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

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 July 1997

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

875-892-5

Cas Number:

1375799-59-9

Molecular formula:

C13 H14 N8 O2 . Na

IUPAC Name:

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

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:(WI)BR (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

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

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

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

Details on exposure:

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

Duration of treatment / exposure:

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

Frequency of treatment:

Once

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw (total dose)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Positive control(s):

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

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

Examinations

Tissues and cell types examined:

Liver cell homogenate

Details of tissue and slide preparation:

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

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

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

Evaluation criteria:

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

Statistics:

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

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

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

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

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

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