Sodium 1-[6-(morpholin-4-yl)pyrimidin-4-yl]-4-(1H-1,2,3-triazol-1-yl)-1H-pyrazol-5-olate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Study report: according to OECD test guideline 471; negative [Herbold, 2009]

Study report: according to OECD guideline 490; positive [Becker, 2009]

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

originally adopted 28 July 2015

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat S9-liver mix
- method of preparation of S9 mix : The S9 fraction of the S9 mix was isolated in house from the livers of Aroclor 1254 induced male Sprague Dawley rats. The used S9 fractions were derived from preparations dated November 17, 2008 (color-code black, protein content 27.2 mg/mL). Prior to first use, the batch was checked for its metabolizing capacity and for possible contaminations. The S9 mix was prepared with 200 mmol/L glucose-6-phosphate, 32 mmol/L NADP and S9 at a ratio of 1:1:2.
Immediately before treatment an appropriate quantity of S9 was thawed and mixed with the cofactors and added to the test cultures at a ratio of 1:20 representing final concentrations of 2.5 mmol/L glucose-6-phosphate, 0.4 mmol/L NADP and 2.5 % S9.

Test concentrations with justification for top dose:

In pre-experiments for cytotoxicity with pulse (3 hours) and continuous (24 hours) treatment
Molidustat, dissolved in DMSO, was tested up to a maximum concentration of 150 or 162 µg/mL, respectively. After pulse treatment moderate cytotoxic effects could be observed at the highest test concentration of 150 µg/mL in the experiment without metabolic activation and at 75 µg/mL and above in the experiment with metabolic activation. After continuous treatment cytotoxicity was observed from 40 µg/mL onwards.
On the basis of these data and under the consideration, that the maximum solubility of Molidustat in DMSO is approximately 17.5 mg/mL at room temperature, the concentration ranges for the main experiments were selected.
Assay 1: 10-175 µg/mL
Assay 2: 10-50 µg/mL (without S9, duration 24 h)
10-100 µg/mL (with S9, duration 3h)

Vehicle / solvent:

- Vehicle/solvent used: DMSO

- Justification for choice of solvent/vehicle: Solubility of the test item

- Justification for percentage of solvent in the final culture medium: 1%

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E+07 cells per culture for 3 h incubation and 5E+06 cells per culture for 24 h incubation
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 h and 24 h
- Harvest time after the end of treatment (sampling/recovery times): After treatment the test item was removed by centrifugation (200 x g, 4 min) and the cells were washed twice with culture medium. After cell counting approximately 6 E+06 cells in 50 mL culture medium (1.2 E+05 cells/mL) were subcultured in tissue culture flasks at 37 °C in a humidified atmosphere with 5 % CO2 for about 2 days.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48 h
- Selection time (if incubation with a selective agent): 10 days
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used, indicate its identity, its concentration and, duration and period of cell exposure: For subsequent mutant selection approximately 2 E+03 cells/well were plated in selective medium (culture medium with 3 µg/mL trifluorothymidine) into 96 well microtiter plates.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: To assess the survival after treatment (1st PE, plating efficiency) and the viability parallel to the
mutant selection (2nd PE) a sample of each culture was diluted to 8 cells/mL in culture medium (horse serum concentration increased to 20 %) and dispensed at 200 µL per well into 96 well microtiter plates (1.6 cells per well). The plates were incubated at 37 °C and 5 % CO2. After at least 6 days the plates were counted manually.
- Criteria for small (slow growing) and large (fast growing) colonies: In a first step the total number of mutant colonies were scored for each dose. In a second step the colonies were evaluated according to their size and morphology (density) and characterized in two classes. Large colonies possessed a totally or partially diffuse nature and overgrew more than one-third of the well. In contrast, small colonies possessed a compact nature (high optical density) and overgrew less than one-third of a well.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG); relative survival (RS)

Evaluation criteria:

The test item is classified as mutagenic if it induces at least in one of the concentrations tested a mutant frequency that is two times higher than the mean of the spontaneous mutant frequency of the corresponding control.
The test item is classified as mutagenic if there is a reproducible concentration-related increase in the mutant frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants. Additionally, the induced mutant frequency have to exceed a value that based on the global distribution of the background mutant frequency for the mouse lymphoma assay and the spontaneous mutant frequency of the performed assay. The value for the global background mutation frequency is called “global evalutation factor” (GEF) and was determined for the microwell version of the mouse lymphoma assay as 126 per 1E+06 cells. Consequently, for a positive call at any test concentration the induced mutant frequency have to exceed the totalized values of the spontaneous mutant frequency of the corresponding control and the GEF. Significant increases in the mutant frequency, which are obtained at extremely high levels of cytotoxicity (RTG value lower than 10 %), are – in accordance with internationally accepted recommendations – usually considered biologically irrelevant and disregarded in evaluation.
However, in the evaluation of the test results scientific plausibility is taken into consideration.

Statistics:

No statistical evaluation is required.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

induced mutant frequencies exceeded the threshold of twice the colony counts of the corresponding solvent controls at test concentrations of 25 µg/mL and above, no concentration-related increase, not exceeding GEF (126E-06)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

32 µg/mL and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

24 h treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 30 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

pulse treatment for 3 hours did not lead to mutant frequencies that exceeded the threshold of twice the number of mutant colonies of the solvent control.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

100 µg/mL and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Evaluation of the data indicates that under the experimental conditions described Molidustat
is a mutagen in the mouse lymphoma assay in the absence and presence of an extrinsic metabolic activation system.

Executive summary:

In a mammalian cell gene mutation assay similar to OECD test guideline 490 (2015), mouse lymphoma L5178Y cells cultured in vitro were exposed to Molidustat in DMSO at concentrations of 0, 10, 25, 50, 100, and 175 µg/mL for 3h in the presence and absence of mammalian metabolic activation and of 0, 10, 20, 30, 40, and 50 µg/mL for 24 h in the absence of mammalian metabolic activation.

Molidustat was tested up to cytotoxic concentrations. In the absence of metabolic activation it induced a strong nearly 15-fold increase in the number of mutant colonies after continuous treatment (24 h) whereas pulse treatment for 3 h did not lead to mutant frequencies that exceeded the threshold of twice the number of mutant colonies of the solvent control. In the presence of metabolic activation it induced mutant frequencies exceeded the threshold of twice the colony counts of the corresponding solvent controls at test concentrations of 25 µg/mL and above. However, a concentration-related increase of the induced mutant frequencies was not observed. The number of mutant colonies remained at a plateau on or above the threshold of twice the mutant frequency of the solvent control, but did not exceed the sum of the spontaneous mutant frequency of the control plus the global evaluation factor (GEF, 126 E-06).

 

The positive controls did induce the appropriate response. There was evidence of induced mutant colonies over background under the conditions of the test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-04-01 to 2009-07-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

His locus

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9 Liver-mix
- method of preparation of S9 mix : S9 mix was used to simulate the mammalian metabolism of the test substance. It was made from the livers of at least six adult male Sprague Dawley rats, of approximately 200 to 300 g in weight. For enzyme induction, the animals received a single intraperitoneal injection of Aroclor 1254, dissolved in corn oil, at a dose of 500 mg/kg body weight, five days prior to sacrifice. The animals were prepared unfasted, following the directions of Ames et al (1975) and Maron and Ames (1983). The rats were terminated. Livers were removed under sterile conditions immediately after sacrifice and kept at 4°C until all animals had been prepared. All the remaining steps were carried out under sterile conditions at 4°C.
The livers were washed with cold (4°C), 0.15 M KCI solution (approximately 1 mLKCI per 1 g liver), and then homogenized in fresh, cold (4°C), 0.15 M KCl (approximately 3 mL KCI per 1 g liver). The homogenate was then centrifuged in a cooling centrifuge at 4°C and 9000 g for 10 minutes. The supernatant (the S9 fraction) was stored at -80°C in small portions.
These portions were slowly thawed before use. The S9 mix was freshly prepared, kept on ice and used only on the same day. Seventy mL of cofactor solution are composed as follows:
MgCl2 x 6H2O 162.6 mg
KCl 246.0 mg
Glucose-6-phosphate (disodium salt) 179.1 mg
NADP (disodium salt) 315.0 mg
Sodium phosphate buffer (100 mM; pH 7.4) 100.0 mM
The S9 mix comprised 10% S9 fraction, 70% cofactor solution and 20% 0.15 M KCl.
The S9 fraction was derived from the preparation dated January 26, 2009 (protein content 24.4 mg per mL). Prior to first use, each batch was checked for its metabolizing capacity by using reference mutagens; appropriate activity was demonstrated. At the beginning of each experiment 4 aliquots of the S9 mix were plated (0.5 mLper plate) in order to assess its sterility. This was repeated after completion of test tube plating. The sterility control plates were then incubated for 48 hours at 37°C. No indication of contamination of S9 mix was found.

Test concentrations with justification for top dose:

0, 16, 50, 158, 500, 1581, 5000 µg/plate, as recommended by the OECD test guideline
Due to toxicity of the substance doses ranging from 5 to 1581 µg/plate were chosen for the repeat test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO and water for one positive control (Mitomycin C)

- Justification for choice of solvent/vehicle: The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether (EGDE), and DMF according to information given by the sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.
No "untreated" negative control was set up for the used solvent, since sufficient evidence was available in the literature (e.g. Maron and Ames, 1983) indicating that this solvent had no influence on the spontaneous mutant counts of the used strains.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

cumene hydroperoxide

mitomycin C

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min at 37°C


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The toxicity of the substance was assessed in three ways. The first method was a gross appraisal of background growth on the plates for mutant determination. Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls.
Thirdly, the titer was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.

Rationale for test conditions:

As recommended by OECD test guideline 471

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity occurred from 158 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity occurred from 158 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity occurred from 158 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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

Remarks:

cytotoxicity occurred from 158 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity occurred from 158 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

As may be seen, there was no indication of a bacteriotoxic effect of the test item at doses of up to and including 158 μg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. Higher doses had a strong, strain-specific bacteriotoxic effect. Therefore they could only be used for assessment purposes up to and including 500 μg per plate.
None of the five strains concerned showed in the plate incorporation test a dose-related and
biologically relevant increase in mutant counts over those of the negative controls. This applied
both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Summary of Mean Values Without S9 Mix
Group [µg/plate]	Strain
	TA1535	TA100	TA1537	TA98	TA102
Plate incorporation method
0	7	98	6	16	205
16	6	122	6	16	211
50	7	121	6	15	240
158	5	112	4	12	115
500	4	100	2	19	105
1581	0	0	0	0	0
5000	0	0	0	0	0
Na-Azide	896
NF		314
4-NPDA			42	81
MMC					834
Preincubation method
0	7	123	5	22	226
5	6	114	6	17	251
16	7	116	5	24	248
50	8	123	6	18	195
158	6	127	6	23	204
500	8	111	6	25	178
1581	1	28	0	2	29
Na-Azide	918
NF		465
4-NPDA			37	71
Cumene					405

Summary of Mean Values With S9 Mix
Group [µg/plate]	Strain
	TA1535	TA100	TA1537	TA98	TA102
Plate incorporation method
0	9	196	8	39	303
16	9	200	7	35	328
50	10	183	8	39	287
158	10	167	7	28	270
500	9	167	6	25	239
1581	3	42	0	0	41
5000	0	0	0	0	0
2-AA	104	2731	281	1902	686
Preincubation method
0	13	180	10	32	292
5	11	161	8	34	315
16	11	170	9	31	317
50	10	171	10	32	281
158	9	162	9	28	271
500	8	143	7	24	230
1581	4	42	0	3	152
2-AA	92	2515	303	1786	553

 

 

 

 

 

 

Conclusions:

The Salmonella/microsome test, employing doses of up to 5000 μg per plate, showed that the test item Molidustat produced bacteriotoxic effects, starting at 500 μg per plate. Therefore,
1581 μg per plate and above could not be used for assessment.
Evaluation of individual dose groups, with respect to relevant assessment parameters ( dose effect, reproducibility) revealed no biologically relevant variations from the respective negative
controls.
In spite of the low doses used, positive controls increased the mutant counts to well over those of the negative controls, and thus demonstrated the system's high sensitivity.
Despite this sensitivity, no indications of mutagenic effects of the test item could be found at assessable doses of up to 500 μg per plate in any of the Salmonella typhimurium strains used.
Due to these results Molidustat has to be regarded as non-mutagenic.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1997), Salmonella typhimurium strains TA98, TA100, TA102, TA1535,and TA1537 were exposed to Molidustat in in DMSO in concentrations of 0 (control), 16, 50, 158, 500, 1581 and 5000 µg/plate in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix)in the plate incorporation assay. Due to the observed toxicity at > 158 µg/plate a preincubation assay was performed with 0 (control), 5, 16, 50, 158, 500 and 1581 µg/plate in the presence and absence of metabolic activation.

No precipitation was observed at any concentration. The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the five tester strains (TA98, TA 100, TA102, TA1535, or TA1537) examined at dose levels up to 1581 µg/plate in the absence and presence of a metabolic activation source (S9). Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strains TA98, TA100, TA102, TA1535, and TA 1537 under the conditions employed (plate incorporation  and preincubation assay).

There was no evidence of induced mutant colonies over background.

Under the conditions of the study, the test substance was negative for mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Study report: OECD test guideline 474; result: inconclusive [Herbold, 2007]

Study report: OECD test guideline 486; result: Molidustat is not correlated with the occurrence of DNA adducts [Herbold, 2010]

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

21 July 1997

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Remarks:

Crl:(WI)BR (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 to 10 weeks
- Weight at study initiation: 182 to 292 g (16 hours interval) and 164 to 259 g (4 hours interval)
- Fasting period before study: 6 h
- Housing: The animals were kept singly in type III h cages. Beeding of soft wood granules, type BK 8/15 (J. Rettenmaier & Sohne, Füllstoff-Fabriken, 73494 Ellwangen-Holzmühle) was used.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5-23.5
- Humidity (%): 18-61
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

ethanol (10%) and HS15 (40%) and drinking water
- Concentration of test material in vehicle: 50 and 100 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was suspended a mixture of 10% ethanol (Merck, order no. 1.00974.1011, batch K38909374830), 40% solutol HS15 (BASF, order no. S10106, batch 39222016Ko) and 50% drinking water using a magnetic stirrer and formed light-yellow milky suspensions. The suspensions were stirred with a magnetic mixer during administration and given orally. The positive controls were also administered orally. For this purpose 2-acetylaminofluorene was dissolved in corn oil (Sigma, batch 058K0070). N,N'-Dimethylhydrazine was dissolved in physiological saline solution (Baxter S.A., batch 12BAS02).

Duration of treatment / exposure:

Administration: once 4 h or 16 h until isolation of liver cells

Frequency of treatment:

Once

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

500 mg/kg bw (total dose)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Positive control(s):

none; no data; 2-acetylaminofluorene; N,N'-Dimethylhydrazine

- Route of administration: gavage
- Doses / concentrations: 100 and 40 mg/kg, respectively

Tissues and cell types examined:

Liver cell homogenate

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Sampling times 4 h and 16 h after treatment, the liver was removed and perfused in order to isolate the hepatocytes. After isolation the hepatocytes were radiolablled with ³H-thymidine for 4h. Thereafter, the cultures in the 6 well dishes were washed twice with PBS. Subsequently, a 1 %
sodiw:n citrate solution was added for 5-10 minutes to swell the nuclei. The cells were then fixed twice by a mixture consisting of one part acetic acid and four parts absolute ethanol for a total time of at least 30 minutes. The coverslips in the wells were then washed 2 to 4 times with deionized water and air dried.

DETAILS OF SLIDE PREPARATION: Air-dried coverslips were mounted cell-side-up on microscope slides. The following autoradiography-procedure was performed in the dark. The slides were dipped in a NTB photographic emulsion (Kodak), which was previously diluted 1: 1 with distilled water. Slides were air-dried overnight. Thereafter, coated slides were stored in light-tight boxes for 10 days at -20°C in the presence of a drying agent (Drierite). This storage was performed to reduce the CG background in order to increase the sensitivity of the test system. The photographic emulsion was then developed for 3-4 minutes ,in Kodak D-19 developer at temperatures below 15°C. Thereafter, slides were rinsed with distilled water, fixed in Kodak Fixer for 7-8 minutes and air dried. Dried slides were stained with hematoxylin and eosin.

METHOD OF ANALYSIS:
Slides were put onto a light microscope using an oil immersion objective with a magnification of 100. This microscope was interfaced via a Sony high resolution TV color camera to a Sony TV color screen. Nuclear grains and cytoplasma grains were counted on this TV color screen by the naked eye.
Slides were read blind and meanderwise. Generally, three slides and 50 cells per slide were
examined per animal. Per cell the nuclear grain count (NG) was determined once. In addition,
the number of grains was determined in 3 cytoplasmic areas of this cell. These areas had the
same size as the corresponding nucleus. The NG was reduced by the mean of the grains in the cytoplasmic areas (CG). This value was referred to as the nuclear net grain (NNG) count of the cell. The mean NNG value per dose group was calculated from the mean NNG value of
each animal of the respective dose group. The number of cells in repair (nuclei with 5 or more net grains) was also determined. Only cells viable at the time of fixation and with nuclei evenly coated with emulsion were scored. Cells with abnormal morphology, s~ch as those with pyknotic or lysed nuclei, as well as isolated nuclei not surrounded by cytoplasm were not read. Cells with heavily labelled nuclei (= S-phase cells) were excluded from scoring.

Evaluation criteria:

A test was considered positive if there was a biologically relevant and statistically significant
increase of NNG in hepatocytes of treated animals in comparison to the negative control. A
test will be considered also positive, if a chemical yields dose-dependently a mean of at least
+2 NNG. Cells in repair have to be considered in addition. A test was considered negative if there was no biologically relevant and statistically significant increase ofNNG. A test was also considered negative if there was a significant increase of NNG, which according to the laboratory's experience was within the range of historical negative controls. A group mean of 0 NNG or lower will also be indicative for a negative response.

Statistics:

Descriptive statistical methods were used for NG, CG and NNG. Means and standard devia-
tions were calculated for each animal and for each group. The standard deviations for an ani-
mal were calculated from the respective means calculated individually for each of its three
coverslips [Windows NT 4.0 based software "UDS-Test" (V02.04) of Bayer AG]. The standard deviations for a group were calculated on the basis of these animal means (Windows XP Professional version 2002). In addition, numbers ofNNG in the BA Y 85-3934 groups and in the positive controls (provided this exceeded 0) were checked by Wilcoxon's non-parametric rank sum test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: A pilot test was performed in the laboratory which conducted the main study using animals of the same source, strain and age. Per dose three male rats, which received no feed for 6 hours prior to treatment, were used. The following single oral doses were tested: 500 mglkg, 1000 mg/kg and 2000 mg/kg of the test item. The following symptoms were recorded for up to 4 days after the application, starting at 500 mg/kg: apathy, reduced motility, roughened fur, bloody nose, spasm, difficulty in breathing, rapid breathing and slitted eyes. In addition, two of three males died in the 2000 mg/kg group.
Based on these results 1000 mg/kg of the test item were chosen as MTD for this study.

- Clinical signs of toxicity in test animals: After single oral administrations of 500 and 1000 mg/kg of the test item, treated animals showed for the 4 hours interval no symptoms. However, for the 16 hours interval the following symptoms were recorded: apathy, roughened fur, spasm and stitted eyes. These symptoms demonstrate relevant systemic exposure of males to the test substance. One of five treated animals died during the test period, due to the acute toxicity of 1000 mg/kg of the test item. This died animal clearly proves 1000 mg/kg to be at least the maximum tolerated dose. No symptoms were recorded for the control groups. No animals died in these groups.

- Evidence of cytotoxicity in tissue analysed: No
- Rationale for exposure: determined MDT

A mean NNG value of - 2.09 (8D= 0.31) was observed for negative control animals which were sacrificed 16 hours after treatment. For negative control animals sacrificed 4 hours after treatment the mean NNG value was -1.44 (SD= 0.25). These values are in a range, which is typical for negative controls. In addition, the average numbers of cells with 5 or more net grains (cells in repair) for these animals were also in a range which is typical for negative controls.
For animals sacrificed 16 hours after treatment with 500 mg/kg of the test item a mean NNG value of - 1.48 (8D= 0.32) was observed. For animals treated with 1000 mg/kg a mean NNG of - 1.30 (SD= 0.17) was recorded. Animals sacrificed 4 hours after treatment with 500 mg/kg of the test item had a mean NNG value of - 0.80 (SD= 0.12) and animals treated with 1000 mg/kg showed a mean NNG of - 0.68 (SD= 0.13). For both sacrifice times no biologically relevant increase was observed. The positive controls 2-acetylaminofluorene and N,N'-dimethylhydrazine increased the mean NNG values to + 4.65 (SD= 0.37) and + 6.26 (8D= 0.68) and demonstrated the sensitive of the test system used.

Conclusions:

Molidustat was tested for genotoxic activity in the in vivo UDS assay after single oral
treatment of rats with doses of 500 mg/kg and 1000 mg/kg.
No treatment related cytotoxic effects could be observed. The availability of a good cell popu-
lation for the in vitro part of the assay was demonstrated.
After treatment with the test substance no biologically relevant increase in nuclear labelling was
induced.
The positive controls 2-acetylaminofluorene and N,N-dimethylhydrazine induced significant
increases in NNG and in the percentage of cells in repair and thus demonstrated the sensitivity
of the test system for the detection of induced DNA-damage.
Based on the results of this study Molidustat is considered negative in the in vivo UDS
Assay with rat liver cells.

Executive summary:

In a test conducted according to OECD guideline 486 (1997), male Wistar rats were exposed orally to Molidustat at 0, 500 and 1000 mg/kg bw. Subsequently, after 4h and 16 h the animals were sacrificed and the occurrence of DNA adducts in liver homogenates was determined. During the administration (4h duration) there were no premature deaths and the animals were in good health with respect to general examination. However, There was only a slight decrease in body weight in females exposed to Estradiol. However, for the 16 hours interval the following symptoms were recorded: apathy, roughened fur, spasm and stitted eyes. These symptoms demonstrate relevant systemic exposure of males to the test substance. One of five treated animals died during the test period, due to the acute toxicity of 1000 mg/kg of the test item. This died animal clearly proves 1000 mg/kg to be at least the maximum tolerated dose. No symptoms were recorded for the control groups. No animals died in these groups. DNA adducts were not observed in male animals after 4 h or 17 h. Thus, it is considered that Molidustat is not correlated with the occurrence of DNA adducts. Based on the results obtained from the present study Molidustat is not considered positive with regard to DNA adduct formation in mammalian cells.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

21 July 1997

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld
- Age at study initiation: 6-12 weeks
- Weight at study initiation: 36-43 g
- Assigned to test groups randomly: yes
- Housing:The animals were kept singly in type II cages. They were identified by cage marks.
Bedding of soft wood granules, type BK 8/15 (1. Rettenmaier & Schne, Fiillstoff-Fabriken, 73494 Ellwangen-Holzmiihle) was used.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 25-53
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

0.5 % aqueous Cremophor

Duration of treatment / exposure:

24 h after last treatment

Frequency of treatment:

The test item and the negative control were administered twice, separated by 24 h, whereas cyclophosphamide was administered only once.

Dose / conc.:

200 mg/kg bw (total dose)

Dose / conc.:

100 mg/kg bw (total dose)

Dose / conc.:

50 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide;
- Route of administration: intraperitoneal
- Doses / concentrations: 20 mg/kg bw

Tissues and cell types examined:

The micronucleus test evaluates the presence of micronucleated polychromatic erythrocytes in a somatic tissue, the bone marrow, in order to assess the clastogenic potential of the test compound. Normally, bone-marrow erythrocytes are anucleated.
An increased incidence of these micronucleated cells in comparison to the negative control indicates that the test compound may cause chromosome breaks or spindle disorders in the erythroblasts.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The selection of the test item doses was based on a pilot test. Groups consisting each of three males received two intraperitoneal injections separated by 24 hours. The following doses were used: 200 mg/kg and 400 mg/kg of the test item. The following symptoms were recorded, starting at 200 mg/kg: apathy, roughened fur, loss of weight, sternal recumbency, spasm, periodically stretching of body, difficulty in breathing and slitted eyes. Symptoms were recorded for up to at least 24 hours after the second application. In addition, 2 of 3 males died in the 400 mg/kg group. Based on these findings, 200 mg/kg of the test item was chosen as MTD.

Male mice treated with the test item received two intraperitoneal administrations of
50, 100 and 200 mg/kg, respectively, separated by 24 hours. The negative control animals received the vehicle of the test item using the same application regimen. Males of the positive control received a single intraperitoneal treatment with 20 mg/kg cyclophosphamide. The femoral marrow of all groups was prepared 24 hours after the last
administration.


DETAILS OF SLIDE PREPARATION: Schmid's method was used to produce the smears.
At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor).
The femur was separated from muscular tissue. The lower-leg stump, including the knee and all attached soft parts, was separated in the distal epiphyseal cartilage by a gentle pull at the distal end. The proximal end of the femur was opened making visible a small opening in the bone-marrow channel. A suitable tube was filled with sufficient fetal calf serum. A small amount of serum was drawn from the tube into a suitable syringe with a thin cannula. The cannula was pushed into the open end of the marrow cavity. The femur was then completely immersed in the calf serum and pressed against the wall of the tube, to prevent its slipping off. The contents were then flushed several times and the bone marrow was passed into the serum as a fine suspension. Finally, the flushing might be repeated from the other end, after it had been opened. The tube containing the serum and bone marrow was centrifuged in a suitable centrifuge at approximately 1000 rpm for five minutes. The supernatant was removed with a suitable pipette (e.g. Pasteur pipette), leaving only a small remainder. The sediment was mixed to produce a homogeneous suspension. One drop of the viscous suspension was placed on a well-cleaned slide and spread with a suitable object, to allow proper evaluation of the smear. The labeled slides were dried overnight. If fresh smears needed to be stained, they needed to be dried with heat for a short period.
The Staining of Smears
The smears were stained automatically with an Ames Hema-Tek Slide Stainer from the Miles Company. The slides were then "destained" with methanol, rinsed with deionized water, and left to dry.

METHOD OF ANALYSIS: Normally, 2000 polychromatic erythrocytes were counted per animal. In addition to the number of normochromatic erythrocytes per 2000 polychromatic
ones, the number of normochromatic erythrocytes showing micronuclei was also esstablished. An increase in the number of micronucleated normochromatic erythrocytes, without a preceding increase in micronucleated polychromatic erythrocytes, is irrelevant to
the assessment of a clastogenic effect, since normochromatic erythrocytes originate from polychromatic ones.

Evaluation criteria:

A test was considered positive ifthere was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of historical negative controls.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. A test was also considered equivocal, if its result was implausible. In both case, normally a second test will be performed.

Key result

Sex:

male

Genotoxicity:

ambiguous

Remarks:

no statistically significant variations existed for males between the negative control and the groups treated intraperitoneally with the test item, however, the result of the 200 mg/kg group exceeded the all-time historical negative controls.

Toxicity:

yes

Remarks:

compound related symtoms in the animals: apathy, rough-ened fur, spasm, periodically stretching of body and difficulty in breathing. Symptoms were recorded until sacrifice.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

In conclusion, the results obtained for Molidustat were inconclusive for the assessment of its clastogenic potential in the micronucleus test on the male mouse, i.e. in a somatic test system in vivo. However, a carcinogenicity study (OECD test guideline 451) in mice revealed no tumorigenic effect of Molidustat. Consequently, the mutagenic risk is considered to be low/not relevant.

Executive summary:

In a NMRI mouse bone marrow micronucleus assay according to OECD test guideline 474, 5 male mice/dose were treated intraperitoneally with Molidustat at doses of 0, 50, 100, and 200 mg/kg bw.  Bone marrow cells were harvested at 24 h post-treatment.  The vehicle was 0.5% aqueous Cremophor.

There were signs of toxicity during the study. After two intraperitoneal administrations of 50,  100 and 200 mg/kg of with the test item treated males showed the following compound-related symptoms: apathy, roughened fur, spasm, periodically stretching of body and difficulty in breathing. Symptoms were recorded until sacrifice. These symptoms demonstrate relevant systemic exposure of males to the test substance. There was no substance-induced mortality. No symptoms were recorded for the control groups. No animals died in these groups.  

The ratio of polychromatic to normochromatic erythrocytes in males was altered by the treatment with the test substance, being 2000:1967 (1 s=607) in the negative control, 2000: 3418 (1 s=898) in the 50 mg/kg group, 2000: 2878 (1s=338) in the 100 mg/kg group and 2000: 4036 (ls=854) in the 200 mg/kg group. Relevant variations were thus noted for 200 mg/kg. The incidence of these micronucleated cells was 4.4/2000 (1s=3.6) in the negative control, and 6.8/2000 (1s=1.5), 6.4/2000 (1s=3.5) and 8.2/2000 (1s=4.1) in the treatment groups. No statistically significant variations existed for males between the negative control and the groups treated intraperitoneally with the test substance, with respect to the incidence of micronucleated polychromatic erythrocytes. However, as compared to the attached historical negative controls, the result of the 200 mg/kg group exceeded the all-time historical negative controls.

Similarly, there could be no biologically significant variation between the negative control and treatment groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones.

The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 25.8/2000 (1s=13.3), which represents biologically relevant increases in comparison to the negative control. There could not have been a biologically relevant effect on the number of micronucleated normochromatic erythrocytes in the positive control since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromatic ones. No further effect of cyclophosphamide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since this ratio did not vary to a biologically relevant degree [2000: 2450 (1s=619), as against 2000:  1967 in the negative control]. This clearly demonstrates that an alteration of the ratio of polychromatic to normochromatic erythrocytes is not necessary for the induction of micronuclei.

 

There was a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after 24 h. However, based on the results presented above Molidustat cannot be clearly identified as clastogenic in the in vivo mammalian micronucleus test. The result is inconclusive.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1997), Salmonella typhimurium strains TA98, TA100, TA102, TA1535,and TA1537 were exposed to Molidustat in in DMSO in concentrations of 0 (control), 16, 50, 158, 500, 1581 and 5000 µg/plate in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix)in the plate incorporation assay. Due to the observed toxicity at > 158 µg/plate a preincubation assay was performed with 0 (control), 5, 16, 50, 158, 500 and 1581 µg/plate in the presence and absence of metabolic activation.

No precipitation was observed at any concentration. The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the five tester strains (TA98, TA 100, TA102, TA1535, or TA1537) examined at dose levels up to 1581 µg/plate in the absence and presence of a metabolic activation source (S9). Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strains TA98, TA100, TA102, TA1535, and TA 1537 under the conditions employed (plate incorporation  and preincubation assay).

There was no evidence of induced mutant colonies over background.

Under the conditions of the study, the test substance was negative for mutagenic potential.

 

 

In a mammalian cell gene mutation assay similar to OECD test guideline 490 (2015), mouse lymphoma L5178Y cells cultured in vitro were exposed to Molidustat in DMSO at concentrations of 0, 10, 25, 50, 100, and 175 µg/mL for 3h in the presence and absence of mammalian metabolic activation and of 0, 10, 20, 30, 40, and 50 µg/mL for 24 h in the absence of mammalian metabolic activation.

Molidustat was tested up to cytotoxic concentrations. In the absence of metabolic activation it induced a strong nearly 15-fold increase in the number of mutant colonies after continuous treatment (24 h) whereas pulse treatment for 3 h did not lead to mutant frequencies that exceeded the threshold of twice the number of mutant colonies of the solvent control. In the presence of metabolic activation it induced mutant frequencies exceeded the threshold of twice the colony counts of the corresponding solvent controls at test concentrations of 25 µg/mL and above. However, a concentration-related increase of the induced mutant frequencies was not observed. The number of mutant colonies remained at a plateau on or above the threshold of twice the mutant frequency of the solvent control, but did not exceed the sum of the spontaneous mutant frequency of the control plus the global evaluation factor (GEF, 126 E-06).

 

The positive controls did induce the appropriate response. There was evidence of induced mutant colonies over background under the conditions of the test.

 

 

Genetic toxicity in vivo:

Supplemental in vivo data are available for Molidustat.

In a NMRI mouse bone marrow micronucleus assay according to OECD test guideline 474, 5 male mice/dose were treated intraperitoneally with Molidustat at doses of 0, 50, 100, and 200 mg/kg bw.  Bone marrow cells were harvested at 24 h post-treatment.  The vehicle was 0.5% aqueous Cremophor.

There were signs of toxicity during the study. After two intraperitoneal administrations of 50,  100 and 200 mg/kg of with the test item treated males showed the following compound-related symptoms: apathy, roughened fur, spasm, periodically stretching of body and difficulty in breathing. Symptoms were recorded until sacrifice. These symptoms demonstrate relevant systemic exposure of males to the test substance. There was no substance-induced mortality. No symptoms were recorded for the control groups. No animals died in these groups.  

The ratio of polychromatic to normochromatic erythrocytes in males was altered by the treatment with the test substance, being 2000:1967 (1 s=607) in the negative control, 2000: 3418 (1 s=898) in the 50 mg/kg group, 2000: 2878 (1s=338) in the 100 mg/kg group and 2000: 4036 (ls=854) in the 200 mg/kg group. Relevant variations were thus noted for 200 mg/kg. The incidence of these micronucleated cells was 4.4/2000 (1s=3.6) in the negative control, and 6.8/2000 (1s=1.5), 6.4/2000 (1s=3.5) and 8.2/2000 (1s=4.1) in the treatment groups. No statistically significant variations existed for males between the negative control and the groups treated intraperitoneally with the test substance, with respect to the incidence of micronucleated polychromatic erythrocytes. However, as compared to the attached historical negative controls, the result of the 200 mg/kg group exceeded the all-time historical negative controls.

Similarly, there could be no biologically significant variation between the negative control and treatment groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones.

The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 25.8/2000 (1s=13.3), which represents biologically relevant increases in comparison to the negative control. There could not have been a biologically relevant effect on the number of micronucleated normochromatic erythrocytes in the positive control since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromatic ones. No further effect of cyclophosphamide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since this ratio did not vary to a biologically relevant degree [2000: 2450 (1s=619), as against 2000:  1967 in the negative control]. This clearly demonstrates that an alteration of the ratio of polychromatic to normochromatic erythrocytes is not necessary for the induction of micronuclei.

 

There was a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after 24 h. However, based on the results presented above Molidustat cannot be clearly identified as clastogenic in the in vivo mammalian micronucleus test. The result is inconclusive.

 

 

In a test conducted according to OECD guideline 486 (1997), male Wistar rats were exposed orally to Molidustat at 0, 500 and 1000 mg/kg bw. Subsequently, after 4h and 16 h the animals were sacrificed and the occurrence of DNA adducts in liver homogenates was determined. During the administration (4h duration) there were no premature deaths and the animals were in good health with respect to general examination. However, There was only a slight decrease in body weight in females exposed to Estradiol. However, for the 16 hours interval the following symptoms were recorded: apathy, roughened fur, spasm and stitted eyes. These symptoms demonstrate relevant systemic exposure of males to the test substance. One of five treated animals died during the test period, due to the acute toxicity of 1000 mg/kg of the test item. This died animal clearly proves 1000 mg/kg to be at least the maximum tolerated dose. No symptoms were recorded for the control groups. No animals died in these groups. DNA adducts were not observed in male animals after 4 h or 17 h. Thus, it is considered that Molidustat is not correlated with the occurrence of DNA adducts. Based on the results obtained from the present study Molidustat is not considered positive with regard to DNA adduct formation in mammalian cells.

 

 

Based on the presented results and a carcinogenicity study (OECD TG 451) in mice which revealved no tumorigenic effect of Molidustat, the mutagenic risk is considered to be low/ not relevant.

 

Justification for classification or non-classification

According to Regulation (EU) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals, Molidustat does not need to be classified with respect to mutagenicity for Registrations according to Regulation (EU) nO. 1907/2006 (REACH) Annex VII.