1-[2-fluoro-6-(trifluoromethyl)benzyl]-5-iodo-6-methylpyrimidine-2,4(1H,3H)-dione

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 25 to December 21, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Repeated Dose 28 Day Oral Toxicity study in Rodents (No. 407, adopted 2008), Ministry of Environmental Protection of the People's Republic of China

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-{[2-fluoro-6-(trifluoromethyl)phenyl]methyl}-5-iodo-6-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

EC Number:

687-893-6

Cas Number:

1150560-54-5

Molecular formula:

C13H9F4IN2O2

IUPAC Name:

1-{[2-fluoro-6-(trifluoromethyl)phenyl]methyl}-5-iodo-6-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rats were the preferred species of choice as historically used for the safety evaluation studies and were specified in the appropriate test guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Beijing Vital River Laboratory Animal Technology Co., Ltd
- 80 rats: 40 females and 40 males, 72 selected: 36 females and 36 males
- Weight at study initiation: 197.42-221.20 g for males and 181.02-208.02 g for females
- Housing: Healthy young adult animals were acclimatized to the laboratory conditions and housed in the facility two per cage for 6 days prior to the test.

Animals were housed two per cage for the convenient of food consumption amount calculation for each animal per day during the test. Animals were provided sterilized diet with SPF Rodent Maintenance Feed. Diet and water were available to the animals ad libitum during test. Animals were fasted overnight prior to necropsy, but water was available.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 40-70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Four groups were designed at different dose levels including the 0 mg/kg-bw/day (control), 100 mg/kg•bw/day (low dose), 300 mg/kg-bw/day (middle dose) and 1000 mg/kg-bw/day thigh dose) groups for males, the 0 mg/kg-bw/day (control), 50 mg/kg•bw/day(iowdose), 150 mg/kg•bw/day (middle dose) and 450 mg/kg•bw/day (high dose) groups for females. There were 6 male and 6 female animals in each group. Besides, additional 6 male and 6 female animals were set in the control and high dose groups to make recovery-period observations. Male animals in the three treatment groups were administered with 10, 30 and 100 mg/mL and female animals in the three treatment groups were administered with 5, 15 and 45 mg/mL of test item solutions once daily for 28 days respectively. Animals in the control groups were administrated with the vehicle (corn oil) once daily for 28 days. Dosing volumes were all 10 mL/kg. All the surviving animals in the low dose and middle dose groups, and half of the animals in the control and high dosegroups (6 male and 6 female animals each group) were necropsied after 28-day administrations. The rest animals in the control and high dosegroups (6 male and o female animals each group) were housed for additional 14 days without administrations to make recovery-period observations so that the reversible, durable and tardive toxic effects could be observed. Surviving animals were necropsied after recovery-period observationshad finished. The clinical symptom observations, body weight measurements, food consumption measurements, nerve function observations, clinical examinations including hematology.coagulation, blood biochemistry and urinalysis, gross autopsy and histopathology were conducted after exposure and recovery periods had finished.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Males

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Males

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Males

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Males

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Females

Doses / concentrations 6

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Females

Doses / concentrations 7

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Females

Doses / concentrations 8

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

Females

No. of animals per sex per dose:

6 males and 6 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Husbandry: Animals were housed in a barrier system of the facility. Animals were raised in suspended, stainless steel cages on cage racks. There were 10 cages per layer, and 4 layers per rack. Animals were housed two per cage for them convenient of food consumption amount calculation for each animal per day during the test.

Environmental Conditions:Temperature and humidity were controlled automatically and recorded daily. The target values in the animal room were 20-25°C for
temperature, and 40%-70% for humidity. A controlled light cycle was 12-hour light/12-hour dark.

Food and Water: Animals were provided sterilized diet with SPF Rodent Maintenance Feed. Analysis reports of diet were supplied by the supplier. All the nutritioncomponents and contaminants were within the permitted limits described in the national standard (GB14924.3-2010 and GB14924.2-2001).

Water was purified by HT-ROIOOO purity system. Water analysis was conducted routinely analyzed (annually), and all parameters were within the permitted limits described in the national drinking water standard (085749-2006). Diet and water were available to the animals ad libitum during test. Animals were
fasted overnight prior to necropsy, but water was available.

Animal Welfare:Animal use complied with national animal welfare laws and regulations (instructive notions with respect to caring for laboratory animals) (2006, PRC Ministry of Science). The animal care and use activities required for conduct of this study were reviewed and approved by the testing facility Animal Care and Use Committee (IACUC). The spare 8 animals were all euthanatized by C02.

Dose design: The study dose designation was based on the toxicity data of the pre-study results (study NO.G1856B002A). Male and female animals were administered by the concentration of 0 mg/ml, 50 mg/ml, 100 mg/mlof test item solution for 10 days. During the study there were no evident symptoms and no animals died. All the female treated animals' bodyweights were lower than the control group, and the male treated animals' bodyweights didn't change when compare with the control group.

Method: The healthy animals were randomly grouped using the Provantis System. Animals were initially allocated into 4 groups: 12 animals each sex in the control group and high dose group, 6 animals each sex in the low and middle dose groups. The weight variation in the same groupwas not exceeding ±20% of the mean body weight for either sex at grouping.

Animal Identification: All animals were marked by ear hole and numbers written on colored cage cards.

Examinations

Observations and examinations performed and frequency:

All of the animals were continuously observed for 28 days during the dosing period. The rest animals in the control, and high dose groups were
observed for another 14 days to detect the reversibility, persistence and delayed toxic effects of toxic reactions.

General Clinical Observations:General clinical observations were made once daily at 60 ± 30 minutes after dosing, preferably at the same time each day, and the health conditions and toxic reactions were recorded after observation.

Sacrifice and pathology:

During the study, animals surviving to the end of the study were euthanized by C02 inhalation followed by exsanguinations from abdominal aorta and subjected to a full necropsy and general observation. The necropsy included careful eye examinations of the external features of the carcass, external body orifices, the abdominal, thoracic, and cranial cavities and their contents of all animals, and the location, size, and hardness and the color of the abnormal findings were recorded.

Other examinations:

Death and Moribund Observations:All animals were inspected for mortality and moribund signs twice daily (only once daily on festival and holidays).

Detailed Clinical Observations: Detailed clinical observations were made for all animals prior to the first exposure (at grouping) and once weekly after dosing during the treatment and recovery periods. The animals were taken outside the home cage for observationand all the findings were detailed recorded. Observations included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activities (e.g., lacrimation, pilo-erection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviors (e.g. self-mutilation, walking backwards).

Body Weights: Animals were weighed with 24 hours after arrival, at grouping and once a week during the exposure and recovery periods. Animals were fasted ovemightprior to necropsy and empty stomach body weights were collected before necropsy.

Food Consumption: The food wasquantitativelyadded weekly. Added food weight was500± 1 Og (including the food box weight). The residual food and food boxes were weighedagain 24hours later (24 h ± 1.5 h) as average food-intake weight daily. Mean food consumption for one animal per day was calculated based on the above data. Calculation formulation: Mean daily food consumption (g) = [ added food weight (g, including weight of food
box)- residual food weight (g, including weight of food box)]/2.

Nerve Function Observation: In the fourth week of the treatment period, nerve function observations for recovery animals in the control and high dose groups were made to assess general behavior,sensory reactivity by different types of stimulation, grip strength and motor activity.

Clinical Examination: Blood Collection:all surviving animals at the ends of treatment and recovery periods were anesthetized by CO2 inhalation, and blood was collected via abdominal aorta for hematology, coagulation and serum biochemistry. All animals were fasted overnight before blood collection. The codes of animals at scheduled blood collection.

Hematology: 1-2 mL blood was drawn from abdominal aorta, and was collected into vacuum tubes containing EDTA-K2 as anticoagulant and stored in broken ice.

Coagulation: About 2.7mL bloods was drawn from abdominal aortaand collected into vacuum tubes containing 3.8% trisodium citrate as anticoagulant and stored at room temperature. Samples were centrifuged within half anhour after blood has been collected and the plasmas were assayed for the following items with STA-Compact automatic coagulometer.

Clinical Chemistry: 2-3mL blood was collected into vacuum tube with accelerator/separated gel. The samples were stored at room temperature for 5-10 minutes and then stored in broken ice. Samples were centrifuged in 1 hour after blood collecting, and the serum was assayed.

Urinalysis: Urine Collection: before necropsies of treatment and recovery periods urine samples were collected from all surviving animals via abdominal extrusion. Urinalysis was conducted to measure following parameters using Uritest 500B automatic urinalyzer and test paper. In addition, the appearance of urine was recorded but not statistically analyzed.

Organ Weights: Organs were trimmed of any adherent tissue, as appropriate, and their wet weights were taken as soon as possible after dissection to avoid drying. Organ-to-body weight ratios were calculated. Calculation formulation (relative organ weight): Organ-to-body weight percentages= organ weight (g)/body weight(g)xl00.

Histopathology:Histopathology was examined on the preserved organs and tissues of all the scheduled necropsy animals in the control, 1000 and 450mg/kg•bw/day groups at the ends of both exposure and recovery periods, and detailed records were made. The examinations including all the tissues and organs necropsied. Histopathology reports were provided.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related abnormal results were found in the clinical observation for all animals.
Nose scabbing was observed for 1female in mid-dose group and no other abnormal symptoms were found in all animals during the study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was 1 animal that died in the females in 150 mg/kg•bw/day group during the exposure period on 15th day, which was considered to be accidental and not caused by the toxicity of the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related abnormal results were found in the bodyweights for all animals. During the administration period, the average body weight and body weight gain was not significantly changed when compared with the control groups for male and female animals. No toxicity-related results were found.
During the recovery period, the average body weight and body weight gain was not significantly changed when compared with the control groups for male and female animals. And no toxicity-related results were found.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related abnormal results were found in the food consumption for all animals. During the administration period, the average food consumption amounts of 1 week males in 100 mg/kg-bw/day and 1000 mg/kg-bw/day were increased evidently as compared with that in the control group (p<=0.05), which didn't have toxicological meaning. During the recovery period, the average food consumption amounts were not significantly changed when compared with the control groups for male and female animals. No toxicity-related results were found.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No abnormal changes related to the toxicity of the test item were found in hematological examinations. At the end of administration, there were not any statistically significant or toxicity-related results for treatments of males and females when compared with the control groups. At the end of recovery, HGB and LYMPH levels of the 1,000 mg/kg group of males significantly decreased as compared with those of the control groups (p<=0.05). The variation ranges were all within the background data ranges, which were not considered as toxicological meanings. There were no other statistically significant or
toxicity-related results for treatments of males and females when compared with the control groups. No significant differences were seen in the rest hematological parameters between the treatment and control groups in both males and females at the ends of exposure and recovery periods.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related abnormal results were found in the coagulation examination for all animals. At the end of administration, there were not any statistically significant or toxicity-related results for treatments of males and females when compared with the control groups. At the end of recovery, there were not any statistically significant or toxicity-related results for treatments of males and females when compared with the control groups.

No test item-related abnormal results were found in the biochemical examination for all animals. At the end of administration, the ALT levels of males in 300 mg/kg-bw/day group and the K+ levels of females in 450 mg/kg-bw/day group all significantly decreased (p<=0.05,p<=0.01) as compared with those in the control groups. The variation ranges were slight and no significant dose-response relationships, which were not considered as toxicological meanings. At the end of recovery, the TP and ALB levels of males in 1000 mg/kg-bw/day groups all significantly decreased (p<=0.01) as compared with those in the control
groups. The Na+ levels of females in 450mg/kg -bw/day groups significantly increased (p<=0.05) as compared with those in the control group. The above variation ranges were all within the background data ranges, which was not considered as toxicological meanings.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No abnormalities related to the toxicity of the test item were found in urinalysis. At the end of administration, there were not any toxicity-related results for treatments of males and females when compared with the control groups. At the end of recovery, there were not any toxicity-related results for treatments of
males and females when compared with the control groups. No change of toxicological significance was found in the results of the rest groups.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Male dosed animals had no findings in the general behavior, motor function and sensory function.
Female dosed animals' average grip strength slightly decreased as compared with that of control animals (p<=0.05),which was considered as a test item-related change. There were no abnormal results on motor activity of motor function, general behavior and sensory function in all female dosed animals. Due to the slight change and no related adverse findings, the decreased strength was considered as a non-harmfuleffect to animals.
Based on above results, the slightly decreased grip strength of high dose female group was possibly related to the test item and was considered a non-harmful effect.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute and relative organ weights of all animals in the treatment groups showed no test item-related changes as compared with those in the control group at the ends of exposure and recovery periods. At the end of exposure period, the absolute weights and relative organ to body weight of the adrenal gland of males in 1000 mg/kg> bw/day significantly increased (p<=0.01, p<=0.05), the absolute weights of testis of males in 100 mg/kg-bw/day significantly decreased (p<=0.05). The absolute weights of kidney of females in 50 and 450mg/kg-bw/day significantly increased (p<=0.05). At the end of recovery period, the absolute and relativeweight of the adrenal gland of males of 1000 mg/kg-bw/day group significantly increased (p<=0.01, p<=0.05). The absolute weight of heart of male of 1000 mg/kg-bw/day group significantly increased (p<=0.05). No evident dose-response effects and consistent trends of changes were found between treatment groups in above indexes which showed significantly statistical differences, and no consistent changes were found between the results of these indexes and the results of gross necropsy and histopathology, so the changes above were considered to be incidental and unrelated to the toxicity of the test item. No change of toxicological significance was found in the results of the rest groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of exposure period, all organs' macroscopic observations were normal for male and female animals. The lesions of histopathological examination included hemorrhage and pericarditis inheart, vacuolationin liver, mineralization in testis and kidney, cystin kidney, inflammatory cells infiltrate in prostate gland. The incidence of lesions was not significantly different between the control group and high dose group.
At the end of recovery period, enlarged heart in macroscopic observation was found in female high dose group. The others organs were not abnormal. The incidence of lesions is not significantly difference between control and high dose group.The lesions of histopathological examination included pericarditis in heart, vacuolation and focal necrosisin liver, mineralization in kidney, pelvis dilationand inflammatory cells infiltrate in kidney, inflammatory cells infiltratein prostate gland. The incidence of lesions is not difference between control and high dose group. The incidence of lesions is not significantly different between the control and high group.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of exposure period, all organs' macroscopic observations were normal for male and female animals. The lesions of histopathological examination included hemorrhage and pericarditis in heart, vacuolation in liver, mineralization in testis and kidney, cystin kidney, inflammatory cells infiltrate in prostate gland. The incidence of lesions was not significantly different between the control group and high dose group.
At the end of recovery period, enlarged heart in macroscopic observation was found in female high dose group. The others organs were not abnormal. The incidence of lesions is not significantly difference between control and high dose group.The lesions of histopathological examination included pericarditis in heart, vacuolation and focal necrosisin liver, mineralizationin kidney, pelvis dilation and inflammatory cells infiltrate in kidney, inflammatory cells infiltratein prostate gland. The incidence of lesions is not difference between control and high dose group. The incidence of lesions is not significantly different between the control and high group.

Histopathological findings: neoplastic:

not specified

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

2

Key result

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

According to the above results, there were no test item related toxicological changes for all the male and female animals during the study. Therefore, the
no-observed-adverse-effect-level (NOAEL) for the test material repeated dose 28-day oral toxicity study in male and female SD rats under the condition of the study were considered to be 1000 mg/kg-bw and 450 mg/kg-bw respectively.