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

Endpoint summary

Description of key information

Study report: oral repeated dose toxicity according to OECD test guideline 407 in rats; adverse effects observed [Schladt, 2010]

Study report: according to OECD test guideline 408 in rats; NOAEL: 1.5 mg/kg bw. [Schladt, 2012]

Study report: according to OECD test guideline 452 in rats; NOAEL females: 1.07 mg/kg bw; NOAEL in males: 0.321 mg/kg bw [Schladt, 2012]

Study report: oral repeated dose toxicity, test item in drinking water, administration for 2 weeks, mice: no adverse effects observed under the consitions of the test [Schladt, 2012]

Study report: according to OECD test guideline 408 in mice; results: LOEL: 3.21 mg/kg bw [Schladt, 2012]

Study report: according to OECD test guideline 451 in mice; results: In a 2-year oncogenicity study in mice, Molidustat is non-carcinogenic. Minor mode of action-related effects on hematopoiesis were observed at 5 mg/kg. Please refer to IUCLID Chapter 7.7. [Popp, 2018]

Study report: according to OECD test guideline 409 (modified for the dog) in dogs; NOAEL: 5 mg/kg bw [Ruf, 2010]

Study report: according to OECD test guideline 409 (modified for the dog) in dogs; NOAEL: 7.5 mg/kg bw.

Study report: according to OECD test guideline 452 (modified for the dog) in dogs; NOAEL: 0.5 mg/kg bw.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Subacute oral toxicity study in CD-1 mice (2 weeks)

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

In a subacute toxicity study 15 male and 15 female mice per dose group were treated with 15 or 150 ppm of the test item in their drinking water for two weeks. 5 males and 5 females each served as control groups. The animals were observed for clinical signs and body weights, food and water consumption was measured. Blood for toxicokinetic investigation was collected at the end of the study.
The treatment resulted in the following mean daily test substance intake (in ascending dosages): 3.3 and 34.7 mg test item/kg body weight in males and 4.2 and 41.1 mg test item /kg body weight in females.

GLP compliance:

yes

Species:

mouse

Strain:

CD-1

Sex:

male/female

Route of administration:

oral: drinking water

Details on route of administration:

2 weeks (Animals received the test substance/tap water mixture until day of scheduled necropsy).

Vehicle:

unchanged (no vehicle)

Remarks:

The micronized test substance was administered in the tap water. The test substance was mixed into the drinking water at the appropriate concentrations at room temperature. The formulations were prepared once per week.

Analytical verification of doses or concentrations:

yes

Remarks:

The suitability of the proposed formulations was confirmed by the analyses of concentration and stability of dosage forms with concentrations of 0.001 mg/mLand 0.4 mg/mL. Analyses were carried out before the start of the study.

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

daily

Dose / conc.:

150 ppm

Dose / conc.:

15 ppm

Dose / conc.:

0 ppm

No. of animals per sex per dose:

5 in the negative control and 15 in the treatment groups

Control animals:

yes, concurrent no treatment

Dose descriptor:

NOAEL

Effect level:

15 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

Dose descriptor:

NOAEL

Effect level:

150 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

water consumption and compound intake

Conclusions:

In a subacute toxicity study 15 male and 15 female mice per dose group were treated with 15 or 150 ppm Molidustat in their drinking water for two weeks. 5 males and 5 females each served as control groups. Survival was not affected by the treatment. With the exception of reddened skin, no further treatment-related clinical findings were recorded. At 150 ppm the body weight development in females was slightly retarded. Food intake was not affected by the treatment. Water intake was in treated females groups and in 150 ppm males was slightly increased. The no-observed-adverse-effect level was 150 ppm in males and 15 ppm in female (due to a slight effect on body weight development).

Executive summary:

In a subacute toxicity study 15 male and 15 female mice per dose group were treated with 15 or 150 ppm of the test item in their drinking water for two weeks. 5 males and 5 females each served as control groups.
The animals were observed for clinical signs and body weights, food and water consumption was measured. The treatment resulted in the following mean daily test substance intake (in ascending dosages): 3.3 and 34.7 mg test item/kg body weight in males and 4.2 and 41.1 mg/kg body weight in females. Survival was not affected by the treatment. With the exception of reddened skin, no further treatment-related clinical findings were recorded.
In all male dose groups and in females at 15 ppm body weight development was comparable to that in the respective control group. At 150 ppm the body weight development in females was slightly retarded.
Food intake was not affected by the treatment. Water intake was in treated females groups and in 150 ppm males was slightly increased. The no-observed-adverse-effect level was 150 ppm in males and 15 ppm in female (due to a slight effect on body weight development). 

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

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:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

27 July 1995

Principles of method if other than guideline:

The objective of the study was to identify any potential toxic effects of Molidustat including the dose-response relationship in a mammalian species after repeated exposure, to establish a no-observed-adverse-effect level, and to provide a basis for dose selection for studies with a longer treatment period.
Animals of recovery groups were first treated for 4 weeks, followed by a treatment free period of 2 weeks and observed for reversibility and for persistence of possible toxic effects.

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: Ethanol/ Solutol HS 15 /Tap water (1/4/5; v/v/v)

Analytical verification of doses or concentrations:

yes

Remarks:

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 was done in the study.

Duration of treatment / exposure:

28 days + 14 days recovery group

Frequency of treatment:

daily

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 in the treatment and recovery groups
6 in the satellite groups
5 for the MNT positive control

Control animals:

yes, concurrent vehicle

Remarks on result:

other: no structural chromosomal aberrations, no increase in unscheduled DNA synthesis, thus, increase of micronuclei is not considered to indicate a genotoxic potential

Remarks on result:

other: Micronucleus frequencies in normochromatic erythrocytes, due to the longer half- life of normochromatic erythrocytes, showed a significant increase of micronuclei

Remarks on result:

other: daily doses up to 50 mg/kg was characterized by signs of exaggerated pharmacology and the sequels thereof. 50 mg/kg clearly exceeded the maximum tolerated dose.

Conclusions:

The objective of the study was to identify any potential toxic effects of Molidustat including the dose-response relationship in a mammalian species after repeated exposure, to
establish a no-observed-adverse-effect level, and to provide a basis for dose selection for
studies with a longer treatment period. Animals of recovery groups were first treated for 4 weeks, followed by a treatment free period of 2 weeks and observed for reversibility and for persistence of possible toxic effects.

Executive summary:

In the present subacute oral repeated toxicity study Molidustat was not tolerated without adverse effects at the lowest dose level tested, 5 mg/kg b.w. by female and male Wistar rats. The effects seen were secondary to the pharmacological mode of action of the test item. They are not interpreted as evidence for an intrinsic organ toxicity of Molidustat. Survial was not affected by the treatment up to and including 15 mg/kg bw. At 50 mg/kg, mortality was increased due to the treatment with the test substance (6 out of 20 males and 1 out of 20 females died unscheduled). The main histopathological findings in decedents were increased erythropoiesis, degenerative changes and thrombosis in the heart, renal tubular necrosis and focal inflammation/necrosis of the intestine. These findings are seen as secondary to altered rheological properties of the blood and resultant impaired organ perfusion caused by the pharmacological effect of the test compound. At histopathological investigation, correlating to the hematological findings, a test substance-related increase in erythropoiesis was observed in both sexes starting at 5 mg/kg. Investigation in female rats showed that there was no biologically relevant indication of a clastogenic effect of Moludistat in the cytogenetic bone-marrow test on the female rat, i.e. in a somatic test system in vivo. Moludistat was also investigated for the induction of micronuclei in erythrocytes of the bone-marrow in males. The results are evaluated as inconclusive since there was no conclusive evidence of a biologically relevant clastogenic effect of orally administered test substance in the micronucleus test on the male rat, i.e. in a somatic test system in vivo.
Furthermore, the test item was tested for genotoxic activity in the in vivo UDS assay with the results that it is considered negative in the in vivo UDS Assay with primary rat liver cells. 

Reference 3

Endpoint:

sub-chronic toxicity: oral

Remarks:

Subcbronic Toxicity Study in CD-1 Mice (13 Weeks Daily Administration by Drinking Water)

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:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

The test chemical was administered dose adjusted in their drinking water to 10 male and 10 female CD-1 mice rats per dose group, in intended daily doses of 0, 3.21, 10.7 and 32.1 mg/kg b.w. for a intended period of at least 92 days, but animals ofthe 32.1 mg/kg group had to be necropsied within week 8.
In order to maintain the afore-mentioned intended doses, drinking water concentrations were adapted on a weekly basis taking body weight development into consideration. The treatment resulted in the following mean effective test substance intake (with ascending dosages): 3.4, 11.6 and 41.9 mg test item/kg/day in males and 3.3, 11.7 and 39.8 mg test item /kg/day in females, corresponding to doses of 3.2, 10.8 and 39.2 mg/kg/day in males and 3.1, 10.9 and 37.2 mg/ kg/day in females, based on the active molecule. Additionally, male and female mice (15 males and 15 females per treatment group and 9 males and 9 females per control group) were treated with the same doses for a comparable period of time and were used for toxicokinetics.

GLP compliance:

yes

Species:

mouse

Strain:

CD-1

Sex:

male/female

Route of administration:

oral: drinking water

Vehicle:

other: 100 mM Sodium hydrogen carbonate buffered (about pH 7.0) demineralized water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was administered orally in pH-adjusted (about pH 7.0) sodium hydrogen carbonate buffered demineralized water. To ensure a constant test compound intake on a mean mg/kg bodyweight basis, the amount oftest compound mixed into the drinking water was adjusted weekly based on the water intake and body weight data of each main dose group and sex separately.

Analytical verification of doses or concentrations:

yes

Remarks:

The suitability of the proposed formulation was confirmed by the analyses of concentration and stability of dosage forms which were prepared using this procedure. Analyses for homogeneity were not necessary as the formulations were solutions.

Duration of treatment / exposure:

101 days

Frequency of treatment:

daily

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

3 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 in the main group and 15 in the satellite group

Control animals:

yes, concurrent vehicle

Key result

Dose descriptor:

LOEL

Effect level:

3.21 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Conclusions:

Continuous oral treatment of mice via drinking water with the test item at intended doses of 0, 3.21, 10.7 or 32.1 mg/kg b.w. for a period of up to 13 weeks resulted in a variety of treatment-related effects. Although most effects are seen from 10.7 mg/kg onwards, expected effects due to to (exaggerated) pharmacological effects of the test substance were already seen at 3.21 mg/kg bw.

Executive summary:

Continuous oral treatment of CD-1 mice via drinking water with the test item at intended doses of 0, 3.21, 10.7 or 32.1 mg/kg b.w. for a period of up to 13 weeks resulted in a variety of treatment-related effects. Survival was not affected by the treatment withthe test item up to and including 10.7 mg/kg. At the end of the week 7, seven males and three females of the 32.1 mg/kg group died unscheduled or had to be killed in moribund condition. Histopathology revealed that cause of death was related to increased hematocrit and the sequelae thereof. Clinically observed skin reddening observed in nearly all animals at 10.7 mg/kg and above is regarded to be related to the treatment with the test substance. Neither body weight gain nor food intake was relevantly affected by the treatment with the test item. There was a slight decrease in water intake at 32.1 mg/kg in males and females at the beginning of the dosing period, but a significant increase in water intake in the following weeks until prescheduled necropsy. Furthermore, in the bone marrow of femur and sternum, erythropoiesis was increased starting at 10.7 mg/kg. Bone marrow adipocytes were reduced in the femur starting at 3.21 mg/kg. Taken all data together, a no observed (adverse) effect level could not be established due to the effect, that replacement of hemopoietic marrow by adipocytes was reduced in the femur starting at 3.21 mg/kg. This effect as well the effects observed at higher doses are related to (exaggerated) pharmacological effects ofthe test substance. 

Reference 4

Endpoint:

sub-chronic toxicity: oral

Remarks:

Repeated Dose Toxicity Study in Wistar Rats (13 Weeks Daily Administration by Gavage Followed by a 4-Weeks Recovery Period)

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:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

21 September 1998

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Remarks:

(Hsd Cpb:WU)

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

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

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The formulations were prepared as needed and taking into account the analytically determined stability. For the preparation of the formulations the content of test item was not taken into account. The test item was administered as a solution in the vehicle.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the start of treatment, the suitability of the low dose formulation (0.15 mg/kg) 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. Analyses were carried out before the start of the study

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

later reduced to 10 mg/kg bw
The high dose group was treated with 15 mg/kg b.w. until day 57 and thereafter with 10 mg/kg b.w. until scheduled necropsy.

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

1.5 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 for main and recovery group and 6 and 8 for satellite group 1 and 2, respectively.

Control animals:

yes, concurrent vehicle

Dose descriptor:

NOAEL

Effect level:

1.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Conclusions:

Daily oral treatment of rats with the test item at doses of 0, 1.5, 5 or 15/10 mg/kg b.w. resulted in a variety of treatment-related findings starting at the low dose of 1.5 mg/kg. At the low dose of 1.5 mg/kg, the observed effects were related to the intended pharmacodynamic effect of increased erythropoiesis, so that this dose level is considered the no-observed-adverse-effect-level (NOAEL). Adverse effects such as mortality, thrombosis, prominent cardiac valvular fibrosis, renal basophilic tubules and iron pigment as well as extravasated blood, pigment and plexiform vascular change in/adjacent to the mesenteric lymph node were observed at 5 mg/kg and above. At 10 (reduced from 15 mg/kg) intercurrent mortality was related to thrombosis/infarction and one decedent for the same reason was present at 5 mg/kg. This is considered to be related to rheological effects after mode of action induced marked increase in hematocrit. Whereas many findings were reversible within the recovery period, pronounced findings related to thrombosis, infarction or hemodynamic overstressing were not reversible within the recovery period. All findings observed are related to increased/exaggerated hemopoietic activity and there was no evidence of a specific toxic effect of the test compound. The no observed-adverse-effect level of 1.5 mg/kg. All findings observed are related to increased/exaggerated hemopoietic activity and there was no evidence of a specific toxic effect of the test compound.

Executive summary:

In the present study conducted according to OECD guideline 408 (1998) in rats the daily oral treatment of rats with the test item  by gavage in doses of 0, 1.5, 5 or 15/10 mg/kg b.w. for a period of up to 13 weeks resulted in a variety of treatment-related effects. Survival was not affected by the treatment with the test item at 1.5 mg/kg. Intercurrent mortality was increased at 5 mg/kg and above due to thrombosis/infarction. The main site of thrombosis was the heart where marked changes of the aortic and atrioventricular valves (vascularization, fibrosis, erosion) could be observed. Thus, mortality is considered to be related to an exaggerated pharmacological response to the test substance. In animals necropsied at the end of treatment, similar findings were encountered and in recovery animals no full reversibility of these severe findings could be attained. In addition, hematological effects, effects on clinical chemistry and organ morphology were observed which are attributed to the mode of action. Under the conditions described the administration of Molidustat via gavage to male and female rats was tolerated without adverse effects at 1.5 mg/kg, the lowest dose level tested, whereas adverse effects such as mortality, thrombosis, prominent cardiac valvular fibrosis, renal basophilic tubules and iron pigment as well as extravasated blood, pigment and plexiform vascular change in/adjacent to the mesenteric lymph node were observed at 5 mg/kg and above. The NOAEL of Molidustat is therefore considered to be 1.5 mg/kg. All findings observed are related to increased/exaggerated hemopoietic activity and there was no evidence of a specific toxic effect of the test compound. 

Reference 5

Endpoint:

chronic toxicity: oral

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:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

adopted 07 September 2009

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

(Hsd Cpb:WU)

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: ethanol/Solutol HS® 15/tap water (1 0/40/50 v/v/v)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The formulations were prepared as needed and taking into account the analytically determined stability. For the preparation of the formulations the content of test item was not taken into account. The test item was administered as a solution in the vehicle.

Analytical verification of doses or concentrations:

yes

Remarks:

Before the start oftreatment, the suitability of the formulations 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. Analyses were carried out before the start.

Details on analytical verification of doses or concentrations:

Homogeneity: As the formulations were solutions no analytical investigation for
homogeneity of formulations was necessary.
Stability: The dosage forms prepared for homogeneity analysis were analyzed shortly after preparation and 4, 8 and 15 days thereafter. The analysis revealed that the test item was stable over this period within the defined limits.
Content checks: For this purpose the concentrations of samples of control and each test item dosage form prepared were determined three times during the study.

Duration of treatment / exposure:

6 month (185 days)

Frequency of treatment:

daily

Dose / conc.:

3.21 mg/kg bw/day (nominal)

Dose / conc.:

1.07 mg/kg bw/day (nominal)

Dose / conc.:

0.321 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Key result

Dose descriptor:

NOAEL

Effect level:

1.07 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

haematology

Key result

Dose descriptor:

NOAEL

Effect level:

0.321 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

haematology

Conclusions:

Under the conditions described the administration of Molidustat via gavage to male and female rats was tolerated without adverse effects at 1.07 mg/kg in females and at 0.321 mg/kg in males. At higher doses adverse effects occur which are considered to be related to rheological effects after mode of action induced marked increase in hematocrit. All findings observed are related to increased/exaggerated hemopoietic activity and there was no evidence of a specific toxic effect of the test compound.

Executive summary:

In the present study conducted according to OECD guideline 452 (2009) in male and female Wistar rats (20 animals per sex and dose) the daily oral treatment with the test item by gavage in doses of 0, 0.321, 1.07 or 3.21 mg/kg b.w. for a period of up to 26 weeks resulted in a variety of treatment-related effects.

Survival, development of body weight development, food and water intake was not affected by the treatment with Molidustat. Based on the pharmacological mode of action, the test item stimulated erythropoiesis. As a consequence, the test item altered parameter of hematology and clinical biochemistry. However, under the conditions described the administration of the test substance via gavage to male and female rats was tolerated without adverse effects at 1.07 mg/kg in females and at 0.321 mg/kg in males. At 3.21 mg/kg adverse effects such as valvular fibrosis and dilation of heart, glomerular enlargement (in males also at 1.07 mg/kg), proteinaceous casts and chronic progressive nephropathy were observed. This is considered to be related to rheological effects after mode of action induced marked increase in hematocrit. All findings observed are related to increased/exaggerated hemopoietic activity and there was no evidence of a specific toxic effect of the test compound. The NOAEL was therefore determined to be 1.07 mg/kg bw in females and 0.321 mg/kg bw in males.

Reference 6

Endpoint:

sub-chronic toxicity: oral

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:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

21 September 1998

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

(modified for the dog)
Directive 67/548/EEC of the Council of the European Communities of June 27,1967 (Official Journal of the European Communities L 19611 of August 16, 1967) in its current version as amended for the twenty-second time (Directive 96/54/EEC of the Commission of the European Communities of September 30, 1996; Official Journal of the European Communities L 248 of September 30,1996).

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: PEG 400/Ethanol (90/10)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The formulations were prepared as needed and taking into account the analytically determined stability. For the preparation of the formulations the content of test item (95 %) was multiplied by 1.053 to achieve 100 % of active ingredient, and the densitiy of the vehicle was not taken into account.
The test item was administered as a solution (low dose)/as a suspension (mid and high dose) in the vehicle. The formulations were stored at room temperature.
Before the start of treatment, the suitability of the formulations was confirmed by the analysis of homogeneity and stability of the test article in the vehicle.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity: Dosage forms were prepared from mid and high dose.
Stability: Dosage forms were prepared from low, mid and high dose. The analyses revealed
that the test item was stable over a period of 10 days.
Content checks: for this purpose the concentrations of samples of control and each test item
dosage form were determined twice during the study to confirm the requested content of the
test article in the different administration formulations.

Duration of treatment / exposure:

28-29 days

Frequency of treatment:

daily

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Main group and recovery

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

Main group

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Main group

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Main group and recovery

No. of animals per sex per dose:

3 in the main group and 2 in the recovery group

Control animals:

yes, concurrent vehicle

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

histopathology: non-neoplastic

Conclusions:

Under the conditions described the oral administration of the test item to male and female
beagle dogs was not tolerated without effects caused by the mode of action of the test
compound. Up to and including 5 mg/kg, corresponding to an AUC of 3240 µgxh/L and Cmax of 2445 µgxh/L, the oral administration of the test item was tolerated without adverse effects.
Therefore, the NOAEL (= No Observed Adverse Effect Level) is set at 5 mg/kg. At the end of
the recovery period, not all findings were fully reversible.

Executive summary:

In the present study conducted according to EU Method B7 (modified for the dog) Directive 67/548/EEC of the Council of the European Communities of June 27,1967 (Official Journal of the European Communities L 19611 of August 16, 1967) in its current version as amended for the twenty-second time (Directive 96/54/EEC of the Commission of the European Communities of September 30, 1996; Official Journal of the European Communities L 248 of September 30,1996) in male and female beagle dogs (3 animals per sex and dose) the daily oral treatment with the test item by gavage in doses of 0, 5, 15 and 50 mg/kg b.w. for a period of 28 days and 28 days plus 14 days recovery, respectively, resulted in a variety of treatment-related effects. Survival was not affected by the treatment with the test item up to and including 50 mg/kg. Tests of the reflexes, body temperature and blood pressure showed no adverse effects up to and including 50 mg/kg. The evaluation of electrocardiograms showed no relevant changes in both sexes up to and including 50 mg/kg. Ophthalmological investigation gave no indication of an oculotoxic effect of the test item up to and including 50 mg/kg.  During the treatment period body weight development was slightly reduced in the highest treatment groups; at the end of the recovery period, no relevant changes were observed in body weight development in group IV-females in comparison to control females. Changes in hematology, clinical chemistry, urinalyses and histopathology were detected in all dose groups. Under the conditions described the oral administration of the test item to male and female beagle dogs was not tolerated without effects caused by the mode of action of the test compound. Up to and including 5 mg/kg, corresponding to an AUC of 3240 µgxh/L and Cmax of 2445 µgxh/L, the oral administration of the test substance was tolerated without adverse effects. Therefore, the NOAEL (= No Observed Adverse Effect Level) is set at 5 mg/kg. At the end of the recovery period, not all findings were fully reversible.

Reference 7

Endpoint:

sub-chronic toxicity: oral

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:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

21 September 1998

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: PEG400/Ethanoi (90/10)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered by gavage in PEG400/Ethanol (90/10). The administration volume was 2 mL/kg body weight in all groups including controls. The formulations were prepared as needed taking into account the stability.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The formulations were stored at room temperature.
Before the start of treatment, the suitability of the formulations was confirmed by the analysis of homogeneity and stability of the test article in the vehicle. For the purpose of content checks, the concentrations of samples of control and each test item dosage form were determined three times during the study to confirm the requested content of the test article in the different administration formulations.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

daily

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

7.5 mg/kg bw/day (nominal)

Dose / conc.:

2.5 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Key result

Dose descriptor:

NOAEL

Effect level:

2.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

haematology

histopathology: non-neoplastic

urinalysis

Conclusions:

Under the conditions described the oral administration of the test item to male and female
beagle dogs was not tolerated without effects due to the mode of action of the compound.
7.5 mg/kg are considered to have been tolerated without adverse effects thus representing the
no observed adverse effect level (= NOAEL).

Executive summary:

In the present study conducted according to OECD test guideline 409 (1998) the test item (in PEG 400/Ethanol (90/10)) was administered orally daily to 4 female and 4 male beagle dogs at doses of 0, 2.5, 7.5 and 25 mg/kg bw for 4 weeks. Treatment with the test substance was generally well tolerated except for findings associated with the pharmacological mode of action of the compound. No effects on survival, nutritional state, reflexes, body temperature, blood pressure, ECG, ophthalmology and urinalysis were observed. One animal  of the control group was sacrificed prematurely in week 11  due to an erroneous intratracheal administration.

Body weight development showed no relevant changes in males up to and including 25 mg/kg and in females up to and including 7.5 mg/kg. In females of group III (25 mg/kg), body weight development was reduced in comparison to control animals due to treatment with the test compound.

Food intake showed no changes up to and including 25 mg/kg in males and 7.5 mg/kg in females whereas in group III (25 mg/kg)-females food intake was reduced due to treatment with the test compound. Changes were observed for hematology, clinical biochemistry and histopathology. These changes are considered to be related to the pharmacological mode of action. Under the conditions described the oral administration of Molidustat to male and female beagle dogs was not tolerated without effects due to the mode of action of the compound. 7.5 mg/kg are considered to have been tolerated without adverse effects thus representing the no observed adverse effect level (= NOAEL).

Reference 8

Endpoint:

chronic toxicity: oral

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:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

21 September 1998

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: PEG400/Ethanol (9+1)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered by gavage in PEG400/Ethanol (9+ 1). The density of the vehicle was not taken into account. The administration volume was 2 mL/kg body weight in all groups including controls. The formulations were prepared as needed and taking into account the analytically determined stability.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The formulations were stored at room temperature.
Before the start of treatment, the suitability of the formulations was confirmed by the analysis
of stability of the test article in the vehicle. For the purpose of content checks, the concentrations of each dosage form were determined five times during the study to confirm the requested content of the test article in the different administration formulations.

Duration of treatment / exposure:

39 weeks

Frequency of treatment:

daily

Dose / conc.:

5.4 mg/kg bw/day (nominal)

Dose / conc.:

1.6 mg/kg bw/day (nominal)

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Key result

Dose descriptor:

NOAEL

Effect level:

0.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

Conclusions:

Under the conditions described the oral administration of the test item to beagle dogs was
not tolerated without effects due to the mode of action ofthe compound. The dose of 0.5 mg/kg b. w. is considered to have been tolerated without adverse effects thus representing the no observed adverse effect level(= NOAEL).

Executive summary:

In the present study conducted according to OECD test guideline 452 (1998) the test item (in PEG 400/Ethanol (90/10)) was administered orally daily to 4 female and 4 male beagle dogs at doses of 0, 0.5, 1.6 and 5.4 mg/kg bw for 39 weeks. No effects were observed on body weight development, reflexes, body temperature, blood pressure, electrocardiograms, ophthalmoscopy and urinalyses.

All with 5.4 mg/kg b.w. dosed-females had to be sacrificed prematurely at different time points due to treatment with the test compound. The only change in food intake was in one 5.4 mg/kg b.w.-female which did not eat any food at the day of sacrificing. Daily observations of animals revealed no treatment-related effects at doses up to 1.6 mg/kg b.w. At the dose of 5.4 mg/kg b.w., males exhibited reddening of the inner part of the snout, uncoordinated gait, bloody/reddish discharge from the muzzle. 5.4 mg/kg b.w. dosed-females showed decreased motility, poor general condition, reddening of the snout, inability to move. The severity of these effects led to premature sacrifice of all high dose females at different time points of the study. Further changes in hematology, clinical biochemistry and histopathology were observed and considered to be based on the pharmacological principle of the compound. In summary, under the conditions described the oral administration of Molidustat to beagle dogs was not tolerated without effects due to the mode of action of the compound. The dose of 0.5 mg/kg b. w. is considered to have been tolerated without adverse effects thus representing the no observed adverse effect level(= NOAEL).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.321 mg/kg bw/day

Study duration:

chronic

Experimental exposure time per week (hours/week):

7

Species:

rat

Quality of whole database:

All studies presented were conducted guideline compliant, thus, the whole database is considered of high quality

System:

haematopoietic

Organ:

blood

heart

kidney

liver

skin

spleen

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Several experimental studies have been conducted during the pharmacological development to evaluate the systemic tolerance of Molidustat after repeated administration (oral) to mice, rats and dogs. Data of these studies can be used for the toxicological assessment of Molidustat. Some of the available information is provided here, especially if the type of study suggests a relevance with regard to REACH. As rodents (rats and mice), according to REACH, are the preferred species in repeated dose studies, and as further the non-rodent studies were conducted with dogs, the focus of the tabulated summary below is on the rodent studies.

 

Based on the studies with Molidustat chronic oral administration led in rats generally does affect survival at higher doses while at lower doses it was not affected. Also, body weight and body weight gain as well as food consumption was not changed by treatment with the test item. Main effects resulted from exaggerated pharmacology and the sequels thereof. Based on the pharmacological action treatment mimicked hypoxia in the interstitial cells of the kidney and triggered the synthesis and release of erythropoietin (EPO) followed by stimulation of intra- and extramedullary erythropoiesis. Secondarily to this function skin reddening was observed which was considered to be related to treatment. Furthermore, alterations in hematology and clinical biochemistry indicating an enhanced erythropoiesis as well as changes in organ perfusion were detected at higher doses. Corresponding changes of red blood cell parameters (increased red blood cell count, hematocrit as well as hemoglobin concentration) were observed. Due to induction of erythropoiesis, parameters describing morphology and hemoglobin content of the erythrocytes were affected: anisochromasia, anisocytosis and microcytosis was observed in both sexes.

Thus, with respect to general population (vulnerable groups) safety the effects on enhanced erythropoiesis and coagulation revealed the most critical effects following repeated systemic exposure to Molidustat.

Rat studies:

In a subacute oral repeated dose toxicity study Molidustat was administered at 0, 5, 15, and 50 mg/kg bw to 10 femal and male Wistar rats for 28 days and was not tolerated without adverse effects at the lowest dose level tested, 5 mg/kg b.w. by female and male Wistar rats. The effects seen were secondary to the pharmacological mode of action of the test item. They are not interpreted as evidence for an intrinsic organ toxicity of the test item. Survial was not affected by the treatment up to and including 15 mg/kg bw. At 50 mg/kg, mortality was increased due to the treatment with the test substance (6 out of 20 males and 1 out of 20 females died unscheduled). The main histopathological findings in decedents were increased erythropoiesis, degenerative changes and thrombosis in the heart, renal tubular necrosis and focal inflammation/necrosis of the intestine. These findings are seen as secondary to altered rheological properties of the blood and resultant impaired organ perfusion caused by the pharmacological effect of the test compound. At histopathological investigation, correlating to the hematological findings, a test substance-related increase in erythropoiesis was observed in both sexes starting at 5 mg/kg. Investigation in female rats showed that there was no biologically relevant indication of a clastogenic effect of the test item in the cytogenetic bone-marrow test on the female rat, i.e. in a somatic test system in vivo. Molidustat was also investigated for the induction of micronuclei in erythrocytes of the bone-marrow in males. The results are evaluated as inconclusive since there was no conclusive evidence of a biologically relevant clastogenic effect of orally administered test substance in the micronucleus test on the male rat, i.e. in a somatic test system in vivo.

Furthermore, the test item was tested for genotoxic activity in the in vivo UDS assay with the results that it is considered negative in the in vivo UDS Assay with primary rat liver cells.

In a study conducted according to OECD guideline 408 (1998) in female and male Wistar rats the daily oral treatment of rats with the test item by gavage in doses of 0, 1.5, 5 or 15/10 mg/kg b.w. for a period of up to 13 weeks resulted in a variety of treatment-related effects. Survival was not affected by the treatment with the test item at 1.5 mg/kg. Intercurrent mortality was increased at 5 mg/kg and above due to thrombosis/infarction. The main site of thrombosis was the heart where marked changes of the aortic and atrioventricular valves (vascularization, fibrosis, erosion) could be observed. Thus, mortality is considered to be related to an exaggerated pharmacological response to the test substance. In animals necropsied at the end of treatment, similar findings were encountered and in recovery animals no full reversibility of these severe findings could be attained. In addition, hematological effects, effects on clinical chemistry and organ morphology were observed which are attributed to the mode of action. Under the conditions described the administration of Molidustat via gavage to male and female rats was tolerated without adverse effects at 1.5 mg/kg, the lowest dose level tested, whereas adverse effects such as mortality, thrombosis, prominent cardiac valvular fibrosis, renal basophilic tubules and iron pigment as well as extravasated blood, pigment and plexiform vascular change in/adjacent to the mesenteric lymph node were observed at 5 mg/kg and above. The NOAEL after subchronic administration of Molidustat is therefore considered to be 1.5 mg/kg. All findings observed are related to increased/exaggerated hemopoietic activity and there was no evidence of a specific toxic effect of the test compound.

 

In a study conducted according to OECD guideline 452 (2009) in male and female Wistar rats (20 animals per sex and dose) the daily oral treatment with the test item by gavage in doses of 0, 0.321, 1.07 or 3.21 mg/kg b.w. for a period of up to 26 weeks resulted in a variety of treatment-related effects.

Survival, development of body weight development, food and water intake was not affected by the treatment with the test item. Based on the pharmacological mode of action, the test substance stimulated erythropoiesis. As a consequence, the test item altered parameter of hematology and clinical biochemistry. However, under the conditions described the administration of Molidustat via gavage to male and female rats was tolerated without adverse effects at 1.07 mg/kg in females and at 0.321 mg/kg in males. At 3.21 mg/kg adverse effects such as valvular fibrosis and dilation of heart, glomerular enlargement (in males also at 1.07 mg/kg), proteinaceous casts and chronic progressive nephropathy were observed. This is considered to be related to rheological effects after mode of action induced marked increase in hematocrit. All findings observed are related to increased/exaggerated hemopoietic activity and there was no evidence of a specific toxic effect of the test compound. The NOAEL after chronic administration was therefore determined to be 1.07 mg/kg bw in females and 0.321 mg/kg bw in males.

Mice studies:

In a subacute toxicity study 15 male and 15 female mice per dose group were treated with 15 or 150 ppm of the test item in their drinking water for two weeks. 5 males and 5 females each served as control groups.

The animals were observed for clinical signs and body weights, food and water consumption was measured. The treatment resulted in the following mean daily test substance intake (in ascending dosages): 3.3 and 34.7 mg test item/kg body weight in males and 4.2 and 41.1 mg/kg body weight in females. Survival was not affected by the treatment. With the exception of reddened skin, no further treatment-related clinical findings were recorded.

In all male dose groups and in females at 15 ppm body weight development was comparable to that in the respective control group. At 150 ppm the body weight development in females was slightly retarded.

Food intake was not affected by the treatment. Water intake was in treated females groups and in 150 ppm males was slightly increased. The no-observed-adverse-effect level was 150 ppm in males and 15 ppm in female (due to a slight effect on body weight development).

 

 

Continuous oral treatment of CD-1 mice via drinking water with the test item at intended doses of 0, 3.21, 10.7 or 32.1 mg/kg b.w. for a period of up to 13 weeks resulted in a variety of treatment-related effects. Survival was not affected by the treatment with the test item up to and including 10.7 mg/kg. At the end of the week 7, seven males and three females of the 32.1 mg/kg group died unscheduled or had to be killed in moribund condition. Histopathology revealed that cause of death was related to increased hematocrit and the sequelae thereof. Clinically observed skin reddening observed in nearly all animals at 10.7 mg/kg and above is regarded to be related to the treatment with the test substance. Neither body weight gain nor food intake was relevantly affected by the treatment with the test item. There was a slight decrease in water intake at 32.1 mg/kg in males and females at the beginning of the dosing period, but a significant increase in water intake in the following weeks until prescheduled necropsy. Furthermore, in the bone marrow of femur and sternum, erythropoiesis was increased starting at 10.7 mg/kg. Bone marrow adipocytes were reduced in the femur starting at 3.21 mg/kg. Taken all data together, a no observed (adverse) effect level could not be established due to the effect, that replacement of hemopoietic marrow by adipocytes was reduced in the femur starting at 3.21 mg/kg. This effect as well the effects observed at higher doses are related to (exaggerated) pharmacological effects of the test substance.

 

IIn a study conducted according to OECD guideline 451 (1981) the carcinogenic potetial of Molidustat was investigated by daily oral administration in drinking water of 0, 0.54, 1.61, and 5.35 mg/kg of the substance over a period of up to 2 years to female and male CD-1 mice. The treatment of mice with the test item was well tolerated.
No test item-related mortality or toxicologically relevant test-item-related signs of systemic toxicity were observed up to the high dose of 5 mg/kg. Histopathological investigation did not show any treatment-related increase in neoplastic and non-neoplastic lesions up to and including 5 mg/kg. Thus, Molidustat, is non-carcinogenic in mice. Based on the pharmacological mode of action of the test item, e1ythrocyte count, hemoglobin concentration and hematocrit value were significantly increased in both sexed dosed at 5.0 mg/kg which was also reflected in a slight increase in medullary and splenic erythropoiesis. In conclusion, in a 2-year oncogenicity study in mice, Molidustat is non-carcinogenic. Minor mode of action-related effects on hematopoiesis were observed at 5 mg/kg. 

 

Dog studies:

In the present study conducted according to EU Method B7 (modified for the dog) Directive 67/548/EEC of the Council of the European Communities of June 27,1967 (Official Journal of the European Communities L 19611 of August 16, 1967) in its current version as amended for the twenty-second time (Directive 96/54/EEC of the Commission of the European Communities of September 30, 1996; Official Journal of the European Communities L 248 of September 30,1996) in male and female beagle dogs (3 animals per sex and dose) the daily oral treatment with the test item by gavage in doses of 0, 5, 15 and 50 mg/kg b.w. for a period of 28 days and 28 days plus 14 days recovery, respectively, resulted in a variety of treatment-related effects. Survival was not affected by the treatment with the test item up to and including 50 mg/kg. Tests of the reflexes, body temperature and blood pressure showed no adverse effects up to and including 50 mg/kg. The evaluation of electrocardiograms showed no relevant changes in both sexes up to and including 50 mg/kg. Ophthalmological investigation gave no indication of an oculotoxic effect of the test item up to and including 50 mg/kg. During the treatment period body weight development was slightly reduced in the highest treatment groups; at the end of the recovery period, no relevant changes were observed in body weight development in group IV-females in comparison to control females. Changes in hematology, clinical chemistry, urinalyses and histopathology were detected in all dose groups. Under the conditions described the oral administration of the test item to male and female beagle dogs was not tolerated without effects caused by the mode of action of the test compound. Up to and including 5 mg/kg, the oral administration of the test substance was tolerated without adverse effects. Therefore, the NOAEL (= No Observed Adverse Effect Level) is set at 5 mg/kg. At the end of the recovery period, not all findings were fully reversible.

 

In the present study conducted according to OECD test guideline 409 (1998) the test item (in PEG 400/Ethanol (90/10)) was administered orally daily to 4 female and 4 male beagle dogs at doses of 0, 2.5, 7.5 and 25 mg/kg bw for 4 weeks. Treatment with the test substance was generally well tolerated except for findings associated with the pharmacological mode of action of the compound. No effects on survival, nutritional state, reflexes, body temperature, blood pressure, ECG, ophthalmology and urinalysis were observed. One animal of the control group was sacrificed prematurely in week 11 due to an erroneous intratracheal administration.

Body weight development showed no relevant changes in males up to and including 25 mg/kg and in females up to and including 7.5 mg/kg. In females of group III (25 mg/kg), body weight development was reduced in comparison to control animals due to treatment with the test compound.

Food intake showed no changes up to and including 25 mg/kg in males and 7.5 mg/kg in females whereas in group III (25 mg/kg)-females food intake was reduced due to treatment with the test compound. Changes were observed for hematology, clinical biochemistry and histopathology. These changes are considered to be related to the pharmacological mode of action. Under the conditions described the oral administration of Molidustat to male and female beagle dogs was not tolerated without effects due to the mode of action of the compound. 7.5 mg/kg are considered to have been tolerated without adverse effects thus representing the no observed adverse effect level (= NOAEL).

In another study conducted according to OECD test guideline 452 (1998) the test item (in PEG 400/Ethanol (90/10)) was administered orally daily to 4 female and 4 male beagle dogs at doses of 0, 0.5, 1.6 and 5.4 mg/kg bw for 39 weeks. No effects were observed on body weight development, reflexes, body temperature, blood pressure, electrocardiograms, ophthalmoscopy and urinalyses.

All with 5.4 mg/kg b.w. dosed-females had to be sacrificed prematurely at different time points due to treatment with the test compound. The only change in food intake was in one 5.4 mg/kg b.w.-female which did not eat any food at the day of sacrificing. Daily observations of animals revealed no treatment-related effects at doses up to 1.6 mg/kg b.w. At the dose of 5.4 mg/kg b.w., males exhibited reddening of the inner part of the snout, uncoordinated gait, bloody/reddish discharge from the muzzle. 5.4 mg/kg b.w. dosed-females showed decreased motility, poor general condition, reddening of the snout, inability to move. The severity of these effects led to premature sacrifice of all high dose females at different time points of the study. Further changes in hematology, clinical biochemistry and histopathology were observed and considered to be based on the pharmacological principle of the compound. In summary, under the conditions described the oral administration of Molidustat to beagle dogs was not tolerated without effects due to the mode of action of the compound. The dose of 0.5 mg/kg b. w. is considered to have been tolerated without adverse effects thus representing the no observed adverse effect level(= NOAEL).

 

 

Based on the presented data Molidustat exerts several effects which can be attributed to the pharmacological action of the substance. The substance mimics hypoxia in the interstitial cells of the kidney and triggered the synthesis and release of erythropoietin (EPO) followed by stimulation of intra- and extramedullary erythropoiesis. As a sequel of its pharmacological action changes of red blood cell parameters (increased red blood cell count, hematocrit as well as hemoglobin concentration) were observed. Furthermore, parameters describing morphology and hemoglobin content of the erythrocytes were affected. The increased mortality at high doses is related to an exaggerated pharmacological response which resulted in increased blood volume or altered blood composition. Similarly, changes observed at necropsy, i.e. adverse effects detected in organs like heart, spleen, liver or kidneys are caused by the excessive pharmacological reaction.

For precautionary reasons the overall NOAEL was set to 0.321 mg/kg bw as determined in the chronic repeated dose study conducted according to OECD 452 in rats for male animals. It was shown that male animals were more susceptible to the effects mediated by Molidustat. Furthermore, in the chronic study performed with beagle dogs the NOAEL was only slightly higher with NOAEL = 0.5 mg/kg bw.

Thus, because the established NOAELs from the presented studies in different species were all below 10 mg/kg bw/day, Molidustat needs to be classified as STOT-RE Category 1 according to Regulation (EU) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Justification for classification or non-classification

The established NOAELs from the presented studies in different species were all below 10 mg/kg bw/day, thus, Molidustat needs to be classified as STOT-RE Category 1 according to Regulation (EU) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).