Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 06, 2016 to January 30, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the 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
Details on test material:
Purity >99.1 %; Appearance: white powder

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for dose range finding study.
Sex:
male/female
Details on test animals and environmental conditions:
Species and strain: Crl:WI Wistar rats
Source: Charles River Laboratories, Research Models and Services, Germany, from SPF colony
Hygienic conditions: standard laboratory conditions
Number of animals: 48 male, 48 female rats, 12 animals/sex/group, 4 groups
Age of animals: young adult rats, approximately 10 weeks old at start and 12 weeks old at mating
Body weight range: males: 378-462 g, females: 236-297 g; did not exceed ±20 % of the mean weight for each sex at onset of treatment
Acclimation period: 5 days

Husbandry
Animal health: only healthy animals were used for the test, as certified by the clinical veterinarian. Females were nulliparous and non-pregnant.
Cage type: type II polycarbonate
Bedding: LIGNOCEL ¾ S certified wooden chips produced by J. Rettenmaier & Söhne GmbH+Co.KG, Germany
Nesting: Arbocel nest building material produced by J. Rettenmaier & Söhne GmbH+Co.KG, Germany
Light: 12 hours daily
Temperature: 21.0 – 24.9 °C (target range: 22±3°C)
Relative humidity: 30 – 60% (target range: 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: rodents were group-housed, up to 4 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed, respectively.

Food and water supply
Animals received ssniff SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, Germany, ad libitum, and tap water from the municipal supply from a 500 mL bottle, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Animal identification
Each parental/adult animal (P Generation) was identified by a unique number within the study, written with indelible ink on the tail.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The dosing solutions were administered to the test substance or vehicle-treated (control) animals daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 5 mL/kg bw were administered to all animals. The actual volume to be administered were calculated and adjusted based on each animal’s most recent body weight.
Vehicle:
methylcellulose
Details on oral exposure:
1% (w/v) aqueous methyl cellulose solution (abbreviated as 1% methyl cellulose or 1% MC in the raw data and report) was selected as vehicle of the study based on the formulation and analytical trials.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test substance formulations for concentration and homogeneity was performed using an HPLC-UV method. Top, middle and bottom duplicate samples were taken and analysed from test substance formulations at three times during the study, one set to analyse (which were collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken on each occasion from the middle of the vehicle control formulation.
Acceptance criteria of the concentration analysis were set at 100 ± 10% of the mean nominal concentration. Acceptance criteria of the homogeneity was that the CV of replicates (top, middle and bottom of test substance formulations) must be less than 10%.
The measured concentrations of the test substance evaluated for each test substance containing formulation varied between 94% and 108% of the nominal value. No test substance was detected in the control samples at any occasion. All test substance formulation samples were found to be homogeneous. Formulations were considered to be adequately stable under the study conditions.
Duration of treatment / exposure:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating) and then euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (4-day post-partum dosing).
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
0 mg/mL
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
6 mg/mL
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
20 mg/mL
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
60 mg/mL
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Dosing scheme, Male animals:
Acclimatisation: 5 days
Pre-mating period: 14 days
Mating/Post-mating period: at least 14 days (Last week of treatment: FOB, Day 24, 5 animals/group, Prior to/at necropsy examinations

Dosing scheme, Female animals:
Acclimatisation period: 5 days
Pre-mating period: 14 days
Mating: up to 5 days
Gestation: 22-24 days
Delivery
Lactation period: at least 4 days, FOB: (PND 4, 5 animals/group); Pups necropsy, (PND 4)
PND 5: Prior to/at necropsy examinations

All F1 offspring were terminated on Day 4 post-partum. In order to allow for overnight fasting of dams prior to urine collection on PPD 5, the offspring were euthanized on PPD/PND 4 and the dams on PPD/PND 5.

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS AND FUNCTIONAL OBSERVATION BATTERY (FOB) for all animals:

Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day.
General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment.
On gestation Day (GD) 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).

All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. More detailed examinations were performed once before the first exposure, then at least weekly, in the morning or before treatment. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Five males and five females/group were selected:

Assessment of potential test substance related neurotoxicity was performed during the last exposure week (males on Day 27; females on PPD 4). Selected animals were subjected to the functional observation battery including quantitative assessment of grip strength and measurement of landing foot splay and fore/hind limb grip strength.

Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.
Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated using a scoring system.Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5.

Body weight measurement
All adult animals were weighed with an accuracy of 1 g for randomisation purposes, then on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD 0, 3, 7, 10, 14 and 20 and on post-partum Days (PPD) 0 (within 24 hours after parturition) and 4 (before termination). The body weight of the female animals measured on gestation Days (GD) 3, 10 and 17 were only additional measurements as aid for the calculation of accurate treatment volumes, thus these data were not evaluated statistically.

Food consumption measurement
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g weekly (on the days of body weight measurements).

CLINICAL PATHOLOGY
All animals selected for blood sampling were fasted (overnight period of food deprivation, after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy. For terminal blood sampling in all selected animals (5 males and 5 females/group), 3 samples were taken from each animal: one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

URINALYSIS
Urine samples were collected for 16 hours during an overnight period of food deprivation during the last week of the study (Day 27-28 for males and PPD 4-5 for female animals, respectively) from each selected animal by placing the animals in metabolic cages. The evaluation of the urine samples was performed by observation (e.g. appearance, colour) and test strips.

Sacrifice and pathology:
Gross necropsy was performed on all animals. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea were recorded in the females as applicable.

Organ weight measurements:
At the time of termination, body weight and the weight of the following organs from all adult animals were determined:
- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported.

Tissue preservation and microscopic evaluation:

The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerves were retained in modified Davidson’s fixative, testes with epididymides in Bouin’s solution, and all other organs in 10% buffered formalin solution. In addition, on completion of the macroscopic examination the following tissues and organs were retained from all animals:

Adrenal gland, Lymph node, Small intestine, Ovary, Spinal cord, Aorta, Oviduct, Spleen, Brain, Pancreas, Sternum with marrow, Epididymis, Pituitary, Stomach, Eye with the optic nerve, Prostate, Testis, Oesophagus, Salivary gland (including mandibular, sublingual and parotid glands), Thymus, Femur with marrow, Thyroid with parathyroid gland, Heart, Tongue, Kidney, Sciatic nerve, Trachea, Large intestine, Seminal vesicle with coagulating gland, Urinary bladder, Extraorbital lachrymal gland, Uterus, Harderian gland, Skin, subcutis with mammary gland (inguinal), Vagina, Liver.

For the adult animals, a detailed histological examination was performed as follows:
- on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
- all macroscopic findings (abnormalities), except of minor order from all animals,
- retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups and additionally of the male (#3011) that failed to sire and the female (#3511) that failed to deliver healthy pups.

Statistics:
Data was recorded on the appropriate forms from the relevant SOPs of the labolatory., and then tabulated using the Microsoft Office Word and/or Excel, or using the software PROVANTIS v.9, as appropriate. Numerical data obtained during the conduct of the study was subjected as appropriate to calculation of group means and standard deviations.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Male animals: No clinical signs were detected for any males during the study.
Female animals: Only minor clinical signs, not related to the test substance treatment were detected for some female animals. Piloerection was detected for one Low dose female for three days just before and after the delivery. Paleness and piloerection were observed for one Mid dose female for three days before delivery.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant changes (decreases or increases at p<0.05 or p<0.01) were seen in the Low, Mid and High dose males (30, 100 and 300 mg/kg bw/day, respectively) at some evaluated periods during treatment. Statistically significant changes (decreases or increases at p<0.05 or p<0.01) were seen in the Mid and High dose females at some evaluated periods during treatment. In both cases there were no tendency or dose response, those facts were considered as not being a test substance related effect.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
When compared to the controls, there were no differences in males and females that could be considered toxicologically significant in the Low, Mid and High dose groups (30, 100 and 300 mg/kg bw/day, respectively).
Slight decreased mean haemoglobin concentration with statistical significance was detected for Mid (p<0.05) and High dose (p<0.01) males. However, both values were within the historical control range.
Statistically significant (p<0.05 or p<0.01) decreased mean prothrombin time value was detected in the Low, Mid and High dose males, but differences were minor. Furthermore, none of the differences observed in females reached statistical significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no significant changes or adverse effects on the serum chemistry that could be ascribed to the test substance administration in the Low and Mid animals (30 and 100 mg/kg bw/day, respectively). Minor but statistically significant test substance effects were seen in the High dose males (300 mg/kg bw/day) of this study.
Increased values for parameters related to protein content (total protein concentration, albumin concentration and albumin/globulin ratio) was observed in High dose males. The difference from control was statistically significant (p<0.01) in all those cases. Similar trends were seen in High dose females, although there the difference was statistically significant (p<0.05) only in case of total protein. Furthermore, in case of females the observed total protein value was also significantly higher (p<0.05) in the Mid dose group when compared to control.
In case of male animals, the observed values in the High dose groups were outside the physiological range and historical control range in case of total protein concentration and outside the physiological range in case of albumin concentration. Therefore, the observed values indicated a test substance effect in High dose males. The female values remained within the physiological and/or historical control range in all cases, thus there no clear test item effect was identified.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test substance related increased liver weights were observed in the Mid and High dose males and High dose females, and test substance related increased kidney weights were noted in the High dose males and females. No test substance related effect on organ weights was identified in the Low dose group. Based on the lack of changes in the functional parameters and histopathology, none of these effect in any dose groups were considered adverse.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No test substance-related macroscopic findings were observed up to the dose level of 300 mg/kg bw/day.
Dilatation of right renal pelvis was observed sporadically (for a total of three males. Bilateral enlargement of kidney was detected for two High dose males. Enlargement of mandibular lymph nodes was recorded for one Mid dose male. Enlargement of left testis was detected in one High dose male. Small testes and epididymides were noted in another High dose male. Depressed pale firm area (2x4 mm) of the kidney was recorded in one Control female. Dilatation of cervical body and horn was observed for one Low dose female. Enlarged spleen was observed for one Mid dose and one High dose males, and for two Low dose, three Mid dose and five High dose females. However, all these findings were considered to be incidental and not being test substance related.
Neuropathological findings:
no effects observed
Description (incidence and severity):
There were no test isubstance related effects in the neurological assessment as there were no observed differences in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or test substance treated groups.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
During the detailed histological examination, treatment related non-adverse changes were seen in the liver of the High dose males and females, and in the kidneys from the High dose females.
The minimal/slight centrilobular hypertrophy was seen in the liver of the High dose males (10/12) and females (6/12), correlated with increased liver weights. Hypertrophy was considered as treatment related, non-adverse adaptive change as liver function remained unaffected as confirmed by the relevant clinical chemistry parameters (AST, ALT and GGT).
In the kidney of the High dose females minimal/moderate tubular vacuolation was present at the corticomedullary junction in 6/12 animals. These histopathological changes corresponded with increased female kidney weights. The vacuolation was not observed in High dose males. However, minimal focal/multifocal tubular basophilia (4/12) and/or casts (3/12) were seen in High dose males. The vacuolation noted in the High dose females was considered as non-adverse changes, supported by the lack of clinical pathology changes. Tubular basophilia and casts in the High dose males were considered as background finding, as it could be seen across control and test substance treated groups.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic effects (kidneys)
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproduction

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the NOAEL for the test substance was considered to be 100 mg/kg bw/day for parental/adult generation for systemic effects, and 300 mg/kg bw/day for reproduction effects and for the F1/pups generation.
Executive summary:

A study was conducted to determine repeated dose toxicity of the test substance when administered to Wistar rats according to OECD Guideline 422, in compliance with GLP. Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including post-partum/lactation Day (PND) 4. The test substance, formulated in methyl cellulose, was administered by oral gavage to test animals. One control group and three treated groups were tested (30 mg/kg bw, 100 mg/kg bw and 300 mg/kg bw), each consisting of 12 males and 12 females. Dose formulations were analysed for test substance concentration and homogeneity on three occasions during the treatment period using a validated HPLC-UV method. Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. In addition, the reproductive performance, pregnancy, parturition and post-partum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND4. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups (5 animals/group) and liver, kidney and spleen samples of all groups. Additionally, all organs showing macroscopic findings as well as retained reproductive organs of those animals in the Control group which failed to sire / deliver pups were examined microscopically. Immunohistochemistry for alpha-2-microglobulin of the kidney samples of 5 Control and 5 High dose males were performed at the Test Site. All test substance formulations were within the range of 94-108 % of nominal concentration and were found to be homogenous. No test substance was detected in the vehicle control samples. Based on these results, test substance formulations were considered suitable for the study purposes. In summary, daily administration of the test substance by oral gavage to Wistar rats at dose levels of 30, 100 and 300 mg/kg bw/day did not result in adverse changes in clinical signs, neurological assessment, body weight, food consumption, haematology, coagulation or urinalysis during the treatment period under the conditions of this study. Minor but statistically significant test substance related changes on some serum chemistry parameters were seen in the High dose males (300 mg/kg bw/day), no such effects were determined in Mid and Low dose groups (100 and 30 mg/kg bw/day, respectively). Minimal/slight centrilobular hypertrophy in the liver was observed in the males and females of the High dose group (300 mg/kg bw/day). Hypertrophy was considered to be a treatment related, adaptive change, correlated with organ weight changes (41-64% increase in liver weight), but liver function parameters relevant for hepatic enzymes remained unaffected. Minimal hypertrophy (organ weight change of approximately 30%) was also observed in the Mid dose males (100 mg/kg bw/day). No hypertrophy was noted in the Low dose group (30 mg/kg bw/day). Taking into account the histopathology finding of non-adverse hypertrophy, the liver weight differences were considered to be non-adverse. Severe tubular vacuolation was present in the kidney of High dose females (300 mg/kg bw/day). This change was not seen in High dose males, although organ weight data (37-51% increase) indicated a test substance related effect in High dose males and females. No tubular vacuolation was recorded in the Low and Mid dose females (30 and 100 mg/kg bw/day) or males. In the absence of clinical pathology changes, the vacuolation noted in the High dose females was considered as a non-adverse change. There were no significant differences between Control and High dose males when α-2u globulin scoring was made using immunohistochemistry. No test substance-related microscopic changes were noted in the reproductive organs in any dose groups. There were no significant differences between Control and High dose males when α-2u globulin scoring was made using immunohistochemistry. No test substance related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period in any dose groups when compared to control. There were no effects on the F1 offspring viability, clinical signs, development or at macroscopic observations in any dose groups. Under the study conditions, the NOAEL for the test substance was considered to be 100 mg/kg bw/day for parental/adult generation for systemic effects, and 300 mg/kg bw/day for reproduction effects and for the F1/pups generation (Hargitai, 2017).