1-ethyl-3-methyl-3H-imidazolium dicyanidoamide(1-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic in bacteria, as determined in an OECD 471 study.

The test substance is not chromosome damaging, as determined in an OECD 487 study.

The test substance is not mutagenic in mammalian cells, as determined in an OECD 476 study.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of the test material (as cited in study report): EMIM Dicyanamid
- Purity/composition: 97.4 g/100 g

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from induced rats

Test concentrations with justification for top dose:

33 µg - 5200 µg/plate (dose levels for Standard Plate Test and Preincubation Test)

Vehicle / solvent:

Due to the good solubility of the test substance in ultrapure water, ultrapure water was used as vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene; N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

Standard Plate Test and Preincubation Test

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No bacteriotoxic effect was observed under all test conditions.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test substance was found with and without S9 mix.

Conclusions:

GHS criteria not met

Reference 2

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:

September, 2014

Qualifier:

according to guideline

Guideline:

other: In vitro Mammalian Cell Micronucleus Test, No B.49; No L 193

Version / remarks:

Commission Regulation (EC) No 640/2012 of 06 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH): In vitro Mammalian Cell Micronucleus Test, No B.49; No L 193

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

- Name of test material (as cited in study report): EMIM Dicyanamid
- Test substance No.: 12/0071-2
- Batch identification: 0013141983
- Purity/composition: 97.4 g/100 g (1H-NMR spectroscopy)
- Homogeneity: the homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.
- Storage stability: the stability of the test substance under storage conditions throughout the study period was guaranteed
- Date of production: 20 Feb 2015
- Physical state, appearance: liquid, colourless to brown, clear
- Storage conditions: room temperature (N2 conditions)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELL LINE AND STORAGE
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 source supplemented 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.

CELL CYCLE AND HARVEST TIME
The cell cycle of the untreated V79 cells lasts for about 12 - 14 hours under the selected culture conditions. Thus, a harvest time of 24 hours is about 2 times the normal cell cycle length. V79 cells are an asynchronous cell population, i.e. at the time of test substance treatment there are different cell stages (G1-, S-, G2-phase and mitosis). Since the effect on these cell stages may vary for different test substances, more than one harvest time after treatment may be appropriate. Furthermore, substance-induced mitotic delay may considerably delay the first post-treatment mitosis. Therefore, delayed harvest times (e.g. 44 hours) and prolonged exposure periods (e.g.
24 hours treatment) may be required for the detection of several substances.

Cytokinesis block (if used):

actin polymerisation inhibitor cytochalasin B

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:

- Without S9 mix experiment I [4 hours exposure, 20 hours recovery time, 24 hours harvest time]: 0 (control), 237.5, 475.0, 950.0 and 1900.0 µg/mL
- Without S9 mix experiment II [24 hours exposure, 0 hours recovery time, 24 hours harvest time]: 0 (control), 237.5, 475.0, 950.0 and 1900.0 µg/mL
- With S9 mix experiment I [4 hours exposure, 20 hours recovery time, 24 hours harvest time]: 0 (control), 237.5, 475.0, 950.0 and 1900.0 µg/mL
- With S9 mix experiment II [4 hours exposure, 40 hours recovery time, 44 hours harvest time]: 0 (control), 237.5, 475.0, 950.0 and 1900.0 µg/mL

In all tests, test groups 0 (control), 475.0, 950.0 and 1900.0 µg/mL were evaluated for cytogenetic damage.

Justification for top dose: a pre-test, following the requirements of current OECD Guideline 487 to find a maximum concentration, was performed to determine a correct top dose. In the pretest the pH value was not influenced by the addition of the test substance preparation to the culture medium at the concentrations tested. In addition, a solution of the test substance in MEM was obtained in the stock preparation (Test group: 1900 μg/mL). Thus, no test substance precipitation in culture medium was observed under any experimental condition. After 24 hours continuous treatment in the absence of S9 mix and after 4 hours treatment in the absence and presence of S9 mix no clear cytotoxicity indicated by reduced RPD of about or below 40 - 50% was observed.

Vehicle / solvent:

- Vehicle used: Minimal Essential Medium (MEM)
- Justification for choice of solvent: due to the good solubility of the test substance in water, culture medium MEM was selected as vehicle.

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:

METHOD OF APPLICATION: in medium
- Test substance preparation: the test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The substance was dissolved in culture medium (MEM). To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance formulations were prepared immediately before administration.
- Preparation of test cultures: 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.
- Cell density at seeding: 3 - 5E+05 cells per culture (dependent on the schedule).
- Treatment of test cultures: after the attachment period, about 20 - 24 hours after seeding, the medium was removed from the flasks and the treatment medium was added. 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.

DURATION: see 'Test concentrations with justification for top dose'.

SPINDLE INHIBITOR: actin polymerisation inhibitor cytochalasin B (CytB)

NUMBER OF REPLICATIONS: At least 2 cultures were prepared per test group. From each test group two slides were prepared and, thus, four slides were available for scoring of each test group, in general.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells were prepared based on the method described by Fenech. 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. 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).

NUMBER OF CELLS EVALUATED: at least 1000 cells per culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: see 'Evaluation criteria'.

DETERMINATION OF CYTOTOXICITY
- Method: relative population doubling (RPD), Proliferation Index (CBPI)

OTHER EXAMINATIONS:
- 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.
- 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).

- OTHER: Dose selection for scoring for cytogenetic damage was based on the results of a previous check on slide and/or cell quality, number of analyzable cells and nuclear fragmentation.

Evaluation criteria:

The analysis of micronuclei was carried out following the criteria of Countryman and Heddle:
− 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 the 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.

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 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).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest applied test substance concentration after 24 hours continuous test substance treatment in the 2nd Experiment without S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH values were not influenced by test substance treatment. No precipitation of the test substance in culture medium was observed.

RANGE-FINDING/SCREENING STUDIES
In the pretest for toxicity based on the purity and the molecular weight of the test substance 1900 μg/mL (approx. 10 mM) EMIM Dicyanamid was used as top concentration. The cells were prepared at a harvest time of 24 hours (about 2 cell cycles) after 4 and 24 hours exposure time without S9 mix and after 4 hours exposure time with S9 mix. The pretest was performed following the method described for the main experiment. As indication of test substance toxicity relative population doubling (RPD) and cell attachment / morphology were determined for dose selection. In the pretest various additional parameters were checked or determined for all or at least some selected doses. The following parameters are available: pH (visual examination by pH-indicator phenol red) and solubility.

GENOTOXICITY
No biologically relevant increase in the number of micronucleated cells wasobserved either without S9 mix or after the addition of a metabolizing system. In both experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.3 – 0.8% micronucleated cells) were close to the concurrent negative control values (0.6 – 0.8% micronucleated cells) and within the range of the 95% control limit of our historical negative control data (0.0 - 1.0% micronucleated cells). The positive control substances EMS (without S9 mix; 500 μg/mL) and CPP (with S9 mix; 0.5 μg/mL) induced statistically significant increased micronucleus frequencies in both independently performed experiments. In this study, in the absence and presence of metabolic activation the frequency of micronucleated cells (2.5 – 3.9% micronucleated cells) was clearly above the range of our historical negative control data (0.1 - 1.5% micronucleated cells) and within the historical positive control data range (2.3 – 13.8% micronucleated cells).

CYTOTOXICITY - RELATIVE POPULATION DOUBLING (important for the pretest)
In both main experiments in the absence and presence of S9 mix no cytotoxicity indicated by reduced RPD of below 50% of control was observed. These values were calculated based on cell numbers determined at the end of each experiment.

CYTOTOXICITY - PROLIFERATION INDEX
Reduced proliferative activity was only observed in the 2nd Experiment after 24 hours continuous test substance treatment in the absence of metabolic activation. Cytostasis was concentration-dependent increased at the test groups scored for cytogenetic damage. At the highest applied concentration of 1900 μg/mL 66.2% cytostasis was obtained which is an indication of relevant cytotoxicity.

CELL MORPHOLOGY
In this study, cell morphology/attachment was not adversely influenced (grade > 2) at any dose tested for the occurrence of micronuclei.

Conclusions:

GHS criteria not met

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

July, 2015

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

May, 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August, 1998

Principles of method if other than guideline:

Increased numbers of seeded cells and enzymatic dissociation of the cells at the end of exposure period.

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 of test material (as cited in study report): EMIM Dicyanamid
- Test item No.: 12/0071-2
- Batch identification: 0013141983
- Content: 97.4 g/100 g determined by 1H-NMR spectroscopy
- Homogeneity: the test item was homogeneous by visual inspection
- Storage stability: the stability of the test item under storage conditions over the study period was guaranteed by the sponsor
- Expiry date: February 19, 2017
- Storage conditions: room temperature, under N2
- Physical state / color: liquid / colorless to brown, clear

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 - 16 hours) and
- 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.

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:

- Experiment 1 without S9 mix [4 hours exposure]: 59.4, 118.8, 237.5, 475.0, 950.0 and 1900 µg/mL
- Experiment 1 with S9 mix [4 hours exposure]: 59.4, 118.8, 237.5, 475.0, 950.0 and 1900 µg/mL
- Experiment 2 without S9 mix [4 hours exposure]: 118.8, 237.5, 475.0, 950.0 and 1900 µg/mL
- Experiment 2 with S9 mix [4 hours exposure]: 118.8, 237.5, 475.0, 950.0 and 1900 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ham's F12 medium
- Justification for choice of solvent/vehicle: due to the good solubility of the test substance in water, the aqueous culture medium was selected 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:

METHOD OF APPLICATION: in medium
- Test substance preparation: the substance was dissolved 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. To achieve a solution of the test substance in the vehicle, the test substance preparation was pipetted 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
- Preincubation period: 20 - 24 hours after seeding
- Exposure duration: 4 hours
- Expression time: 7 - 9 days
- Selection time: 6 - 7 days
- Fixation time: from day 16

SELECTION AGENT: TG Medium

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

OTHER EXAMINATIONS:
- pH, osmolality, solubility, cell morphology

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. 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 influenced by test substance treatment. In this study, in the absence and the presence of S9 mix, no precipitation in culture medium was observed up to the highest applied test substance concentration.

RANGE-FINDING/SCREENING STUDIES:
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 purity and the molecular weight of the test substance 1900 μg/mL (approx. 10 mM) 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. In addition, a solution of the test substance in HAM´s F12 was obtained in the stock preparation (Test group: 1900 μg/mL). Thus, no test substance precipitation in culture medium was observed under any experimental condition. After 4 hours treatment in the absence and presence of S9 mix no cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20%.

CELL MORPHOLOGY
After 4 hours treatment either in the absence or presence of metabolic activation, the cell morphology and attachment of the cells was not adversely influenced (grade > 2) in any test group tested for gene mutations.

MUTANT FREQUENCY
In this study, no relevant increase in the number of mutant colonies was observed with or without S9 mix. In both experiments after 4 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 0.69 – 11.61 per E+06 cells) were close to or within the respective vehicle control values (MFcorr.: 2.74 – 9.93 per E+06 cells) and close to the range of the 95% control limit of the historical negative control dat (MFcorr.: 0.00 – 7.19 per E+06 cells). However, in the 1st Experiment in the presence of S9 mix the value for the corrected mutation frequency in test group 950.0 μg/mL (MFcorr.: 11.61 per E+06 cells) was slightly above the range of the 95% control limit and slightly above the concurrent vehicle control (MFcorr.: 9.93 per 106 cells). Nevertheless, the value was neither statistically significant nor dose-related increased. In addition, the results obtained in the 1st Experiment in the presence of S9 mix could not be confirmed in the 2nd Experiment.

In the 1st Experiment in the absence of S9 mix the value for the corrected mutation frequency in test group 237.5 μg/mL (MFcorr.: 7.04 per E+06 cells) was statistically significant compared to the respective vehicle control (MFcorr.: 2.74 per E+06 cells). Nevertheless, the value obtained for the corrected mutation frequency of this experimental part was clearly within the range of the 95% control limit and well within our historical negative control data range (MFcorr.: 0.00 – 11.96 per +E06 cells). Therefore, this finding has to be regarded as biologically irrelevant.

In all experiments, no dose-related increase in the mutant frequency was found in cells after 4 hours of treatment either in the absence or presence of S9 mix. 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.: 139.53 – 166.83 per E+06 cells; with
S9 mix: MFcorr.: 82.72 – 146.43 per E+06 cells) were clearly within our historical positive control data range (without S9 mix: MFcorr.: 49.16 – 242.55 per E+06 cells; with S9 mix: MFcorr.: 41.99 – 674.75 per E+06 cells).

Conclusions:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No further details available.

Additional information

The potential of the test substance to induce genetic toxicity was assessed on three endpoints: the induction of gene mutations in bacteria (Ames test), its chromosome damaging potential (Micronucleus test) and the potential to induce gene mutations in mammalian cells.

The potential to induce mutagenicity in bacteria was assessed in a study according to OECD 471 performed in compliance with GLP criteria. In this study the bacterial strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 were exposed to test substance concentrations of 33 to 5200 µg/plate in the standard plate test and the preincubation test, in the presence and absence of a metabolic activation system (liver S9 mix from rats). Ultrapure water was used as vehicle. In the test no bacteriotoxic effects were observed and no precipitation was observed. No mutagenic effects were observed in the tested strains. Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium / Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Additionally, the substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) in a study according to OECD 487 and in compliance with GLP criteria. In this study, two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested. The test groups printed in bold type were evaluated for cytogenetic damage:

1st Experiment: 4 hours exposure, 24 hours harvest time, +/- S9 mix: 0 (control); 237.5; 475.0; 950.0; 1900.0 μg/mL

2nd Experiment: 24 hours exposure, 24 hours harvest time, without S9 mix; 4 hours exposure, 44 hours harvest time, with S9 mix: 0 (control); 237.5; 475.0; 950.0; 1900.0 μg/mL

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. The negative controls gave frequencies of micronucleated cells within the historical negative control data range for V79 cells. Both positive control substances, ethyl methanesulfonate (EMS) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei. In this study, cytotoxicity indicated by clearly reduced proliferation index (CBPI) was observed only at the highest applied test substance concentration after 24 hours continuous test substance treatment in the 2nd Experiment without S9 mix. 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, under the experimental conditions described, 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.

Moreover, the test 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 in a study according to OECD 476 and in compliance with GLP. Two 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). 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 the current historical control database obtained in the period from January 2014 to December 2015. According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment: +/- S9 mix: 0 (control), 59.4, 118.8, 237.5, 475.0, 950.0 and 1900 µg/mL

2nd Experiment: +/- S9 mix:0 (control), 118.8; 237.5; 475.0; 950.0; 1900.0 μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance 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 (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected increase in the frequencies of forward mutations. In this study in the absence and the presence of metabolic activation, no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

The Ames, micronucleus and mammalian gene mutation tests were negative and therefore no classification is warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.