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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

875-892-5

Cas Number:

1375799-59-9

Molecular formula:

C13 H14 N8 O2 . Na

IUPAC Name:

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

Method

Target gene:

His locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 5

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.

Any other information on results incl. tables

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

 

 

 

 

 

 

Applicant's summary and conclusion

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.