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

Toxic effect type:

dose-dependent

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Study report: ICH guideline S5A: Detection of Toxicity to Reproduction for Medicinal Products; EU: CPMP/ICH/386/95, adopted September 1993; conducted with male and female Wistar rats; result: NOAEL = 2 mg/kg bw based on postimplantation losses and general toxicity in males.

Study report: ICH guideline S5A: Detection of Toxicity to Reproduction for Medicinal Products; EU: CPMP/ICH/386/95, adopted September 1993; conducted with inseminated female Wistar rats; result: NOAEL = 10 mg/kg bw based on postimplantation losses.

Study report: ICH guideline S5A: Detection of Toxicity to Reproduction for Medicinal Products; EU: CPMP/ICH/386/95, adopted September 1993; conducted with female Himalayan rabbits; result: NOAEL = 20 mg/kg bw based on absence of effects up to the highest dose.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ICH guideline S5A: Detection of Toxicity to Reproduction for Medicinal Products; EU: CPMP/ICH/386/95, adopted September 1993

Version / remarks:

1993

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

(Hsd Cpb:WU)

Route of administration:

oral: gavage

Vehicle:

other: ethanol/Solutol ® HS 15/demineralized water (1:4:5 v/v/v)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test item was administered by gavage in ethanol/Solutol® HS 15/demineralized water (1:4:5 v/v/v). The purity was taken into account. The administration volume was 10 mL/kg body weight. The formulations were prepared as needed taking into account the analytically determined stability. The test substance was administered as a suspension in the vehicle. The formulation was stored at room temperature. It was stirred with a magnetic stirrer for several minutes (until an obviously homogenous distribution occurred) before administration. It was also stirred during administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before start of treatment, the suitability of the formulation was confirmed by the analysis of concentration, homogeneity, and stability of dosage forms prepared in the same way as it was done in the study (0.5 and 5 mg/mL, covering the concentration range used). Analyses were carried out before the start of the study. Homogeneity tests and content checks of the active ingredient in samples with concentrations of 0.3, 1 (content checks, only), and 3 mg/mL were carried out twice during the study.

Details on mating procedure:

The animals were mated by placing one or two females overnight into a Type IIIh cage together with one male rat. If sperm was detected in the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation.

Duration of treatment / exposure:

The females were treated daily from days 6 to 17 of gestation.

Frequency of treatment:

daily

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

3 mg/kg bw/day (actual dose received)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

22 females in the main groups and 4 females in the satellite groups

Control animals:

yes, concurrent vehicle

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

pre and post implantation loss

Abnormalities:

effects observed, treatment-related

Localisation:

placenta

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

external malformations

Abnormalities:

effects observed, treatment-related

Localisation:

external: eye

Developmental effects observed:

not specified

Executive summary:

In the present study 22 inseminated female Wistar rats were exposed to 0, 3, 10, and 30 mg/kg bw of the test item in ethanol/Solutol ® HS 15/demineralized water (1:4:5 v/v/v) as vehicle daily from day 6 to day 17 p.c. The fetuses were delivered by cesarean section on day 21 of gestation. Investigation was performed on general tolerance of the test substance by the females as well as on its effect on intrauterine development. Four additional females per group were treated with the doses of 0, 3, 10, and 30 mg/kg from days 6 to 17 p.c. for determination of test substance in the plasma of the females. Appearance, behavior, and mortality were unaffected at dose levels up to 30 mg/kg bw/day. Mean body weight development was decreased at dose level of 30 mg/kg bw/day. Food intake was transiently decreased at the dose levels of 30 mg/kg bw/day. No treatment related gross pathological findings occurred at dose levels up to 30 mg/kg bw/day.

The gestation rate was unaffected by treatment at dose levels up to 30 mg/kg bw/day. Morphological appearance and weight of placentas were only affected by treatment at dose level 30 mg/kg bw/day. Postimplantation loss was increased in dose level 30 mg/kg. Fetal sex distribution was unaffected by treatment at dose levels up to 30 mg/kg bw/day. Weights of fetuses were statistically significantly decreased at dose levels of 30 mg/kg bw/day. Dosing with Molidustat at 30 mg/kg revealed an increased incidence of ocular malformations. A treatment related effect on skeletal deviations was not seen up to a dose level of 30 mg/kg bw/day.

In summary, treatment-related maternal toxicity and developmental toxicity characterized mainly by ocular malformations, reduced fetal weight and increased postimplantation loss were seen at a daily dose of 30 mg/kg of the test item. Consequently, the following no-observed-adverse-effect levels (NOAELs) were determined:

Maternal toxicity: 10 mg/kg bw/day

Developmental toxicity: 10 mg/kg bw/day

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ICH guideline S5A: Detection of Toxicity to Reproduction for Medicinal Products; EU: CPMP/ICH/386/95, adopted September 1993

Version / remarks:

1993

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Route of administration:

oral: gavage

Vehicle:

other: ethanol/Solutol® HS 15/tap water (1:4:5 v/v/v)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test item was administered by gavage in ethanol/Solutol ® HS 15/tap water (1:4:5 v/v/v).
The administration volume was 10 mL/kg body weight. The formulations were prepared as needed taking into account the analytically determined stability.
The test substance was administered as a suspension in the vehicle. The formulation was stored at room temperature. It was stirred with a magnetic stirrer for several minutes (until an obviously homogenous distribution occurred) before administration. It was also stirred during administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the start of treatment, the suitability of the formulation was confirmed by the analysis of concentration, homogeneity, and stability of dosage forms prepared in the same way as it were done in the study (0.15, 0.5, and 5 mg/mL, covering the concentration range used).
Analyses were carried out before the start of the study

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: Pairing was performed overnight (from approximately 15.30 to 08.00 CET; on weekends from approximately 12.00 to 08.00 CET), by placing one female rat together with one male rat into a type IIIh Makrolon ® cage. Allocation of the female animals to the relevant male animals was based on the current animal numbers, details of allocation could be found in the raw data. During the two-week mating period each female animal was paired daily. Females with positive sperm detection were not mated again. If sperm was detected in the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation.
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug/sprem in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol:

Duration of treatment / exposure:

The male rats were treated daily for 4 weeks prior to mating, during the following mating
period, and up to the day before necropsy. The female rats were treated daily for 2 weeks
prior to mating and during the subsequent mating period. After insemination had been verified (day 0 of gestation), treatment of the females was continued up to and including day 7 p.c. The animals were treated daily between 06.00 and 12.00 CET and received the administration formulations orally by gavage.

Frequency of treatment:

daily

Duration of test:

4 weeks prior mating until 16th day of gestation

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

6 mg/kg bw/day (nominal)

Dose / conc.:

2 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were selected on the basis of the results of the 4-week repeat-dose study in rats with doses up to and including 50 mg/kg. The 50 mg/kg dose exceeded the MTD and was associated with mortality and degenerative organ changes due to the strong increase in red blood cell parameters and the impaired hemodynamics.
Thus, 20 mg/kg was chosen as high dose and should result in margins of exposure of about
30-fold of the expected human therapeutic exposure. The low dose of 2 mg/kg should correspond to MoEs of about 3-fold.

Key result

Dose descriptor:

NOAEL

Effect level:

2 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other:

Abnormalities:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Abnormalities:

not specified

Developmental effects observed:

not specified

Executive summary:

In the present study 24 male and female Wistar rats were exposed to 0, 2, 6, and 20 mg/kg bw of the test item. Males were exposed 4 weeks prior to mating, during mating and up to the days before necropsy. Female rats were treated 2 weeks prior to mating, during mating and after insemination up to and including days 7 p.c. Cesarean sections were performed on days 14 to 16 p.c.

Salivation after administration occurred in males and females of all dose levels, which is most likely caused by the vehicle, since males and females of the current control groups also showed this finding. Four males, 3 of the 20 mg/kg bw group and 1 in the 6 mg/kg bw group were found dead.

Mean food intake and body weight gain was decreased in animals of the 20 mg/kg bw level in males and during the first week of treatment in males and females at 6 mg/kg bw. Generalized enlarged blood vessels occurred in males and females at dose levels of 6 mg/kg and 20 mg/kg. Testes weights and ovary weights were unaffected by treatment. Insemination, fertility, and gestation indices were not affected by treatment with the test item at dose levels up to 20 mg/kg bw/day. The mean number of corpora lutea and thus the mean number of implantation sites were unaffected by treatment with Molidustat at dose levels up to 20 mg/kg.

The mean postimplantation loss was increased at the 20 mg/kg level and an effect cannot be excluded for 6 mg/kg bw treatment due to slight post-implantation losses. The mean number of viable embryos was statistically significantly decreased at the 20 mg/kg level, corresponding to the increased postimplantation loss at this dose level. Thus, the following no-observed-adverse-effect levels (NOAELs) were determined:

Systemic tolerability: 2 mg/kg bw/day

Fertility and early embryonic development: 2 mg/kg bw/day

 

 

 

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ICH guideline S5A: Detection of Toxicity to Reproduction for Medicinal Products; EU: CPMP/ICH/386/95, adopted September 1993

Version / remarks:

1993

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

Himalayan

Remarks:

(CRL:CHBB:HM)

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous Tylose MH 300 P2 (methylhydroxyethylcellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered by gavage in 0.5 % aqueous Tylose MH 300 P2 (methylhydroxyethylcellulose). The administration volume was 5 mL/kg body weight. The formulations were prepared as needed taking into account the analytically determined stability. The test substance was administered as a suspension in the vehicle. The formulation was stored at room temperature. It was stirred with a magnetic stirrer for several minutes (until an obviously homogenous distribution occurred) before administration. It was also stirred during administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the start of treatment, the suitability of the formulation was confirmed by the analysis
of concentration, homogeneity, and stability of dosage forms prepared in the same way as it
were done in the study (0.1, 0.5, and 100 mg/mL, covering the concentration range used).
Analyses were carried out before the start of the study. Homogeneity tests and content checks of the active ingredient in samples with concentrations of 0.4, 1.2 (content checks, only), and 4 mg/mL were carried out twice during the study.

Details on mating procedure:

The male animals were used for mating only and were not treated. After copulation was ascertained, 80 females were allocated to four experimental groups according to a computer generated randomization plan (random number generator, Dell PC, data are filed by Bayer Pharma AG, 42096 Wuppertal).
The mating was performed between 06.00 and 10.00 CET. One male rabbit was mated with one female rabbit under observation. About one hour after the first mating had occurred, the same animals were mated again. It was recorded which female was mated with which male.
The day on which the copulation was observed was considered as day 0 of gestation.

Duration of treatment / exposure:

The females were treated daily from days 6 to 20 of gestation.

Frequency of treatment:

daily

Duration of test:

females were sacrificed 21 or 29 days p.c.

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Dose / conc.:

6 mg/kg bw/day (actual dose received)

Dose / conc.:

2 mg/kg bw/day (actual dose received)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 in the main groups and 3 in the satellite groups

Control animals:

yes, concurrent vehicle

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other:

Abnormalities:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Abnormalities:

no effects observed

Developmental effects observed:

no

Executive summary:

In the present study 20 inseminated female Himalayan rabbits were exposed to 0, 2, 6, and 20 mg/kg bw of the test item in 0.5 % aqueous Tylose MH 300 P2 (methylhydroxyethylcellulose) as vehicle daily from day 6 to day 20 p.c. On day 29 of gestation the fetuses were delivered by cesarean section. Investigation was performed on general tolerance of the test substance by the females as well as on its effect on intrauterine development. Three additional females per group were treated with the doses of 0, 2, 6, and 20 mg/kg from days 6 to 20 p.c. for determination of test substance in the plasma of the females.

All parameter measured, such as

 

in females:

• body weight development


• corrected body weight gain


• appearance, behavior, mortality and gross morphology


• gross pathological findings

 

in intrauterine development:

• gestation rate


• weight and appearance of Placentas


• postimplantation loss, number of fetuses


• sex of fetuses


• fetal weight


• fetal malformations


• fetal external and visceral deviations


• fetal skeletal deviations

 

revealed no influence on general tolerance of the test substance by the females as well as on intrauterine development through dosing with Molidustat up to 20 mg/kg bw/day. Summarizing and evaluating all data investigated the following no-observed-adverse-effect levels (NOAELs) were determined:

Maternal toxicity: 20 mg/kg bw/day

Intrauterine development: 20 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

7

Species:

rat

Quality of whole database:

The studies were conducted accoring to ICH guidelines for testing of medical products, thus, the quality of the database is considered high.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

In one study according to ICH guideline S5A: Detection of Toxicity to Reproduction for Medicinal Products; EU: CPMP/ICH/386/95, adopted September 1993, 24 male and female Wistar rats were exposed to 0, 2, 6, and 20 mg/kg bw of the test item. Males were exposed 4 weeks prior to mating, during mating and up to the days before necropsy. Female rats were treated 2 weeks prior to mating, during mating and after insemination up to and including days 7 p.c. Cesarean sections were performed on days 14 to 16 p.c.

Salivation after administration occurred in males and females of all dose levels, which is most likely caused by the vehicle, since males and females of the current control groups also showed this finding. Four males, 3 of the 20 mg/kg bw group and 1 in the 6 mg/kg bw group were found dead.

Mean food intake and body weight gain was decreased in animals of the 20 mg/kg bw level in males and during the first week of treatment in males and females at 6 mg/kg bw. Generalized enlarged blood vessels occurred in males and females at dose levels of 6 mg/kg and 20 mg/kg. Testes weights and ovary weights were unaffected by treatment. Insemination, fertility, and gestation indices were not affected by treatment with the test item at dose levels up to 20 mg/kg bw/day. The mean number of corpora lutea and thus the mean number of implantation sites were unaffected by treatment with Molidustat at dose levels up to 20 mg/kg.

The mean postimplantation loss was increased at the 20 mg/kg level and an effect cannot be excluded for 6 mg/kg bw treatment due to slight post-implantation losses. The mean number of viable embryos was statistically significantly decreased at the 20 mg/kg level, corresponding to the increased postimplantation loss at this dose level. Thus, the following no-observed-adverse-effect levels (NOAELs) were determined:

Systemic tolerability: 2 mg/kg bw/day

Fertility and early embryonic development: 2 mg/kg bw/day

 

In a second study according to ICH guideline S5A: Detection of Toxicity to Reproduction for Medicinal Products; EU: CPMP/ICH/386/95, adopted September 1993, 22 inseminated female Wistar rats were exposed to 0, 3, 10, and 30 mg/kg bw of the test item in ethanol/Solutol ® HS 15/demineralized water (1:4:5 v/v/v) as vehicle daily from day 6 to day 17 p.c. The fetuses were delivered by cesarean section on day 21 of gestation. Investigation was performed on general tolerance of the test substance by the females as well as on its effect on intrauterine development. Four additional females per group were treated with the doses of 0, 3, 10, and 30 mg/kg from days 6 to 17 p.c. for determination of test substance in the plasma of the females. Appearance, behavior, and mortality were unaffected at dose levels up to 30 mg/kg bw/day. Mean body weight development was decreased at dose level of 30 mg/kg bw/day. Food intake was transiently decreased at the dose levels of 30 mg/kg bw/day. No treatment related gross pathological findings occurred at dose levels up to 30 mg/kg bw/day.

The gestation rate was unaffected by treatment at dose levels up to 30 mg/kg bw/day. Morphological appearance and weight of placentas were only affected by treatment at dose level 30 mg/kg bw/day. Postimplantation loss was increased in dose level 30 mg/kg. Fetal sex distribution was unaffected by treatment at dose levels up to 30 mg/kg bw/day. Weights of fetuses were statistically significantly decreased at dose levels of 30 mg/kg bw/day. Dosing with Molidustat at 30 mg/kg revealed an increased incidence of ocular malformations. A treatment related effect on skeletal deviations was not seen up to a dose level of 30 mg/kg bw/day.

In summary, treatment-related maternal toxicity and developmental toxicity characterized mainly by ocular malformations, reduced fetal weight and increased postimplantation loss were seen at a daily dose of 30 mg/kg of the test item. Consequently, the following no-observed-adverse-effect levels (NOAELs) were determined:

Maternal toxicity: 10 mg/kg bw/day

Developmental toxicity: 10 mg/kg bw/day

 

In the last and third study according to CH guideline S5A: Detection of Toxicity to Reproduction for Medicinal Products; EU: CPMP/ICH/386/95, adopted September 1993, 20 inseminated female Himalayan rabbits were exposed to 0, 2, 6, and 20 mg/kg bw of the test item in 0.5 % aqueous Tylose MH 300 P2 (methylhydroxyethylcellulose) as vehicle daily from day 6 to day 20 p.c. On day 29 of gestation the fetuses were delivered by cesarean section. Investigation was performed on general tolerance of the test substance by the females as well as on its effect on intrauterine development. Three additional females per group were treated with the doses of 0, 2, 6, and 20 mg/kg from days 6 to 20 p.c. for determination of test substance in the plasma of the females.

All parameter measured, such as

 

in females:

• body weight development


• corrected body weight gain


• appearance, behavior, mortality and gross morphology


• gross pathological findings

 

in intrauterine development:

• gestation rate


• weight and appearance of Placentas


• postimplantation loss, number of fetuses


• sex of fetuses


• fetal weight


• fetal malformations


• fetal external and visceral deviations


• fetal skeletal deviations

 

revealed no influence on general tolerance of the test substance by the females as well as on intrauterine development through dosing with Molidustat up to 20 mg/kg bw/day. Summarizing and evaluating all data investigated the following no-observed-adverse-effect levels (NOAELs) were determined:

Maternal toxicity: 20 mg/kg bw/day

Intrauterine development: 20 mg/kg bw/day

 

Justification for classification or non-classification

Based on the presented data, which show effects on development without other toxic effects in rats, Molidustat is classified as reproductive toxicant Category 1B according to Regulation (EU) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).