Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 30 to July 17, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and experience and background data is large.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy; supplied by Envigo Netherlands.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: approximately 190 g for males and 160 g for females (at the start of dosing)
- Fasting period before study: no
- Housing: up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages
- Diet: ad libitum
- Water: ad libitum to each cage via water bottles.
- Acclimation period: 6 days prior to the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2° C
- Humidity 55 ± 15%
- Air changes: 15 to 20 per hour
- Photoperiod: the rooms were lit by artificial light for 12 hours each day.

IN-LIFE DATES: from: 30 May 2017 to: 17 July 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the formulations prepared in weeks 1 and 4 of the current study were analysed to check the concentration. Results of the analyses were within the acceptability limits stated in RTC SOPs for concentration of solutions (90-110 %)
Duration of treatment / exposure:
Minimum of 4 consecutive weeks
Frequency of treatment:
All animals were dosed once a day, 7 days a week.
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Each group comprised 5 male and 5 female rats.
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and public holidays, a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day.

Clinical signs and neurotoxicity assessment
All clinical signs were recorded for individual animals. Once before commencement of treatment and once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.Once before commencement of treatment and at least once per week from the start of treatment, each animal was given a detailed clinical examination. Each animal was observed in an open arena. The test included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded.
Once during week 4 of treatment and once during week 2 of recovery, an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength were also performed.

Motor activity assessment (MA)
The motor activity (MA) of all animals was measured once during week 4 of treatment and once during week 2 of recovery by an automated activity recording.
Measurements were performed using a computer generated random order.

Body weight
Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

Food consumption
The weight of food consumed by each cage of rats was recorded at weekly intervals during the treatment and recovery periods. The group mean daily intake per rat was calculated.

Clinical pathology investigations
Prior to necropsy, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava, under conditions of food deprivation. Since possible treatment-related changes were observed, further blood samples were taken from all surviving animals, under identical conditions, at the end of the recovery period for clinical chemistry tests. Blood samples were collected and analysed in the same order. The blood samples collected were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– heparin anticoagulant for biochemical tests
– citrate anticoagulant for coagulation tests
Sacrifice and pathology:
Euthanasia
Animals that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia. All animals were subjected to necropsy, supervised by a pathologist.

Necropsy
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

Organ weights
From all animals completing the scheduled test period, the organs indicated in the protocol were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved
Samples of all the tissues listed in Annex of the protocol were fixed and preserved in 10 % neutral buffered formalin (except eyes, testes and epididymides, which were fixed in Modified Davidson’s fluid and preserved in 70 % ethyl alcohol).

Histopathological examination
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 µm thickness and stained with haematoxylin and eosin.
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance.
Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If data was found to be inhomogeneous, a modified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathological finding was carried out by means of a nonparametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs were observed in animals of control groups and in those of Groups 2 and 3. Rales, ataxia and dyspnoea were individually observed in male animals of Group 4 during the treatment or the first days of the recovery period.

2 animals of the high dose group (Group 4) showed rales on day 19-20 (animal no. A2787052) and from day 21 to 29 (animal no. A2787058). One animal of the same group (no. A2787056) showed ataxia and dyspnoea between days 12 and 17 of the dosing phase.
Rales continued to be noted in one animal (no. A2787058) during the first three days of the recovery period. This sign was no longer apparent from day 4 up to the end of the observation period. No clinical signs were recorded in control animals and in the other animals of Group 4.

No changes of toxicological significance were found at the weekly clinical examination during treatment and recovery periods, which included an evaluation of neurotoxicity.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male animal of the control group was found dead on day 23 of the treatment phase. No clinical signs were observed before the death and the macroscopic observation performed at necropsy suggested that it could be related to a mis-dosing.

Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
From day 8 to day 29 of the dosing period, a very slight reduction in body weight (ranging from -8 % to -4 %), statistically significant only on day 22, was recorded in males of the high dose group. No remarkable differences in body weights were noted during the recovery period, when compared to controls. A very slight, but statistically significant, decrease in body weight (included in a range of -12 % to -7 %) was noted in female animals of the low and mid-dose groups, on days 22 and 29 of the treatment period, when compared to females of the control group. A very slight and statistically significant, decrease in body weight (ranging from -8 % to -6 %) was also recorded in females of the high dose group from day 8 up to the end of the treatment period.
Body weights remained lower than controls also during the recovery period, with a slight, but statistically significant, reduction in body weight of -11 % and -10 % on days 8 and 15, respectively.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The statistically significant difference of leucocytes recorded between controls and males dosed at 10 mg/kg/day (37 % below mean control data) was not dose-related, therefore it was considered to be incidental.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Dosing phase: compared to animals of the control groups, statistically significant fluctuations of some biochemical parameters were recorded, as follows: decrease of alkaline phosphatase in males dosed at 20 mg/kg/day (-21 %), decrease of aspartate aminotransferase in males receiving 20 and 40 mg/kg/day (-23 % and -20 %, respectively), decrease of triglycerides in males dosed at 40 mg/kg/day (-45 %) and increase of calcium in those receiving 20 mg/kg/day (+12 %), decrease of aspartate aminotransferase in females dosed at 20 mg/kg/day (-14 %), increase of glucose in those receiving 10 mg/kg/day (+19 %) and 40 mg/kg/day (+26 %) and decrease of chloride in females treated at 40 mg/kg/day (-3 %). Some of these changes (alkaline phosphatase and calcium in males, aspartate aminotransferase in females) were not dose-related, therefore considered unrelated to treatment. The other findings were not considered to be suggestive of tissue/organ injury, due to their slight severity.
Recovery phase: cholesterol and chloride showed a complete reversibility in females. In male animals triglycerides were lower than controls (-33 %), although this change was not considered to be adverse.The other statistically significant changes recorded (sodium in males, urea, triglycerides and potassium in females) were considered incidental, since not observed during the dosing phase.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No differences of toxicological relevance were observed on terminal body weight of treated animals, when compared to controls. Male animals of mid- (Group 3, 20 mg/kg/day body weight) and high (Group 4, 40 mg/kg/day body weight) dose groups showed a statistically significant increase in absolute and relative mean liver weights (approximately 14 % and 22 %, respectively), when compared to controls.
A statistically significant increase in relative liver weight was recorded in females of the high dose group (approximately 15 %) and in kidneys relative weight (approximately 13 %) of the same group, when compared to animals of the control group.
No other remarkable changes in absolute and relative organ weights were noted in the animals of the other treated groups, when compared to the control data.
No changes of toxicological relevance were observed on terminal body weight, absolute and relative organ weights in the animals of the recovery phase.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted.
Neuropathological findings:
no effects observed
Description (incidence and severity):
Functional tests and motor activity
No differences between treated animals and controls, which could be considered of toxicological relevance, were observed at functional tests (sensory reactivity, landing footsplay, grip strength) performed at the end of treatment and recovery periods.
Motor activity measurements performed at the end of treatment and recovery periods did not show any toxicologically significant differences between treated animals and controls.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related changes, consisting in moderate to marked hyaline droplets accumulation, were noted in kidneys of male animals of Group 4. In addition, multifocal centrilobular hepatocellular hypertrophy from minimal to mild degree was observed in the liver of all males of this group.
The histopathological lesions involving the liver and kidneys of male animals of the high dose group showed a complete reversibility at the end of the recovery period.
Details on results:
Macroscopic observations
Final and recovery sacrifice: no relevant macroscopic alterations that could be considered treatment-related were noted in animals sacrificed at the end of the treatment and recovery period. The lesions observed (multiple dark areas of the stomach, enlarged ovaries or distended uterus) were considered spontaneous and/or incidental, commonly observed in animals of this species and age under our experimental conditions.

Microscopic observations
Final sacrifice: treatment-related changes were noted in male animals of the high dose group (Group 4) receiving 40 mg/kg/day bw. These alterations consisted in a moderate to marked increase in hyaline droplets accumulation in kidneys (consisting in eosinophilic homogeneous cytoplasmatic droplets, that could represent low molecular weight protein accumulation within lysosomes, as a result of either increased filtered protein loads or decreased catabolism), observed in males of Group 4, when compared to males treated at 20 mg/kg/day body weight or with the vehicle. All male animals of the high dose group showed a minimal to mild multifocal centrilobular hepatocellular hypertrophy, that could be considered as adaptative changes associated to a microsomal enzyme induction, deriving from the exposure to test item.
The remaining lesions observed in control and treated animals were considered spontaneous and/or incidental, commonly seen in animals of this species and age under our experimental conditions.
Recovery sacrifice: pathological changes involving the liver showed a complete reversibility. The hyaline droplets observed in the kidneys of the high dose group males were comparable to those seen in the kidneys of the control animals.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
clinical signs
histopathology: non-neoplastic
Remarks on result:
other: reversible effects after the recovery period
Dose descriptor:
NOAEL
Effect level:
35 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
histopathology: non-neoplastic
Remarks on result:
other: reversible effects after the recovery period

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL = 40 mg/kg bw/day.
Executive summary:

Method

Repeated dose toxicity was tested in rats dosed daily by oral route for 4 consecutive weeks; a 2 -week recovery period, followed dosing.

Three groups, each of 5 male and 5 female Sprague Dawley rats, were dosed at 10, 20 and 40 mg/kg/day. A fourth group received vehicle alone (water) and acted as a control. Control and high dose groups included 5 additional animals per sex to be sacrificed after 2 weeks of recovery.

Observations were: daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli, assessment of grip strength and motor activity, body weight, food consumption, clinical pathology investigations, terminal body weight, organ weights, macroscopic observations and histopathological examination.

Results

Mortality

One male animal of the control group, likely due to mis-dosing.

Daily clinical signs

No clinical signs were observed in animals of the control groups and in those of Groups 2 and 3. Rales, ataxia and dyspnoea were individually observed in male animals of Group 4 during the treatment or the first days of the recovery period.

Weekly detailed clinical signs

No changes of note were found at the weekly clinical examination which included an evaluation of neurotoxicity during treatment and recovery periods.

Neurotoxicity assessment (functional tests and motor activity)

No treatment related effects were noted.

Body weight

Very slight reductions in body weight were noted in animals of treated groups compared to controls. These decrements were statistically significant on day 22 for males of Group 4, on days 22 and 29 for females of Groups 2 and 3 and on day 8 up to the end of treatment and recovery periods for females of Group 4.

Food consumption

No changes in food consumption were noted.

Haematology

No treatment-related changes were recorded.

Clinical chemistry

Alterations of some biochemical parameters observed during the treatment or recovery phase were considered unrelated to the treatment or, due to their slight entity, they were not deemed to be suggestive of tissue/organ injury or adverse.

Terminal body weight and organ weights

No alterations of toxicological relevance were observed on terminal body weights. Male animals of Groups 3 and 4 showed a statistically significant increase in absolute and relative liver weights. In addition, a statistically significant increase in relative liver weight was noted in females of Group 4, in relative kidneys weight for males of the same treated group, when compared to controls.

No remarkable changes in terminal body weight and in absolute/relative organs weight were noted in animals of the recovery phase.

Macroscopic observations

No treatment-related changes were noted.

Microscopic observations

Final sacrifice

Treatment-related changes, consisting in moderate to marked hyaline droplets accumulation, were noted in kidneys of male animals of Group 4. In addition, multifocal centrilobular hepatocellular hypertrophy from minimal to mild degree was observed in the liver of all males of this group.

Recovery sacrifice

The histopathological lesions involving the liver and kidneys of male animals of the high dose group showed a complete reversibility at the end of the recovery period.

Conclusion

Histopathological alterations observed in kidneys and liver of male animals treated at the high dose level showed a complete reversibility at the end of the recovery period. Therefore, the dose level of 40 mg/kg/day, equivalent to 35 mg/kg a.i.) could be considered as a NOAEL.