Reaction mass of Sodiumbis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and Sodium [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and Sodiumbis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is mutagenic in bacteria, as determined in an OECD 471 study (BASF SE, 2016).

The test substance is not mutagenic in mammalian cells, as determined in an OECD 476 study (BASF SE, 2016).

The test substance is not chromosome damaging, as determined in an OECD 487 study (BASF SE, 2016).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

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)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS/TRP

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

33 μg - 6300 μg/plate (SPT, all tester strains)
3.3 μg - 1000 μg/plate (Prival; TA 100, TA1537 and TA 98)

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/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:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

congo red

other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine

Remarks:

S9-mix from rats and hamsters was used

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DETERMINATION OF CYTOTOXICITY
- Method: relative total 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 10E9 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 historical negative control
range under all experimental conditions in at least two experiments carried out
independently of each other.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

MUTAGENICITY

Standard plate test

Tests without S9 mix

TA 1535: No relevant increase in the number of his+ revertants.

TA 100: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 2.1 and 2.1, respectively).

TA 1537: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 3.7 and 4.3, respectively).

TA 98: Increase in the number of his+ revertants at 33, 100, 333, 1000 and 3150 μg/plate (factors 4.0, 7.8, 12.8, 18.7 and 23.8,

respectively).

E. coli WP2 uvrA: No relevant increase in the number of trp+ revertants.

Tests with S9 mix

TA 1535: No relevant increase in the number of his+ revertants.

TA 100: Increase in the number of his+ revertants at 100, 333 and 1000 μg/plate (factors 2.2, 2.5 and 2.3, respectively).

TA 1537: Slight increase in the number of his+ revertants at 333 μg/plate (Factor 2.9).

TA 98: Increase in the number of his+ revertants at 33, 100, 333, 1000 and 3150 μg/plate (factors 5.0, 7.3, 13.1, 13.7 and 17.1,

respectively).

E. coli WP2 uvrA: No relevant increase in the number of trp+ revertants.

Prival preincubation test

Tests without S9 mix

TA 100: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 1.9 and 2.6, respectively).

TA 1537: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 6.0 and 7.8, respectively).

TA 98: Increase in the number of his+ revertants at 10, 33, 100, 333 and 1000 μg/plate (factors 3.3, 5.0, 8.7, 12.5 and 33.6,

respectively).

Tests with S9 mix

TA 100: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 2.1 and 2.1, respectively).

TA 1537: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 4.0 and 4.1, respectively).

TA 98: Increase in the number of his+ revertants at 33, 100, 333 and 1000 μg/plate (factors 2.1, 4.3, 8.9 and 14.5, respectively).

TOXICITY

A bacteriotoxic effect (decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 1000 μg/plate onward.

In the prival preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was not observed up to the highest required concentration.

SOLUBILITY

Test substance precipitation was found from 333 μg/plate onward with and without S9 mix.

DISCUSSION

According to the results of the present study the test substance led to 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 (standard plate and prival preincubation test) carried out independently of each other. Based on the recent assessment criteria the test substance has to be considered positive.

Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.

In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

CONCLUSION

Thus, under the experimental conditions chosen here, it is concluded that Eukesolar Black ER Liquid - dried is a potent mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-30 to 2016-05-04

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)

Type of assay:

other: in vitro gene mutation test in mammalian cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch identification: Dye powder sample 14/103 from Material no. 52629782; batch no. M-R/G
- Purity: Purity: 99.11 area-% (HPLC, 260 nm), 99.71 area-% (HPLC, 375 nm)
- Composition: 79.0 g/100 g of Cr-Complex (C32H18CrN6NaO8) is postulated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under argon
- Storage stability: The stability of the test substance under storage conditions was guaranteed until 09 Jun 2017 as indicated by the sponsor.
- Stability of the test substance in the vehicle:The stability of the test substance at room temperature in the vehicle DMSO over a period of 4 hours was determined analytically.

OTHER SPECIFICS:
- Date of production: 09 Jun 2014
- Physical state, appearance: Solid, black
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.

Target gene:

HPRT locus

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)
- 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) and 1 % (v/v) amphotericine B (stock solution: 250 μg/mL)
- Culture medium for the 1st Experiment: Ham's F12 medium containing stable glutamine and hypoxanthine (Biochrom; Cat. No. FG 0815) supplemented with 10 % (v/v) fetal calf serum (FCS).
- Culture medium for the 2nd Experiment: 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 1E-3 mg/mL), aminopterin (0.18 x 1E-3 mg/mL), thymidine (3.88 x 1E-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 ≥ 9 0 % relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm² plastic flasks).

ROUTINE PASSAGE (preparation of a single cell suspension in 75 cm² flask)
- Cell medium was removed and cells were washed with 5 mL PBS or HBSS (both Ca-Mg-free).
- Cells were trypsinized with 2 mL HBSS (Hanks balanced salt solution; Ca-Mg-free) and 2 mL trypsin (0.25 % [w/v]) to remove the cells from the bottom of the plastic flasks.
- This reaction was stopped by adding 6 mL culture medium incl. 10 % (v/v) FCS.
- Cells were pipetted up and down to separate them and to prepare a homogeneous single cell suspension.
- Cells were counted in a counting chamber or using a cell counter.
- Cell suspensions were diluted with complete culture medium to the desired cell count.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from phenobarbital (i.p.) and β-naphthoflavone (oral) induced Wistar rat livers

Test concentrations with justification for top dose:

PRETEST:
- Justification: Following the requirements of the current international guidelines and the ICPEMC Task Group (5) 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 of the test substance 6500.0 μg/mL was used as top concentration.
- concentrations with and without S9 mix [4 hour exposure]: 25.4, 50.8, 101.6, 203.1, 406.3, 812.5, 1625.0, 3250.0, 6500.0 µg/mL

MAIN TESTS
- justification: Doses were based on the data and the observations from the pretest and current guidelines were taking into account.
- concentrations Experiment 1, with and without S9 mix [4 hour exposure]: 2.5, 5.0, 10.0, 20.0, 40.0, 80.0, 160.0 µg/mL
- concentrations Experiment 2, with and without S9 mix [4 hour exposure]: 6.3, 12.5, 25.0, 50.0, 100.0, 160.0 µg/mL

Vehicle / solvent:

PRETEST
- Vehicle: culture medium
- Justification for choice of vehicle: it was the only feasible vehicle to reach a maximum concentration of 6500 μg/mL (homogeneous suspension) due to physico-chemical properties.

MAIN TEST
- Vehicle: DMSO
- Justification for choice of vehicle: in the pretest, precipitates were found in all exposed test groups from the lowest applied concentration of 25.4 μg/mL onward. DMSO was chosen based on an additional solubility test with a lower top concentration.

Untreated negative controls:

yes

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 shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance solutions were prepared immediately before administration.
- 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. 3 – 5E+5 cells were seeded per flask (75 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+6 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 the respective exposure period at 37 °C, 5 % (v/v) CO2 and ≥ 90 % relative humidity.
- Expression period: The exposure period was completed by rinsing several times with HBSS. This was directly followed by the 1st passage in which 2E+6 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 2E+6 cells. After the expression period the cells were transferred into selection medium (3rd passage).
- Selection period: For selection of the mutants, two 175 cm² flasks with 2E+6 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. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

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

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

CYTOTOXICITY DETERMINATION
- Cloning efficiency (CE) (pre-experiment): 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 1E+6 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. Due to technical reasons in the pretest no cytotoxicity data were obtained.
- Cloning efficiency 1 (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 2 (CE2; 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 flasks (25 cm2) 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. The absolute and relative cloning efficiencies (%) were calculated for each test group

CHECK OR DETERMINATION OF FURTHER PARAMETERS
- pH: the pH was measured at least for the top concentrations and for the vehicle controls with and without S9 mix.
- Osmolality: Osmolality was measured in at least the top concentrations and the vehicle 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 the laboratory’s historical negative control data (95 % control limit).
Isolated increases of mutant frequencies above our 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 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:

no cytotoxicity, but tested up to precipitating 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.

RANGE-FINDING/SCREENING STUDIES:
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 homogeneous suspension of the test substance in the vehicle HAM´s F12 was obtained in the stock preparation (Test group: 6500 μg/mL) as well as in all applied concentrations down to 25.4 μg/mL. Thus, no soluble test substance concentration was obtained. Due to techniqual reasons, only the results of the cell counting during the 1st passage after test substance incubation could be used for dose selection.

CELL MORPHOLOGY
Only in the 1st Experiment in the absence of S9 mix, after 4 hours treatment in the morphology and attachment of the cells was adversely influenced (grade > 2) in the highest applied concentration (160.0 μg/mL).

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.: 0.00 – 9.79 per E+6 cells) were close to the respective vehicle control values (MFcorr.: 1.44 – 8.29 per E+6 cells) and close to or within the range of the 95% control limit of our historical negative control data (MFcorr.: 0.00 – 6.84 per E+6 cells). However, in the 1st Experiment in the presence of S9 mix the values for the corrected mutation frequencies in test group 40.0 μg/mL (MFcorr.: 8.89 per E+6 cells) and 160 μg/mL (MFcorr.: 9.79 per E+6 cells) were slightly above the range of the 95% control limit and slightly above the concurrent vehicle control (MFcorr.: 8.29 per E+6 cells). Nevertheless, the values were 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 all experiments, no statistically significant 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.
However, in the 1st Experiment in the absence of S9 mix the values for the corrected mutation frequencies in test group 40.0 μg/mL (MFcorr.: 6.27per E+6 cells) and 80 μg/mL (MFcorr.: 7.75 per E+6 cells) were statistically significant compared with the respective vehicle control (MFcorr.: 1.44 per E+6 cells). Nevertheless, the values obtained for the corrected mutation frequency of this experimental part were close to the range of the 95% control limit and well within our historical negative control data range (MFcorr.: 0.00 – 9.16 per E+6 cells). Therefore, this finding has 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.: 42.47 – 264.00 per E+6 cells; with S9 mix: MFcorr.: 96.88 – 189.14 per E+6 cells) were within our historical positive control data range (without S9 mix: MFcorr.: 62.02 – 268.09 per E+6 cells; with S9 mix: MFcorr.: 41.99 – 736.50 per E+6 cells).

Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20 % of the respective negative control values were not observed in both experiments in the presence and absence of S9 mix, up to the highest applied concentrations.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-17 to 2016-05-18

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:

26 Sep 2014

Deviations:

no

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test substance: Eukesolar Black ER Liquid - dried
- Batch identification: Dye powder sample 14/103 from Material no. 52629782; batch no. M-R/G
- Content: 79.0 g/100 g of Cr-Complex is postulated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Room temperature
- Storage stability: The stability of the test substance under storage conditions throughout the study period was guaranteed until 09 Jun 2017 as indicated by the sponsor.

OTHER SPECIFICS:
- Date of production: 09 Jun 2014
- Physical state, appearance: Solid, black
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

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 cytochalasin B.
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 (last measurement based on the BrdU method of Speit et al.: 12 hours; May 2014). 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:

S9 fraction from phenobarbital and β-naphthoflavone induced Wistar rat livers

Test concentrations with justification for top dose:

PRETEST
- Justification: Following the requirements of the current OECD Guideline 487 a test substance with defined composition should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. When the test substance is not of defined composition, e.g. substance of unknown or variable composition, complex reaction products or biological materials (socalled UVCBs), or environmental extracts, the top concentration should be higher to increase the concentration of each of the components (e.g. 5 mg/mL). In case of toxicity, the top concentration should produce 55 ± 5 % cytotoxicity: reduction of the proliferation index (CBPI) to 45 ± 5 % of the concurrent vehicle control. For relatively insoluble test substances only one concentration should be tested showing turbidity or precipitation in culture medium at the end of exposure period. In the pretest for toxicity based on the content of the Cr-complex of the test substance in the 2600 μg/mL total substance was used as top concentration.
- Concentrations, with and without S9 mix: 20.3, 40.6, 81.3, 162.5, 325.0, 650.0, 1300.0, 2600.0 μg/mL

MAIN TEST
- Justification: The 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 (experimental conduct with records and documentation in general accordance with the GLP principles).
- Concentrations 1ST Experiment, with and without S9 mix, 24 hour preparation interval: 5.00, 10.00, 20.00, 40.00, 80.00, 160.00 μg/mL
- Concentrations 2ST Experiment, without S9 mix, 24 hour preparation interval: 10.00, 20.00, 40.00, 80.00, 160.00 μg/mL
- Concentrations 2ST Experiment, with and without S9 mix, 44 hour preparation interval: 10.00, 20.00, 40.00, 80.00, 160.00 μg/mL

Vehicle / solvent:

PRETEST
- Vehicle used: culture medium
- Justification for choice of vehicle: Culture medium was the only feasible vehicle to reach a maximum concentration of 2600 μg/mL (homogeneous suspension) due to physico-chemical properties.

MAIN TEST
- Vehicle used: DMSO
- Justification for choice of vehicle: In the pretest, precipitates were found in all exposed test groups from the lowest applied concentration of 20 μg/mL onward. Following an additional solubility test with a lower top concentration it was decided to use dimethyl sulfoxide (DMSO) as vehicle in the main experiments of this study.

Untreated negative controls:

yes

Remarks:

without vehicle and S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

EXPERIMENTAL PROCEDURE
- Method of application: in medium
- 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.
- Seeding of the cells: A single cell suspension with the required cell count (3 - 5 E+5 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.
- Definition of test cultures: A test group consists of two separately treated flasks (Culture A and B). Each, two slides were prepared and, thus, four slides were available for scoring of each test group, in general.
- 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
Exposure-, Recovery- and Harvest time: see Table 1.

SPINDLE INHIBITOR
actin polymerisation inhibitor cytochalasin B (CytB)

STAIN
4’,6-diamidino-2-phenylindole dihydrochloride and propidium iodide

NUMBER OF REPLICATIONS
2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED
- Cell harvest and preparation of slides: The cells were prepared based on the method described by Fenech, 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, 5xE+4 cells per slide were centrifuged at 600 rpm for 7 minutes onto labelled 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 10x1E4 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).

NUMBER OF CELLS EVALUATED
At least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group

CRITERIA FOR MICRONUCLEUS IDENTIFICATION
see 'Evaluation criteria'.

DETERMINATION OF CYTOTOXICITY
- Method: relative population doubling (RPD), Proliferation Index (CBPI)
- Any supplementary information relevant to cytotoxicity (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.

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

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 analysable 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 the 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).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: Osmolality and pH values were not influenced by test substance treatment.
- Precipitation: Test substance precipitation in culture medium at the end of exposure period was observed in the absence of S9 mix at 80 μg/mL and above in the 1st Experiment and at 160 μg/mL in the 2nd Experiment. In addition, in the presence of metabolic activation precipitates in culture medium occurred at 10 μg/mL and above in both experiments (macroscopical assessment).

RANGE-FINDING/SCREENING STUDIES
The test substance was poorly soluble in all commonly used vehicles. Thus, test substance suspensions in culture medium were used in the pretest. Precipitation in culture medium was observed from the lowest applied concentration of 20.3 μg/mL onward 4 and 24 hours after start of exposure. No cytotoxicity was observed in the pretest when tested up to the highest required concentration of 2600 μg/mL. Based on the experience on solubility of the test substance in other in vitro test systems (internal data) the vehicle DMSO was used in both main experiments. However, precipitates in culture medium were still observed in both experiments. Therefore, in the main experiments of this micronucleus study concentrations at the border of solubility in culture medium were tested for cytogenetic damage.

GENOTOXICITY - MICRONUCLEUS ANALYSIS
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 both experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.3 – 0.9 % micronucleated cells) were close to the concurrent vehicle/negative control values (0.3 - 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).
Besides, in the 1st Experiment in the absence of S9 mix the micronucleus rates were concentration-related increased at 40, 80 and 160 μg/mL (0.5 %, 0.6 % and 0.9 % micronucleated cells, respectively). However, all values were clearly within the range of the 95 % control limit of our historical negative control data range and, therefore, this finding has to be regarded as biologically irrelevant.
The positive control substances ethyl methanesulfonate and cyclophosphamide induced statistically significant increased micronucleus frequencies in both independently performed experiments in at least one positive control group each. In this study, in the absence and presence of metabolic activation the frequency of micronucleated cells (1.6– 5.9 % micronucleated cells) was above the range of our historical negative control data (0.1 - 1.5 % micronucleated cells) and close to or within our historical positive control data range (2.3 – 13.8 % micronucleated cells). Unfortunately, in the 1st Experiment in the absence of S9 mix the positive control EMS did not show the expected increase at 500 μg/mL (1.3 % micronucleated cells). But the second positive control group of 600 μg/mL EMS led to the expected statistically significant increase of the micronucleus rate (2.3 % micronucleated cells).

CYTOTOXICITY - RELATIVE POPULATION DOUBLING
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 up to the highest applied test substance concentrations. These values were 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 cytochalasin B to the cultures. But, it also gives an useful information on cell loss due to test substance exposure.

CYTOTOXICITY - PROLIFERATION INDEX
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. However, slightly elevated cytostasis indicated by reduction of the CBPI was obtained in the absence of S9 mix at 160 μg/mL (25.4 % of control) in the 2nd Experiment.
Due to the use of DMSO either as vehicle for the test substance or as solvent for cytochalasin B a final concentration of 1.5 % (v/v) DMSO was reached in the treatment medium in the 2nd Experiment at 24 hours continuous treatment in the absence of S9 mix. Therefore, a negative control culture supplemented with cytochalasin B (0.5 % [v/v] DMSO) only was run in parallel. This culture showed only a slightly higher CBPI (absolute value: 2.19) than the vehicle control (absolute value: 2.12). Thus, based on these data it was confirmed that the applied DMSO concentration had no detrimental impact on the outcome of this experimental part.

CELL MORPHOLOGY
Cell attachment/morphology was adversely influenced (grade > 2) only in the 1st Experiment in the presence of metabolic activation at 160 μg/mL.

Table 2. Historical negative control data, Cytochalasin B Method, Period: December 2013 - December 2014

	Without S9 mix - all vehicles*	With S9 mix - all vehicles*
	Micronucleated Cells [%]	Micronucleated Cells [%]
Exposure / Sampling period	4 h / 24 h & 24 h / 24 h	4 h / 24 h & 4 h / 44 h
Mean	0.5	0.6
Minimum	0.2	0.1
Maximum	0.9	1.5
Standard Deviation	0.2	0.3
95% Lower Control Limit	0.1	0.0
95% Upper Control Limit	0.8	1.1
No. of Experiments	43	42

* = culture medium, DMSO 1% (v/v), acetone 1% (v/v), ethanol 1% (v/v)

 

Table 3. Historical positive control data, Cytochalasin B Method, Period: December 2013 - December 2014

	Without S9 mix Ethyl methanesulfonate (EMS) 300 - 500 μg/mL	With S9 mix Cyclophosphamide (CPP) 0.5 - 1.0 μg/mL
	Micronucleated Cells [%]	Micronucleated Cells [%]
Exposure / Sampling period	4 h / 24 h & 24 h / 24 h	4 h / 24 h & 4 h / 44 h
Mean	3.0	5.4
Minimum	2.3	2.6
Maximum	6.4	13.8
Standard Deviation	0.8	2.3
95% Lower Control Limit	1.4	0.7
95% Upper Control Limit	4.7	10.1
No. of Experiments	43	42

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study planned

Study period:

not yet defined

Justification for type of information:

- Name of the substance on which testing is proposed to be carried out: substance under registration EC: 915-756-5

CONSIDERATIONS:
- Available GLP studies:
The test substance is mutagenic in bacteria, as determined in an OECD 471 study (BASF SE, 2016).
The test substance is not mutagenic in mammalian cells, as determined in an OECD 476 study (BASF SE, 2016).
The test substance is not chromosome damaging, as determined in an OECD 487 study (BASF SE, 2016).
- Available non-GLP studies: Not Available
- Grouping and read-across: the direct-acting mutagenic activity of nitroreductase was confirmed on one component (CAS 57206-81-2) and played a significant role in the mutagenic properties of the substance.
- Historical human data: Not Available
- (Q)SAR: Not Available
- in vitro methods:
The test substance is mutagenic in bacteria, as determined in an OECD 471 study (BASF SE, 2016).
The test substance is not mutagenic in mammalian cells, as determined in an OECD 476 study (BASF SE, 2016).
The test substance is not chromosome damaging, as determined in an OECD 487 study (BASF SE, 2016).
- Weight of evidence: Not Available
For more details, please see the attached justification

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Route of administration:

oral: gavage

Sex:

not specified

Genotoxicity:

other: to be performed

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames-Test

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (Ames standard plate test and Prival preincubation test). The modified Bacterial Reverse Mutation Test according to Prival facilitates azo reduction and is therefore the most appropriate method for the investigation of azo-dyes and diazo compounds. The test was conducted according to OECD 471 guideline and GLP (BASF SE, 2016).

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 6300 μg/plate (SPT, all tester strains) 3.3 μg - 1000 μg/plate (Prival; TA 100, TA1537 and TA 98)

TEST CONDITIONS: Standard plate test (SPT) and Prival preincubation test (Prival) both with and without metabolic activation. SOLUBILITY: Precipitation of the test substance was found from about 333 μg/plate onward with and without S9 mix.

TOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 1000 μg/plate onward (for details see item 4.2.).

MUTAGENICITY: A biologically relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test either with or without S9 mix using tester strains TA 1535 and E.coli WP2 uvrA (both strains not tested in the Prival preincubation test).

A distinct, reproducible and dose dependent increase in the number of his+ revertants exceeding a factor of 2 compared to the concurrent vehicle control was observed with TA 100 and TA 98 with and without metabolic activation in the standard plate and in the prival preincubation test.

TA 100

without S9 mix:

Increase of revertants at concentrations of 333 and 1000 μg/plate in the standard plate and at a concentration of 1000 μg/plate in the prival preincubation test.

with S9 mix:

Increase of revertants at concentrations of 100, 333 and 1000 μg/plate in the standard plate test and at concentrations of 333 and 1000 μg/plate in the prival preincubation test.

TA 98

without S9 mix:

Increase of revertants at concentrations of 33 up to 3150 μg/plate in the standard plate test and at concentrations of 10 up to 1000 μg/plate in the prival preincubation test

with S9 mix:

Increase of revertants at concentrations of 33, 100, 333 and 1000 μg/plate in the standard plate test and in the prival preincubation test.

Using tester strain TA 1537 a dose depending increase of revertants exceeding a factor of 3 compared to the concurrent vehicle control was observed in the standard plate test without metabolic activation and in the prival preincubation test with and without metabolic activation.

TA 1537

without S9 mix:

Increase of revertants at concentrations of 333 and 1000 μg/plate in the standard plate test and in the prival preincubation test.

with S9 mix:

Increase of revertants at concentrations of 333 and 1000 μg/plate in the prival preincubation test.

CONCLUSION: Thus, under the experimental conditions of this study, the test substance is strongly mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

HPRT assay

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 GLP compliant study according to OECD Guideline 476, EU Method B. 17 and EPA OPPTS 870.5300 (BASF SE, 2016). 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 2013 to December 2014.

In the 1st experiment the following concentrations were tested with and without S9 mix: 0; 2.5; 5.0; 10.0; 20.0; 40.0; 80.0; 160.0 μg/mL. In the 2nd experiment the following concentrations were tested with and without S9 mix: 0; 6.3; 12.5; 25.0; 50.0; 100.0; 160.0 μg/mL. The 6.3 μg/mL was not tested for gene mutations.

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 statistically significant and dose-dependent 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.

Micronucleus Assay

The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) in a GLP compliant study according to OECD Guideline 487 and EU method B. 49 (BASF SE, 2016). 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.

1st 4 hours exposure, 24 hours harvest time, without S9 mix

- Tested concentrations: 0; 5.00; 10.00; 20.00; 40.00; 80.00; 160.00 μg/mL

- Evaluated test groups: 0; 40.00; 80.00; 160.00 μg/mL

1st Experiment_4 hours exposure, 24 hours harvest time, with S9 mix

- Tested concentrations: 0; 5.00; 10.00; 20.00; 40.00; 80.00; 160.00 μg/mL

- Evaluated test groups: 0; 5.00; 10.00; 20.00μg/mL

2nd Experiment, 24 hours exposure, 24 hours harvest time, without S9 mix

- Tested concentrations:0; 10.00; 20.00; 40.00; 80.00; 160.00 μg/mL

- Evaluated test groups:0; 40.00; 80.00; 160.00 μg/mL

2nd Experiment, 4 hours exposure, 44 hours harvest time, with S9 mix

- Tested concentrations: 0; 2.50; 5.00; 10.00; 20.00; 40.00 μg/mL

- Evaluated test groups: 0; 5.00; 10.00; 20.00 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 vehicle 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.

No cytotoxicity, indicated by reduced cell count (indicated by relative population doubling) or proliferation index (CBPI), was observed up to the highest applied test substance concentration. The highest applied test substance concentration was clearly precipitating in cell culture medium under all experimental conditions.

On the basis of the results of this 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.

General conclusion

Here, the substance was mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay (Ames test) in the absence and the presence of metabolic activation. On the contrary, 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 and non-mutagenic in the in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations.

Currently, the assessment of the genotoxic potential of the substance under registration is inconclusive; in order to trace the most reliable as possible genotoxic profile, deeper investigations, even involvingin vivo experiments, would be required. Based on the available information on gene mutation, a Comet Assay, OECD 489, is poposed. The dossier will be promptly updated when reliable and pertinent results for the substance will be available.

Justification for classification or non-classification

Currently, the assessment of the genotoxic potential of the substance under registration is inconclusive; in order to trace the most reliable as possible genotoxic profile, deeper investigations, even involving in vivo experiments would be required. Different tests are in progress or planned on other metal-complexes, but now it is not possible to establish a common mechanism/adverse pathway leading to a definitive conclusion on the assessment of genotoxic potential. The dossier will be promptly updated when reliable and pertinent results for the substance will be available.