Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data of a whole battery of robust high quality in vitro studies show that the test item does not possess any mutagenic or genotoxic properties.
The test substance was not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation. In addition, an HPRT assay and an in vitro micronucleus assay were negative with and without metabolic activation.

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 Jan 2016 to 21 Jan 2016
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:
Jul 21, 1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Aug, 1998
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: Ra-He 2014-054
- Test substance No.: 15/0531-1
- Purity test date: 100 % UVCB
- Homogeity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Appearance: Solid, beige

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Storage stability: The stability of the test substance under storage conditions is guaranteed until Oct 2016

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in dimethyl sulfoxide (DMSO). To achieve a clear solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly. The further concentrations were diluted from the stock solution according to the planned doses. All test substance formulations were prepared immediately before administration.
Target gene:
- S. typhimurium: his
- E. coli: trp
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 induces rat liver S9 mix
Test concentrations with justification for top dose:
- Standard plate test: 33; 100; 333; 1000; 2500 and 5000 μg/plate
- Preincubation test: 33; 100; 333; 1000; 2500 and 5000 μg/plate

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:
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
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
- EXPERIMENT 1:
METHOD OF APPLICATION: in agar (plate incorporation) (SPT)

DURATION
- Exposure duration: 48-72 h at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)


- EXPERIMENT 2:
METHOD OF APPLICATION: preincubation (PIT)

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48-72 h at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
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 109 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.
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100
Remarks:
SPT
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
SPT
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100
Remarks:
PIT
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Remarks:
PIT
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TOXICITY
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was observed in the standard plate and preincubation test depending on the strain and test conditions from about 2500 μg/plate onward.

SOLUBILITY
Test substance precipitation was found at 5000 μg/plate with and without S9 mix.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Feb 2016 to 10 Jun 2016
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:
28 Jul, 2015
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
Aug, 1998
Principles of method if other than guideline:
As follow-up on the revision of the OECD Guideline No. 476 minor changes in test procedure were implemented in this study (e.g. increased numbers of seeded cells and enzymatic dissociation of the cells at the end of exposure period). Although validated inhouse no robust dataset on this setup recently exist, these changes may have a minor impact on the data. However, it was concluded to use for data interpretation of this study our current historical control database obtained in the period from January 2014 to December 2015.
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro gene mutation study in mammalian cells
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: Ra-He 2014-054
- Purity test date: 100 % UVCB
- Homogeity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Appearance: Solid, beige

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Storage stability: The stability of the test substance under storage conditions is guaranteed until Oct 2016

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The substance was suspended in culture medium (Ham’s F12). The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. Due to the pH shift observed at the dose group 5000.0 μg/mL (stock solution) in the pretest, in the main experiments the pH of the stock solutions were determined and adjusted case-bycase to a physiological value using small amounts of 1N NaOH. To achieve homogeneity of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and shaken thoroughly. To keep the test substance homogeneously in the vehicle, the test substance preparation was carefully pipetted before removal. The further concentrations were diluted according to the planned doses. All test substance solutions were prepared immediately before administration.
Target gene:
hprt
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELL LINE
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 - 16 hours)
- 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.

MEDIA
All media were supplemented with 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL) and 1% (v/v) amphotericine B (stock solution: 250 μg/mL)
- Culture medium: Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) 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) and 10% (v/v) FCS
- Selection medium: ("TG" medium): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 6-thioguanine (10 μg/mL) and 10% (v/v) fetal calf serum (FCS)

CELL CULTURE
For cell cultivation, deep-frozen cell suspensions were thawed at 37°C in a water bath, and volumes of 0.5 mL were transferred into 25 cm2 plastic flasks containing about 5 mL Ham's F12 medium including 10% (v/v) FCS. Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm2 plastic flasks).
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from induced rats (phenobarbital; i.p. and β-naphthoflavone; oral)
Test concentrations with justification for top dose:
1st Experiment
- Without S9 mix (discontinued due to insufficient cell number of the positive control): 6.3; 12.5; 25.0; 50.0; 100.0; 200.0; 400.0 μg/mL
- With S9 mix (discontinued due to failure in achieving the expected cytotoxicity): 6.3; 12.5; 25.0; 50.0; 100.0; 200.0; 400.0 μg/mL

2nd Experiment
- Without S9 mix: 12.5; 25.0; 50.0; 100.0; 200.0; 400.0; 600.0 μg/mL
- With S9 mix: 12.5; 25.0; 50.0; 100.0; 200.0; 400.0; 600.0μg/mL

3rd Experiment
- Without S9 mix: 9.4; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 μg/mL
- With S9 mix: 9.4; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 μg/mL

The doses/concentrations tested in this study were selected in accordance with the requirements set forth in the test guidelines and based on the results of a preliminary range finding test.
Vehicle / solvent:
In comparison to other commonly used vehicles (e.g. DMSO, acetone etc.), water was the most suitable. Therefore, culture medium was used as vehicle.
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:
PRETEST FOR DOSE SELECTION
Following the requirements of the current international guidelines and the ICPEMC Task Group,a test substance should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. In case of toxicity, the top dose should result in approximately 10 - 20% relative survival (relative cloning efficiency), but not less than 10%. For relatively insoluble test substances at least one concentration should be scored showing no precipitation in culture medium at the end of the exposure period. In the pretest for toxicity based on the composition, 5000.0 μg/mL of 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. 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.

However, a slight pH shift was observed at the highest required concentration prior to testing. Therefore, the pH of the stock solution was adjusted to a physiological value prior to application using small amounts of 1N NaOH. In addition, a homogeneous suspension of the test substance in HAM´s F12 was obtained in the stock preparation (Test group: 5000 μg/mL). In culture medium test substance precipitation occurred at 625 μg/mL and above 4 hours after start of treatment in the absence and presence of S9 mix. After 4 hours treatment, cytotoxicity was observed as indicated by a distinctly reduced cell number during the 1st passage of the treated cells at 1250.0 μg/mL and above in the absence and presence of S9 mix. Furthermore, subsequent cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20% relative survival at 312.5 μg/mL and above in the presence and absence of S9 mix.

DURATION
- Attachment period: For each test group, about 20x106 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 - 24 hours.
- Exposure period: After the attachment period, the medium was removed from the flasks and the treatment medium was added. The cultures were incubated for 4 hours at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity.
- Expression time: The exposure period was completed by rinsing several times with HBSS. This was directly followed by the 1st passage in which 2x106 cells were seeded in 20 mL medium (in 175 cm2 flasks). The flasks were left to stand in the incubator for about 3 days at 37°C, relative humidity of ≥ 90% and 5% (v/v) CO2 atmosphere. After about 3 days, the cells were passaged a 2nd time in 175 cm2 flasks with 2x106 cells. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (3rd passage).
- Selection period: For selection of the mutants, two 175 cm2 flasks with 2x106 cells each from every treatment group, if possible, were seeded in 20 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 – 7 days. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

DETERMINATION OF CYTOTOXICITY
- Cloning efficiency (pre exiperiment): The determination of the cloning efficiency in the pre-experiment was similar to that described for the determination of the cloning efficiency 1 (CE1) in the main experiments, excepting that 1x106 cells were seeded in 25 cm2 flasks coated with 5 mL Ham´s F12 medium incl. 10% (v/v) FCS. After test substance incubation, 200 cells were transferred into new Ham´s F12 medium incl. 10% (v/v) FCS.
- Cloning efficiency (CE1; survival): For the determination of the influence of the test substance after the exposure period, about 200 cells per concentration were reserved from the treated cells and were seeded in 25 cm2 flasks and coated with 5 mL Ham's F12 medium incl. 10% (v/v) FCS in parallel to the 1st passage directly after test substance incubation.
- Cloning efficiency (CE1; viability): For the determination of the mutation rate after the expression period, two aliquots of about 200 cells each were reserved from the transfer into selection medium (after 7 – 9 days) and seeded in two 25 cm2 flasks containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS. In all cases, after seeding the flasks were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted.

MUTANT FREQUENCY
The number of colonies in each flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized per every 1E+06 cells seeded. The uncorrected mutant frequency per 1E+06 cells was calculated for each test group. The uncorrected mutant frequency was corrected with the absolute cloning efficiency 2 for each test group to get the corrected mutant frequency

OTHER PARAMETERS MEASURED
- pH: The pH was measured at least for the top concentrations and for the negative controls with and without S9 mix.
- Osmolality: Osmolality was measured in at least the top concentrations and the negative controls with and without S9 mix.
- Solubility: Test substance precipitation was assessed immediately after dosing the test cultures and at the end of treatment.
- Cell morphology: The test cultures of all test groups were examined microscopically for cell morphology and cellular attachment at the end of the exposure period, which is a further indication for cytotoxicity.
Evaluation criteria:
ACCEPTANCE CRITERIA
The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
- The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
- The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range

ASSESSMENT 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 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 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 our 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 used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. 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 carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. 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
Remarks:
at least at the highest concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
MUTANT FREQUENCY
In this study, no biologically relevant increase in the number of mutant colonies was observed with or without S9 mix.

In the 2nd and 3rd Experiment after 4 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 0 .00 – 8.31 per 1E+06 cells) were within or above the respective vehicle control values (MFcorr.: 0.66 – 2.25 per 1E+06 cells) and within or close to the range of the 95% control limit of our historical negative control data (without S9 mix: MFcorr.: 0.00 – 7.37 per 1E+06 cells; with S9 mix: MFcorr.: 0.00 – 7.11 per 1E+06 cells).

In detail, in the 3rd Experiment in the absence of S9 mix the value for the corrected mutation frequency in test group 150.0 μg/mL (MFcorr.: 8.31 per 1E+06 cells) was slightly above the range of the 95% control limit and above the concurrent negative control (MFcorr.: 2.25 per 1E+06 cells). In addition, in the presence of S9 mix, the values for the corrected mutation frequencies in test group 75.0 μg/mL (MFcorr.: 4.44 per 1E+06 cells) and 150.0 μg/mL (MFcorr.: 3.81 per 1E+06 cells) were slightly above the respective negative control values (MFcorr.: 0.66 – 2.25 per 1E+06 cells), but well within the range of the 95% control limit of our historical negative control data (without S9 mix: MFcorr.: 0.00 – 7.37 per 1E+06 cells). Furthermore, these values were statistically significant but not dose-related increased compared to the concurrent negative control. All values obtained for the corrected mutation frequencies of this study were well within our historical negative control data range (MFcorr.: 0.00 – 11.96 per 1E+06 cells) and therefore, these findings have to be regarded as biologically irrelevant.

The positive control substances EMS (without S9 mix; 400 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 146.72 – 157.76 per 1E+06 cells; with S9 mix: MFcorr.: 110.37 – 144.31 per 1E+06 cells) were within our historical positive control data range (without S9 mix: MFcorr.: 49.16 – 242.55 per 1E+06 cells; with S9 mix: MFcorr.: 41.99 – 674.75 per 1E+06 cells).

ADDITIONAL INFORMATION ON CYTOTOXICITY
In the 1st Experiment in the presence of metabolic activation, the expected cytotoxicity (CE1 level between 10-20% survival) at the highest applied concentration of 400.0 μg/mL was not achieved. For this reason, the experimental part was discontinued. Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were observed in the 2nd and 3rd Experiment in the presence of S9 mix, at least at the highest applied concentrations. In contrast, in the absence of S9 mix, there was a decrease in the number of colonies at 400.0 μg/mL and above in the 2nd Experiment and at 600.0 μg/mL (CE1 relative: 0.6%) in the 3rd Experiment.

CELL MORPHOLOGY
In all experimental parts, after 4 hours treatment in the 2nd and 3rd Experiment the morphology and attachment of the cells was adversely influenced (grade > 2) at concentrations of 300.0 μg/mL and above.

TREATMENT CONDITIONS
The pH value of the test substance preparation was adjusted by adding small amounts of NaOH. Osmolality and pH values were not influenced by test substance treatment. In the 2nd and 3rd Experiment, in the absence and the presence of S9 mix, precipitation in culture medium was observed at the highest applied test substance concentration of 600.0 μg/mL.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Jan 2016 to 08 Jun 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
26 Sep, 2014
Qualifier:
according to
Guideline:
other: EU Method B.49 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
6 July 2012
Principles of method if other than guideline:
Due to some differences between the above cited guidelines at diverging points (e.g. dose selection) the recommendations of the most recent OECD Guideline were followed.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: Ra-He 2014-054
- Test substance No.: 15/0531-1
- Purity test date: 100 % UVCB
- Homogeity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Appearance: Solid, beige

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Storage stability: The stability of the test substance under storage conditions is guaranteed until Oct 2016

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The substance was suspended in culture medium (MEM). To achieve homogeneity of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly. In the main experiments, except 3rd Experiment, the pH of the stock solutions was adjusted to a physiological value using small amounts of 2 M NaOH. The further concentrations were diluted according to the planned doses. All test substance formulations were prepared immediately before administration.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
The V79 cell line is a permanent cell line derived from the Chinese hamster and has a
− high proliferation rate (doubling time of about 12 - 14 hours),
− high plating efficiency (≥ 90%),
− stable karyotype (modal number of 22 chromosomes).
The V79 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 V79 cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% (v/v) dimethyl sulfoxide (DMSO) in culture medium as a cryoprotectant. Each batch used for the cytogenetic experiments was checked for
− mycoplasma contamination,
− karyotype stability,
− plating efficiency (=colony forming ability) incl. vital staining.

CULTURE MEDIA
MEM (minimal essential medium with Earle's salts) containing a L-glutamine sourcesupplemented with
− 10% (v/v) fetal calf serum (FCS)
− 1% (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL)
− 1% (v/v) amphotericine B (250 μg/mL)

CELL CULTURE
Deep-frozen cell stocks were thawed at 37°C in a water bath, and volumes of 0.5 mL were transferred into 25 cm2 plastic flasks containing about 5 mL MEM supplemented with 10% (v/v) FCS. Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity and subcultured twice weekly. Cell monolayers were suspended in culture medium after detachment with 0.25% (w/v) trypsin solution.

CULTURE PREPARATION
The stocks of cells (1.0-mL portions) were thawed at 37°C in a water bath. 0.5 mL were pipetted into 25 cm2 cell culture flasks containing 5 mL MEM (incl. 10% [v/v] FCS). The flasks were subsequently incubated at 37°C, 5% (v/v) CO2 and relative humidity of ≥ 90% until they have reached confluency of at least 50% (duration about 2 – 4 days). The medium was replaced after about 24 - 30 hours to remove any dead cells. Prior to the preparation of the final test cultures, the cells may run through max. 15 routine passages. After the "last" routine passage, there was another passage to prepare test cultures.

SEEDING
A single cell suspension with the required cell count (3 - 5x105 cells per culture, depending on the schedule) was prepared in MEM incl. 10% (v/v) FCS. 5 mL cell suspension was transferred into 25 cm² cell culture flasks using a dispenser. Subsequently, the test cultures were incubated at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity. The cultures were visually checked for attachment and viability before treatment of the test cultures.
Cytokinesis block (if used):
actin polymerisation inhibitor cytochalasin B (CytB) was used as cytokinesis block
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from induced rats (phenobarbital; i.p. and β-naphthoflavone; oral)
Test concentrations with justification for top dose:
1st Experiment
- 4 hours exposure, 24 hours harvest time, without S9 mix: 31.3; 62.5; 125.0; 250.0; 500.0; 1000.0 μg/mL
- 4 hours exposure, 24 hours harvest time, with S9 mix: 31.3; 62.5; 125.0; 250.0; 500.0; 1000.0 μg/mL

2nd Experiment
- 4 hours exposure, 24 hours harvest time, without S9 mix: 6.3; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL
- 4 hours exposure, 44 hours harvest time, with S9 mix: 25.0; 50.0; 100.0; 200.0; 400.0; 800.0 μg/mL

3rd Experiment
- 24 hours exposure, 24 hours harvest time, without S9 mix: 6.3; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL

After 4 hours treatment in the absence and presence of S9 mix and after 24 hours continuous test substance treatment cytotoxicity indicated by reduced RPD of about or below 40 - 50% was observed at 312.5 μg/mL and above. Therefore, the top dose set to 200 µg/mL in the 3rd Experiment.
Vehicle / solvent:
The test substance was suspended in culture medium (MEM). To achieve homogeneity of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
TREATMENT
After the attachment period, about 20 - 24 hours after seeding, the medium was removed from the flasks and the treatment medium was added (see 'Any other information on materials and methods incl. tables'). The cultures were incubated for the respective exposure period at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity. At the end of the 4-hour exposure period, the medium was removed and the cultures were rinsed twice with 5 mL HBSS (Hanks Balanced Salt Solution). Subsequently, 5 mL MEM (incl. 10% [v/v] FCS) supplemented with CytB (final concentration: 3 μg/mL; stock: 0.6 mg/mL in DMSO; AppliChem, Cat.No. A7657) was added and the cultures were 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 was added to the treatment medium at start of treatment, and cell preparation was started directly at the end of exposure. At 44 hours preparation interval in the presence of S9 mix CytB was added 24 hours before preparation of the cultures.

CELL HARVEST AND SLIDE PREPARATION
The cells were prepared based on the method described by Fenech, M. (Mut. Res. 285, 35-44; 1993).
Just before preparation the culture medium was completely removed. Single cell suspensions were prepared from each test group by enzymatic dissociation. Then, the cell number per flask of each cell suspension was determined using a cell counter (CASY®, Roche Applied Science, Mannheim, Germany). Subsequently, 5x104 cells per slide were 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 were prepared. In the case of strongly reduced cell numbers below 10x104 cells per flask no slides were prepared. After drying, the slides were fixed in 90% (v/v) methanol for 10 minutes.

STAINING
Before scoring, the slides were 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).

CRITERIA FOR MICRONUCLEUS IDENTIFICATION
The cytospin slides were scored by fluorescence microscopy (Axio Imager.Z2, Zeiss, Göttingen, Germany). As a rule, at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei. The analysis of micronuclei was carried out following the criteria of Countryman, P.I., Heddle, J.A. (Mut. Res. 41, 321-332. 1976):

− The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of he cell.
− Only binucleated cells clearly surrounded by a membrane were scored.

Slides were coded before microscopic analysis with an appropriate computer program. Cultures with only few isolated cells were not analysed for micronuclei. Since the absolute values shown were rounded but the calculations were made using the unedited values, there may be deviations in the given relative values.

CYTOTOXICITY DETERMINATION (Relative population doubling; RPD)
The RPD takes into account either cytotoxicity or cell proliferation over the whole incubation period until slide preparation when determination of cell numbers occurs. However, in the main experiments supplementation of culture medium with CytB blocks cell division. Thus, under the experimental conditions described, RPD is an indication of cell viability mainly for the time period before addition of CytB. Before preparing the cytospin slides the cell count was determined from trypsinized cultures. The relative population doubling will be calculated based on the following formula:

Population doubling (PD) = log (Post-treatment cell number / Initial cell number) / log 2

RPD = ((No. of PD in treated cultures) / (No. of PD in vehicle control cultures)) * 100

CYTOTOXICITY DETERMINATION (Proliferation Index)

The cytokinesis-block proliferation index (CBPI) is a direct measure of the proliferative activity of the cells and it was determined in at least 1000 cells per culture (at least 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. The number of mononucleated, binucleated and multinucleated cells was recorded and the CBPI was calculated using the following formula:

CBPI = ((No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)) / (Total number of cells)

The CBPI can be used to calculate the % cytostasis (relative inhibition of cell growth compared to the respective vehicle control group) - a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

% Cytostasis = 100 - 100 {(CBPIT - 1) / (CBPIC - 1)}
T = test substance treated culture C = vehicle control culture

CELL MORPHOLOGY
At the end of the treatment period, all test groups were examined microscopically with regard to cell morphology, which is a further indication for cytotoxicity.

OTHER PARAMETERS MEASURED
- pH: Changes in the pH were apparent by a color change of the indicator in the culture medium (phenol red: normal range: about pH 6.7 - 8.3). The pH was measured at least for the top concentration and for the vehicle control with and without S9 mix.
- Osmolality: Osmolality was measured at least for the top concentration and for the vehicle control with and without S9 mix
- Solubility: Test substance precipitation was checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically / microscopically).
Evaluation criteria:
ACCEPTANCE CRITERIA
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed the evaluation of a sufficient number of analyzable cells both in the control groups (vehicle/positive) and in at least three exposed test groups.
- Sufficient cell proliferation was demonstrated in the vehicle control.
- The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
- The positive control substances both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range

ASSESSMENT CRITERIA
A test substance is considered to be clearly positive if the following criteria are met:
- A statistically significant increase in the number of micronucleated cells was obtained.
- A dose-related increase in the number of cells containing micronuclei was observed.
- The number of micronucleated cells exceeded both the value of the concurrent vehicle control 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 criterion is met:
- Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
- The number of micronucleated cells in all treated test groups was close to the concurrent
vehicle control value and within the range of our laboratory’s historical negative control data
(95% control limit).
Statistics:
The statistical evaluation of the data was carried out using an appropriate statistical analysis. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE).
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at least at the highest concentration in the presence of metabolic activation in Exp. 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
GENOTOXICITY
In this study, no biologically relevant increase in the number of micronucleated cells was observed either without S9 mix or after the addition of a metabolizing system. In all reliable experiments according to the criteria defined in the current OECD Guideline No. 487 in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.4 – 1.1% micronucleated cells) were close to the concurrent negative control values (0.3 – 0.8% micronucleated cells) and close to the range of the 95% control limit of our historical negative control data (0.0 - 1.0% micronucleated cells.

In the 3rd Experiment in the absence of S9 mix a single value (1.1% micronucleated cells) at an intermediate concentration of 100 μg/mL slightly exceeded the range of the 95% control limit of our historical negative control data (0.0 - 1.0% micronucleated cells). However, this value was close to the respective negative control value (0.8% micronucleated cells) and, therefore, no statistical significance was obtained. Thus, this observation has been regarded as biologically irrelevant.

In deviation to the current OECD Guideline No. 487 in both experimental parts with metabolic activation more than one test group showing clear test substance precipitation were scored for cytogenetic damage. Additionally, these test groups were strongly cytotoxic. Unfortunately, in the 2nd Experiment with metabolic activation the test substance concentration 400 μg/mL led to a statistically significant increase in the number of micronucleated cells (2.4%) which strongly exceeded the range of the 95% control limit of our historical negative control data (0.0 - 1.0% micronucleated cells). Nevertheless, at this test group RPD was strongly reduced to 30.2% compared to the respective negative control. Thus, this finding has to be regarded as biologically irrelevant due to severe cytotoxicity and strong test substance precipitation in culture medium.

The positive control substances EMS (without S9 mix; 500 or 600 μg/mL) and CPP (with S9 mix; 0.5 μg/mL) induced statistically significant increased micronucleus frequencies in all independently performed and reliable experiments in at least one positive control group each. In this study, in the absence and presence of metabolic activation in at least one positive control group each the frequency of micronucleated cells (1.9 – 3.4% micronucleated cells) was clearly above the range of our historical negative control data (0.1 - 1.5% micronucleated cells) and close to our historical positive control data range (2.3 – 13.8% micronucleated cells).

CYTOTOXICITY - RELATIVE POPULATION DOUBLING
In the presence of S9 mix cytotoxicity indicated by reduced RPD of below 50% of control was observed at the two highest applied test substance concentrations each. The 1st Experiment in the absence of S9 mix showed severe cytotoxicity which was not in concordance with the data of the preliminary range finding experiment. This experimental part did not fulfill the requirements of the current OECD Guideline. In the absence of metabolic activation, in the 2nd and 3rd Experiment no relevant cytotoxicity was obtained when tested up to the border of test substance solubility. The RPD was calculated based on cell numbers determined at the end of each experiment. In the pretest the parameter RPD is a valuable indicator of test substance toxicity. However, in the main experiments due to the use of the cytokinesis block method it is a measure of cell proliferation only until addition of CytB to the cultures. But, it also gives an useful information on direct cell loss due to test substance exposure. In detail, in the absence of S9 mix weakly reduced RPD was obtained at 200 μg/mL (65.7% of control) after 24 hours exposure in the 3rd Experiment. Besides, in the presence of S9 mix RPD was decreased from 500 μg/mL (-64.0%) onward in the 1st Experiment at 24 hours preparation interval and from 400 μg/mL (30.2%) onward in the 2nd Experiment at 44 hours preparation interval. In addition, in the 1st Experiment with S9 mix severe cytotoxicity indicated by strongly reduced cell count was observed at 1000 μg/mL (RPD: -247.6%). Thus, no slides were prepared for this test group.

CYTOTOXICITY - PROLIFERATION INDEX
In this study, no clearly reduced proliferative activity was observed either after 4 hours exposure interval in the absence and presence of S9 mix or after 24 hours continuous test substance treatment in the test groups scored for cytogenetic damage with regard to the recommendations on dose selection of the current OECD Guideline No. 487. Besides, elevated cytostasis indicated by pronounced reduction of the CBPI was obtained in the presence of S9 mix in the 1st Experiment at 500 μg/mL (49.2%). But, at this test group also clear test substance precipitates were observed.

CELL MORPHOLOGY
In this study, cell morphology/attachment was not adversely influenced (grade > 2) at any dose tested for micronuclei. In this study, cell morphology/attachment was adversely influenced (grade > 2) from 125 μg/mL onward in the 1st Experiment in the absence of metabolic activation. Besides, in the presence of metabolic activation cell attachment/ morphology was adversely influenced at 1000 μg/mL in the 1st Experiment and at 800 μg/mL in the 2nd Experiment. No slides were prepared due to strongly reduced cell numbers in the presence of S9 mix at 1000 μg/mL in the 1st Experiment.

TREATMENT CONDITIONS
Osmolality and pH values were not influenced by test substance treatment. However, the pH of the stock solutions was adjusted to a physiological value using small amounts of NaOH in the 1st and 2nd Experiment. The test substance was poorly soluble in several commonly used vehicles. Although, culture medium was the most appropriate vehicle as demonstrated in a preliminary solubility trial. All stock preparations and several preparations of the dilution series were homogeneous suspensions. Test substance precipitation in culture medium at the end of exposure period was observed in all experimental parts from about 200 μg/mL onward (macroscopical assessment) in the absence and presence of S9 mix.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro

Bacterial reverse mutation tests

A test for bacterial gene mutagenicity was conducted with the test substance according to the OECD TG 471 under GLP conditions with the following bacterial strains: Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA (BASF, 2016). The test concentrations were 0 (vehicle control), 33, 100, 333, 1000, 2500, and 5000 µg/plate for the standard plate test with and without S9-mix, and for the preincubation test with and without S9-mix, respectively. Negative (sterility and solvent) and positive controls were considered. Under the experimental conditions chosen, the test substance was not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation. Precipitation of the test substance was found at 5000 µg/plate in the absence and presence of metabolic activation. A bacteriotoxic effect was observed depending on the strain and test conditions from about 2500 μg/plate onward. All negative and positive controls were as expected and confirmed the validity, suitability and sensitivity of the test method and system used.

HPRT

A HPRT assay was conducted with the test item according to OECD TG 476 under GLP conditions to assess its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro (BASF, 2016). Three independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Based on an initial range-finding test, the test concentrations were 0 (vehicle control; 6.3; 12.5; 25.0; 50.0; 100.0; 200.0; 400.0 μg/mL for Experiment 1, 0 (vehicle control); 12.5; 25.0; 50.0; 100.0; 200.0; 400.0; 600.0 μg/mL for Experiment 2 and 0 (vehicle control); 9.4; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 μg/mL for Experiment 3. Experiment 1 was discontinued due to insufficient cell density in the positive control. In the 2nd and 3rd Experiment, at least the highest concentrations evaluated for gene mutations were clearly cytotoxic in the absence and the presence of metabolic activation. The test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two valid experiments performed independently of each other. All negative and positive controls of the valid tests fulfilled the acceptance criteria of this study.

In vitro micronucleus test

An in vitro micronucleus test was conducted with the test item according to OECD TG 487 under GLP conditions to assess its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) (BASF, 2016). Three

independent experiments were carried out with and/or without the addition of liver S9 mix from induced rats (exogenous metabolic activation). Based on an initial range-finding test, the test concentrations were 0 (vehicle control);31.3; 62.5; 125.0; 250.0; 500.0; 1000.0 for Experiment 1, 0 (vehicle control); 6.3; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL for Experiment 2 without metabolic activation, 0 (vehicle control); 25.0; 50.0; 100.0; 200.0; 400.0; 800.0 μg/mL for Experiment 2 with metabolic activation and 0 (vehicle control); 6.3; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL for Experiment 3. Experiment 1 was considered not valid due to severe cytotoxicity. All negative and positive controls of the valid tests fulfilled the acceptance criteria of this study. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. Thus, the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

Based on the available data, which are reliable and suitable for classification, classification for genotoxicity is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.