(1R,2R)-1-Amino-2-(difluoromethyl)-N-(1-methylcyclopropylsulfonyl)cyclopropanecarboxamide hydrochloride

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:

05 July - 29 October 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

The objective of this study was to investigate and identify the toxic target organs, to determine the no observed effect level, and to provide information for the selections of dose and endpoints of subchronic and chronic toxicity tests.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(1R,2R)-1-amino-2-difluoromethyl)-N- (1-methylcyclopropylsulfonyl)cyclo propanecarboxyamide hydrochloride

EC Number:

943-460-6

Cas Number:

1360828-80-3

Molecular formula:

C9H15CIF2N203S

IUPAC Name:

(1R,2R)-1-amino-2-difluoromethyl)-N- (1-methylcyclopropylsulfonyl)cyclo propanecarboxyamide hydrochloride

Test material form:

solid: particulate/powder

Details on test material:

White solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rats were the preferred species as historically they were 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:

Receipt:
At first 86 rats were purchased (43 males and 43 females) and 72 were selected and later 56 rats were purchased (28 males and 28 females) and 48 were selected. Body weight ranged from 138.66-166.18 g for males firstly purchased and 137.53-165.27 g for females firstly purchased; 185.21-206.52 g for males firstly purchased and 162.23-177.86 g for females firstly purchased at grouping. 142.52-162.28 g for males secondly purchased and 147.87-172.83 g for females secondly purchased; 177.68-205.64 g for males secondly purchased and 153.36-172.14 g for females secondly purchased at grouping.

Environmental Acclimation:
Healthy young adult animals were acclimatized to the lab conditions and housed in the facility two per cage for 6 days prior to the test. Clinical observations were performed daily until treatment.

Husbandry
Housing:
Animals were housed in a room in the barrier system of the facility in suspended, stainless steel cages on cage racks. There were 10 cages per layer and 4 layers per rack. Animals were house two per cage. All animals were weighed and marked by special animal markers on their hair and numbers were written on cage cards within 24 hours after arrival.

Environmental Conditions:
Temperature and humidity were controlled automatically and daily recorded. The target values in the animal room were 20-25 degrees 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 with sterilized diet with complete nutrition. Diet and water were available to the animals ad libitum during the test. Animals were fasted overnight prior to necropsy, but water was available.

Animal Welfare:
Animal use complied with national animal welfare laws and regulations. The spare 14 animals were all euthanized by CO2.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral mute (gavage) was selected for admipistration. The dosing volumes were adjusted according to the recent body
weights. Formulations were administrated to animals' stomachs using standard gavage tubes attached to disposal syringes.

Vehicle:

water

Details on oral exposure:

The test item and vehicle were administered by gavage once daily for 7 days, respectively, and there were 3 males and 3 females for each group and 2 males and 2 females in the control group.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Four groups were initially designed at different dose levels including the 0 mg/kg-bw/day control, 60 mg/kg-bw/day, 200 mg/kg-bw/day and 600
mg/kg-bw/day groups.There were 6 male and 6 female animals in each group. An additional 6 male and 6 female animals were set in the control and 600 mg/kg-bw/day groups to make recovery-period observations. Animals in the three treatment groups were administered with 60, 200, and 60 mg/mL of test item solutions once daily for 28 days, respectively. Animals in the control group were administrated the vehicle once daily for 28 days.
Dosing volumes were all 10 mL/kg. All the surviving animals in the 60 and 200 mg/kg-bw/day groups, half of the animals in the control, and 600
mg/kg-bw/day groups (6 male and 6 female animals each group) were necropsied after 28-day administrations. The rest of the animals in the control and the 600 mg/kg-bw/day groups (6 male andfemale animals each group) were housed for an 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 the recovery-period observations had 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. The male and female animals in 600 mg/kg-bw/day groups began to die intensively from the 2nd week of exposure period, and 5 males and 2 females died before the beginning of the 3rd week of exposure period. Additionally, some of the surviving animals showed significant toxic reactions such as loss of body weight, emaciation, perineum filth and humidity and decrease in locomotor activity. Based on the above situation, another 0 mg/kg-bw/day group (control group) and a 450 mg/kg-bw/day group were supplemented. There were 24 animals (12 males and 12 females) in each group and 6 male and 6 female animals were set in each group
to make recovery-period observations. Animals in these two groups were administered with vehicle and 45 mg/ml of the test item solution once daily, respectively. All the test methods including grouping, dosing, observations and detections and performances of these two groups were the same
as the initially designed control and dosing groups, respectively.

Duration of treatment / exposure:

7 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

60 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 6

Dose / conc.:

450 mg/kg bw/day (nominal)

Doses / concentrations 7

Dose / conc.:

600 mg/kg bw/day (nominal)

Doses / concentrations 8

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3 per sex per dose and 2 per sex for the control group

Control animals:

yes, concurrent vehicle

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

Observation Period: All of the animals were continuously observed for 28 days during the dosing period. The animals in the control, 6 per sex 450 and 600 mg/kg bw/day groups were observed for an additonal 14 days to determine reversibility, persistence, and delayed toxic effects.

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.

Death and Moribund Observations: all animals were inspected for mortality and moribund signs twice daily.

Detailed Clinical Observations: detailed clinical observations were made for all animals prior to the first exposure and once weekly after dosing during the treatment and recovery periods. Observations included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activities, changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes or odd behaviors.

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 overnight prior to necropsy and empty stomach body weights were collected before necropsy.

Food Consumption: The food was quantificationally added weekly. Added food weight was 550 ± 20 g. The residual food and food boxes were weighed
again 24 hours later as average food-intake weight daily. Mean food consumption for one animal per day was calculated based on the above data.

Nerve Function Observation:
In the fourth week of the treatment period, nerve function observations for recovery animals in the control, 450 and 600 mg/kg bw/day groups were made to assess general behavior, sensory reactivity by different types of stimulation, grip strength and motor activity.

Sacrifice and pathology:

Blood Collection: all surviving animals at the ends of treatment and recovery periods were anesthetized by C02 inhalation, and blood was collected via abdominal aorta for hematology, coagulation and serum biochemistry. All animals were fasted overnight before blood collection.

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

Coagulation: About 2.7 ml of blood was drawn from abdominal aorta and collected into vacuum tubes containing 3.8% trisodium citrate as anticoagulant and stored at room temperature. Samples were centrifuged within half an hour after blood was collected and the plasmas were assayed.

Clinical Chemistry: 2-3 mL of blood were 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.

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 parameters using Uritest 5008 automatic urinalyzer and test paper.

Gross Necropsy: 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.

Other examinations:

Organ Weights: Organs were trimmed of any adherent tissue, as appropriate, and their wet weights taken as soon as possible after dissection to avoid drying. Care was exercised when trimming the prostate complex to avoid puncture of the fluid filled seminal vesicle. Organ-to-body weight ratios were calculated.

Statistics:

Statistical results for body weight, food consumption, food utilization rate, serum biochemistry, hematology, coagulation, urinalysis, pathological data, and histopathology were determined.

Statistical Method: Above parameters were analyzed by Bartlett test for variance homogeneity. In case of heterogeneity of variance (p<0.05), a Kruskal-Wallis Non-parametric analysis of variance pairwise comparison was used to evaluate the difference between each treatment and the control group. If the test was significant, non-parametric Dunnett's test was then used for pairwise comparisons between each treatment group and the control group. Statistic analysis were finished when the test was non-significant (p>0.05). In the case of homogeneity of variance, one-way analysis of variance was used to analyze above parameters. If the test was significant and the number of animals of each group was same, the Dunnett's test conducted pairwise comparisons between each treated group and the control group. If the test was significant and the number of animals of each group was different, Duncan's test was conducted. Statistic analysis was finished when the test was non-significant. Neurotoxicity was analyzed by Mann-Whitney U -test, The data of clinical sign was analyzed by X2, and the incidences of pathological findings were analyzed by the Fisher's exact test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Significant toxic reactions were observed during the whole test period in the males and females in 600 mg/kg-bw/day group which were considered to be related to the toxicity of the test item.

There were 7 and 4 accidental deaths that occurred in the males and females in the 600 mg/kg-bw/day groups. Decreases in locomotor activity were commonly seen in the dead males and females in 600 mg/kg-bw/day groups, and other abnormal symptoms such as perineum filth and humidity, emaciation, filthy and fluffy fur were also observed in the dead animals of these two groups; besides, obvious toxic symptoms such as ocular hemorrhage, scab, lateral position and hunchback were found in the dead males in 600 mg/kg-bw/day group. The above symptoms were all considered to be related to the toxicity of the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

The mortality results indicated that the large number of deaths observed in both of the males and females in 600 mg/kg-bw/day groups during both of the exposure and recovery periods were all related to the toxicity of the test item.

There were 6 animals who died during the exposure period and 1 animal who died during the recovery period in the males in the 600 mg/kg-bw/day group. The mortalities before the scheduled necropsies at the ends of exposure and recovery periods were 50% and 58%, respectively. There were 4 animals who died during the exposure period and no animal died during the recovery period in the females in the 600 mg/kg-bw/day group, and the mortalities before the scheduled necropsies at the ends of exposure and recovery periods were both 33%. It was considered that the high mortalities were caused by the toxicity of the test item. One animal died during the exposure period in the females in 60 mg/kg-bw/day group and the mortality was 17% before the scheduled necropsy at the end of treatment period. No deaths were observed in the rest of the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The statistical results of body weight indicated that the body weight gains of the males in 450 and 600 mg/kg-bw/day groups and of the females in 600
mg/kg-bw/day group were significantly inhibited by the test item.

The average body weight during the 4th week of the exposure period and the bodyweight gains of the exposure period all significantly decreased as compared to those in the control group in the males in 450 and 600 mg/kg-bw/day groups. The average body weight each week and the body weight gain of the exposure period all significantly decreased as compared to those in the control group in males in 600 mg/kg-bw/day group. All above changes were considered to be caused by the toxicity of the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The statistical results of food consumption indicated that the food consumption of females in the 200, 450 and 600 mg/kg-bw/day groups were significantly inhibited by the test item.

The average food consumption amounts of females in 200 mg/kg-bw/day group from 2nd week to 4th week of exposure period all evidently decreased as compared to those in the control group, and the decreases in 2nd week and 3rd week had statistical significance. Additionally, the total food consumption amount of the exposure period also significantly decreased as compared with that in the control group. The average food consumption amount of females in 450
mg/kg-bw/day group at each week were all lower than that of the control group during the exposure period. The decreases at 1st and 2nd week of exposure period as well as at 2nd week of recovery period had statistical significance as compared to those in the control group. The total food consumption amount of exposure and the whole test period also significantly decreased as compared to those in the control group. The food consumption amount for females in 600 mg/kg- bw/ day group significantly increased at the 1st week of exposure, which was of no toxicological significance. The food consumption of females in
600 mg/kg-bw/day group from Z'" week to 4th week during exposure period and the total food consumption amount of exposure period all significantly decreased compared to those in the control group. All the decreases showed above were considered to be related to the toxicity of the test item.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The statistical results of food utilization rates showed that the food utilization rates of males in the 450 mg/kg-bw/day group and of females in the 60 mg/kg-bw/day group were both significantly inhibited by the test item.

As compared with those in the control group, the food utilization rate each week for males in the 450 mg/kg-bw/day group during the exposure period showed a decrease trend, and the food utilization rates of the 4th week exposure period and of the whole test both significantly decreased. These changes were considered to be related to the toxicity of the test item. The food utilization rates of females in the 60 mg/kg-bw/day group for 1st week to the 3rd week all decreased as compared to those in the control group, and statistical differences were found in the 1st and 3rd week. The average food utilization rate exposure period of this group also significantly decreased. All the changes were considered to be caused by the test item.

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

not examined

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.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The statistical results of serum biochemical examinations showed that the test item caused significant decreases in the K+ levels of males in the 60, 200 and 600 mg/kg­bw/day groups.

The results showed that the K+ levels of males in 60, 200 and 600 mg/kg-bw/day groups all significantly decreased (p<0.05 or p<0.01) as compared with those in the control group at the end of exposure period. Because consistent changes were observed in these three groups, it was considered that these changes were caused by the test item.

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.

Behaviour (functional findings):

not specified

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.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Massive and dark red pleural effusion, lung adhesion and thymus enlargement was found in the accidentally dead animal in 60 mglkg-bw/day group and no abnormalities were found in the rest of the animals. There were 7 male animals and 4 female animals who died in the 600 mg/kg-bw/day groups and the death times were mainly concentrated within two weeks. Because a large number of deaths in both males and females occurred in a short time period only in 600 mg/kg-bw/day groups, under the conditions of this test, it is considered that the test item at this dose had a lethal effect on rats.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No nerve toxicity related to the toxicity of the test item was found in the nerve function statistical results at the end of exposure period.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Single hepatocyte necrosis, hepatic vacuolation, renal tubular epithelial cell necrosis and Hadashi gland pigmentation occurred at a higher rate, with mild to moderate level and with no gender difference were found in both of male and female died animals in 600 mg/kg-bw/day groups. In the females in the 60 mg/kg-bw/day group, 1 case of pulmonary pleural necrosis and cardiac necrosis was found. Because all of the above lesions were not lethal the cause of death was unknown. Because of the high incidences of these-lesions, it was considered that under the conditions of this study, the test item at this dose led to single liver cell necrosis of liver, liver vacuolar degeneration, renal tubular epithelial cell necrosis, and Hadashi gland pigmentation in dead animals.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

mortality

neuropathology

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Due to significant deaths and observable toxic changes in both male and female animals at a dose of 600 mg/kg-bw/day for a 28-day treatment period, the NOAEL for DIfluorosulfonamide HCl repeat dose 28-day oral tox study in SD rats was determined ot be 450 m/kg/ bw/day for both males and females.