Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Apr 2014 - 08 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
(1998)
Deviations:
yes
Remarks:
higher relative humidity were noted on several occasions during the study period
GLP compliance:
yes (incl. QA statement)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Limit test:
no

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): Y-15099
- Physical state: pale yellow liquid
- Analytical purity: 95.4%
- Lot/batch No.: 13NSUN161
- Expiration date of the lot/batch: 18 Dec 2017
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the original container protected from light under nitrogen headspace

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: 178 - 218 g (males), 147 - 180 g (females)
- Housing: The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- Diet: pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 15 (deviations are not considered to affect study integrity)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 Apr 2014 To: 13 Aug 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: desiccated Arachis oil BP
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the required concentrations as a solution in desiccated Arachis oil BP. The stability and homogeneity of the test item formulations showed the formulations to be stable for at least twenty days when stored at 4 °C, in the dark, under nitrogen headspace. The test item formulations were therefore prepared fortnightly and stored at approximately 4 °C in the dark, under nitrogen headspace.

VEHICLE
- Concentration in vehicle: 0, 4, 20 and 100 mg/mL
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak. The formulations investigated during the study were found to comprise test item in the range of 96 to 102%, and thus, the required content limit of ±10% with reference to the nominal content was met. The test item was found to be stable in the formulations when kept for 20 days in the refrigerator (4 °C) in the dark under nitrogen headspace due to results which the variation limit of 15% from the time-zero mean. In conclusion, the results indicate the accurate use of the test item and dried Arachis oil BP as vehicle during the study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 20, 100 and 500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 (main study)
10 (recovery control and high-dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels and treatment volume were chosen in consultation with the Sponsor based on the available data from a 28 Day study (RCC study Number: 808378, November 2001).
- Rationale for selecting satellite groups: Ten additional animals per sex per dose were included as recovery group for analysis of possible post-exposure reversibility of intoxication symptoms.
- Post-exposure recovery period in satellite groups: 28 days
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. During the treatment-free period, animals were observed daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed individual clinical observations were performed for each non-recovery animal using a purpose built arena. The following parameters were observed: gait, hyper/hypothermia, tremors, skin color, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behavior, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation, lachrymation.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY: Yes
- Time schedule for examinations: Weekly food efficiency was also recorded retrospectively.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all non-recovery control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 0.5% tropicamide solution detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.
- Dose groups that were examined: All non-recovery control and high dose animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological investigations were performed on all surviving non-recovery animals from each test and control group at the end of the study (Day 90) and on all recovery group animals at the end of the treatment-free period (Day 118). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 91 and 119.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All animals
- Parameters examined: erythrocyte count (RBC), haematocrit (Hct), erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), total leukocyte count (WBC), differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), platelet count (PLT), reticulocyte count (Retic) (methylene blue stained slides were prepared but reticulocytes were not assessed), prothrombin time (CT) and activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood chemical investigations were performed on all surviving non-recovery animals from each test and control group at the end of the study (Day 90) and on all recovery group animals at the end of the treatment-free period (Day 118). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 91 and 119.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All animals
- Parameters examined: urea, inorganic phosphorus (P), glucose aspartate aminotransferase (ASAT), total protein (Tot. Prot.), alanine aminotransferase (ALAT), albumin, alkaline phosphatase (AP), albumin/globulin (A/G) ratio, creatinine (Creat), sodium (Na+), total cholesterol (Chol), potassium (K+), total bilirubin (Bili), chloride (Cl-), bile acids and calcium (Ca++).

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalytical investigations were performed on all non-recovery test and control group animals during Week 12 and on all recovery group animals during the final week of the recovery period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood and appearance.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performance tests were performed on all animals together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: All non-recovery animals
- Battery of functions tested: Sensory activity, grip strength and motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- On completion of the dosing period or in the case of recovery group animals, at the end of the treatment-free period all surviving animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes
- Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated: adrenals, aorta (thoracic), bone/bone marrow (femur including stifle joint/sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides, esophagus, eyes, gross lesions, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (mandibular and mesenteric), mammary glands, muscle (skeletal), ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, mid thoracic and lumbar), spleen, stomach, testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus (with cervix) and vagina. All tissues were dispatched to the histology processing Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU). All tissues from non-recovery control and 500 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the liver and kidneys (male and female), thyroid gland (female only) and thymus (males only) from animals in the low, medium and recovery dose groups.
Other examinations:
ORGAN WEIGHT:
- The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period (recovery group), were dissected free from fat and weighed before fixation: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus and uterus.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Urine volume and specific gravity were statistically analyzed using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pairwise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test. Probability values (p) are presented as follows: p<0.01 **; p<0.05 *; p>0.05 (not significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
20 mg/kg bw/day: one male was killed on Day 27 of the study due to a decline in clinical condition. Clinical signs prior to death included tiptoe/splayed gait, laboured respiration, decreased respiratory rate, hunched posture, diuresis and ataxia (non-adverse)
Mortality:
mortality observed, treatment-related
Description (incidence):
20 mg/kg bw/day: one male was killed on Day 27 of the study due to a decline in clinical condition. Clinical signs prior to death included tiptoe/splayed gait, laboured respiration, decreased respiratory rate, hunched posture, diuresis and ataxia (non-adverse)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
500 mg/kg bw/day: statistically significant lower body weight gain in males (adverse); 100 mg/kg bw/day: statistically significant lower body weight gain (occasionally) in males (non-adverse)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
500 mg/kg bw/day: decreased food consumption in males (non-adverse)
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
all dose levels: statistically, significant decrease in mean cell hemoglobin concentrations in females; 500 mg/kg bw/day: lower platelet counts in females (non-adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
500 mg/kg bw/day: statistically, significant increase in ALAT and decreased Chol in males; statistically, significant decrease in plasma levels of AP, A/G ratio (and at 100 mg/kg bw/day), urea and increased albumin, Tot. Prot., Creat and Ca (non-adverse)
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
500 mg/kg bw/day: statistically, significant increase in specific gravity in males; statistically, significant increase in urine volume and statistically, significant decrease in specific gravity in recovery females (non-adverse)
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
500 mg/kg bw/day: statistically, significant increase in kidney weights (adverse) and liver weights (non-adverse) in males/females; 100 mg/kg bw/day: statistically, significant increase in liver weights in females (non-adverse)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
500 mg/kg bw/day: histopathology findings in kidneys (adverse) and liver (non-adverse) in males/females, thyroid (non-adverse) in females and thymus (non-adverse) in males
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One male animal of the low dose group was killed on Day 27 of the study due to a decline in clinical condition. Clinical signs prior to death included tiptoe/splayed gait, laboured respiration, decreased respiratory rate, hunched posture, diuresis and ataxia. At necropsy, macroscopic findings for this animal were confined to dark spleen and reddened/enlarged/fluid-filled bladder. At histopathology examination, the bladder was observed to be dilated with signs of inflammation but no cause of death was established although there may have been a blockage in the urinary tract which was not identified. There were no further deaths until the end of the study period and due to the isolated occurrence of unscheduled death, it was considered not to be related to treatment with the test material. There were no clinical observations throughout the study that were considered as test material-related. At the high dose group, animals of either sex showed sporadic instances of increased post-dose salivation from the second week of dosing, which persisted throughout the treatment period. A few instances of increased post-dose salivation were also observed in males and females of the medium dose group from Week 10 of the study. Individual control animals of either sex showed similar clinical signs on one day during Week 4 of the study. Observations of this nature are commonly observed following the oral administration of an unpalatable test material formulation and, were therefore, considered to be of no toxicological importance. There were no clinical observations for any of the surviving animals of the low dose group throughout the treatment period. With the exception of one control male showing sporadic episodes of staining around eyes during the treatment and treatment-free periods, there were no clinical signs for any of the recovery animals during the treatment-free period.

BODY WEIGHT AND WEIGHT GAIN
During the first week of dosing, group mean body weight gains in males of the medium and high dose groups were slightly but statistically significantly lower than controls (p<0.05 and p<0.01, respectively). Thereafter, the mean body weight gains in males of the high dose group generally remained lower than controls up to and including Week 10 of the study, achieving statistical significance in most instances; body weight gains in these males over the remaining dosing period were similar to controls. Males of the medium dose group also showed some instances of slightly lower weight gains up to and including Week 8 of dosing, achieving statistical significance in Week 8 (p<0.05); subsequent weekly body weight gains in these animals were similar to controls. Male animals of the low dose group showed some instances of slightly lower mean weight gains albeit without achieving statistical significance. Overall body weight gains in non-recovery males at the end of the treatment period were slightly lower than controls; 6%, 6% and 13% in the low, medium and high dose groups, respectively. Following the cessation of dosing, males previously given the high dose showed slightly higher body weight gains over the first two weeks when compared with controls. Subsequent body weight gains in these animals were similar to controls with the overall mean body weight gain being 11% lower than controls. Taking into consideration the overall effect on body weight development in males of the high dose group during the treatment period together with the lack of a substantial recovery over the treatment-free period, this may indicate an adverse effect of test material administration. Throughout the study, there was no effect of treatment with the test material at any dose level on body weight development in females. Any intergroup differences achieving statistical significance were considered incidental and unrelated to treatment with the test material (please refer to table 1 and 2).

FOOD CONSUMPTION AND COMPOUND INTAKE
Throughout the treatment and treatment-free periods, food intake in males of the high dose group was generally marginally lower than controls. Food intake in males of the low and medium dose groups was generally similar to controls throughout the treatment period. Food consumption in females of all dose groups appeared to be similar to controls throughout the study.

FOOD EFFICIENCY
Food efficiency in animals of either sex treated with the test material at all dose levels was generally comparable with controls throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE
Visual inspection of water bottles did not reveal any intergroup differences.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmoscopic examination did not indicate any differences in control and test item treated animals.

HAEMATOLOGY
At the end of the treatment period, females receiving the test material at all dose levels showed marginally but statistically significantly lower mean cell haemoglobin concentrations when compared with controls (p<0.01 at the high dose and p<0.05 at the low and medium dose). Slightly higher mean platelet counts were also apparent in animals of either sex receiving the high dose in comparison with the respective controls, but statistical significance was only achieved in females (p<0.05). At the end of the treatment-free period, the mean cell haemoglobin concentration in females previously given the high dose was marginally but statistically significantly lower than controls (p<0.05) with these females also showing statistically significantly higher mean haematocrit value when compared with controls (p<0.05). Group mean platelet counts in animals of either sex were also slightly higher than controls, achieving statistical significance in females (p<0.05). The majority of individual values for the above parameters were within the historical background data ranges and in the absence of any histopathology correlates, these findings were considered not to represent an adverse effect of treatment (please refer to table 3).

CLINICAL CHEMISTRY
At the end of the dosing period, group mean plasma concentrations of alanine aminotransferase (ALAT) in males at all dose levels were slightly higher than controls achieving statistical significance in the high dose group (p<0.05). A dose-relationship was apparent and some individual values from all test material-treated groups were slightly higher than the background data ranges. At the end of the recovery period, the mean alanine aminotransferase (ALAAT) value in males previously given the high dose was similar to controls. Group mean plasma levels of cholesterol (Chol) in males of the medium and high dose groups were slightly lower than controls at the end of the treatment period, achieving statistical significance in the high dose males (p<0.01). Although a dose-relationship was apparent, all individual values were within the background data ranges and the group mean value from recovery males at the end of the treatment-free period was similar to controls. Females of the high dose group showed statistically significantly lower plasma levels of alkaline phosphatase (AP) when compared with controls (p<0.05). There was, however, no dose-relationship and, with the exception of one female, all individual values were within the background data ranges. The mean plasma concentrations of albumin and total protein (Tot. Prot.) in these females were also statistically significantly higher than controls (p<0.05 and p<0.01, respectively) resulting in statistically significantly lower mean A/G ratio in these animals (p<0.05); the majority of individual values for the total protein and some individual values for the albumin and A/G ratio were outside the background data ranges. The mean A/G ratio in females of the medium dose group was also statistically significantly lower than controls (p<0.05); however, all individual values from these animals were within the background data ranges. At the end of the treatment-free period, the corresponding values in recovery females previously treated with the high dose were similar to controls. At the end of the dosing period, group mean plasma level of urea in females of the high dose group was slightly but statistically significantly lower than controls (p<0.05) whilst the mean creatinine concentration in these animals was statistically significantly higher than controls (p<0.05); the majority of individual values were within the background data ranges. Group mean plasma level of calcium in non-recovery females treated with the high dose was also statistically significantly higher than controls (p<0.01) with 4/10 test material-treated individuals showing values higher than the background data ranges. At the end of the treatment-free period, the corresponding values in females previously given the high dose were similar to controls. Taking into account the increased liver weights in animals of either sex of the high dose and in females of the medium dose together with the centrilobular hypertrophy observed in the liver from some of the animals of the high dose (likely to be of an adaptive nature and showing complete reversibility in the recovery animals), the above findings were considered not to represent an adverse effect of treatment with the test material. These parameters may have been influenced by liver changes and variations in plasma markers and hepatic changes may well be indicative of fluctuations in metabolic processes associated with test material detoxification rather than representing any adverse effect of treatment. At the end of the treatment-free period, females previously receiving the high dose showed slightly but statistically significantly higher group mean concentration of bilirubin (Bili) when compared with controls (p<0.05). All individual values were, however, within the background data ranges and taking into consideration the mean plasma concentration of bilirubin at the end of the treatment period in high dose females, which was similar to controls, this finding was considered not to be of any toxicological significance (please refer to table 4 and 5).

URINALYSIS
Towards the end of the 90 day treatment period, a high degree of individual variation was apparent for urine volume and specific gravity values and any intergroup differences between animals of either sex receiving the test material and controls were not statistically significant or dose-related; the only exception was non-recovery males treated with the high dose showing statistically significantly higher group mean specific gravity when compared with controls (p<0.05). Towards the end of the recovery period, group mean urine volume in recovery females was statistically significantly higher than controls (p<0.05) whilst specific gravity was statistically significantly lower than controls (p<0.05). Towards the end of the treatment period, urine samples from the majority of non-recovery males treated with the high dose tested positive for the presence of protein and ketones (9/10 and 7/10 animals, respectively) with one male each also showing positive results for the presence of erythrocytes or hemoglobin. Of the corresponding females, urine samples from 5/10 animals showed the presence of protein whilst the presence of ketones was not detected for these animals; it should be noted that urine samples from 3/10 non-recovery control females also tested positive for the presence of protein. Of recovery males previously given the high dose, 5/10 animals showed the presence of protein in urine samples whilst one male also showed the presence of hemoglobin; urine samples from the corresponding recovery females no longer showed the presence of protein. Increased kidney weights were evident at the end of the treatment period in animals of either sex receiving the high dose whilst histopathology findings observed in the kidneys from these animals included multifocal basophilic tubules (degenerating/regenerating) in both sexes and single cell necrosis in females. At the end of the treatment-free period, increased kidneys weights were still apparent in recovery females previously given the high dose, whilst histopathological evaluation of kidneys from recovery animals of either sex identified multifocal basophilic tubules, tubular fibrosis with fibrous tissue surrounding the affected groups of tubules, lymphocytic infiltration in the intestinal surrounding of the affected tubules and tubular atrophy in some females. In the light of the increased kidney weights and associated histopathology findings, these observations were considered to be of toxicological significance (please refer to table 6).

NEUROBEHAVIOUR
There were no changes in the behavioral parameters considered to be related to treatment with the test material at any dose level. There were no treatment-related changes in functional performance at any dose level. The group mean overall activity in males of the low and medium dose groups was statistically significantly higher than controls (p<0.05); however, there was no dose-relationship and the corresponding value in males of the high dose group was similar to controls, and therefore, these differences were considered unrelated to administration of the test material. Sensory reactivity scores across all test material-treated groups were similar to controls.

ORGAN WEIGHTS
At the end of the treatment period, animals of either sex of the high dose group and females of the medium dose group showed statistically significantly higher values for absolute and body weight-related liver weights when compared to controls (p<0.01 at the high dose and p<0.05 at the medium dose in females); a dose-relationship was apparent in females. All individual absolute and body weight-related values in females from the high dose group and the majority of individual body weight-related values in the corresponding males were outside the background data ranges. In recovery animals of either sex previously given the high dose, group mean absolute and body weight-related liver weights were similar to controls. The increases in liver weights were consistent with adaptive histopathological changes (centrilobular hypertrophy) observed at the high dose level and, were therefore, considered not to represent an adverse effect of treatment. Group mean absolute and body weight-related kidney weights in males, and in particular females, of the high dose group were statistically significantly higher than controls (p<0.01). The majority of individual absolute and body weight-related values in these females and the majority of individual body weight-related values in the corresponding males were outside the background data ranges. At the end of the treatment-free period, the group mean absolute and body weight-related kidneys weights in males previously treated with the high dose were similar to controls, but the values in the corresponding females were statistically significantly higher than controls (p<0.01). In the light of the results from histopathology of the kidneys, the increases in kidney weights were considered to represent an adverse effect of test material administration at the high dose. At the end of the treatment period, absolute and body weight-related spleen weights in females of the medium and high dose groups were statistically significantly higher than controls (p<0.01) with an apparent dose-relationship. Whilst the majority of individual values from the intermediate dose group were within the background data ranges, 5/10 high dose females showed values that were outside these ranges. At the end of the recovery period, the corresponding group mean absolute and body weight-related spleen weights in females previously treated with the high dose were similar to controls. In the absence of any histopathology correlates, these observations were considered not to represent an adverse effect of test material administration at these dose levels. At the high dose, females showed statistically significant increases in absolute and body weight-related heart and ovaries weights when compared with the respective controls (p<0.05). The majority of individual values from these animals were within the background data ranges. At the end of the treatment-free period, the corresponding values from the recovery females previously given the high dose were similar to controls. Due to the lack of any associated histopathology observations, these findings were considered not to be toxicologically significant (please refer to table 7 and 8).

GROSS PATHOLOGY
The incidence and distribution of macroscopic necropsy findings in treated animals did not indicate any adverse systemic effect of treatment. The following findings were noted: At the end of the treatment period, a small number of individual animals of either sex from different dose groups showed reddened lungs and one female of the high dose group were observed with white fibrous adhesion in the heart and the thymus and a fluid-filled mass in the lungs. At the end of the treatment-free period, macroscopic findings were confined to reddened lungs or mandibular lymph nodes in one control male and one high dose female, respectively. Macroscopic findings in the early decedent (one male of the low dose grou) that was killed in extremis on Day 27 of dosing were confined to the spleen (dark) and urinary bladder (reddened, enlarged and fluid-filled with red color fluid). The histopathology examination showed that the bladder was dilated with signs of inflammation but no cause of death was established although there may have been a blockage in the urinary tract which was not identified. No other findings were considered to have contributed to death.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related findings were recorded in the kidney and liver of non-recovery males and females, the thyroid gland of non-recovery females and the thymus of non-recovery males of the high dose group. The changes in the kidneys persisted after the recovery period but all other changes had resolved. Multifocal basophilic tubules (degenerating/regenerating) were present in the kidneys at a mild level in 8/10 non-recovery high dose males and at a mild or moderate level in all corresponding females. In 8/10 non-recovery high dose females, single cell necrosis at a minimal or mild level was also notable within the tubular cells. Hyaline droplets were increased in male animals receiving the test material at all dose levels in a dose-dependent manner. After a 28 day treatment-free period, multifocal basophilic tubules at a minimal to moderate level were still present in all animals previously treated with the test material at the high dose. Tubular fibrosis at a minimal to mild level was also apparent with fibrous tissue surrounding the affected groups of tubules in these animals. Several animals of either sex also showed lymphocytic infiltration in the intestinal surrounding of the affected tubules. Tubular atrophy at a minimal level was also present in 2/10 of these females. The hyaline droplets within the tubules in males were consistent with the accumulation of alpha-2u-globulin, a common finding in untreated male rats and as such of no relevance to humans. As this finding was more apparent in recovery control males, it is not clear whether the increase in males of the low and medium dose groups was treatment related. The remaining histopathology findings in the kidneys from the high dose group were considered to be toxicologically significant. Centrilobular hypertrophy in the liver was present in 2/10 non-recovery high dose males at a minimal level and in 7/10 corresponding females at a minimal to mild level. Mild pleomorphism (variable size of nuclei/cells) was also present in 2/10 non-recovery high dose females. No treatment-related changes were noted in any other groups and centrilobular hypertrophy in animals of either sex previously treated with the high dose had resolved after a 28 day treatment-free period. These changes were considered to be adaptive resulting from the administration of the test material and as such did not represent an adverse effect of treatment with the test material. Minimal follicular hypertrophy (enlarged follicles with hypertrophic lining cells) of the thyroid gland was present in 2/10 non-recovery females of the high dose group. This change was considered to be associated with liver enzyme induction, and had resolved following the four week recovery period. The single incidence of minimal change seen in a female of the low dose group may be attributed to individual variation and not to the test material. Involution in the thymus was present in 2/10 non recovery high dose males at a minimal to mild level. This finding was considered likely to be a secondary stress-related effect and had resolved following the recovery period. It was also noted in 1/10 males of the medium dose group, however, at such a low incidence, it was considered to be sporadic and unrelated to treatment with the test material.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no treatment-related observations at 20 and 100 mg/kg bw/day
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: non-adverse effects on body weight development, blood chemistry, organ weights
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced body weight in males and adverse effects on kidneys (increased kidney weights, histopathological findings) in animals of either sex at 500 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Group mean body weight gains (males)

 

Increase in Body Weight (g) - Day Numbers Relative to Start Date

Abs. Gain

Gain (%)

Group

From:

To:

1

8

8

15

15

22

22

29

29

36

36

43

43

50

50

57

57

64

64

71

71

78

78

85

85

91

85

92

92

99

99

106

106

113

113

119

1

119

1

119

1

Mean

SD

N

39.5

4.5

20

30.7

3.9

20

23.5

4.7

20

23.1

3.8

20

17.6

4.2

20

14.5

4.0

20

11.4

2.8

20

13.4

3.5

20

12.6

3.7

20

9.4

4.2

20

12.1

3.8

20

1.3

4.5

20

4.4

5.1

10

6.8

3.3

10

5.2

2.8

10

6.9

3.3

10

9.2

3.4

10

-3.2

2.2

10

235.5

24.2

10

119.8

9.9

10

2

Mean

SD

N

38.6

6.0

10

27.2

6.6

10

21.8

6.8

10

20.0

6.6

9

17.4

3.8

9

13.1

3.7

9

10.1

3.7

9

11.0

4.6

9

10.8

3.5

9

9.3

4.6

9

10.8

2.8

9

-2.2

3.0

9

6.9

2.4

9

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

3

Mean

SD

N

36.0*

3.7

10

28.7

4.4

10

22.5

4.8

10

20.3

5.0

10

16.8

2.8

10

12.4

3.3

10

12.4

1.6

10

9.7*

4.3

10

11.2

1.8

10

11.9

3.9

10

10.6

3.3

10

0.9

2.6

10

5.3

2.7

10

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

4

Mean

SD

N

34.9**

4.1

20

25.0**

7.1

20

22.9

8.6

20

19.0*

5.3

20

14.8*

3.5

20

11.5*

3.4

20

10.7

4.4

20

10.9*

3.5

20

9.8*

3.6

20

7.0

4.0

20

12.0

5.0

20

1.4

5.2

20

6.7

3.4

10

5.6

3.2

10

7.0

3.0

10

10.1*

2.3

10

8.2

4.9

10

-3.5

2.5

10

209.6

29.5

10

106.6

11.6

10

Group 1, 2, 3 and 4: 0, 20, 100 and 500 mg/kg bw/day

N: number of animals

SD: standard deviation

*: p<0.05

**: p<0.01

Table 2: Group mean body weight gains (females)

 

Increase in Body Weight (g) - Day Numbers Relative to Start Date

Abs. Gain

Gain (%)

Group

From:

To:

1

8

8

15

15

22

22

29

29

36

36

43

43

50

50

57

57

64

64

71

71

78

78

85

85

91

85

92

92

99

99

106

106

113

113

119

1

119

1

119

1

Mean

SD

N

13.6

6.6

20

12.4

5.7

20

8.1

7.6

20

7.0

4.9

20

4.5

5.4

20

5.6

6.7

20

2.8

4.4

20

4.0

4.0

20

1.9

3.9

20

4.0

5.1

20

4.6

4.3

20

-1.5

4.1

20

4.3

4.9

10

1.3

2.7

10

3.5

5.0

10

2.9

5.4

10

1.7

3.1

10

-5.6

4.6

10

75.0

16.3

10

44.3

9.9

10

2

Mean

SD

N

13.2

7.0

10

10.3

6.8

10

9.6

4.9

10

4.2

5.3

10

8.9

4.8

10

3.5

5.2

10

4.0

4.2

10

1.4

4.5

10

4.8

5.5

10

5.2

5.1

10

3.3

4.1

10

-2.6

5.4

10

6.6

5.1

10

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

3

Mean

SD

N

13.5

5.4

10

12.6

5.6

10

6.1

4.7

10

5.2

4.6

10

7.7

5.0

10

5.9

3.2

10

3.1

3.5

10

2.6

5.7

10

3.1

5.8

10

5.1

2.4

10

2.4

4.8

10

-2.9

5.5

10

5.4

4.4

10

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

4

Mean

SD

N

13.0

6.9

20

11.7

7.3

20

11.0

7.7

20

5.5

7.0

20

6.1

7.6

20

5.0

6.7

20

5.8*

3.4

20

3.0

4.3

20

2.5

5.2

20

4.5

5.2

20

4.1

4.5

20

-0.7

4.8

20

4.9

5.2

10

-3.0**

2.3

10

1.3

3.5

10

4.9

2.7

10

4.5

3.8

10

-7.8

3.7

10

73.0

14.8

10

43.7

9.1

10

Group 1, 2, 3 and 4: 0, 20, 100 and 500 mg/kg bw/day

N: number of animals

SD: standard deviation

*: p<0.05

**: p<0.01

Table 3: Group mean hematological values (females)

 

Group

 

Non-Recovery Group

Recovery Group

MCHC

(g/dL)

PLT

(10^9/L)

Hct

(%)

MCHC

(g/dL)

PLT

(10^9/L)

 

1

Mean

SD

N

35.07

0.42

10

611.3

61.7

10

43.25

2.28

10

35.01

0.32

10

585.8

70.7

10

 

2

Mean

SD

N

34.75*

0.26

10

614.8

84.0

10

-

-

-

-

-

-

-

-

-

 

3

Mean

SD

N

34.73*

0.25

10

611.1

72.7

10

-

-

-

-

-

-

-

-

-

 

4

Mean

SD

N

34.45*

0.27

10

678.4*

56.9

10

45.03*

1.26

10

34.61*

0.34

10

659.6*

75.8

10

Group 1, 2, 3 and 4: 0, 20, 100 and 500 mg/kg bw/day

N: number of animals

SD: standard deviation

*: p<0.05

**: p<0.01

Table 4: Group mean blood chemical values (males)

 

Group

 

Non-Recovery Group

ALAT

(IU/L)

Chol

(mg/dL)

 

1

Mean

SD

N

57.4

13.0

10

101.9

13.4

10

 

2

Mean

SD

N

64.7

13.0

9

97.6

18.1

9

 

3

Mean

SD

N

67.5

25.4

10

91.9

7.0

10

 

4

Mean

SD

N

70.6*

11.0

10

79.8**

12.8

10

Group 1, 2, 3 and 4: 0, 20, 100 and 500 mg/kg bw/day

N: number of animals

SD: standard deviation

*: p<0.05

**: p<0.01

Table 5: Group mean blood chemical values (females)

 

Group

 

Non-Recovery Group

Recovery Group

Urea (mg/dL)

Tot. Prot. (g/dL)

Albumin (g/dL)

A/G Ratio

Ca

(mmol/L)

AP

(IU/L)

Creat (mg/dL)

Bili

(mg/dL)

 

1

Mean

SD

N

43.7

7.0

10

7.529

0.612

10

4.53

0.38

10

1.503

0.109

10

2.792

0.133

10

90.4

26.7

10

0.817

0.090

10

0.066

0.037

10

 

2

Mean

SD

N

43.7

5.7

10

7.535

0.652

10

4.47

0.45

10

1.459

0.162

10

2.857

0.103

10

88.7

16.7

10

0.894

0.114

10

-

-

-

 

3

Mean

SD

N

42.8

7.9

10

7.485

0.601

10

4.36

0.35

10

1.403*

0.065

10

2.821

0.086

10

94.3

19.2

10

0.896

0.123

10

-

-

-

 

4

Mean

SD

N

36.4*

6.9

10

8.568**

0.498

10

4.98*

0.35

10

1.382*

0.082

10

3.047**

0.180

10

61.7*

31.4

10

0.954*

0.200

10

0.094*

0.010

10

Group 1, 2, 3 and 4: 0, 20, 100 and 500 mg/kg bw/day

N: number of animals

SD: standard deviation

*: p<0.05

**: p<0.01

Table 6: Group mean urinalysis values (males and females)

 

Group

 

Non-Recovery Group

Recovery Group

Males

Females

Specific gravity

Volume

Specific gravity

 

1

Mean

SD

N

1.011

0.003

10

12.5

6-6

10

1.017

0.009

10

 

2

Mean

SD

N

1.012

0.004

9

-

-

-

-

-

-

 

3

Mean

SD

N

1.010

0.003

10

-

-

-

-

-

-

 

4

Mean

SD

N

1.016*

0.006

10

20.0*

8.0

10

1.009*

0.004

10

Group 1, 2, 3 and 4: 0, 20, 100 and 500 mg/kg bw/day

N: number of animals

SD: standard deviation

*: p<0.05

**: p<0.01

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of Y-15099 to male and female Wistar HanTM-RccHanTM:WIST strain rats, at dose levels of 20, 100 or 500 mg/kg bw/day was well tolerated. Based on a reduction in body weight gain and adverse effects on the kidney, a dose level of 100 mg/kg bw/day is considered to be the No Observed Adverse Effect Level (NOAEL) for systemic toxicity.
Executive summary:

A subchronic oral repeated dose toxicity study was performed according to OECD Guideline 408 (Rashid, 2015). Y-15099 was administered daily by oral gavage to 10 SPF-bred Wistar rats of both sexes at dose levels of 20, 100 and 500 mg/kg bw/day for a period of 90 consecutive days. A control group was treated similarly with the vehicle, desiccated Arachis oil BP, only. Ten additional animals per sex per dose were included as recovery group for analysis of possible post-exposure reversibility of intoxication symptoms. These animals were treated with 0 and 500 mg/kg bw/day of the test material for 90 days followed by a 28-day treatment-free recovery period prior to sacrifice. No treatment-related deaths or clinical signs were observed at any dose level considered to be test material-related. Neurobehavioural examination revealed no treatment-related effects at any dose level in both sexes. There was no effect of treatment on food consumption in females at all dose levels and males treated with 20 or 100 mg/kg bw/day. At 500 mg/kg bw/day, food consumption in males was generally marginally lower than controls throughout the treatment and treatment-free periods. Food efficiency and water consumption in animals of either sex treated with the test item at all dose levels was generally comparable with controls throughout the study period. Ophthalmoscopic examination of non-recovery animals of both sexes from control and high dose groups did not reveal any differences. There was no effect of test material administration on body weight development in females at any dose level. Statistically significant lower body weight gain was recorded in males of the 500 mg/kg bw/day dose group and considered to be adverse. Haematology and blood chemistry evaluations did not reveal any toxicologically significant effects in animals of either sex. Urinalysis revealed treatment-related findings of toxicological significance in animals of either sex administered with 500 mg/kg bw/day such as statistically significant increased specific gravity in non-recovery males, statistically significant increased urine volume and statistically significant decreased specific gravity in recovery females. There were no treatment-related observations at 20 and 100 mg/kg bw/day. Macroscopic examination did not reveal any treatment-related finding. Increased kidney weights in animals of either sex treated with 500 mg/kg bw/day at the end of the treatment period and adverse histopathological findings in the kidney, along with adverse effects previously noted in urine samples, were considered to be treatment-related. At the end of the treatment period, multifocal basophilic tubules (degenerating/regenerating) were present in male and female animals at 500 mg/kg bw/day. Single cell necrosis within the tubular cells was observed in females. At all dose levels, hyaline droplets were noted in kidneys from males with a dose-related increase. Following the four week recovery period, multifocal basophilic tubules were still present in all animals previously treated with the test item at 500 mg/kg bw/day; tubular fibrosis was also apparent with fibrous tissue surrounding the affected groups of tubules in these animals. Lymphocytic infiltration in the intestinal surrounding the affected tubules was observed in several animals with 2/10 females showing minimal tubular atrophy. Hyaline droplets observed within the tubules of males were consistent with the accu-2u-globulin, a common finding in untreated male rats and as such of no relevance to humans. In recovery control males, hyaline droplets were more apparent so the relationship between the increase in hyaline droplets observed in males receiving 20 or 100 mg/kg bw/day and treatment with the test item was not clear. The remaining histopathology findings in the kidneys from the high dose group were considered to represent an adverse effect of treatment with the test item.

 

Based on the adverse effects on body weight development in males and toxicologically significant kidney findings (increased kidney weights, histopathological findings) in animals of either sex at 500 mg/kg bw/day, a subchronic NOEL of 20 mg/kg bw/day and a subchronic NOEAL of 100 mg/kg bw/day was derived for Y-15099 in male and female Wistar rats.