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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From NOvember 17, 2017 to February 9, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Reproduction/Developmental Toxicity Screening Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy.
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation:
♂ 200 to 225 g
♀ 175 to 200 g
- Fasting period before study:
- Housing: From arrival to mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor ( Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary and changed at least 2 times a week.
- Diet: A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
- Water: Drinking water was supplied ad libitum to each cage via water bottles
- Acclimation period: approximately 22 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:

- DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

- VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage): 5 mL/kg body weight
The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
- Lot/batch no. (if required):
- Purity:
-Other: The required amount of test item was suspended in the vehicle. The formulations were prepared weekly (concentrations of 30, 100 and 200 mg/ml)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A 28 hour stability at room temperature and an 8 day stability at +5 °C ± 3 °C were verifi ed in the range from 10 to 200 mg /ml.
Duration of treatment / exposure:
Males: Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter for a total of 46/47 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females: Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifi ce( for at least 51 days). Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.
Recovery groups: (Groups 5 and 6)
Animals were dosed once a day, 7 days a week, for 4 consecutive weeks. Dose volumes were adjusted once per week for each animal according to the last recorded body weight. No treatment was given during the recovery period.
Frequency of treatment:
Animals were dosed once a day, 7 days a week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Main group
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Main group
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Main group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Main group
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Recovery group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Recovery group
No. of animals per sex per dose:
Each main group comprised 10 male and 10 female rats. Two groups (control and high dose levels) included 6 animals per sex to be sacrificed after 4 weeks of recovery.
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: dose volumes were calculated according to the last recorded body
weight.

Examinations

Observations and examinations performed and frequency:
MORTALITY: Yes
Throughout the study, all animals were checked early in each working day in the morning
and in the afternoon. At weekends and Public Holidays a similar procedure was followed
except that the fi nal check was carried out at approximately mid-day. This allowed post
mortem examinations to be carried out during the working period of that day.

DETAILED CLINICAL OBSERVATIONS: Yes
Once before commencement of treatment and at least 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.

BODY WEIGHT: Yes
Main groups:
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identifi cation of mating and on
Days 0, 7, 14 and 20 post coitum.
Dams and pups were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.
Recovery groups:
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:
Main groups:
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post
coitum and on Days 7 and 13 post partum starting from Day 1 post partum.
Recovery groups:
The weight of food consumed by each cage of rats was recorded at weekly intervals following Day 1 of dosing.
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
Males
Blood samples were collected under isofl uorane anaesthesia from the retro-orbital sinus.
The order of collection was equalised between groups.
Females
As part of the sacrifi cial procedure, samples of blood were withdrawn under isofl uorane
anaesthesia from the abdominal vena cava. The order of collection was equalised between
groups.
Blood collection was performed only for animals at termination.

CLINICAL CHEMISTRY: Yes
Bioanalysis:
Thyroid hormone determination (T3, T4 and TSH)
Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay, using Luminex Magpix system and the MILLIPLEX MAP Rat Thyroid Magnetic Bead Panel kit (Merk Millipore, cat. no. RTHYMAG-30K).
In the fi rst instance the determination was restricted as detailed below:
– Samples from all parental males from all main groups
– Samples from pups on Day 14 post partum
The results of these analyses are presented as individual data, mean and standard deviations. Since no treatment related effects were seen in the determination performed in parental males and in pups on Day 14 post partum, samples obtained from females and from pups on Day 4 post partum were not analysed and will be destroyed after the fi nalisation of the report. When the serum levels for Total triidothyronine (total T3) were BLOQ (below the
limit of quantifi cation - 3.75 ng /mL), the values were not included in the mean calculation.

OTHER:
VAGINAL SMEARS AND OESTROUS CYCLE:
Stock females:
Oestrous cycle was monitored by vaginal smears in all stock females for 1 week before allocation in order to exclude from the study females with irregular cycle.
Females allocated to groups (Main groups)
Vaginal smears were taken in the morning from Day 1 of dosing up to positive identifi cation of mating.
The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Vaginal smears were also taken from all females, before despatch to necropsy. No vaginal smears were taken from females sacrifi ced for humane reasons.


Sacrifice and pathology:
Euthanasia (All groups)
Parental animals were killed by exsanguination under isofl uorane anaesthesia. Pups were euthanised by intraperitoneal injection of Thiopenthal.
Parental males (Main groups)
The males were killed after the mating of all females, after a total of 46/47 days of treatment.
Parental females (Main groups)
The females with live pups were killed on Day 14 post partum, after a total of 51 days of treatment.
Males and females (Recovery groups)
Animals were killed after 4 weeks of recovery.

The clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifi ces). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.
Females (Main groups)
All females were examined also for the following:
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).
Uteri of apparently non-pregnant females were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

Organ weights (Main and Recovery groups)
Parental animals
From all animals, the organs indicated in section 4.5.8 were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Histopathological examination (Main and Recovery groups)
The tissues required for histopathological examination are listed below:
Adrenal glands
Brain (cerebrum, cerebellum, medulla/pons)
Caecum
Clitoral gland
Colon
Duodenum
Epididymides
One epididymal cauda
Heart
Ileum
Jejunum (including Peyer’s patches)
Kidneys
Liver
Lymph nodes – mandibular
Lymph nodes – mesenteric
Oesophagus
Mammary gland - Females
Mammary gland - Males
Ovaries with oviducts
Parathyroid glands
Pituitary gland
Penis
Prostate gland (dorsolateral and ventral)
Rectum
Sciatic nerve
Seminal vesicles with coagulating glands
Spleen
Stomach
Testes
Thymus (where present)
Thyroid
Uterus – cervix
Vagina

After dehydration and embedding in paraffi n wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS).
The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
In the first instance, the examination was restricted as detailed below:
i) Tissues specified above from all males and all females in the control and high dose groups killed at term.
ii) All abnormalities in all groups.



Statistics:
Standard deviations were calculated as appropriate. For continuous variables the signifi cance of the differences amongst group means was assessed by Dunnett’s test or a modifi ed t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test, if n was more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical signifi cance wasp <0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No signs of toxicity were observed throughout the study, both in main and recovery groups. The staining of the tail observed in all animals receiving 500 and 1000 mg/kg/day and in animals after 4 weeks of recovery, was related to the colour of the test item.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred throughout the study, both in main and recovery groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Main groups:
No relevant differences in body weight and body weight gain were noted between control and treated males and females throughout the study.
Recovery groups:
Means of body weight and body weight gain were comparable between controls and the treated groups both in males and females throughout the treatment phase. During the recovery period, no relevant differences in body weight and body weight gain were recorded in animals of both sexes, when compared to the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Main and recovery groups
No effects on food consumption were observed in either males or females throughout the study, both for main and recovery groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid stimulating hormone was decreased in males of all treated groups. Compared with controls, changes were 39% to 46%, with no dose-relation. Due to the absence of related changes of the other thyroid hormones and/or histopathological fi ndings, these were considered to be of no toxicological relevance.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Main and recovery groups
The most relevant change observed at necropsy in the animals sacrifi ced at the end of treatment and recovery periods was brown staining of the tail, which was considered likely due to the colour of the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Enlarged liver observed respectively in one mid- (no. X0850047) and one high dose female (no. X0850067) and a subcutaneous mass observed in one mid-dose female (no. X0850053) were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Main and recovery groups
No treatment-related changes were noted in animals sacrifi ced at the end of the treatment period. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
The sporadic lesions reported in control and/or treated animals such as epithelial hyperplasia of non glandular region of the stomach in one high dose male (no. X0850080) or luminal dilatation of the uterus of one high dose female (no. X0850079), were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age. In particular, the M-Adenocarcinoma in the mammary glands of one mid-dose female (no. X0850053), which correlated to the subcutaneous mass observed during necropsy, was considered spontaneous.
Histopathological findings: neoplastic:
not examined
Details on results:
During the in vivo phase of the study, no signifi cant clinical signs were observed in all treated main animals of both sexes. Body weight, as well as body weight gain of treated males and females did not show differences of toxicological signifi cance throughout the study. The food consumption in male and female animals of treated groups was generally comparable with the control group.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no releated effects observed
Remarks on result:
other: higher dose

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general and reproduction/developmental toxicity was considered to be 1000 mg/kg/day.
Executive summary:

The toxic effects on rats of both sexes after repeated oral dosing with test substance, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and early lactation of the offspring were investigated. A 4 week treatment-free period was allowed in order to assess any delayed toxicity or recovery from any adverse effects observed during the dosing phase.

The vehicle was softened water. All doses were administered at a constant volume of 5 ml/kg body weight.

The study design was as follows:

Main groups
Group  Treatment
(mg/kg/day)
Number of animals
1 0 10♂ + 10♀
2 150 10♂ + 10♀
3 500 10♂ + 10♀
4 1000 10♂ + 10♀
Recovery groups
Group  Treatment
(mg/kg/day)
Number of animals
5 0 6♂ + 6♀
6 1000 6♂ + 6♀

Main groups

Males of the main groups were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 46/47 days. Females were treated for 2 weeks prior to pairing, and thereafter during pairing, post coitum and post partum periods

until Day 13 post partum (for at least 51 days).

The following investigations were performed in both sexes of all groups: mortality check, clinical signs, body weight, food consumption, oestrous cycle, mating performance, litter data, sex ratios, macroscopic observations and organ weights. Sperm analysis was performed in all control and high dose males. Histopathological examination was performed in control and high dose groups, as well as on all abnormalities detected during post mortem observation. The identification of the stages of the spermatogenic cycle was also performed

in five randomly selected males of the control and high dose groups. Thyroid hormone determination was performed in all males. Clinical signs were performed in all pups. Thyroid weight and thyroid hormone determination of 1/pup/sex/litter (where possible) at Day 14

post partum were evaluated. All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection. All live

pups sacrificed on Day 14 post partum were examined for external abnormalities and sex confirmation by gonadal inspection. The anogenital distance in all pups and the presence of nipples/areolae in male pups were also verified.

Recovery groups

Recovery animals were treated for 4 consecutive weeks and killed after 4 weeks of recovery period.

The following parameters were evaluated in these animals: mortality check, clinical signs, body weight, food consumption, macroscopic observations and organ weights.

Mortality and fate of females

No mortality occurred throughout the study. Mating was not detected in one female of the mid-dose group. However, this female gave

birth and was sacrificed with the litter on Day 14 post partum.

One female in the low dose group and one female in the high dose group were found not pregnant at necropsy. In addition, one female in the high dose group had unilateral implantation.

The number of females with live pups on Day 14 post partum was: 10 in the control, 9 in the low dose, 10 in the mid-dose and 9 in the high dose group.

Clinical signs

Main and recovery groups. No signs of toxicity were observed throughout the study, both sexes, both for main and recovery groups.

Body weight and body weight gain

Main and recovery groups. No differences of toxicological relevance were recorded in body weight and body weight gain, both for main and recovery groups respect to the control groups.

Food consumption

Main and recovery groups. No effects on food consumption were observed in either males or females throughout the study, both for main and recovery groups.

Thyroid hormone determination

No treatment-related differences were noted in hormones levels in parental males and in pups at Day 14 post partum, when compared to control group animals.

Oestrous cycle, reproductive parameters, pairing combination and mating performance

Oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show any differences that could be related to treatment.

Implantation, pre-birth loss data and gestation length of females

Corpora lutea, implantations, litter size and pre-natal loss (percentage) were similar in control and treated groups. Gestation periods were similar between treated and control females. All pregnant dams gave birth on Day 22 post coitum (mean value).

Litter data and sex ratio of pups

Litter data at birth on Days 1, 4 and 13 post partum did not show differences of toxicological relevance between groups. Sex ratios at birth, on Days 4 and 14 post partum did not show relevant differences between groups, when calculated as the percentage of males.

Clinical signs of pups

Pre-weaning clinical signs, pups found dead and missing pups were comparable between treated and control groups.

Anogenital distance

No relevant differences were seen between the control and treated pups in anogenital distance.

Necropsy findings in pups

Necropsy findings in deceased pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect.

Pup thyroid weight

No significant differences were noted in mean thyroid weight of pups in control and treated groups.

Terminal body weight and organ weights

Main and recovery groups

No relevant changes were observed on terminal body weight, absolute and relative organ weight of treated animals that completed the treatment or recovery period, when compared with controls.

Sperm analysis

No relevant differences were observed at sperm analysis including sperm motility and concentration, and cauda weight between the control and the high dose group at the end of treatment.

Macroscopic observations

Main and recovery groups. No remarkable differences were noted at post mortem examination in treated animals sacrificed at the end of treatment and recovery periods, when compared with controls.

Microscopic observations

No treatment-related changes were noted in animals sacrificed at the end of the treatment period. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular

layering in the germinal epithelium was noted.

Conclusion

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general and reproduction/developmental toxicity was considered to be 1000 mg/kg/day.