Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Remarks:
13 weeks plus 4 week recovery
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 6, 2019 - March 9, 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 408 (July 2018), Test method B.26

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
TG July 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
TG 2008
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Details on test material:
Polyol TD, batch no. 3977901, was described as a clear liquid, and stored at ambient temperature in the dark. Stability purity and identity were confirmed by the sponsor in a material safety data sheet (not available).

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The animals were male and female Sprague-Dawley Crl:CD(SD) rats (60 males, 60 females). The animals were supplied by Charles River Italia S.p.A, Calco (Lecco), Italy.
Sex:
male/female
Details on test animals or test system and environmental conditions:
The rats were aged 27-29 days old on arrival, and weighed 75-99 g. They were allowed to acclimatise to the laboratory conditions for 18 days prior to dosing, during which time the health status of the animals was assessed by thorough observations. After arrival, on 24 of October 2019, the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. Body weights were in the range of 76.4-84.4 g for males and 66.2-72.9 g for females. A health check was then performed by a veterinarian.

The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C±2°C and 55%±15%, respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day. The animals were housed up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages as indicated in the relevant SOP. Nesting material was provided inside suitable bedding bags and changed at least twice a week. Drinking water was supplied ad libitum to each cage via water bottles, except when overnight urine samples were collected in the last week of treatment from main phase groups. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei,4, 20019, Settimo Milanese (MI), Italy), with certified dietary levels of phytoestrogen (genistein less than 350μg/gram of diet), was offered ad libitum throughout the study,except in cases when water was restricted as mentioned above. There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at the test facility. Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.

On the day of allocation (5 days prior to the start of treatment) all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. The rats were allocated to the 4 groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed up to 3 of one sex per cage. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery(Figure 1) to minimise possible environmental effects. No replacements occurred after the first dose was administered.

Procedures and facilities were compliant with the requirements of the Directive 2010/63/EUon the protection of animals used for scientific purposes. The national transposition of theDirective is defined inDecreto Legislativo 26/2014.The test facility is fully accredited by AAALAC. Aspects of the protocol concerning animal welfare have been approved by the animal-welfare body.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was dissolved in the vehicle and administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight. Preparations were made weekly (concentrations of 10,30, and 100 mg/mL) according to stability data obtained from a previous study (Study No. A3648).
Vehicle:
water
Remarks:
softened water (by reverse osmosis)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verification available via a previous non-GLP stability study (ERBC Study no. A3648).
Duration of treatment / exposure:
13 consecutive weeks, followed by a recovery period of 4 weeks for 5 males and 5 females from groups 1 and 4. Animals in the main phase were dosed up until the day before necropsy.
Frequency of treatment:
All animals were dosed once a day, 7 days a week.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1; includes recovery 4 wk phase
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4; includes recovery 4 wk phase
No. of animals per sex per dose:
10 male and 10 female rats, control and high dose groups (group 1 & 4) included 5 additional animals per sex for 4 weeks of recovery
Control animals:
yes, concurrent vehicle
Details on study design:
The Sprague Dawley SD rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.

The oral route was selected as it is a possible route of exposure of the test item in man and in agreement with authority’s decision.

Dose levels were selected in consultation with the Sponsor, based on information from aprevious study (non GLP ERBC Study No. E0419). In this study, the test item was admin-istered at the dose levels of 100, 300 and 1000 mg/kg/day, for two consecutive weeks and notreatment-related clinical signs nor differences in body weight, body weight gain and foodconsumption were recorded. At clinical pathology evaluations, non adverse alterations in some clinical chemistry parameters (such as urea, potassium and sodium) were recorded in females of the high dose group; however, the correlation with the test item was doubtful since no other changes were noted. No changes were observed in treated males. The post mortem macroscopic observations showed no relevant changes that could be related to treatment. For these above reasons, the high dose level tested of 1000 mg/kg/day was considered suitable for a toxicity study of longer duration.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
Throughout the study, all animals' clinical signs 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. From Day 1 to Day 71 of the treatment period, observations were performed approximately 2 hours postdose. Starting from Day 72, since clinical signs were noticed, observations were performed at approximately 30 minutes from administration.

Each animal was weighed on the day of allocation to treatment group, on the day that treatment commenced, weekly there after and just prior to necropsy.

The weight of food consumed by each cage of rats was recorded at weekly intervals followingallocation. The group mean daily intake per rat was calculated.
Sacrifice and pathology:
During the last week of treatment, individual overnight urine samples were collected fromall male and female animals from each main phase group under conditions of food and water deprivation.

At the same time interval, blood samples ofapproximately 0.5 mL for thyroid hormone determination (T3, T4, TSH) were collected fromthe sublingual vein, under light isoflurane anaesthesia and condition of food deprivation.

Prior to necropsy, blood samples were collected from all animals in main phase and recovery groups under isoflurane anaesthesia from the abdominal vena cava for haematology,coagulation and clinical chemistry, under conditions of food deprivation. Further blood and urine samples were taken under identical conditions at the end of the recovery period.

Animals that had completed the scheduled test period were killed by exsanguination under isoflurane anaesthesia. All animals were subjected to necropsy, supervised by a pathologist. Requisite organs, according to TG 408, were weighed and the required tissue samples preserved in fixative. Necessary organ weights to body weights were calculated. Samples of all the tissues observed were fixed and preserved in 10% neutralbuffered formalin (except eyes, optic nerves, Harderian glands, testes and epididymideswhich were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Histopathological examinations as outlines by TG 408 were performed on all animals in the control and high dose groups killed at the end of the 13 weeks of treatment and all specimens showing abnormalities in all main phase groups. Liver analysis was performed on all groups as possible treatment-related changes were observed. One cauda from one epididymis of each animal completing the scheduled test period in the high dose and the control group was taken for sperm count and evaluation of motility and morphology.
Other examinations:
Once before commencement of treatment and at least once per week during the study from the start of treatment, each animal was given a detailed clinical examination. Observations included gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypiesor 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. Once during Weeks 12 of treatment and once during Week 4 of recovery an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptivestimuli), motor activity, and an assessment of grip strength was also performed.

Food consumption per cage was recorded weekly. The group mean daily intake per rat was calculated.

Both eyes of all animals were examined prior to the commencement of treatment by means of an ophthalmoscope, and by a slit-lamp microscope, after the instillation of 0.5% Tropic-amide (Visumidriatic®, Visufarma, Rome, Italy). The eyes of all animals from high dose and control groups were re-examined during Week 13 of treatment.

At the end of the treatment and recovery periods, just prior to necropsy, vaginal smears were taken from all females.
Statistics:
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 the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. 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, non-treatment-related
Description (incidence and severity):
Salivation was noticed starting from Week 11, in both genders treated at 1000 mg/kg/day. Almost all males (14 out of 15) and females (11 out of 15) showed salivation after 30 minutesfrom dosing. Some males treated with 300 mg/kg/day, at the end of Week 11 and/or beginning of Week 12 of the treatment period.Other signs, such as abrasion and scab on the tail, hairloss and tooth/teeth missing, were recorded during the study in single animals, with no correlation with the dose and were considered as minor clinical signs not related to the test item. Salivation was no longer observed during the recovery period and other signs recorded, such as swelling and hairloss, were considered as incidental and not relevant since the animals affected did not show any sign of toxicity during the treatment period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The only statistically significant increase found (+127%), that was not considered related to treatment, was the mean absolute body weight gain of males treated at 1000 mg/kg/day, on Day 22 of the recovery period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Dosing phase:
Slight leucocytosis was recorded in males dosed at 300 and 1000 mg/kg/day. Compared with controls, changes were 31% and 57%, respectively, and involved mostly lymphocytes. Mean corpuscular volume was statistically significantly decreased in males dosed at 1000 mg/kg/day (3% below controls). Due to the minimal severity (values were withinthe range of historical data), this finding was considered to be not adverse. The other statistically significant differences between control and treated animals (haemoglobin in males, reticulocytes and lymphocytes in females) were recorded in animals dosed with 100 mg/kg/day, therefore they were considered to be unrelated to treatment.

Recovery phase:
In males, leucocytes were comparable with controls, confirming complete reversibility. In females, leucocytes were higher than controls. However, this was mostly due to the low control data; leucocytes values of the treated females were comparable with those recorded at end of the the dosing phase.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Many of the findings were mostly due to changes in one male dosed at 1000 mg/kg/day (animal no. A3649082): compared with mean control data, this animal showed increases of alanine aminotransferase (3.2 fold), aspartate aminotransferase (2.4 fold), total bilirubin (2fold), total cholesterol (2.1 fold, both HDL and LDL) and triglycerides (2.8 fold). Due to the minimal incidence, these findings cannot be conclusively attributed to treatment. Cholesterol and triglycerides changes were also recorded in a number of males dosed at 1000 mg/kg/day.The other statistically significant differences between control and treated animals (BUN/ureain both sexes, protein and albumin in males) were not dose-related, therefore they were considered to be incidental. All findings recorded at Week 13 showed a complete reversibility after 4 weeks of recovery.One treated male (no. A3649096) showed an increase of aspartate aminotransferase (2.4fold). Similar finding was not recorded at dosing phase (with the exception of animal no.A3649082), therefore this was considered to be incidental.

Alanine aminotransferase was still higher than controls in males (1.4 fold). However, the severity of this finding was insufficient to represent an hepatic disease, therefore thischange was considered to be not adverse.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean values of thyroid stimulating hormone were lower than mean control data in all malegroups. Changes were 21% to 53% with no clear dose-relation. Since low value was also recorded in one control male (no. A3649012), no dose-relation or statistically significance were achieved and no concurrent changes of the other hormones were recorded, the above finding was considered to be of no toxicological relevance.The statistically significant difference of thyroxine recorded between control and females dosed at 100 mg/kg/day (42% above controls) was not dose-related, therefore it was considered to be incidental.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant mild increase in mean liver weight was recorded in males treated at 300 mg/kg/day (relative weight only) and 1000 mg/kg/day (absolute and relative weights), compared to controls. This increase was dose-related (+10% and+24% in mid- and high dose groups for relative weights) and accompanied by corroborative histopathological changes, such as centrilobular hypertrophy. A statistically significant increase in mean relative kidneys weight (approximately +11% of relative weight) and in absolute and relative mean spleen weights (approximately +17% of relative weight) was recorded in males treated at 1000 mg/kg/day, when compared to controls. However, since these weight variations were not accompanied by corroborating histopathological changes in both kidneys and spleen, these were considered not toxicologically relevant. The minimal statistically significant increase (+11%), still observed in the liver of males previously dosed at 1000 mg/kg/day, was considered not toxicologically relevant since, at histomorphology, the liver was comparable to control animals.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All observed changes were considered spontaneous and incidental, having a comparableincidence in control and treated groups and/or are characteristically seen in untreatedSprague Dawley SD rats of the same age. In particular, the mass observed at necropsy in the prostate of one high dose treated male (no. A3649082) corresponded microscopicallyto a benign prostatic adenoma in the ventral lobe and it was considered as a spontaneous pathology.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, a slight but statistically significant increase in mean grip strength was recorded in females treated at 100 mg/kg/day (+16%). At the end of recovery, this parameter was found increased in males (+31%), and decreased in females (-22%) treated at 1000 mg/kg/day. No toxicological significance was attributed to these findings, since almost all values obtained were comparable to the other values recorded during the study and were there therefore considered to be incidental.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination revealed treatment-related changes in the liver, consisted in centrilobular hepatocytic hypertrophy, morphologically represented by an enlarged size of the hepatocytes, of males treated at 1000mg/kg/day. This change was no longer observed at the end of the 4-week recovery period. This change correlated with the increased liver weight observed in mid- and high dose males and was most likely considered related to microsomal enzyme induction.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
All other reported findings, including the benign prostatic adenoma in the prostate ventrallobe of a single high dose male (no. A3649082), were considered spontaneous and incidental, having a comparable incidence in control and treated groups. In particular, the nephropathy observed was not considered treatment-related because it was observed in male rats of all groups, including control animals; the nephropathy was characterised by tubular degeneration/regeneration and/or necrosis and/or granular casts, in some cases associated with mononuclear cell infiltration, localised in the cortex and/or in the cortical/medullary junction.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The effects seen in males, slight leucocytosis, increased cholesterol and triglycerides levels and centrilobular hepatocytocytic hypertrophy in the liver, were treatment related, but reversible and therefore the not defined as adverse effects.
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: The slight leucocytosis and increased cholesterol and triglycerides and the histopathological effects (centrilobular hepatocytic hypertrophy) in males were considered to be related to treatment but, reversible and therefore not defined as adverse effects.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
A repeated dose 90-day oral toxicity study with exposyre to Polyol TD by daily gavage in the rat was performed according to OECD guideline and GLP principles. No Observed Adverse Effect Level (NOAEL) for this study was 1000 mg/kg/day and the No Observed Effect Level(NOEL) was 100 mg/kg/day. There was slight leucocytosis in males treated at 300 and 1000 mg/kg/day, increased cholesterol and triglycerides in some males dosed at 1000 mg/kg/day and centrilobular hepatocytic hypertrophy in the liver of males dosed at 1000 mg/kg/day. The above findings were treatment related, but were no longer observed at the end of the recovery period and therefore were not defined as adverse effects.
Executive summary:

The toxicity of Polyol TD in rats after oral administration for 13 consecutive weeks and recovery from any treatment-related effects during a period of 4 weeks were investigated in accordance to OECD guideline 408 and GLP principles. Three groups, each of 10 male and 10 female Sprague Dawley rats, received the test item by gavage at dosages of 100, 300 and 1000 mg/kg body weight/day for 13 consecutive weeks. A fourth similarly constituted group received the vehicle alone (softened water) and acted as a control. Five additional animals for each sex were included in the high dose and controlgroups for recovery assessment. The following investigations were performed: daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli and motor activity, body weight, food consumption, ophthalmoscopy, oestrous cycle, clinical pathology investigations, sperm analysis, terminal body weight, organweights, macroscopic observations and histopathological examination.

Signs of effects related to treatment were mainly observed in animals from the high dose group. Most of these effects were recorded at clinical pathology evaluations (slight leucocytosis inmales treated at 300 and 1000 mg/kg/day, with a dose-relation trend and increased cholesterol and triglycerides in some males dosed at 1000 mg/kg/day) and at histopathology(centrilobular hepatocytic hypertrophy in the liver of males dosed at 1000 mg/kg/day). These findings were considered to be related to treatment but, since they were no longer observed at the end of the recovery period, they were not defined as adverse effects.

Based on the above mentioned results, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 1000 mg/kg/day and the No Observed Effect Level(NOEL) was 100 mg/kg/day.