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Administrative data

Endpoint:
acute toxicity: other routes
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2009-04-21 to 2009-07-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-04-21 to 2009-07-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
17 December 2001
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Species:
mouse
Strain:
NMRI
Remarks:
Hsd WIN:NMRI
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan GmbH, 5960 AD Horst, Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 21-25g
- Fasting period before study: 3-4 h
The animals were group caged conventionally in polycarbonate cages on low dust wood granulate bedding (Lignocel BK 8-15, Finna Rettenmaier, Germany).
- Diet (e.g. ad libitum): ad libitum, “Provimi Kliba 3883 PM S15 Maus/Ratte Haltung, Kaiseraugst Switzerland”,
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
polyethylene glykol 400
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 0.5, 10, 30 and 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw


CLASS METHOD (if applicable) Acute toxic class
- Rationale for the selection of the starting dose: as described in the OECD test guideline
Doses:
5, 50, 300 and 2000 mg/kg bw
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Duration of observation period following administration: 21 days
- Frequency of observations and weighing: Clinical signs and mortality rates were determined several times on the day of administration and subsequently at least once daily for an observation period of at least 14 to 21 days. Mortality and in the event of symptoms occurring, nature, duration and intensity were recorded individually. The day of administration is defined as day 1. Times after administration until the following day were recorded either in minutes or in hours, depending on what was appropriate.
- Necropsy of survivors performed: yes
- Clinical signs including body weight: The weight gain of the animals was checked weekly until the end of the studies.
- Other examinations performed: clinical signs, body weight
Statistics:
The LD50 were estimated according to OECD - Guideline for Testing of Chemicals No. 423 - "Acute Oral Toxicity - Acute Toxic Class Method"; adopted: December 17, 2001
Key result
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
1 000 mg/kg bw
Based on:
test mat.
Mortality:
Two animals of the 2000 mg/kg bw group died
Clinical signs:
other: In the 2000 mg/kg bw testgroup the following clinical signs occurred: decreased motility, narrowed palpebral fissure, piloerection, uncoordinated gait, hunched posture, sunken flanks, high legged gait, decreased food and water intake, poor general condition
Gross pathology:
In one animal treated with 2000 mg/kg bw that died during the observation period
the following gross pathological changes were detected: liver, pale; lung,
hemorrhagic; spleen, pale and kidneys, pale. In the second mouse that died only
autolysis was detected.
The necropsies performed at the end of the study in the surviving animals treated
with 2000 mg/kg bw and animals treated with 300 mg/kg bw revealed no particular
findings



























































Dose mg/kg bw



Toxicological result*



Occurrence of signs



Time of death



Mortality (%)



female



(1st) 2000



2



3



3



5d -12d



6d-8d



67



(2nd) 2000



0



3



3



4d – 19d**



--



0



(1st) 300



0



0



3



--



--



0



(2nd) 300



0



0



3



--



--



0



* number of animals which died spontaneously and/or were sacrificed in moribund state / number of animals with signs of toxicity / total number of animals used per group



** (because of the severity and duration of symptoms the post treatment observation phase of the acute oral toxicity in rats was extended to three weeks)



LD50 cut-off: 1000 mg/kg bw ((adapted to OECD guideline 423 using the 2000 mg/kg bw groups 1 and 2 in an inverse order)


Interpretation of results:
Category 4 based on GHS criteria
Remarks:
according to Annex 2d of OECD guideline 423
Conclusions:
In the present study which was conducted equivalent to OECD test guideline 423, female NMRI mice (3/group) were orally administered a single dose Molidustat and were observed for the following 21 days. The doses applied were. 5, 50, 300, and 2000 mg/kg bw. Two animals died after the administration of 2000 mg/kg bw Molidustat. The following clinical signs were observed at 2000 mg/kg bw: decreased motility, narrowed palpebral fissure, piloerection, uncoordinated gait, hunched posture, sunken flanks, high legged gait, decreased food and water intake, poor general condition, tachypnea, labored breathing and closed eyelids. There was a significant loss of body weight on day 8 of the study in all surviving mice treated with 2000 mg/kg bw and in one mouse again on day 15 of the study. In one animal treated with 2000 mg/kg bw that died during the observation period the following gross pathological changes were detected: liver, pale; lung, hemorrhagic; spleen, pale and kidneys, pale. In the second mouse that died only autolysis was detected. In the necropsy performed at the end of the post-treatment observation period no gross pathological changes were detected. Based on these results the LD50 cut-off value for acute oral toxicity is considered 1000 mg/kg bw.
Executive summary:

In an acute oral toxicity study similar to OECD test guideline 423, groups of fasted, 5-6 weeks old female NMRI mice (3/group) were given a single oral dose of Molidustat in ethylene glycol at doses of 5, 50, 300,and 2000 mg/kg bw and observed for 21 days.


Oral LD50 cut-off Females = 1000 mg/kg bw


 


The following clinical signs were observed at 2000 mg/kg bw: decreased motility, narrowed palpebral fissure, piloerection, uncoordinated gait, hunched posture, sunken flanks, high legged gait, decreased food and water intake, poor general condition, tachypnea, labored breathing and closed eyelids. There was a significant loss of body weight on day 8 of the study in all surviving mice treated with 2000 mg/kg bw and in one mouse again on day 15 of the study. In one animal treated with 2000 mg/kg bw that died during the observation period the following gross pathological changes were detected: liver, pale; lung, hemorrhagic; spleen, pale and kidneys, pale. In the second mouse that died only autolysis was detected. In the necropsy performed at the end of the post-treatment observation period no gross pathological changes were detected.


Molidustat is of moderate Toxicity based on the LD50 cut-off in female NMRI mice.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-04-21 to 2009-07-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
17 December 2001
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Species:
rat
Strain:
Wistar
Remarks:
HsdCpb:Wu
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan GmbH, 5960 AD Horst, Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 - 12 weeks
- Weight at study initiation: 160 g - 197 g
Fasting period before study: 16-24 h
- Housing: The animals were group caged conventionally in polycarbonate cages on low dust
wood granulate bedding (Lignocel BK 8-15, Finna Rettenmaier, Germany).
- Diet (e.g. ad libitum): ad libitum, “Provimi Kliba 3883 PM S15 Maus/Ratte Haltung, Kaiseraugst Switzerland”,
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 5 days
- Method of randomisation in assigning animals to test and control groups

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
polyethylene-glycol 400
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 30 and 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw


CLASS METHOD (if applicable) Acute toxic class
- Rationale for the selection of the starting dose: as described in the OECD test guideline
Doses:
Three animals are used for each step. The dose level to be used as the starting dose is selected from one of four fixed levels, 5, 50, 300 and 2000 mg/kg body weight.
The starting dose level should be that which is most likely to produce mortality in some of the dosed animals. Absence or presence of compound-related mortality of the animals dosed at one step will determine the next step, i.e.:
- no further testing is needed,
- dosing of three additional animals, with the same dose,
- dosing of three additional animals at the next higher or the next lower dose level.
The substance is tested using a stepwise procedure, each step using three animals of a single sex (normally females). The procedure is described in the flow charts of Annex 2, OECD guideline 423.
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Duration of observation period following administration: 21 days
- Frequency of observations and weighing: Clinical signs and mortality rates were determined several times on the day of administration and subsequently at least once daily for an observation period of at least 14 to 21 days. Mortality and in the event of symptoms occurring, nature, duration and intensity were recorded individually. The day of administration is defined as day 1. Times after administration until the following day were recorded either in minutes or in hours, depending on what was appropriate.
- Necropsy of survivors performed: yes
- Clinical signs including body weight: The weight gain of the animals was checked weekly until the end of the studies.
- Other examinations performed: clinical signs, body weight,
Statistics:
The LD50 were estimated according to OECD - Guideline for Testing of Chemicals No. 423 - "Acute Oral Toxicity - Acute Toxic Class Method"; adopted: December 17, 2001
Key result
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
2 500 mg/kg bw
Based on:
test mat.
Mortality:
One animal died during observation period after administration of 2000 mg/kg bw
Clinical signs:
other: piloerection, blood-crusted snout, decreased motility, spasmodic state, high legged gait, sunken flanks, decreased food intake, decreased water intake, tachypnea, emaciation, poor general condition, hunched posture, staggering gait and narrowed palpebral
Gross pathology:
In the animal that died during the observation period, no other gross pathological changes than cannibalism were detected.
In the necropsy performed at the end of the post-treatment observation period in the surviving animals, no gross pathological changes were detected.









































Dose mg/kg bw



Toxicological result*



Occurrence of signs



Time of death



Mortality (%)



female



(1st) 2000



0



3



3



3d -17d**



--



0



(2nd) 2000



1



3



3



3d – 16d**



10d



33



* number of animals which died spontaneously and/or were sacrificed in moribund state / number of animals with signs of toxicity / total number of animals used per group



** (because of the severity and duration of symptoms the post treatment observation phase of the acute oral toxicity in rats was extended to three weeks)



LD50 cut-off: 2500 mg/kg bw (according to OECD guideline 423)


Interpretation of results:
Category 5 based on GHS criteria
Remarks:
according to Annex 2d of OECD guideline 423
Conclusions:
In the present study which was conducted equivalent to OECD test guideline 423, female Wistar rats (3/group) were orally administered a single dose Molidustat and were observed for the following 21 days. The doses applied were. 5, 50, 300, and 2000 mg/kg bw. One animal died after the administration of 2000 mg/kg bw Molidustat. The following clinical signs were observed: piloerection, blood-crusted snout, decreased motility, spasmodic state, high legged gait, sunken flanks, decreased food intake, decreased water intake, tachypnea, emaciation, poor general condition, hunched posture, staggering gait and narrowed palpebral fissure. There was a significant loss of body weight on day 8 of the study in the rats treated with 2000 mg/kg bw. In the animal that died during the observation period no other changes than cannibalism was detected. In the necropsy performed at the end of the post-treatment observation period no gross pathological changes were detected . Based on these results the LD50 cut-off value for acute oral toxicity is considered 2500 mg/kg bw.
Executive summary:

In an acute oral toxicity study similar to OECD test guideline 423, groups of fasted, 10-12 weeks old female Wistar rats (3/group) were given a single oral dose of Molidustat in ethylene glycol  at doses of 5, 50, 300,and 2000 mg/kg bw and observed for 21 days.


Oral LD50 cut-off Females = 2500 mg/kg bw


 


One animal treated with 2000 mg/kg bw died during the observation period. The following clinical signs were observed: piloerection, blood-crusted snout, decreased motility, spasmodic state, high legged gait, sunken flanks, decreased food intake, decreased water intake, tachypnea, emaciation, poor general condition, hunched posture, staggering gait and narrowed palpebral fissure. There was a significant loss of body weight on day 8 of the study in the rats treated with 2000 mg/kg bw. In the animal that died during the observation period no other changes than cannibalism was detected. In the necropsy performed at the end of the post-treatment observation period no gross pathological changes were detected .


 


Molidustat is of low Toxicity based on the LD50 cut-off in female Wistar rats, thus, no classification is needed according to Regulation (EU) No. 1272/2008 (CLP).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD test guideline 423: Acute toxic class
Version / remarks:
17 December 2001
Principles of method if other than guideline:
- Principle of test: similar to OECD test guideline 423 except the substance was administered intravenously
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
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

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan GmbH, 5960 AD Horst, Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 21-25g
- Fasting period before study: 3-4 h
The animals were group caged conventionally in polycarbonate cages on low dust
wood granulate bedding (Lignocel BK 8-15, Finna Rettenmaier, Germany).
- Diet (e.g. ad libitum): ad libitum, “Provimi Kliba 3883 PM S15 Maus/Ratte Haltung, Kaiseraugst Switzerland”,
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intravenous
Vehicle:
polyethylene glycol
Remarks:
poly ethylene glycol 400
Details on exposure:
For intravenous administration the administration volume was 10 mL/kg body weight administered into one of the tail veins. The duration of the injection was approx. 30 seconds.
VEHICLE
- Concentration in vehicle: 0.5, 10, 30 and 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw


CLASS METHOD (if applicable) Acute toxic class
- Rationale for the selection of the starting dose: as described in the OECD test guideline
Doses:
5, 50, 300 and 2000 mg/kg bw
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Duration of observation period following administration: 21 days
- Frequency of observations and weighing: Clinical signs and mortality rates were determined several times on the day of administration and subsequently at least once daily for an observation period of at least 14 to 21 days. Mortality and in the event of symptoms occurring, nature, duration and intensity were recorded individually. The day of administration is defined as day 1. Times after administration until the following day were recorded either in minutes or in hours, depending on what was appropriate.
- Necropsy of survivors performed: yes
- Clinical signs including body weight: The weight gain of the animals was checked weekly until the end of the studies.
- Other examinations performed: clinical signs, body weight
Statistics:
The LD50 were estimated according to OECD - Guideline for Testing of Chemicals No. 423 - "Acute Oral Toxicity - Acute Toxic Class Method"; adopted: December 17, 2001

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
other: LD50 cut-off
Effect level:
50 mg/kg bw
Based on:
test mat.
Mortality:
All animals of the 100 mg/kg bw treatment group and each one of the two 30 mg/kg bw treatment groups died subsequent to administration.
Clinical signs:
100 mg/kg bw: clonical cramps and labored breathing.
30 mg/kg bw: abdominal position, palmospasm, clonical cramps, tachypnea, labored breathing, decreased motility, temporary tremor, narrowed palpebral fissure, blue colored and lost tail
Body weight:
There were no toxicological effects on body weights or on body weight development in surviving mice treated with 30 mg/kg bw. The slight decrease in body weight of one animal on day 15 is regarded as due to chance.
Gross pathology:
In all animals that died during the observation period and in the necropsies performed at the end of the post-treatment observation period no gross pathological changes were detected beside the local effects ofthe formulation on the injection site (blue colored tail 1/6 and lost tail 3/6 animals).

Any other information on results incl. tables



















































Dose mg/kg bw



Toxicological result*



Occurrence of signs**



Time of death



Mortality (%)



female



100



3



3



3



0‘



0‘



100



(1st) 30



1



3



3



0‘ – 1h



0‘



33



(2nd) 30



1



3



3



0‘ – 1h



0‘



33



* number of animals which died spontaneously and/or were sacrificed in moribund state / number of animals with signs of toxicity / total number of animals used per group



** excluding the local effects of the formulation at the injection site



LD50 cut-off: 50 mg/kg bw (adapted to OECD guideline 423, for calculation of LD50 cut-off the highest applicable dose of 100 mg/kg bw was used instead of 200 mg/kg)


Applicant's summary and conclusion

Conclusions:
In the present study which was conducted equivalent to OECD test guideline 423, female NMRI mice (3/group) were intravenously administered a single dose Molidustat and were observed for the following 21 days. The doses applied were. 30 and 100 mg/kg bw. All animals died after the administration of 100 mg/kg bw Molidustat. The following clinical signs were observed at 100 mg/kg bw: clonical cramps and labored breathing and at 30 mg/kg bw: abdominal position, palmospasm, clonical cramps, tachypnea, labored breathing, decreased motility, temporary tremor, narrowed palpebral fissure, blue colored and lost tail. There were no toxicological effects on body weights or on body weight development in surviving mice treated with 30 mg/kg bw. The slight decrease in body weight of one animal on day 15 is regarded as due to chance.In all animals that died during the observation period and in the necropsies performed at the end of the post-treatment observation period no gross pathological changes were detected beside the local effects of the formulation on the injection site (blue colored tail 1/6 and lost tail 3/6 animals) Based on these results the LD50 cut-off value for acute i.v. toxicity is considered 50 mg/kg bw.
Executive summary:

In an acute toxicity study similar to OECD test guideline 423, groups of fasted, 5-6 weeks old female NMRI mice (3/group) were given a single intravenous dose of Molidustat in ethylene glycol at doses of 30 and 100 mg/kg bw and observed for 21 days.


Oral LD50 cut-off Females = 50 mg/kg bw


 


The following clinical signs were observed at 100 mg/kg bw: clonical cramps and labored breathing and at 30 mg/kg bw: abdominal position, palmospasm, clonical cramps, tachypnea, labored breathing, decreased motility, temporary tremor, narrowed palpebral fissure, blue colored and lost tail. There were no toxicological effects on body weights or on body weight development in surviving mice treated with 30 mg/kg bw. The slight decrease in body weight of one animal on day 15 is regarded as due to chance. In all animals that died during the observation period and in the necropsies performed at the end of the post-treatment observation period no gross pathological changes were detected beside the local effects of the formulation on the injection site (blue colored tail 1/6 and lost tail 3/6 animals).