calf serum (FCS).
- Treatment medium is similar to culture medium, except for the absence of FSC in incubations in presence of S9.
- All incubations occur with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity.
Cytokinesis block (if used):
cytochalasin B (CytB)
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
In the pretest the pH value was not relevantly influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, precipitation of the test substance in the vehicle DMSO was not observed in the stock solution (Test group: 2100.00 μg/mL). In culture medium, test substance precipitation occurred at 262.50 μg/mL and above 4 and 24 hours after start of treatment in the absence of S9 mix. In the presence of S9 mix precipitation of the test substance in culture medium at the end of 4 hours exposure occurred at 65.63 μg/mL and above at 24 and 48 hours preparation interval.
Due to the use of a suspension cell line the measurement of cell numbers for the determination of cytotoxicity may have been interferred by test substance precipitation. After 4 hours treatment in the absence of S9 mix cytotoxicity indicated by reduced RPD of about or below 40 - 50% was observed at 65.63 μg/mL and above. In contrast, in the pretest with 24 hours continuous treatment in the absence of S9 mix, the cell numbers were not markedly reduced. Besides, in the presence of S9 mix, no clearly reduced cell numbers were observed up to the highest applied test substance concentration.

On the basis of the data and the observations on test substance solubility in culture medium from the pretest and taking into account the current guidelines, the following concentrations were selected for the 1st Experiment both in presence and absence of S9: 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/mL
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: Due to insolubility of the test substance in water, dimethyl sulfoxide (DMSO) was selected as vehicle, which has been demonstrated to be suitable in the in vitro cytogenetic assay and for which historical control data are available.
- Stability of the test substance in vehicle: The stability of the test substance at room temperature in the vehicle DMSO for 4 hours was verified analytically. The study was carried out in compliance with the Principles of Good Laboratory Practice.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
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
- Preparation of test substance: The test substance is weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution.
- Preparation of test cultures: From the routine passage, cells in culture medium are transferred in tubes and centrifuged at 1000 rpm (173 g) for 5 minutes. Subsequently, the medium (supernatant) is removed and the cells are resuspended in fresh 20 - 50 mL RPMI 1640 (incl. 10% [v/v] FCS). A homogeneous single cell suspension is obtained after pipetting gently. The number of cells in this suspension is determined using a cell counter. Finally, a single cell suspension with the required cell count (2.5 E5 cells per mL) is prepared in RPMI 1640 incl. 10% (v/v) FCS. The cultures are visually checked for viability before treatment of the test cultures.
- Definition of test cultures: A test group consists of two separately treated flasks. Each, two slides are prepared and, thus, 4 slides are available for scoring of each test group.
- Treatment of test cultures: Following centrifugation and resuspension 3 mL cell suspension are dispensed into each 25 cm² flask (7.5 E5 cells per flask). Two cultures are treated in parallel for each test group. Subsequently the treatment medium are added. The cultures are incubated for the respective exposure period at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity.

DURATION
- Exposure duration: 4h (without and with S9 mix experiment I, with S9 mix experiment II) and 24 h (without S9 in experiment II)
- Recovery time: 20 hours (without and with S9 mix experiment I) and 40 hours (with S9 in experiment II)
- Harvest time: 24 hours (without and with S9 mix experiment I, with S9 mix experiment II) and 44 hours (with S9 in experiment II)

SPINDLE INHIBITOR (cytogenetic assays):
At the end of the exposure period, the cells are transferred in tubes, centrifuged for 5 minutes at 1000 rpm (173 g) and are resuspended in HBSS (Hanks Balanced Salt Solution) with Mg2+/Ca2+. Washing of the cells will be repeated at least once. Then the cells are centrifuged at 1000 rpm (173 g, 5 min) and are resuspended in RPMI 1640 medium with 10% (v/v) FCS and transferred into 25 cm² cell culture flasks. CytoB (final concentration: 3 μg/mL; stock: 0.6 mg/mL in DMSO; AppliChem, Cat.No. A7657) is added and incubated at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity for the respective recovery time. In the case of 24-hour continuous exposure, CytB is added to the treatment medium at start of treatment, and cell preparation starts directly at the end of exposure after washing of the cells. At 44 hours preparation interval in the presence of S9 mix the supplementation of CytB is 24 hours before preparation of the cultures.

STAIN (for cytogenetic assays): a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/mL; Sigma-Aldrich, Cat.No. D9542) and propidium iodide (stock: 5 mg/mL; Sigma-Aldrich, Cat.No. P4170) in Fluoroshield™ (Sigma-Aldrich, Cat.No. F6182) at a concentration of 0.25 μg/mL each.

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- Single cell suspensions are prepared from each test group by resuspending. Then, the cell number per flask of each cell suspension is determined using a cell counter (CASY®, Roche Applied Science, Mannheim, Germany). Subsequently, 5 E4 cells per slide are centrifuged at 600 rpm for 7 minutes onto labeled slides using a Cytospin centrifuge (Cellspin I, Tharmac, Waldsolms, Germany). At least two slides per flask are prepared. In the case of strongly reduced cell numbers no slides are prepared. After drying, the slides are fixed in 90% (v/v) methanol for 10 minutes.
- Before scoring, the slides are stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/mL; Sigma-Aldrich, Cat.No. D9542) and propidium iodide (stock: 5 mg/mL; Sigma-Aldrich, Cat.No. P4170) in Fluoroshield™ (Sigma-Aldrich, Cat.No. F6182) at a concentration of 0.25 μg/mL each. By the use of the combination of both fluorescence dyes it can be differentiated between DNA (DAPI; excitation: 350 nm, emission: 460 nm) and cytoplasm (PI; excitation: 488 nm, emission: 590 nm).

NUMBER OF CELLS EVALUATED:
1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, are evaluated for the occurrence of micronuclei.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
- The diameter of the micronucleus is less than 1/3 of the main nucleus
- The micronucleus and the main nucleus retain the same color.
- The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
- Only binucleated cells will be scored.

DETERMINATION OF CYTOTOXICITY
The cytokinesis-block proliferation index (CBPI) is a direct measure of the proliferative activity of the cells and it is determined in 1000 cells per culture (2000 cells per test group). This value indicates the average number of cell cycles per cell during the period of exposure to the actin polymerisation inhibitor cytochalasin B.

OTHER EXAMINATIONS:
- pH value: Changes in the pH will be apparent by a color change of the indicator in the culture medium (phenol red: normal range: about pH 6.7 - 8.3). The pH is measured at least for the top concentration and for the vehicle control, each.
- Osmolality: Osmolality is measured at least for the top concentration and for the vehicle control, each.
- Solubility: Test substance precipitation is checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically/ microscopically).
Evaluation criteria:
A test substance is considered to be clearly positive if all following criteria are met:
- A statistically significant increase in the number of micronucleated cells is obtained.
- A dose-related increase in the number of cells containing micronuclei is observed.
- The number of micronucleated cells exceeds both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).

A test substance is considered to be clearly negative if the following criteria are met:
- Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei is observed under any experimental condition.
- The number of micronucleated cells in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).
Statistics:
- In case of inconclusive data an appropriate statistical analysis is performed. The proportion of cells containing micronuclei is calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group is carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test).
- In case of clearly negative/positive findings, a statistical evaluation may not be carried out.
Key result
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in presence of S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for TK6 cells. In all valid experiments, both positive control substances, mitomycin C (MMC) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei.
Cytotoxicity indicated by clearly reduced cell count (indicated by relative population doubling) or proliferation index (CBPI) was dose-dependent observed only with metabolic activation in all experimental parts of this study.

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

OECD 471 (Ames test)

In an Ames test performed according to GLP and OECD 471 the mutagenic potential of the test substance was evaluated in the bacterial strains S. typhimurium TA 1535, TA 100, TA 1537 and TA 98 and E. coli WP2 uvrA with and without metabolic activation (S9 mix from phenobarbital and β-naphthoflavone induced rats livers). The test substance was tested in triplicate at 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate in the standard plate test using DMSO as vehicle. In the second experiment this procedure was repeated for strains TA 100 (0; 0.3; 1; 3.3; 10; 33 and 100 μg/plate), TA 1537 (same doses) and TA 98 (0; 0.1; 0.3; 1; 3.3; 10 and 33 μg/plate) because mutagenicity was observed in the first experiment. Therefore, the standard plate test was repeated instead of the prival modification. Test substance precipitation was found from about 100 μg/plate onward. A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was only observed using the tester strains TA 1535 and E. coli WP2 uvrA, depending on the strain and test conditions about 1000 μg/plate onward. An evident and dose dependent increase in the number of his+ revertants with the tester strains TA 100, TA 1537 and TA 98, all with and without S9 mix. The increase of revertants was reproducible in two experiments carried out independently of each other. According to the results of the present study, the test substance is mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen (BASF 2016).

Several Ames tests with constituents of the substance

Several Ames tests are available which are performed with constituents of the substance in tester strains TA 98, 100, 1537 and 1538 in absence and presence of metabolic activation. Most of the tests in TA 98 and TA 100 appear positive. However, the dose-dependent effect was found at dose levels at which the substance precipitated on the agar plates. Therefore, impurities as the cause for the increase in the number of mutant colonies cannot be ruled out. Consequently the results might be considered as false positive. The assays performed in TA 1537 and 1538 are negative.

OECD 476 (HPRT assay in CHO cells)

In a GLP compliant study performed according to OECD 476, the substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out with and/or without the addition of liver S9 mix from phenobarbital- and ß-naphthoflavone induced rats. According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

- 1st Experiment without S9 mix:0;31.3; 62.5; 125.0; 250.0; 500.0; 1000 μg/mL

- 1st experiment with S9 mix:0; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0 μg/mL

- 2nd Experiment without S9 mix:0;50.0; 100.0; 200.0; 400.0; 800.0;1000.0 μg/mL

- 2nd Experiment with S9 mix: 0; 12.5; 25.0; 50.0; 100.0; 200.0; 400.0 μg/mL

- 3rd Experiment with S9 mix:0;12.5; 25.0; 50.0; 100.0; 200.0; 400.0 μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance (using DMSO as vehicle) for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate and 7,12-dimethylbenz[a]- anthracene, led to a statistically significant increase in the frequencies of forward mutations. However, in the 2nd Experiment in the presence of metabolic activation the positive control response was lower than expected, which triggered the performance of the third test. In all experiments in the absence and presence of S9 mix at least the highest concentrations tested for gene mutations were clearly cytotoxic. Statistical significances observed in several test groups were regarded as biologically irrelevant. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in three experiments performed independently of each other.Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation (BASF 2017).

OECD 487 in vitro micronucleus assay in TK6 cells

The test substance was assessed for its potential to induce micronuclei in TK6 cells in vitro (clastogenic or aneugenic activity). Three independent experiments were carried out. According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested.

1st Experiment:

4 hours exposure, 24 hours harvest time, without S9 mix          
Invalid experiment

4 hours exposure, 24 hours harvest time, with S9 mix    
0;
3.91;7.81; 15.63; 31.25;62.5; 125µg/mL

2nd Experiment:

4 hours exposure, 24 hours harvest time, without S9 mix          
0;
3.91;7.81; 15.63; 31.25;62.5; 125µg/mL

3rd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix       
0; 3.91; 7.81; 15.63;
31.25; 62.5; 125; 250, 375µg/mL

4 hours exposure, 44 hours harvest time, with S9 mix    
0;
3.91; 7.81;15.63; 31.25; 62.5;125µg/mL

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.

The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for TK6 cells. In all valid experiments, both positive control substances,mitomycin C(MMC) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei.

Cytotoxicity indicated by clearly reduced cell count (indicated by relative population doubling) or proliferation index (CBPI) was dose-dependent observed only with metabolic activation in all experimental parts of this study.

On the basis of the results of the present study, the test substance did not cause any statistically significant or biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Thus, under the experimental conditions described,the test item is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in human TK6 cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.