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Description of key information

Oral repeated dose toxicity (OECD 422 and OECD 408), rat (m/f): NOAEL systemic = 300 mg/kg bw/day

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Jul 2020 - 17 Aug 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 2016
Deviations:
yes
Remarks:
several examinations/observations not performed for single animals at some occasions; triglycerides not determined in clinical chemistry; limited reporting on historical controls
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories, Margate, United Kingdom
- Age at dosing: 9 – 11 weeks (males) and 7 – 8 weeks (females)
- Weight at study initiation: 268.8 – 397.8 g (males) and 154.0 – 236.2 g (females)
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes. Aspen wood chips were used as bedding. Before pairing, the rats were housed in groups of up to 3/sex. During pairing, males and females were housed on a 1:1 basis. After pairing, males were housed in groups of 3 as before pairing and females were housed individually or with their litter (during gestation).
- Diet: SDS Rat and Mouse breeder diet VRF1 (Special Diets Services Ltd., Witham, United Kingdom), ad libitum
- Water: Tap water from the main supply, ad libitum
- Acclimation period: 2 weeks prior to initiation of dosing (males) or 1 week prior to smearing (females)

DETAILS OF FOOD AND WATER QUALITY: Each batch of diet and the water was periodically analysed for specific constituents and contaminants. No contaminants were present in the diet and in the water at levels that might have interfered with achieving the objective of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 40 – 70%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 Jul 2020 To: 23 Sep 2020
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose formulations were pepared weekly and stored at room temperature (15 - 25 °C) in a sealed container protected from light. The test article was formulated as a solution in purified water: The test substance was slowly added to purified water while being stirred for approximately 10 minutes. Water solubility was approximately 290 g/L, at which point the test article resulted in a gel. As a result, formulations were not required to be stirred before or throughout dosing. No correction factor was applied.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity of the test item in the vehicle were confirmed for formulations of 1 and 250 mg/mL at 15 - 25°C for 14 days as part of a previous study (Covance, study no. 8422999). In the present study, formulations prepared for the use during weeks 1, 2 and 6 were analysed for the achieved concentration. Triplicate samples were collected from the middle of the test article formulation. The mean achieved concentrations were 111 – 112% (week 1), 96 – 102% (week 2) and 99 – 105% (week 6) of the nominal concentration with relative standard deviations ranging from 0.18 – 0.35% (week 1), 0.21 – 0.91% (week 2) and 0.11 – 0.77% (week 6). The values of weeks 2 and 6 fell into the accepted range of 90 – 110% of the nominal concentration with a relative standard deviation of ≤ 5.0% and were therefore considered acceptable. The values of 111 - 112% of the nominal concentration observed for test item formulations of week 1 were slightly above the accepted range, but due to the small magnitude of deviation considered not to impact the study outcome.
Duration of treatment / exposure:
Males were dosed for 42 consecutive days: 2 weeks prior to pairing, 2 weeks during the pairing phase, and 2 weeks post-pairing.
Females were dosed for up to 57 days: 2 weeks prior to pairing, during pairing, throughout gestation, and up to lactation Day 13. 3/10 females at 0 mg/kg bw/day, 4/10 females at 100 mg/kg bw/day, 3/10 females at 300 mg/kg bw/day and 2/10 females at 1000 mg/kg bw/day were not dosed on gestation Day 21/22 due to signs of parturition.
Frequency of treatment:
daily, 7 days/week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were chosen based on the results of a preliminary dose range-finding study in rats o the same strain (Covance study no. 8421688), in which dose levels of 100 – 1000 mg/kg bw/day were administered daily for up to 14 days. Dose levels up to 1000 mg/kg bw/day were well tolerated with only marginal reductions in body weight and food consumption, as well as observations of jaw chomping, mouth rubbing, paddling and excessive salivation.
A second dose range-finding study was performed in the pregnant rat using the same dose levels (Covance study no. 8417090). Again, dose levels up to 1000 mg/kg bw/day were well tolerated with only transiently lower food consumption following administration of 1000 mg/kg bw/day during early pregnancy. No foetal variations or malformations were observed.
Based on the observations of the previous studies, the limit dose level was set to 1000 mg/kg bw/day. The intermediate dose level of 300 mg/kg bw/day was selected based on guideline requirements of a 2- to 4-fold increase and anticipated to be a NOAEL. The low-dose level of 100 mg/kg bw/day was anticipated to be an alternate NOAEL in case of adverse effects noted at the intermediate dose level.
- Fasting period before blood sampling for clinical biochemistry: The animals were fasted overnight prior to necropsy. Accordingly, blood samples for haematology and clinical chemistry and terminal body weights were collected from fasted animals.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Health observations were made twice daily (beginning and end (nominal) of the working day). Clinical examinations were made once daily from the first day of dosing until necropsy.
- Cage side observations included: signs associated with dosing, clinical signs of ill-health and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males were assessed once prior to treatment and once weekly thereafter. Females were assessed once prior to treatment, once weekly during pre-pairing and pairing phases and on gestation Day 0, 7, 14 and 20, as well as on lactation Days 1, 7 and 13.
- Detailed clinical observations included: Detailed physical examinations and observations within the home cage, in the hand and in the arena.

BODY WEIGHT: Yes
- Time schedule for examinations in males: Twice prior to dosing (4 and 1 days prior to dosing), on the first day of dosing and at weekly intervals thereafter, on the day prior to necropsy (Day 42) and at necropsy (Day 43, fasted body weight).
- Time schedule for examinations in females: Twice prior to dosing (4 and 1 days prior to dosing), on the first day of dosing, weekly prior to pairing, weekly until confirmation of mating, on gestation Days 0, 7, 14 and 20, on lactation Days 0, 1, 4, 7 and 13 and at scheduled necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations in males: Weekly prior to pairing and during the post-pairing phase until the day prior to necropsy.
- Time schedule for examinations in females: Weekly prior to pairing, from gestation Days 0 – 7, 7 – 14 and 14 – 20 and from lactation Days 1 – 4, 4 – 7 and 7 – 13.

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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination (study Day 43 in males and lactation Day 14 in females), blood samples were collected from the abdominal aorta.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals.
- Parameters examined: hemoglobin, red blood cell count, packed cell volume, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, reticulocyte count, red cell distribution width, hemoglobin distribution width, total and differential white cell count, platelet count, platelet crit, mean platelet volume, platelet distribution width, fibrinogen, prothrombin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination (study Day 43 in males and lactation Day 14 in females), blood samples were collected from the abdominal aorta.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals.
- Parameters examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, total bilirubin, bile acids, urea, creatinine, glucose, total cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, total protein, albumin, globulin and albumin/globulin ratio.

THYROID HORMONE ANALYSIS: Yes
- Time schedule for collection of blood: At termination (on study Day 43 in males and on lactation Day 14 in females), blood samples were withdrawn from the jugular vein in males and from the abdominal aorta in females.
- Anaesthetic used for blood collection: In adult animals, blood was collected under isoflurane anesthesia.
- Animals fasted: Yes
- How many animals: All animals.
- Parameters examined: thyroxine (T4) and thyroid stimulating hormone (TSH).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups examined: Qualitative assessments were undertaken for 5 selected animals/sex/group (the first 5 males/group and the first 5 littered females/group) during Week 6 of the dosing period for males (post pairing Day 9) and on lactation Day 13 for females. Motor activity was assessed in an automated photocell activity recorder (Kinder Motor Monitor system) for 60 min for males and 30 min for females for 5 selected animals/sex/group (the first 5 males/group and the first 5 littered females/group) during Week 6 of the dosing period for males and on lactation Days 10 ± 2 for females.
- Battery of functions tested: sensory activity, hindlimb foot splay, grip strength (forelimb and hindlimp grip strength) and motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE:
Under isoflurane anesthesia, the major blood vessels were severed to exsanguinate the animals. Males were sacrificed on Day 43 of the dosing phase (post-pairing day 14). Females were sacrificed on lactation Day 14. Females that failed to produce a litter were sacrificed on Day 26 post-coitum.

GROSS PATHOLOGY:
All adult animals were subject to a full macroscopic examination, and all lesions were recorded.

ORGAN WEIGHTS:
The following organ weights were collected for the first 5 F0 males and for the first 5 lactating F0 females: adrenals, brain (including cerebrum, cerebellum and pons), epididymides, heart, kidneys, liver, ovary (weighed together with oviducts), pituitary, prostrate, seminal vesicles with coagulating glands, spleen, testes (including tunica albuginea, tissue weighed as a pair), thymus, thyroid with parathyroids and uterus with cervix.
The following organ weights were collected from all remaining adult animals, excluding the decedent and non-pregnant females: epididymides, ovary, pituitary, prostate, seminal vesicle with coagulating glands, testis (including tunica albuguinea), thyroids with parathyroids and uterus with cervix.

HISTOPATHOLOGY:
Tissues were retained in 10% neutral-buffered formalin. Epididymides and testes were fixed in modified Davidson’s fixative; eyes were fixed in Davidson’s fluid fixative. After fixation, the tissues listed below were embedded in paraffin wax (block stage), sectioned at 5 µm and stained with hematoxylin and eosin.

The following tissues were examined histopathologically for the first 5 F0 males and the first 5 F0 females that produced a litter in the control and high dose group: adrenals, aorta, brain (including cerebrum, cerebellum and pons), cecum, colon, duodenum, eyes, ), epididymides, femur with bone marrow (including femerotibial joint, gut-associated lymphoid tissue (GALT)/Peyer’s patch, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs with main stem bronchi and bronchioles, lymph node (mandibular and mesenteric) mammary gland, muscle (biceps femoris), optic nerve, sciatic nerve, oesophagus, ovary, oviduct, pituitary, prostrate, rectum, seminal vesicles with coagulating glands, spinal cord (cervical, lumbar and thoracic), spleen, sternum with bone marrow, stomach, testes (including tunica albuginea, tissue weighed as a pair), thymus, thyroid with parathyroids, trachea, urinary bladder, uterus with cervix and vagina.

The following tissues were examined histopathologically for all remaining F0 animals of the control and high dose group: epididymides, gross lesions, mammary gland, ovary, oviduct, prostate, seminal vesicle with coagulating glands, testis (including tunica albuguinea), thyroid with parathyroid, uterus with cervix and vagina.
In addition, the stomach was histopathologically examined from the first 5 F0 males and the first 5 F0 females that produced a litter in the low and intermediate dose group.
Statistics:
Please refer to the attached document "Statistical analysis_OECD 422" under "Attached background material".
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Excessive salivation was observed in males of the 1000 mg/kg bw/day group pre-pairing, during pairing and post-pairing. The same observations were made in males of the 300 mg/kg bw/day group post-pairing. The observation made immediately after dose administration and thereafter throughout the whole study period.
In addition, 1/10 males of the 1000 mg/kg bw/day group was recorded with audible respiration, decreased body tone and thin appearance during the pre-pairing phase. Audible respiration was also observed in 1/9 females on lactation Days 10 and 11.
On gestation Day 23, 1/10 females of the high dose group showed decreased activity, red staining around the urogenital region, piloerection and pallor of extremities. The observations were consistent with dystocia and the animal was sacrificed for humane reasons.
Post-dose observations upon return to the home cage comprised incidences of head burrowing in males at ≥ 300 mg/kg bw/day and in females at 1000 mg/kg bw/day during the pre-pairing period only. Mouth rubbing was observed in males at 100, 300 and 1000 mg/kg bw/day and in females at 300 and 1000 mg/kg bw/day. In addition, incidences of paddling were observed in males and females of the high dose group. The observations of mouth rubbing and paddling were made throughout the whole study period, except for paddling, which was noted in females from pre-pairing throughout gestation, but not during lactation. During gestation, excessive salivation was also noted for females of the high dose group.
The findings of salivation, mouth rubbing and paddling behaviour were attributed to the unpalatable nature or consistency of the test article formulation rather than systemic toxicity and considered non-adverse.
For details on clinical signs of toxicity, please refer to Table 1 under “Any other information on results incl. tables”.

Detailed clinical examination:
During the first 2 study weeks (pre-pairing phase), there was a decrease in activity noted for males (9/10) and females (4/10) at 1000 mg/kg bw/day. A decrease in activity was further noted for 1/10 males at 100 and for 2/10 males at 300 mg/kg bw/day. During pairing, decreased activity was observed among males of all dose groups (2/10, 3/10, 3/10 and 4/10 at 0, 100, 300 and 1000 mg/kg bw/day). After pairing, activity remained decreased in 1/10 males at 300 and in 3/10 males at 1000 mg/kg bw/day. The observations in males were attributed to treatment with the test item and accompanied by incidences of piloerection, mouth rubbing, brown discharge from the anus (pre-pairing phase only), hunched posture (pre-pairing phase only) and salivation.
In addition, squinting of the eyes was observed pre-pairing in males at 300 (1/10) and 1000 mg/kg bw/day (2/10). In females, incidences of vocalization were noted throughout the whole study period among all dose and control groups. During gestation, females of the highest dose level showed increased incidences of mouth rubbing (4/10 animals). For details, please refer to Table 7 under “Any other information on results incl. tables”.
Mortality:
mortality observed, treatment-related
Description (incidence):
10/10, 9/10, 9/10 and 9/10 females at 0, 100, 300 and 1000 mg/kg bw/day survived until the end of the study.
At 1000 mg/kg bw/day, 1/10 females was sacrificed on GD 23 due to decreased activity, red coloration of the urogenital regions, piloerection, and pallor of the extremities. These findings were consistent with dystocia. In combination with other findings in animals of this group (i.e. a marginal increase in gestation length and a higher than expected incidence of stillborn pups), the finding of dystocia was attributed to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no statistically significant changes in absolute body weight noted for any sex at any dose level throughout the study period.
At 1000 mg/kg bw/day, there was a statistically significant reduction in mean body weight gain noted for males during the first study week, although body weight gain was comparable with controls during Week 2. This resulted in a -49% reduction in mean body weight gains during the pre-pairing phase when compared to control animals. The observation was consistent with a reduction in food consumption in the high dose group males during the first study week. Thereafter, during pairing and post-pairing, the body weight gain in the animals of this group recovered and was only slightly reduced (-12%, statistically not significant) at the end of the study period.
In females, mean body weight gain at 1000 mg/kg bw/day was -8% reduced in the first study week when compared to control animals. The finding gained no statistical significance, therefore mean body weight gain in females was considered unaffected at any dose group during the pre-pairing phase.
However, females of the 100 and 1000 mg/kg bw/day group showed a statistically significant reduction in body weight gain during the first week of gestation (-22% and -27% at 100 and 1000 mg/kg bw/day, respectively). At the end of the gestation phase, the body weight of the test item-treated groups was comparable to those of control animals. Females of the 1000 mg/kg bw/day group further showed a statistically significant increase in body weight gain during lactation Days 7 – 13 (+162 %). Body weight gain was still increased in the animals of this group at study termination (+35%, statistically not significant).
The findings in body weight development noted for the high dose group animals were considered treatment-related, but non-adverse and of lower toxicological relevance. There were no treatment-related changes in body weight gain at the lower dose groups.
For details, please refer to Tables 2 and 3 under “Any other information on results incl. tables”.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the pre-pairing period (first week of the study), there was a reduction in food consumption noted for males at ≥ 300 mg/kg bw/day (-17% and -27% at 300 and 1000 mg/kg bw/day, respectively) and in females of the high dose group (-15%) when compared to control animals. The findings were statistically not significant. From the second study week onwards, food consumption was comparable to those of control animals. The findings were related to treatment, but as all animals recovered and no effects were observed during gestation and lactation, the finding was considered non-adverse. For details on food consumption please refer to Table 4 under “Any other information on results incl. tables”.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, there was a statistically significant increase in mean platelet volume (MPV, +4%) and in mean corpuscular volume (MCV, +4%) in male animals. The values obtained for MCV were within the historical control range of the testing laboratory. The values for MPV were all outside the historical control range, including the value for the control group. In the absence of any associated changes in red cell parameters and due to the low magnitude, the changes in MPV and MCV were not attributed to treatment.
At ≥ 300 mg/kg bw/day in males (+5 and + 7%) and at 1000 mg/kg bw/day in females (+15%) there was a statistically significant increase in red cell distribution width (RDW) when compared to control animals. The values remained within the historical control range of the testing laboratory and were therefore considered to have arisen incidentally. For details, please refer to Table 5 under “Any other information on results incl. tables”.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, there was a statistically significant increase in alanine aminotransferase in male animals (+46%) when compared with control animals. In the absence of any other findings, the observation was reported as an adaptive response and not considered to represent an adverse effect.
In females of the high dose group, there was a statistically significant increase in calcium (+5%). The increase was considered to have arisen due to two control values which were lower than the historical control data range (2.48 to 2.81 mmol/L), and all females administered 1000 mg/kg bw/day were with this range. As such, this intergroup was considered to have arisen incidentally.
There were no findings in clinical chemistry parameters at the low and intermediate dose group. For details please refer to Table 6 under “Any other information on results incl. tables”.
Endocrine findings:
not specified
Description (incidence and severity):
The following ED-related parameters were investigated in the study: T4 and TSH level, anogenital distance, epididymides weight and histopathology, oestrus cyclicity, genital abnormalities, liver weight, mammary gland histopathology, ovary weight and histopathology, oviduct histopathology, prostate weight and histopathology, seminal vesicles with coagulating gland weight and histopathology, testes weight and histopathology, thyroid weight and histopathology, uterus (with cervix) weight and histopathology, vagina histopathology, adrenals weight and histopathology, brain weight, pituitary weight and histopathology, fertility, foetal development, gestation length, litter size, litter/pup weight, number of implantations/ corpora lutea, number of live births, presence of foetal anomalies, reproduction and sex ratio.
For details, please refer to the respective result fields in sections 7.5.1 and 7.8.1 and the endpoint summaries of sections 7.5 and 7.8..
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Quantitative assessment:
At 1000 mg/kg bw/day, there was an increase in latency of first step noted for males during pre-pairing Day 3 and on post-pairing Day 9 (4.0 ± 7.20 and 7.4 ± 22.37, statistically not significant) when compared to control animals (1.2 ± 0.63 on pre-pairing Day 3 and 0.0 ± 0.00 on post-pairing Day 9). In addition, males of the all dose groups and females of the high dose group showed reduction in the incidences of rearing on most occasions. The decrease in rearing was statistically significant for males and females of the high dose group only during the early pre-pairing phase (-70% and -79% in males on pre-pairing Days 3 and 9 and -46% in females on pre-pairing Day 2) and during pairing in males (-58% on pairing Day 2), and on gestation Days 0 and 20 in females (-39% and -58%).
In addition, urine pools were statistically significantly increased in males at 300 and 1000 mg/kg bw/day the first days after pairing (1.1 ± 0.99 at 300 mg/kg bw/day and 0.6 ± 0.70 at 1000 mg/kg bw/day vs. 0.0 ± 0.00 in control animals, respectively). An increase in urine pools was also noted for males of other dose groups and during pre-pairing and post-pairing, however, the effects did not reach statistical significance.
On lactation Day 13, Fore limb grip strength was increased in females at ≥ 100 mg/kg bw/day in 2/3 tests and at ≥ 300 mg/kg bw/day in 3/3 tests. In the high dose group, the increase in fore limb grip strength was +31%, +38% and +28% in the first, second and third test, respectively, reaching statistical significance in the first test only.
The quantitative assessment findings observed for the high dose group animals were attributed to treatment, however, as the observations were only transiently observed, not considered as adverse. There were no findings on the number of faecal boli, hind limb grip strength or hind limb foot splay observed for any sex of any dose group. For details please refer to Tables 8, 9 and 10 under “Any other information on results incl. tables”.

Motor activity:
At 300 and 1000 mg/kg bw/day, there was a statistically significant increase in basic movement (+26% and +28%), total ambulations (+57% and +69%), total rears (+28% and +42%) and total distance travelled (+36% and +48%) in males during the first 10 minutes of observation when compared to control animals. The number of total ambulations in the high dose group males remained high during the first 30 minutes, but was no longer statistically significant. Increases were also noted for fine movement of males at ≥ 300 mg/kg bw/day (+16% at 300 and at 1000 mg/kg bw/day), during the first 10 minutes of observation, but the values reached not statistical significance. An increase in total distance travelled was further noted for females of the high dose group during the first 10 minutes of observation (+26%) when compared to control animals, but the increase was not statistically significant. The findings in males of the intermediate and high dose group were considered treatment-related but non-adverse.
There were no treatment-related findings on motor activity in females at any dose group. Within the first 10 minutes of observation, the females of the low dose group showed a statistically significant decrease in basic movements, fine movements, total ambulations, total rears and total distance travelled, but in the absence of a dose response relationship the findings were considered as incidental. There were no effects on habituation to the test environment. For details please refer to Tables 11 and 12 under “Any other information on results incl. tables”.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no changes in organ weights noted for any sex at any dose level.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, 7/8 males showed an irregular surface of the stomach. In addition, an isolated occurrence of thick cardia/limiting ridge was noted in 1/5 males at 300 mg/kg bw/day and in 1/8 males administered 1000 mg/kg bw/day. A thick gelatinous cardia was also noted in 1/5 males administered 300 mg/kg bw/day. The findings correlated with histopathological abnormalities and were attributed to the test item. There were no remarkable findings in females. For details please refer to Table 13 under “Any other information on results incl. tables”.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At 300 and 1000 mg/kg bw/day, microscopical abnormalities were noted in the stomach of male and female animals. 4/5 males at 300 mg/kg bw/day, 8/8 males at 1000 mg/kg bw/day, 2/5 females at 300 mg/kg bw/day and 5/5 females at 1000 mg/kg bw/day showed squamous cell hyperplasia of mild to marked degree. Squamous cell hyperplasia was characterised by generally diffusely increased thickness of the forestomach epithelium, that sometimes led to a rugose appearance.
Minimal to moderate hyperkeratosis was observed in 3/5 males at 300 mg/kg bw/day, 8/8 males at 1000 mg/kg bw/day, 1/5 females at 300 mg/kg bw/day and 3/5 females at 1000 mg/kg bw/day. Hyperkeratosis generally accompanied such hyperplasia, and was characterised by diffuse, prominent thickening of the superficial keratin layer.
In addition, inflammation of the forestomach of minimal to slight degree was observed in 4/5 males at 300 mg/kg bw/day, 7/8 males at 1000 mg/kg bw/day, 0/5 females at 300 mg/kg bw/day and 1/5 females at 1000 mg/kg bw/day. Forestomach inflammation was characterised by minor submucosal mixed inflammatory cell infiltrate, sometimes with accompanying edema and hyperemia.
The findings were dose-related, increased in severity at higher dose levels and correlated with macroscopic abnormalities observed in these animals. The findings were attributed to the irritant effect of the test item. The non-glandular stomach is a feature of the rodent stomach and serves as a reservoir for continual digestion. The low-grade changes noted were considered consistent with minor ongoing irritation, possibly due to prolonged exposure to a minor irritant. No evidence of similar irritation was noted in other regions of the gastrointestinal tract, and no evidence of major irritation, such as ulceration, hemorrhage, or severe inflammation, was noted. Furthermore, no evidence of accompanying stress was noted in affected animals. The forestomach has no counterpart in humans or most other species; as such, these findings were considered of unlikely significance to humans.
For details please refer to Table 14 under “Any other information on results incl. tables”.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone analysis:
There was a statistically significant increase in T4 levels (+35%) in females at 1000 mg/kg bw/day when compared to control animals. The values were within the range of the laboratory's historical control range and therefore considered not treatment-related. For details please refer to Table 15 under "Any other information on results incl. tables".
Key result
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
clinical signs
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Critical effects observed:
no

Table 1: Incidences of clinical findings in adults

Dose group (mg/kg bw/day) Males Females
0 100 300 1000 0 100 300 1000
Clinical observations pre-pairing: Discharge, excessive salivation
mild, clear 0/10 0/10 0/10 4/10 0/10 0/10 0/10 0/10
Clinical observations during pairing: Discharge, excessive salivation
mild, clear 0/10 0/10 0/10 4/10 0/10 0/10 0/10 0/10
Clinical observations: Discharge, excessive salivation post-pairing in males, during gestation (G) and lactation (L) in females
mild, clear 0/10 0/10 3/10 6/10 G: 0/10
L: 0/10
G: 0/10
L: 0/9
G: 0/10
L: 0/9
G: 1/10
L: 0/9
Post-dose observations upon return to home cage after dosing: pre-pairing
head burrowing 0/10 0/10 1/10 3/10 0/10 0/10 0/10 5/10
mouth rubbing 0/10 5/10 10/10 10/10 0/10 0/10 10/10 10/10
paddling 0/10 0/10 0/10 1/10 0/10 0/10 0/10 2/10
Excessive salivation: mild, clear 0/10 0/10 0/10 3/10 0/10 0/10 0/10 3/10
Excessive salivation: moderate, clear 0/10 0/10 0/10 2/10 0/10 0/10 0/10 1/10
Post-dose observations upon return to home cage after dosing: during pairing
mouth rubbing 0/10 1/10 9/10 10/10 0/10 0/10 1/10 3/10
paddling 0/10 0/10 0/10 4/10 0/10 0/10 0/10 1/10
Excessive salivation: mild, clear 0/10 0/10 3/10 8/10 0/10 0/10 0/10 0/10
Excessive salivation: mild, colorless 0/10 0/10 0/10 3/10 0/10 0/10 0/10 0/10
Excessive salivation: moderate, colorless 0/10 0/10 0/10 1/10 0/10 0/10 0/10 0/10
Post-dose observations upon return to home cage after dosing: post-pairing in males, during gestation (G) and lactation (L) in females
mouth rubbing 0/10 2/10 10/10 10/10 G: 0/10
L: 0/10
G: 0/10
L: 1/9
G: 8/10
L: 4/9
G: 10/10
L: 3/9
paddling 0/10 0/10 0/10 9/10 G: 0/10
L: 0/10
G: 0/10
L: 0/9
G: 2/10
L: 0/9
G: 4/10
L: 0/9
Excessive salivation: mild, colorless 0/10 0/10 0/10 2/10 G: 0/10
L: 0/10
G: 0/10
L: 0/9
G: 0/10
L: 0/9
G: 4/10
L: 0/9
Excessive salivation: mild, translucent 0/10 0/10 2/10 5/10 G: 0/10
L: 0/10
G: 0/10
L: 0/9
G: 0/10
L: 0/9
G: 2/10
L: 0/9

Table 2: Summary of body weight change (mean ± standard deviation) in adult males

Dose group (mg/kg bw/day) Males
0 100 300 1000
PRP 1 - 8 18.6 ± 6.38 27.4 ± 5.48 16.4 ± 10.21 -0.4 ± 11.30 ***
% control 100 147 88 -102
PRP 1 - 15 34.4 ± 9.94 44.9 ± 8.70 34.0 ± 10.91 17.6 ± 10.93 **
% control 100 131 99 51
PRP 15 - PR 14 14.2 ± 8.68 20.6 ± 8.86 22.2 ± 8.03 22.1 ± 6.35
% control 100 145 156 156
PR 14 - PP 13 19.9 ± 7.43 25.2 ± 8.91 21.8 ± 9.39 20.4 ± 5.04
% control 100 127 110 103
PRP 1 - PP 13 68.5 ± 18.76 90.7 ± 18.28* 77.9 ± 17.96 60.0 ± 15.53
% control 100 132 114 88
PRP: pre-pairing; PR: pairing; PP: post-pairing; *, ** and ***: statistical significance at p < 0.05, p < 0.01 and p < 0.001 level

Table 3: Summary of body weight change (mean ± standard deviation) in adult females

Dose group (mg/kg bw/day) Females
0 100 300 1000
PRP 1 - 8 13.1 ± 5.47 16.1 ± 3.91 14.9 ± 3.85 12.1 ± 12.37
% control 100 123 114 92
PRP 1 - 15 22.8 ± 6.79 26.2 ± 4.04 22.2 ± 7.56 26.2 ± 9.20
% control 100 115 97 115
GD 0 - 7 26.7 ± 4.92 20.7 ± 6.49* 21.5 ± 3.13 19.6 ± 5.43*
% control 100 78 81 73
GD 7 - 14 26.7 ± 3.95 25.0 ± 8.10 22.4 ± 4.84 28.4 ± 7.19
GD 14 - 20 54.0 ± 11.95 59.1 ± 11.79 60.9 ± 9.93 56.1 ± 8.91
GD 0 - 20 107.5 ± 11.71 104.8 ± 16.26 104.9 ± 11.19 104.0 ± 13.48
% control 100 97 98 97
LD 0 - 1 -2.9 ± 6.43 2.0 ± 4.55 3.1 ± 7.56 4.5 ± 5.02
LD 1 - 4 11.6 ± 5.53 8.9 ± 6.97 10.3 ± 7.90 13.9 ± 8.31
LD 4 - 7 10.3 ± 5.00 12.4 ± 7.87 9.2 ± 6.14 7.1 ± 7.92
LD 7 - 13 6.6 ± 8.90 12.2 ± 8.45 11.9 ± 2.35 17.3 ± 7.65**
% control 100 185 180 262
LD 1 - 13 28.4 ± 13.19 33.5 ± 9.95 31.4 ± 6.40 38.4 ± 6.20
% control 100 118 111 135
PRP: pre-pairing; GD: gestation day; LD: lactation day *, ** and ***: statistical significance at p < 0.05, p < 0.01 and p < 0.001 level

Table 4: Food consumption findings in adults

Dose group (mg/kg bw/day) Males Females
0 100 300 1000 0 100 300 1000
PRP 1 - 8 23.2 ± 1.19 26.0 ± 2.71 19.3 ± 9.24 17.1 ± 2.96 15.3 ± 0.76 15.0 ± 1.22 15.2 ± 0.67 13.0 ± 1.58
% control 100 112 83 73 100 98 99 85
PRP 8 - 15 23.2 ± 1.24 25.3 ± 2.54 23.7 ± 0.94 23.1 ± 3.34 16.1 ± 0.75 15.4 ± 1.18 15.9 ± 0.63 16.4 ± 1.32
% control 100 109 102 100 100 96 99 102
PR 1 - 15 23.2 ± 1.19 25.6 ± 2.61 21.5 ± 5.06 20.1 ± 2.59 15.7 ± 0.74 15.2 ± 1.20 15.6 ± 0.63 14.7 ± 1.41
% control 100 110 93 87 100 97 99 94
PP 1 - 12 22.5 ± 1.34 25.4 ± 2.19 24.3 ± 1.58 23.6 ± 1.29 not applicable
% control 100 113 108 105
GD 0 - 20 not applicable 20.9 ± 1.24 20.0 ± 2.30 20.6 ± 1.18 20.2 ± 1.76
% control 100 95 98 97
LD 1 - 13 45.3 ± 7.50 46.8 ± 5.70 46.9 ± 3.86 43.0 ± 9.44
% control 100 103 103 95
PRP: pre-pairing; PR: pairing; PP: post-pairing; GD: gestation day; LD: lactation day

Table 5: Findings in haematology parameters in adults

Dose group (mg/kg bw/day) Males Females
0 100 300 1000 0 100 300 1000
MPV (fL) 9.64 ± 0.292 9.88 ± 0.238 9.92 ± 0.140 10.03 ± 0.287** 8.77 ± 0.298 8.70 ± 0.296 8.89 ± 0.387 8.93 ± 0.411
% control 100 102 103 104 100 99 101 102
HCD 7.5 - 9.3 not reported
MCVG (fL) 52.45 ± 1.640 52.88 ± 1.244 52.62 ± 1.638 54.50 ± 1.198* 57.67 ± 1.811 57.53 ± 1.865 57.40 ± 1.677 59.03 ± 1.728
% control 100 101 100 104 100 100 100 102
HCD 50.7 - 58.0 not reported
RDWG (%) 12.63 ± 0.427 12.73 ± 0.249 13.28 ± 0.565* 13.49 ± 0.610** 12.46 ± 1.374 12.60 ± 0.781 12.88 ± 0.520 14.30 ± 1.365*
% control 100 101 105 107 100 101 103 115
HCD 11.6 - 19.7 11.8 - 15.3
HCD: historical control data generated in the testing facility from 2013 - 2020
MPV: mean platelet volume; MCVG: mean corpuscular volume gated; RDWG: red cell distribution width gated
* and **: statistical significance at p < 0.05 and p < 0.01 

Table 6: Clinical chemistry findings in adults

Dose group (mg/kg bw/day) Males Females
0 100 300 1000 0 100 300 1000
ALTP (IU/L) 48.4 ± 7.95 47.0 ± 15.39 46.6 ± 9.05 . 64.4 ± 9.00 68.2 ± 11.18 75.7 ± 18.45 81.8 ± 20.42
% control 100 97 96 146 100 106 118 127
HCD 22 - 63 45 - 95
CAL (mmol/L) 2.441 ± 0.0619 2.398 ± 0.0777 2.466 ± 0.1127 2.427 ± 0.0776 2.580 ± 0.1324 2.594 ± 0.0527 2.583 ± 0.1022 2.705 ± 0.0878*
% control 100 98 101 99 100 101 100 105
HCD not reported not reported
HCD: historical control data generated in the testing facility from 2013 - 2020
ALTP: alanine aminotransferase; CAL: calcium
* and **: statistical significance at p < 0.05 and p < 0.01 

Table 7: Findings at detailed clinical examination of adults

Dose group (mg/kg bw/day) Males Females
0 100 300 1000 0 100 300 1000
Pre-pairing phase
Decreased activity, mild 1/10 1/10 2/10 7/10 0/10 0/10 0/10 4/10
Decreased activity, moderate 0/10 0/10 0/10 2/10 0/10 0/10 0/10 0/10
Decreased activity, severe 0/10 0/10 0/10 1/10 0/10 0/10 0/10 0/10
Increased activity, mild 0/10 0/10 0/10 0/10 1/10 1/10 1/10 0/10
Piloerection, mild 0/10 0/10 0/10 6/10 0/10 0/10 0/10 1/10
No response to stimuli 0/10 0/10 0/10 2/10 0/10 0/10 0/10 0/10
Mouth rubbing 0/10 0/10 0/10 4/10 0/10 0/10 0/10 0/10
Brown discharge from the anus 0/10 0/10 0/10 4/10 0/10 0/10 0/10 0/10
Squinting eyes 0/10 0/10 1/10 2/10 0/10 0/10 0/10 0/10
Hunched posture, mild 0/10 0/10 0/10 1/10 0/10 0/10 0/10 0/10
Hunched posture, moderate 0/10 0/10 0/10 2/10 0/10 0/10 0/10 0/10
High stepping gait 0/10 0/10 0/10 0/10 0/10 2/10 0/10 1/10
Salivation, mild 0/10 0/10 0/10 3/10 0/10 0/10 0/10 0/10
Vocalisation, mild or moderate 1/10 0/10 0/10 1/10 1/10 3/10 3/10 2/10
Pairing phase
Decreased activity, mild 2/10 3/10 3/10 4/10 0/10 0/10 0/10 0/10
Decreased activity, moderate 0/10 0/10 0/10 1/10 0/10 0/10 0/10 0/10
No response to stimuli 0/10 0/10 0/10 1/10 0/10 0/10 0/10 0/10
Mouth rubbing 0/10 0/10 0/10 2/10 0/10 0/10 0/10 0/10
Piloerection, mild 0/10 0/10 0/10 2/10 0/10 0/10 0/10 0/10
Salivation, mild 0/10 0/10 0/10 3/10 0/10 0/10 0/10 0/10
Vocalisation, mild or moderate 1/10 1/10 1/10 1/10 1/10 1/10 2/10 0/10
Post pairing phase in males, gestation (G) and lactation (L) phase in females
Decreased activity, mild 2/10 0/10 1/10 3/10 G: 0/10
L: 0/10
G: 1/10
L: 0/9
G: 0/10
L: 0/9
G: 0/10
L: 0/9
Decreased activity, moderate 0/10 0/10 0/10 1/10 G: 0/10
L: 0/10
G: 0/10
L: 0/9
G: 0/10
L: 0/9
G: 0/10
L: 0/9
Mouth rubbing 0/10 0/10 0/10 1/10 G: 0/10
L: 0/10
G: 0/10
L: 0/9
G: 0/10
L: 0/9
G: 0/10
L: 3/9
Piloerection, mild 0/10 1/10 2/10 3/10 G: 0/10
L: 0/10
G: 0/10
L: 0/9
G: 1/10
L: 0/9
G: 0/10
L: 0/9
Salivation, mild 0/10 0/10 2/10 6/10 G: 0/10
L: 0/10
G: 0/10
L: 0/9
G: 0/10
L: 0/9
G: 2/10
L: 0/9
Salivation, moderate 0/10 0/10 0/10 1/10 G: 0/10
L: 0/10
G: 0/10
L: 0/9
G: 0/10
L: 0/9
G: 0/10
L: 0/9
Vocalisation, mild or moderate 1/10 0/10 0/10 2/10 G: 1/10
L: 0/10
G: 3/10
L: 0/9
G: 1/10
L: 1/9
G: 2/10
L: 0/9

G: gestation; L: lactation

Table 8: Findings of quantitative assessment in adult males

Dose group (mg/kg bw/day) Males
0 100 300 1000
Quantitative assessment
Latency to first step
PRP 3 1.2 ± 0.63 1.1 ± 0.74 1.1 ± 0.32 4.0 ± 7.20
PRP 10 0.2 ± 0.42 0.1 ± 0.32 1.0 ± 2.83 0.4 ± 0.97
PR 8 0.5 ± 0.97 0.1 ± 0.32 0.3 ± 0.67 0.1 ± 0.32
PP 9 0.0 ± 0.00 0.7 ± 1.25 0.6 ± 1.07 7.4 ± 22.37
Rears
PRP 3 8.1 ± 4.93 4.4 ± 2.67 5.0 ± 3.09 2.4 ± 2.84**
% control 100 54 62 30
PRP 10 6.8 ± 5.39 4.0 ± 3.43 4.1 ± 4.01 1.4 ± 1.78**
% control 100 59 60 21
PR 1 11.6 ± 10.21 9.1 ± 3.76 8.0 ± 3.43 6.9 ± 4.91
% control 100 78 69 59
PR 2 9.3 ± 4.92 9.3 ± 6.29 4.4 ± 3.10* 3.9 ± 2.47*
% control 100 100 47 42
PR 8 6.5 ± 5.58 4.2 ± 3.01 3.7 ± 2.06 3.5 ± 2.95
% control 100 65 57 54
PP 9 6.5 ± 3.69 5.0 ± 2.45 3.9 ± 2.42 3.3 ± 3.02
% control 100 77 60 51
Urine pools
PRP 10 0.1 ± 0.32 0.3 ± 0.48 0.4 ± 0.70 0.4 ± 0.70
PR 8 0.5 ± 0.97 0.1 ± 0.32 0.3 ± 0.67 0.1 ± 0.32
PP 2 0.0 ± 0.00 0.4 ± 0.70 1.1 ± 0.99** 0.6 ± 0.70*
PP 9 0.0 ± 0.00 0.2 ± 0.63 0.3 ± 0.95 0.2 ± 0.42
PRP: pre-pairing; PR: pairing; PP: post-pairing; * and **: statistical significance at p < 0.05 and p < 0.01 level

Table 9: Findings of quantitative assessment in adult females

Dose group (mg/kg bw/day) Females
0 100 300 1000
Quantitative assessment
Rears
PRP 2 10.8 ± 3.36 13.5 ± 4.20 10.7 ± 6.07 5.8 ± 3.05**
% control 100 125 99 54
PRP 9 7.8 ± 4.76 9.3 ± 6.24 10.6 ± 4.88 5.6 ± 3.95
% control 100 119 136 72
PR 1 11.6 ± 10.21 9.1 ± 3.76 8.0 ± 3.43 6.9 ± 4.91
% control 100 78 69 59
GD 0 10.4 ± 3.89 10.3 ± 2.31 10.2 ± 4.64 6.3 ± 2.54*
% control 100 99 98 61
GD 7 9.9 ± 6.15 9.3 ± 3.68 8.9 ± 5.63 7.6 ± 2.99
% control 100 94 90 77
GD 14 7.0 ± 2.91 8.2 ± 2.66 7.4 ± 5.08 6.1 ± 1.35
% control 100 117 106 87
GD 20 6.5 ± 3.06 6.9 ± 2.47 6.8 ± 2.25 2.7 ± 1.16**
% control 100 106 105 42
LD 7 12.7 ± 4.03 15.9 ± 4.59 14.0 ± 8.31 9.1 ± 4.68
% control 100 125 110 72
LD 13 10.1 ± 4.38 12.3 ± 2.78 9.1 ± 6.68 8.4 ± 4.59
% control 100 122 90 83
PRP: pre-pairing; PR: pairing; PP: post-pairing; GD: gestation day; LD: lactation day; * and **: statistical significance at p < 0.05 and p < 0.01 level


Table 10: Findings on fore limp grip strength in adult males on post-pairing Day 9 and in adult females on lactation Day 13

Dose group (mg/kg bw/day) Males Females
0 100 300 1000 0 100 300 1000
Forelimb grip strength
Test 1 (kg) 1.4014 ± 0.22127 1.4826 ± 0.20770 1.5000 ± 0.10490 1.3000 ± 0.24652 0.8878 ± 0.11960 0.8066 ± 0.15506 1.0408 ± 0.22477 1.1608 ± 0.07030*
% control 100 106 107 93 100 91 117 131
Test 2 (kg) 1.4270 ± 0.13200 1.4134 ± 0.19263 1.4260 ± 0.08987 1.2964 ± 0.18747 0.8034 ± 0.25050 1.0132 ± 0.22645 0.9730 ± 0.28845 1.1086 ± 0.12226
% control 100 99 100 91 100 126 121 138
Test 3 (kg) 1.3936 ± 0.14629 1.3734 ± 0.12651 1.3974 ± 0.07662 1.3520 ± 0.15491 0.9052 ± 0.13453 1.1136 ± 0.31880 1.0474 ± 0.06652 1.1574 ± 0.07619
% control 100 99 100 97 100 123 116 128

*: statistical significance at p < 0.05 level

Table 11: Findings in motor activity in adult males

Dose group (mg/kg bw/day) Males
0 100 300 1000
Basic movement
1-10 min 2460 ± 312.2 2254 ± 271.9 3092 ± 532.4* 3158 ± 392.6*
20-30 min 357 ± 126.6 589 ± 150.7 607 ± 168.6 557 ± 267.6
50-60 min 165 ± 93.8 117 ± 65.7 107 ± 61.7 169 ± 124.4
Overall 703 ± 72.1 740 ± 64.5 909 ± 114.5 890 ± 135.0
Fine movement
1-10 min 1869 ± 224.8 1673 ± 184.9 2161 ± 311.2 2159 ± 233.3
20-30 min 290 ± 100.5 461 ± 112.5 481 ± 115.2 398 ± 174.6
50-60 min 137 ± 76.7 91 ± 43.4 97 ± 55.9 130 ± 93.4
Overall 545 ± 50.3 563 ± 35.5 666 ± 58.0 632 ± 93.7
Total ambulations
1-10 min 597 ± 107.3 587 ± 98.1 939 ± 225.7* 1006 ± 174.8*
20-30 min 66 ± 26.2 128 ± 51.5 126 ± 70.4 159 ± 93.8
50-60 min 28 ± 17.2 26 ± 23.6 10 ± 5.9 38 ± 31.1
Overall 159 ± 24.8 177 ± 32.6 244 ± 56.6 259 ± 47.2
Total rears (events)
1-10 min 53 ± 6.3 46 ± 7.3 68 ± 10.4* 75 ± 14.3*
20-30 min 5 ± 2.1 7 ± 2.5 11 ± 4.0 14 ± 8.2
50-60 min 1 ± 0.9 2 ± 1.2 1 ± 1.0 2 ± 1.8
Overall 13 ± 2.1 14 ± 1.2 19 ± 3.4 19 ± 2.8
Total distance travelled (cm)
1-10 min 5181 ± 467.5 5049 ± 585.0 7047 ± 1239.7* 7660 ± 1188.9*
20-30 min 714 ± 254.8 1433 ± 417.6 1424 ± 487.5 1393 ± 781.7
50-60 min 291 ± 166.0 269 ± 179.0 221 ± 130.8 391 ± 307.3
Overall 1475 ± 131.7 1735 ± 231.1 2102 ± 328.7  2106 ± 348.8
*: statistically significant at p < 0.05 level

Table 12: Findings in motor activity in adult females

Dose group (mg/kg bw/day) Females
0 100 300 1000
Basic movement
1-5 min 856 ± 173.5 360 ± 62.0* 677 ± 74.5 933 ± 155.0
15-20 min 322 ± 170.9 343 ± 138.7 217 ± 110.1 426 ± 155.3
25-30 min 204 ± 177.5 96 ± 54.3 424 ± 169.4 306 ± 100.3
Overall 347 ± 111.6 257 ± 30.9 356 ± 59.2 498 ± 121.8
Fine movement
1-5 min 644 ± 113.1 301 ± 55.3* 482 ± 47.7 689 ± 95.4
15-20 min 240 ± 120.4 264 ± 102.0 154 ± 72.2 346 ± 127.0
25-30 min 147 ± 121.9 72 ± 34.0 323 ± 122.8 284 ± 94.9
Overall 263 ± 76.6 206 ± 20.5 265 ± 51.8 398 ± 93.9
Total ambulations
1-5 min 217 ± 61.9 64 ± 17.6* 201 ± 28.2 251 ± 69.0
15-20 min 82 ± 50.9 79 ± 41.0 63 ± 40.7 81 ± 36.5
25-30 min 56 ± 55.5 23 ± 20.5 101 ± 52.4 22 ± 12.9
Overall 85 ± 36.1 52 ± 13.2 92 ± 11.2 101 ± 36.1
Total rears (events)
1-5 min 16 ± 4.0 5 ± 1.5* 15 ± 4.1 20 ± 4.2
15-20 min 5 ± 3.0 6 ± 2.2 5 ± 3.1 6 ± 2.5
25-30 min 3 ± 3.2 1 ± 0.6 6 ± 3.0 2 ± 1.3
Overall 6 ± 1.6 4 ± 1.1 6 ± 1.2 8 ± 2.2
Total distance travelled (cm)
1-5 min 1827 ± 384.5 985 ± 287.3* 1533 ± 189.7 2298 ± 292.3
15-20 min 788 ± 474.1 820 ± 341.8 512 ± 283.9 900 ± 336.9
25-30 min 475 ± 429.1 191 ± 117.3 975 ± 422.4 603 ± 224.7
Overall 783 ± 261.8 615 ± 45.0 797 ± 118.5 1124 ± 250.6
*: statistically significant at p < 0.05 level

Table 13: Gross macroscopical findings in adults

Dose group (mg/kg bw/day) Males Females
0 100 300 1000 0 100 300 1000
Stomach
Gelatinous 0/6 0/5 1/5 0/8 0/5 0/6 0/5 0/5
Irregular surface 0/6 0/5 0/5 7/8 0/5 0/6 0/5 0/5
Red 0/6 0/5 0/5 1/8 0/5 0/6 0/5 0/5
Red area 0/6 0/5 0/5 0/8 0/5 1/6 0/5 0/5
Red focus 1/6 0/5 0/5 0/8 0/5 0/6 2/5 0/5
Striation 0/6 0/5 0/5 0/8 1/5 0/6 0/5 0/5
Thick 0/6 0/5 1/5 1/8 0/5 0/6 0/5 0/5

Table 14: Histopathological findings in adults

Dose group (mg/kg bw/day) Males Females
0 100 300 1000 0 100 300 1000
Stomach
Squamous cell hyperplasia
Grade 1  1/6 0/5 3/5 0/8 1/5 0/5 1/5 3/5
Grade 2  0/6 0/5 1/5 1/8 0/5 0/5 1/5 2/5
Grade 3  0/6 0/5 0/5 5/8 0/5 0/5 0/5 0/5
Grade 4  0/6 0/5 0/5 2/8 0/5 0/5 0/5 0/5
Hyperkeratosis
Grade 1  0/6 0/5 3/5 0/8 0/5 0/5 0/5 2/5
Grade 2  0/6 0/5 0/5 1/8 1/5 0/5 1/5 1/5
Grade 3  0/6 0/5 0/5 7/8 0/5 0/5 0/5 0/5
Inflammation forestomach
Grade 1  0/6 0/5 3/5 3/8 0/5 0/5 0/5 0/5
Grade 2  0/6 0/5 1/5 4/8 0/5 0/5 0/5 1/5
1 = minimal; 2 = slight; 3 = moderate; 4 = marked

Table 15: Findings in thyroid hormone T4 in adult males on post-pairing Day 14 and in adult females on lactation Day 14

Dose group (mg/kg bw/day) Males Females
0 100 300 1000 0 100 300 1000
Thyroxin (T4, nmol/L) 62.0 ± 14.92 77.4 ± 17.69 73.5 ± 13.00 62.2 ± 18.60 43.0 ± 11.75 43.8 ± 11.13 41.6 ± 8.23 57.9 ± 15.87*
% control 100 125 119 100 100 102 97 135
HCD not reported < 26 - 79 nmol/L
* statistically significant at p < 0.05 level; HCD: historical control data generated in the testing laboratory from 2013 - 2020
Conclusions:
Under the conditions of the study, the NOAEL for systemic toxicity was 300 mg/kg bw/day for male and female rats. The NOAEL was chosen based on the effects on physical health of the animals including effects on behavior due to the irritancy following administration of 1000 mg/kg bw/day.
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 May 2020 - 26 Jul 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted in 2018
Deviations:
yes
Remarks:
pituitary gland, prostate and thyroid weights not recorded, skeletal muscle not histopathologically examined, water consumption not recorded, no HDL + LDL at clinical chemistry, estrous cycles not determined and vaginal cytology not performed
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Margate, United Kingdom
- Age at dosing: 6 – 8 weeks
- Weight at study initiation: 198.8 – 266.7 g (males) and 148.8 – 188.6 g (females)
- Housing: In groups of 3/sex in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes. Aspen wood chips were used as bedding.
- Diet: 5LF2 EU Rodent Diet (International Product Supplies Ltd., London, United Kingdom), ad libitum. A few pellets of diet (removed from the food hopper) were moistened with water for Animal R0109 (Group 2 male) on Days 64 through 67 of the dosing phase (due to clinical observations of missing and/or broken upper teeth) and Animal R0503 (Group 2 female) on Day 91 of the dosing phase (due to body weight loss).
- Water: Main supply water, ad libitum
- Acclimation period: 16 days

DETAILS OF FOOD AND WATER QUALITY:
Each batch of diet and the water was periodically analysed for specific constituents and contaminants. No contaminants were present in the diet and in the water at levels that might have interfered with achieving the objective of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 – 70%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 Jun 2020 To: 25 Sep 2020
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was formulated as a solution in purified water. The test article was slowly added to purified water while stirring, then stirred for approximately 10 minutes. Formulations were prepared weekly and stored at room temperature (15 - 25°C) in a sealed container.

VEHICLE
- Concentration in vehicle: 1, 10 and 30 mg/mL
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity of the test item in the vehicle were confirmed for formulations of 1 and 250 mg/mL at 15 - 25°C for 14 days as part of a previous study (Covance study no. 8422999). In the present study, formulations prepared for the use during weeks 1 and 13 of the dosing phase were analysed for the achieved concentration. Triplicate samples were collected from the middle of the test article formulation. The mean achieved concentrations were 99 – 108% (week 1) and 99 – 103% (week 13) of the nominal concentration with relative standard deviations ranging from 0.35 – 1.68% (week 1) and 0.64 – 1.76% (week 13). The values fell into the accepted range of 90 – 110% of the nominal concentration with a relative standard deviation of ≤ 5.0% and were therefore considered acceptable.

Duration of treatment / exposure:
90 days
Frequency of treatment:
daily, 7 days/week
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of two preliminary 14-day oral repeated dose toxicity studies and on the results of a preliminary 4 week dietary administration study.
In the first preliminary 14-days oral repeated dose toxicity study (no study reference given), the animals were administered 62.5, 250 or 1000 mg/kg bw/day. No toxicologically significant changes were observed for clinical chemistry, hematological, or coagulation parameters; organ weights; or macroscopic findings in animals up to and including 1000 mg/kg bw/day.
In the second study of this type (2 weeks dose-range finding study, Covance study no. 8422999), oral gavage of 100, 300 and 1000 mg/kg bw/day resulted in reduced body weight gain in males at ≥ 300 mg/kg bw/day and in reduced food consumption at 1000 mg/kg bw/day, however, no overall body weight loss was noted. Clinical observations, such as mouth rubbing, paddling, and jaw chomping, were noted in both sexes administered 1000 mg/kg bw/day, and these animals were provided with a moist diet from Day 5 of the dosing phase. Clinical observations were alleviated after addition of a moist diet, and the findings observed were considered attributable to the taste of the test article and not toxicological effects.
In the 4-week daily administration study (ECHA, 2020), the animals were administered 100, 300 or 1000 mg/kg bw/day. No adverse clinical examination, neurotoxicity assessment, body weight, or food consumption observations were noted. Macroscopically and microscopically, test article-related findings were restricted to changes in the forestomach of animals (mainly males) administered 1000 mg/kg bw/day. An increase in white blood cell count was noted, especially in males administered 1000 mg/kg bw/day, and this was considered secondary to inflammatory changes due to the observed forestomach findings in high-dose animals. The NOAEL obtained in the 4-weeks oral toxicity study was ≥ 1000 mg/kg bw/day.
Based on these initial findings, 300 mg/kg bw/day was considered to represent an acceptable high dose for a longer term study.

- Fasting period before blood sampling for clinical biochemistry: The animals were fasted overnight.


Reference: The European Chemicals Agency https://echa.europa.eu/registration-dossier/-/registered-dossier/14336/7/6/1 (accessed 15 May 2020).
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: After dosing, the animals were observed 0.5, 1, 2 and 4 hours upon return to their home cage during weeks 1 and 2 and 3 times a week during weeks 3 - 13. During the course of the study, all animals were observed at the beginning and end (nominal) of each working day for signs of ill health or overt toxicity. Each animal was given a detailed physical examination once daily at the time of dosing and on the day of terminal necropsy.
- Examinations included: clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during the pre-dose phase and once weekly during the dosing phase.
- Examinations included: behaviour, gait, posture, repiration, secretion, excretion, involuntary movements, skin, tail, eyes, pelage and activity.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of all animals were recorded on Day 1 of the pre-dose phase, 3 times weekly during Weeks 1 and 2 of the dosing phase, once weekly from Weeks 3 - 13 of the dosing phase and before scheduled necropsy. Animals scheduled for necropsy on Day 93 of the dosing phase were subjected to overnight fasting in error from Day 91 to 92 of the dosing phase; therefore, the same animals needed to be subjected to overnight fasting on a second occasion from Day 92 to Day 93 of the dosing phase prior to necropsy on Day 93 of the dosing phase. This meant that affected animals were subjected to 2 consecutive days of fasting.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-dosing and during study week 12.
- Dose groups that were examined: control and high dose group animals only.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from the abdominal aorta at scheduled necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, the animals were fasted overnight.
- How many animals: all animals in all groups
- Parameters examined: haemoglobin, red blood cell count, packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, reticulocyte count, red cell distribution width, total and differential white cell count, platelet count, prothrombin time, fibrinogen and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from the abdominal aorta at scheduled necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, the animals were fasted overnight.
- How many animals: all animals in all groups
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, alkaline phosphatase, total cholesterol, triglycerides, bilirubin, total protein, albumin, globulin, albumin:globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, creatinine, urea, creatine kinase, glucose

URINALYSIS: Yes
- Time schedule for collection of urine: during week 12 of the dosing phase.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined: volume, color, turbidity, specific gravity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood and microscopy of sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 12 of the dosing phase.
- Dose groups that were examined: all groups.
- Battery of functions tested: locomotor activity

IMMUNOLOGY: No

OTHER: Thyroid Hormone Analysis
- Time schedule for collection of blood: Blood samples were collected from the abdominal aorta at scheduled necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, the animals were fasted overnight.
- How many animals: all animals in all groups
- Parameters examined: triiodothyronine (T3), thyroxin (T4) and thyroid stimulating hormone (TSH)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals were subjected to necropsy on study Days 92 – 93 of the dosing phase after an overnight period without food. The animals were administered isoflurane anaesthesia and exsanguinated by severing the major blood vessels. All organs and tissues were observed macroscopically.
The following organs were weighed: adrenals, brain, epididymides, heart, kidneys, liver, ovaries with oviducts, spleen, testes, thymus and uterus with cervix.


HISTOPATHOLOGY: Yes
The following organs and tissues were collected from all animals of all groups and preserved in 10% neutral-buffered formalin. Bone designated for microscopic examination was decalcified using Kristenson’s fluid and epididyides and ovaries were preserved in modified Davidson’s fluid fixative and in Davidson fluid fixative, respectively.
Adrenals, aorta, brain, cecum, colon, duodenum, epididymides, esophagus, eyes, femur with bone and marrow and femorotibial joint, gut-associated lymphoid tissue, Peyer’s patches, gross lesions, head (not processed), heart, ileum, jejunum, kidneys, liver, lungs with main stem bronchie and bronchioles, lymph nodes (mandibular and mesenteric), mammary glands, mandibular salivary glands, optic nerve, sciatic nerve, ovaries, pancreas, pituitary, prostate, rectum, seminal vesicle with coagulating glands, skin and subcutis, spinal cord (cervical, lumbar and thoracic), spleen, sternum with marrow, stomach, testis, thymus, thyroid with parathyroids, trachea, urinary bladder, uterus with cervix and vagina.
For the control and high dose groups, all tissues and organs were embedded in paraffin wax BP (block stage), sectioned at 5 µm and stained with haematoxylin and eosin for histopathological examination. For the low and mid dose groups, only the gross lesions, kidneys, livers and spleens were processed, but only gross lesions were examined microscopically.
Other examinations:
Seminology:
For each scheduled sacrifice male, sperm number, motility, and velocity were recorded by Computer Assisted Sperm Analysis (CASA) from a sample of sperm from one epididymis and epididymis cauda. Each sperm sample was prepared for microscopic evaluation of sperm morphology. The slides prepared for the first 10 surviving males from each group were read for morphological changes.
Statistics:
For details on statistical analysis please refer to the document "Statistical analysis" under "Attached background material".
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Post-dosing, mouth rubbing was observed in 0/10, 0/10, 10/10 and 10/10 males and in 0/10, 1/10, 6/10 and 10/10 females at 0, 10, 100 and 300 mg/kg bw/day. In addition, excessive salivation was observed in 7/10 males and 6/10 females of the high dose group. During the course of the study, excessive salivation remained within the animals of both sexes (8/10 males and 4/10 females at 300 mg/kg bw/day, respectively). Increased incidences of mild or moderate vocalization were observed predominantly in females of all treatment groups, but occurred also in females of the control group. Further observations included jaw chomping post-dosing and decreased activity, red discharge from the nose, piloerection, hunched posture and audible respiration in animals among all groups. Jaw chomping, mouth rubbing and excessive salivation were attributed to the palatability of the test article. Without a dose-response relationship, the findings were considered to be incidental and non-adverse. For details please refer to Table 1 under “Any other information on results incl. tables”.
At detailed clinical examination, mild to moderate decreases in activity or mild increases in activity, high stepping gait, escape attempts, vocalization, eye squinting or closure, constricted pupil status and/or no or slow pupillary responses were occasionally observed among animals of all treatment and control groups. In the absence of any dose-response relationship, the observations were not attributed to treatment.
For details please refer to Table 2 under “Any other information on results incl. tables”.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Fluctuations in body weight and body weight gain were occasionally observed during the course of the study in males and females of all treatment and control groups. However, the changes were transient, of small magnitude and did not reach statistical significance. As overall body weight gain was noted for all animals, the observations were not considered to be of any biological relevance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Fluctuations in food consumption was occassionally observed during the course of the study in male and female animals of all groups. On study Days 50 - 53, males of the high dose group showed a statistically significant increase in food consumption (+9% when compared to control animals). Females of the high dose group showed a statistically significant increase in food consumption on study Days 8 - 11 (+15% when compared to control animals). The findings were transient, of small magnitude and not correlated to any changes in body weight development and therefore considered non-adverse. For details please refer to Table 3 under "Any other information on results incl. tables".
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no remarkable observations noted in either sex.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
When compared to control animals, males showed increases in white blood cell count (+18, +18 and +35%) and in lymphocytes (+21, +24 and +45%) at 10, 200 and 300 mg/kg bw/day, reaching statistical significance at the highest dose level. In females, there were slight increases in lymphocytes (+14, +12 and +18%, statistically not significant) and in platelets (+6, +7 and +17%, statistically significant at the highest dose level) at 10, 200 and 300 mg/kg bw/day. In addition, an increase in absolute reticulocytes (+5, +6 and +12% at 10, 100 and 300 mg/kg bw/day, respectively, statistically not significant) was noted in females. The changes were considered indicative of an infection response, but as no organ weight or macroscopic or microscopic changes occurred, these changes were, therefore, considered non adverse. For details please refer to Table 4 under “Any other information on results incl. tables”.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
An increase in inorganic phosphate was noted in males at all dose levels (+18.8%, +12.5% and + 31.3% at 10, 100 and 300 mg/kg bw/day, respectively), reaching statistical significance for the high dose group only. In the absence of any macroscopic or microscopic findings, the observation was considered to be non-adverse. For details please refer to Table 5 under “Any other information on results incl. tables”.
Endocrine findings:
not specified
Description (incidence and severity):
The following ED-related parameters were investigated in the study: T3, T4 and TSH level, epididymides weight and histopathology, liver weight, mammary gland histopathology, ovary weight and histopathology, prostate histopathology, seminal vesicles with coagulating gland histopathology, sperm morphology, motility and numbers, testes weight and histopathology, thyroid histopathology, uterus (with cervix) weight and histopathology, vagina histopathology, adrenals weight and histopathology, brain weight and pituitary histopathology.
For details, please refer to the respective result fields and the endpoint summary.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
When compared to control animals, an increased urine volume was noted in males (+38%, statistically not significant) and females (+76%, statistically significant) administered 300 mg/kg/day. In the absence of any findings in the kidneys the increase was considered non-adverse. For details please refer to Table 6 under “Any other information on results incl. tables”.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Changes in motor activity were made in males and females of all dose groups and were evident as increased basic movements, changes in fine movements, total ambulations, total rears and total distance travelling. Although statistical significance was reached for several intervals during the observation period, the observations were mainly noted at early intervals soon after handling and there was no dose-response relationship. Therefore, the findings were attributed to handling rather than to treatment. For details please refer to Table 7 under “Any other information on results incl. tables”.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There was a statistically significantly increased relative liver to brain weight (+13%) in males of the high dose group when compared to control animals and a statistically significantly increased relative adrenals to body weight (+19%) in females of the high dose group when compared to control animals. The findings were attributed to normal biological variation, of small magnitude, inconsistent between both sexes and, in the absence of any correlating histopathological findings, not related to treatment with the test item. For details please refer to Table 8 under “Any other information on results incl. tables”.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings that were related to treatment. All macroscopic findings were generally consistent with the usual background pattern of findings in rats of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no histopathological findings that were related to treatment. All microscopic findings were generally consistent with the usual background pattern of findings in rats of this strain and age and therefore considered to be incidental.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormone level:
In males and females there was an increase in T4 levels at 100 mg/kg bw/day (+20% and +14%, statistically not significant). There was further an increase in TSH levels in males at 10, 100 and 300 mg/kg bw/day (+75, +144 and +69%). In the absence of any thyroid weight organ weight changes, without any dose-response relationship and without any histopathological findings, the observations were considered to be non-adverse. For details please refer to Table 9 under "Any other information on results incl. tables".

Seminology:
There were no adverse findings observed at any dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Critical effects observed:
no

Table 1: Clinical findings

Dose group (mg/kg bw/day) Males Females
0 10 100 300 0 10 100 300
Decreased activity, mild 0/10 0/10 0/10 1/10 0/10 0/10 0/10 0/10
Increased activity, mild 0/10 0/10 0/10 0/10 0/10 0/10 1/10 0/10
Vocalisation, mild to moderate 1/10 0/10 0/10 1/10 3/10 2/10 4/10 5/10
Excessive salivation 0/10 0/10 0/10 8/10 0/10 0/10 0/10 4/10
Red discharge from the nose 0/10 0/10 0/10 1/10 0/10 0/10 0/10 0/10
Pelage 0/10 1/10 0/10 0/10 1/10 1/10 0/10 0/10
Piloerection 0/10 0/10 0/10 0/10 0/10 1/10 0/10 0/10
Hunched posture 0/10 0/10 0/10 0/10 0/10 1/10 0/10 0/10
Audible respiration 0/10 0/10 1/10 0/10 0/10 0/10 0/10 1/10
Scabs, dorsal cervical 2/10 1/10 1/10 0/10 1/10 0/10 1/10 0/10

Table 2: Findings at detailed clinical examination

Dose group (mg/kg bw/day) Males Females
0 10 100 300 0 10 100 300
Decreased activity, mild to moderate 3/10 4/10 1/10 3/10 1/10 2/10 1/10 2/10
Increased activity, mild 0/10 1/10 0/10 1/10 5/10 4/10 4/10 3/10
Gait, high stepping 0/10 0/10 0/10 0/10 3/10 2/10 2/10 1/10
Escape attempt 0/10 0/10 0/10 1/10 8/10 7/10 7/10 6/10
No approach response 1/10 2/10 1/10 1/10 0/10 0/10 0/10 0/10
Vocalisation, mild 1/10 1/10 0/10 2/10 2/10 1/10 3/10 4/10
Squiniting eyes 0/10 1/10 0/10 0/10 0/10 0/10 0/10 0/10
Eye closure 0/10 1/10 1/10 1/10 0/10 0/10 0/10 0/10
Constricted pupils 0/10 2/10 0/10 0/10 0/10 0/10 0/10 0/10
No or slow pupillary response 1/10 0/10 1/10 0/10 0/10 0/10 0/10 0/10

Table 3: Food consumption findings

Dose group (mg/kg bw/day) Males Females
0 10 100 300 0 10 100 300
Days 1-4 21.0 ± 0.52 20.1 ± 0.83 20.7 ± 0.99 21.9 ± 1.23 13.6 ± 1.35 13.3 ± 1.25 13.4 ± 0.61 14.1 ± 0.62
% control 100 96 99 104 100 98 99 104
Days 8-11 22.2 ± 0.87 21.5 ± 1.00 21.8 ± 1.62 23.6 ± 1.66 16.6 ± 1.32 15.9 ± 1.29 15.4 ± 0.59 19.1 ± 1.30*
% control 100 97 98 106 100 96 93 115
Days 32-36 19.8 ± 0.66 19.5 ± 0.71 19.6 ± 0.96 20.9 ± 2.28 14.5 ± 1.25 14.0 ± 0.95 13.5 ± 0.47 14.0 ± 1.13
% control 100 98 99 106 100 97 93 97
Days 50-53 19.3 ± 0.96 19.3 ± 0.52 20.1 ± 0.55 21.1 ± 0.83* 14.4 ± 1.32 13.8 ± 0.42 14.3 ± 0.40 14.3 ± 0.32
% control 100 100 104 109 100 96 99 99
Days 85-88 21.6 ± 2.00 20.7 ± 0.67 22.2 ± 0.71 22.2 ± 1.74 16.5 ± 0.94 15.7 ± 0.74 15.7 ± 1.43 16.5 ± 0.49
% control 100 96 103 103 100 95 95 100
*: p < 0.05% statistically significant when compared to control animals

Table 4: Haematology findings

Dose group (mg/kg bw/day) Males Females
0 10 100 300 0 10 100 300
WBC (10E9/L) 3.4 ± 0.68 4.0 ± 1.03 4.0 ± 0.89 4.6 ± 1.07* 2.4 ± 0.71 2.7 ± 1.33 2.6 ± 1.11 2.8 ± 0.74
% control 100 118 118 135 100 113 108 117
L (10E9/L) 2.55 ± 0.961 3.08 ± 0.774 3.17 ± 0.723 3.70 ± 0.920* 1.91 ± 0.641 2.17 ± 1.157 2.16 ± 1.038 2.25 ± 0.724
% control 100 121 124 145 100 114 113 118
PLT (10E9/L) 815 ± 67.8 860 ± 77.5 805 ± 106.1 858 ± 79.4 761 ± 70.9 803 ± 98.7 822 ± 49.5 889 ± 66.2*
% control 100 106 99 105 100 106 108 117
RETG (10*9/L) 194.8 ± 25.94 186.8 ± 16.39 188.7 ± 28.69 207.9 ± 22.95 204.8 ± 36.25 215.8 ± 18.22 216.9 ± 28.25 228.9 ± 30.53
% control 100 96 97 107 100 105 106 112
WBC: white blood cell count; L: lymphocytes; PLT: platelets; RETG: absolute reticulocytes gated from the retic method, *: p < 0.05

Table 5: Clinical chemistry findings

Dose group (mg/kg bw/day) Males Females
0 10 100 300 0 10 100 300
P (mmol/L) 1.6 ± 0.34 1.9 ± 0.37 1.8 ± 0.23 2.1 ± 0.35* 1.7 ± 0.17 1.6 ± 0.35 1.7 ± 0.20 1.8 ± 0.27
% control 100 119 113 131 100 94 100 106
P: Inorganic phosphate, *: p < 0.05

Table 6: Urinalysis findings

Dose group (mg/kg bw/day) Males Females
0 10 100 300 0 10 100 300
Volume 17.2 ± 4.98 13.2 ± 4.62 17.3 ± 6.49 23.7 ± 9.81 10.2 ± 5.28 9.5 ± 5.44 9.7 ± 5.53 18.0 ± 7.26*
% control 100 77 101 138 100 93 95 176
*: p < 0.05% statistically significant when compared to control animals

Table 7: Findings in motor activity

Dose group (mg/kg bw/day) Males Females
0 10 100 300 0 10 100 300
Basic movements, 10 Min Intervals
1-10 min 2532 ± 245.3 2998 ± 174.9* 2831 ± 226.0 3006 ± 215.0* 1946 ± 191.1 2693 ± 167.7* 2098 ± 179.7 2836 ± 143.0*
% control 100 118 112 119 100 138 108 146
10-20 min 1090 ± 200.8 1062 ± 125.7 899 ± 113.6 1281 ± 184.2 848 ± 91.8 1438 ± 129.3* 1031 ± 143.0 1497 ± 128.8*
% control 100 97 82 118 100 170 122 177
40-50 min 304 ± 118.7 373 ± 120.2 499 ± 134.0 402 ± 174.2 724 ± 189.1 337 ± 109.4 308 ± 148.4 200 ± 71.7*
% control 100 123 164 132 100 47 43 28
Overall 895 ± 125.0 981 ± 98.2 849 ± 83.2 991 ± 92.0 785 ± 90.1 967 ± 77.7 756 ± 89.5 960 ± 69.8
% control 100 110 95 111 100 123 96 122
Fine movements, 10 Min Intervals
1-10 min 1922 ± 176.4 2174 ± 128.5 2062 ± 153.0 2168 ± 152.0 1233 ± 114.1 1671 ± 99.9* 1281 ± 92.8 1669 ± 84.9*
% control 100 113 107 113 100 136 104 135
10-20 min 866 ± 156.3 823 ± 97.6 712 ± 92.3 965 ± 126.4 600 ± 71.9 991 ± 82.6* 705 ± 94.8 1000 ± 93.4*
% control 100 95 82 111 100 165 118 167
20-30 min 674 ± 209.7 530 ± 162.7 317 ± 72.6* 552 ± 136.8 319 ± 93.2 600 ± 121.1 374 ± 62.9 492 ± 86.2
% control 100 79 47 82 100 188 117 154
40-50 min 255 ± 55.5 222 ± 82.6 139 ± 54.9 98 ± 68.7 504 ± 125.6 233 ± 68.5 217 ± 96.4 164 ± 58.7*
% control 100 87 55 38 100 46 43 33
Overall 706 ± 101.7 736 ± 74.0 641 ± 60.7 732 ± 62.5 538 ± 56.1 648 ± 52.6 500 ± 57.2 621 ± 42.9
% control 100 104 91 104 100 120 93 115
Total Ambulations, 10 Min Intervals
1-10 min 617 ± 78.0 831 ± 51.8* 775 ± 86.8* 846 ± 94.0* 718 ± 89.4 1029 ± 79.8* 822 ± 97.8 1173 ± 77.9*
% control 100 135 126 137 100 143 114 163
10-20 min 223 ± 47.0 238 ± 31.0 188 ± 28.0 315 ± 64.0 248 ± 23.7 448 ± 51.1* 326 ± 51.7 496 ± 43.7*
% control 100 107 84 141 100 181 131 200
40-50 min 49 ± 30.0 93 ± 32.4 107 ± 29.5 102 ± 49.6 220 ± 66.7 105 ± 42.7 92 ± 53.7 36 ± 19.1*
% control 100 190 218 208 100 48 42 16
Overall 189 ± 28.2 246 ± 25.8 209 ± 25.0 260 ± 35.2 248 ± 35.4 320 ± 29.3 257 ± 35.0 340 ± 30.1*
% control 100 130 111 138 100 129 104 137
Total Rears, 10 Min Intervals
1-10 min 57 ± 6.8 68 ± 4.7 57 ± 5.8 76 ± 9.5* 43 ± 5.6 72 ± 6.9* 50 ± 5.9 66 ± 6.1*
% control 100 119 100 133 100 167 116 153
10-20 min 20 ± 5.7 17 ± 2.6 15 ± 3.4 26 ± 6.0 16 ± 2.4 30 ± 3.9* 15 ± 3.4 30 ± 4.7*
% control 100 85 75 130 100 188 94 188
Overall 17 ± 2.9 19 ± 2.6 16 ± 3.0 24 ± 4.2 15 ± 1.9 22 ± 3.0 14 ± 2.0 20 ± 2.5
% control 100 112 94 141 100 147 93 133
Total Distance Travelled, 10 Min Intervals
1-10 min 5352 ± 539.4 6590 ± 377.1* 6327 ± 477.9 7080 ± 474.0* 4777 ± 502.6 6826 ± 517.4* 5386 ± 524.5 7416 ± 383.1*
% control 100 123 118 132 100 143 113 155
10-20 min 2375 ± 468.8 2172 ± 229.6 2014 ± 258.4 3112 ± 424.5 1987 ± 205.3 3452 ± 333.5* 2538 ± 383.6 3761 ± 320.3*
% control 100 91 85 131 100 174 128 189
40-50 min 592 ± 242.5  848 ± 286.0 1271 ± 350.4 1044 ± 478.7 1831 ± 512.8 816 ± 289.3 720 ± 371.6* 429 ± 167.3*
% control 100 143 215 176 100 45 39 23
Overall 1922 ± 207.9 2149 ± 224.0 1958 ± 218.9 2386 ± 235.7 1876 ± 237.0 2354 ± 214.8 1866 ± 242.0 2402 ± 191.8
% control 100 112 102 124 100 125 99 128
*: p < 0.05% statistically significant when compared to control animals

Table 8: Organ weight changes

Dose group (mg/kg bw/day) Males Females
0 10 100 300 0 10 100 300
Liver
Absolute (g) 10.152 ± 1.0200 9.563 ± 0.9217 9.775 ± 1.0839 11.160 ± 1.1386 6.531 ± 0.9934 6.150 ± 0.3832 6.205 ± 0.9053 6.812 ± 0.9205
% control 100 94 96 110 100 94 95 96
Rel. to BW (%) 2.4064 ± 0.16248 2.4029 ± 0.16263 2.3299 ± 0.18324 2.5596 ± 0.14997 2.6251 ± 0.30243 2.6264 ± 0.31503 2.5585 ± 0.25556 2.7243 ± 0.21435
% control 100 100 97 94 100 100 97 104
Rel. to BrW (%) 482.8346 ± 53.13543 473.2668 ± 50.03644 470.5565 ± 49.10419 544.5306 ± 47.94408* 348.6549 ± 57.73961 329.5487 ± 21.97858 323.4173 ± 47.06962 356.4685 ± 48.70617
% control 100 98 97 113 100 95 93 102
Adrenals
Absolute (g) 0.060 ± 0.0076 0.054 ± 0.0056 0.058 ± 0.0084 0.058 ± 0.0071 0.060 ± 0.0109 0.068 ± 0.0104 0.057 ± 0.0098 0.061 ± 0.0076
% control 100 91 97 97 100 113 95 102
Rel. to BW (%) 0.0142 ± 0.00186 0.0135 ± 0.00191 0.0139 ± 0.00224 0.0133 ± 0.00144 0.0243 ± 0.00420 0.0288 ± 0.00454* 0.0236 ± 0.003700.0247 ± 0.00378
% control 100 95 98 94 100 119 97 102
Rel. to BrW (%) 2.8469 ± 0.37682 2.6883 ± 0.30918 2.7967 ± 0.41218 2.8338 ± 0.31064 3.1990 ± 0.51918 3.6225 ± 0.47282 2.9690 ± 0.46550 3.2105 ± 0.41703
% control 100 94 98 100 100 113 93 100
*: p < 0.05% statistically significant when compared to control animals; BW: body weight; BrW: brain weight

Table 9: Effects on thyroid hormone levels

Dose group (mg/kg bw/day) Males Females
0 10 100 300 0 10 100 300
T3 (nmol/L) < 0.60 ± 0.143 < 0.66 ± 0.142 0.73 ± 0.154 < 0.63 ± 0.161 0.98 ± 0.085 1.02 ± 0.087 1.03 ± 0.120 1.07 ± 0.154
% control 100 110 122 105 100 104 105 109
T4 (nmol/L) 56 ± 11.3 57 ± 15.7 67 ± 14.8 57 ± 14.1 42 ± 14.8 45 ± 13.3 48 ± 12.0 42 ± 4.2
% control 100 102 120 102 100 107 114 100
TSH (µIU/mL) 0.16 ± 0.051 0.28 ± 0.185 0.39 ± 0.327 0.27 ± 0.202 0.21 ± 0.125 0.17 ± 0.085 0.25 ± 0.167 0.21 ± 0.081
% control 100 175 244 169 100 81 119 100
Conclusions:
Based on the results of the study, the NOAEL for systemic toxicity was ≥ 300 mg/kg bw/day in male and female rats.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises adequate and reliable studies (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.
System:
other: Based on the irritating properties of the test item, adverse findings on neurobehavior, clinical signs of toxicity and macroscopic and microscopic findings in the stomach were observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral repeated dose toxicity:

Subacute:

The test item was investigated in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD guideline 422 and in compliance with GLP (Covance, 2021).

Groups of 10 male and 10 female Crl:WI(Han) rats were exposed daily to the test item by oral gavage at doses of 100, 300 and 1000 mg/kg bw/day. A similar constituted group of 10 male and 10 female rats received the vehicle (purified water) and served as control. The dose levels for the main study were selected based on the results of a preliminary 14-days oral repeated dose range-finding study, as well as on a second preliminary range-finding study performed in pregnant rats.

In the present study, male rats were treated for 42 consecutive days, beginning 2 weeks prior to pairing, 2 weeks during the pairing phase and 2 weeks post-pairing.

Females that delivered were treated daily for up to 57 days: 2 weeks prior to pairing, during pairing, throughout gestation, and up to lactation Day 13. Females that failed to produce viable litter were treated until 25 days after mating. 3/10 females at 0 mg/kg bw/day, 4/10 females at 100 mg/kg bw/day, 3/10 females at 300 mg/kg bw/day and 2/10 females at 1000 mg/kg bw/day were not dosed on gestation Day (GD) 21/22 due to signs of parturition.

Observations and examinations of the animals included mortality, clinical signs, detailed physical examination and arena observations, functional neurobehavioral observations (for 5 selected animals/sex/group), body weight, food consumption, haematology, clinical chemistry, measurement of thyroid hormones TSH and T4, gross necropsy, organ weights and histopathology.

Achieved concentrations of the test item formulations in purified water were confirmed by analytical methods. Stability and homogeneity of the formulations in the solvent were confirmed as part of a previous toxicity study.

Treatment-related mortality was restricted to 1/10 females of the high dose group, which was sacrificed on GD 23 due to decreased activity, red coloration of the urogenital regions, piloerection, and pallor of the extremities. These findings were consistent with dystocia. In combination with other findings in animals of this group (i.e. a marginal increase in gestation length and a higher than expected incidence of stillborn pups), the finding of dystocia was attributed to treatment. 10/10, 9/10, 9/10 and 9/10 females at 0, 100, 300 and 1000 mg/kg bw/day survived until the end of the study.

Clinical signs of toxicity immediately after dosing comprised mouth rubbing in males at all dose levels and in females at ≥ 300 mg/kg bw/day and incidences of paddling behaviour in both sexes of the high dose group. In addition, excessive salivation was observed in males of the 1000 mg/kg bw/day group pre-pairing, during pairing and post-pairing and in males of the 300 mg/kg bw/day group post-pairing. The findings of salivation, mouth rubbing and paddling behaviour were attributed to the unpalatable nature or consistency of the test article formulation rather than systemic toxicity and considered non-adverse.

At detailed clinical examination, treatment-related signs of toxicity were predominantly observed in males of the 300 and 1000 mg/kg bw/day group. The effects were observed predominantly during pre-pairing and to a lesser extent throughout the pairing and post-pairing phase in a dose-related manner and comprised decreased activity, piloerection, no response to stimuli, hunched posture, indicating a compromised physical condition. In females of the high dose group, decreased activity was only noted during the pre-pairing phase. 1/10 males of the 1000 mg/kg bw/day group further showed audible respiration, decreased body tone and thin appearance during the pre-pairing phase.

The findings in males were consistent with behavioural changes noted during the FOB evaluations. Quantitative assessment revealed an increase in latency of first step in males at 1000 mg/kg bw/day when compared to control animals at different occasions during the study period, a reduction of rearing in high dose group males and females at most occasions during the course of the study, increased urine pools in males at 300 and 1000 mg/kg bw/day during the first days after pairing and increased grip strength in females of all dose groups during lactation Day 13, reaching statistical significance for the high dose group only. The quantitative assessment findings observed for the high dose group animals were attributed to treatment, however, as the observations were only transiently observed, not considered as adverse.

Statistically significant increases in motor activity basic movements, total ambulations and total distance travelled were noted for males administered 300 and 1000 mg/kg bw/day during the first 10 minutes of testing, indicating increased exploratory behavior. However, the observation was made in males only, females and males habituated to the test environment were unaffected, therefore the changes seen were considered non-adverse and did not represent neurotoxicity.

At 1000 mg/kg bw/day, there was a statistically significant reduction in mean body weight gain noted for males during the first study week, although body weight gain was comparable with controls during Week 2. This resulted in a -49% reduction in mean body weight gains during the pre-pairing phase when compared to control animals. During pairing and post-pairing, the body weight gain in the animals of this group recovered and was only slightly reduced (-12%, statistically not significant) at the end of the study period.

The findings in body weight impairment were consistent with a reduced food intake during the first week of the study, which was observed for males at ≥ 300 mg/kg bw/day (-17% and -27% at 300 and 1000 mg/kg bw/day, respectively) and in females of the high dose group (-15%) when compared to control animals (statistically not significant). From the second study week onwards, food consumption was comparable to those of control animals. The findings were related to treatment, but as all animals recovered and no effects were observed during gestation and lactation, the finding was considered non-adverse.

Haematology findings comprised a statistically significant increase in mean platelet volume (+4%) and in mean corpuscular volume (+4%) in male animals of the high dose group and a statistically significant increase in red cell distribution width in males at 300 and 1000 mg/kg bw/day (+5 and +7%) and in females at 1000 mg/kg bw/day (+15%). All findings were either within the range of the laboratory’s historical control data or of low magnitude. In the absence of any correlating histopathological findings, the changes were considered to be incidental.

Clinical chemistry findings revealed a statistically significant increase in alanine aminotransferase in male animals (+46%) at 1000 mg/kg bw/day when compared with control animals. In the absence of any other findings, the observation was reported as an adaptive response and not considered to represent an adverse effect. In females of the high dose group, there was a statistically significant increase in calcium (+5%) and a statistically significant increase in thyroxin (T4) levels (+35%) when compared to control animals. The findings were both within the range of the laboratory’s historical control data and therefore not attributed to treatment.

At macroscopical examination, 7/8 males of the high dose group were found to have an irregular surface of the stomach. In addition, an isolated occurrence of thick cardia/limiting ridge was noted in 1/5 males at 300 mg/kg bw/day and in 1/8 males administered 1000 mg/kg bw/day. A thick gelatinous cardia was also noted in 1/5 males administered 300 mg/kg bw/day. Organ weights remained unaffected by treatment. At microscopical examination, the findings in the stomach were confirmed to be irritating effects caused by the test item and observed for males and females at ≥ 300 mg/kg bw/day. Squamous cell hyperplasia of mild to marked degree was observed in 4/5 males at 300 mg/kg bw/day, 8/8 males at 1000 mg/kg bw/day, 2/5 females at 300 mg/kg bw/day and 5/5 females at 1000 mg/kg bw/day. Minimal to moderate hyperkeratosis was observed in 3/5 males at 300 mg/kg bw/day, 8/8 males at 1000 mg/kg bw/day, 1/5 females at 300 mg/kg bw/day and 3/5 females at 1000 mg/kg bw/day. In addition, inflammation of the forestomach of minimal to slight degree was observed in 4/5 males at 300 mg/kg bw/day, 7/8 males at 1000 mg/kg bw/day, 0/5 females at 300 mg/kg bw/day and 1/5 females at 1000 mg/kg bw/day.

The findings were dose-related and increased in severity at higher dose. The low-grade changes noted were considered consistent with minor ongoing irritation, possibly due to prolonged exposure to a minor irritant. No evidence of similar irritation was noted in other regions of the gastrointestinal tract, and no evidence of major irritation, such as ulceration, hemorrhage, or severe inflammation, was noted. Furthermore, no evidence of accompanying stress was noted in affected animals.The microscopic forestomach changes were considered of no relevance to humans.
B
ased on the extent of the effects on the physical health of animals, which included effects on behaviour due to irritancy following administration of 1000 mg/kg/day, the no observed adverse effect level (NOAEL) for systemic toxicity was established at 300 mg/kg bw/day for both sexes.

 

 

Subchronic:

The test item was investigated in a subchronic oral repeated dose toxicity study similar to OECD 408 and compliant with GLP (Covance, 2021). The test substance was dissolved in purified water and administered daily to male and female Crl:WI(Han) rats by oral gavage for up to 90 consecutive days. Dose levels were selected based on the results of a two preliminary 2-week (Covance study no. 8422999, second study reference not given) and one 4-week repeated dose range-finding studies (ECHA, 2020).

In the main study, groups of 10 male and 10 female rats were exposed to test item doses of 10, 100 and 300 mg/kg bw/day. A similar constituted group of 10 males and 10 females received the vehicle and served as control. During the 90 days of treatment, the animals were observed twice daily for mortality and clinical signs of toxicity. Body weights were recorded on Day 1 of the pre-dose phase, 3 times weekly during Weeks 1 and 2 of the dosing phase, once weekly from Weeks 3 - 13 of the dosing phase and before scheduled necropsy. Food consumption was recorded twice weekly and ophthalmoscopy was conducted on all animals prior to treatment and during study Week 12. In addition, urinalysis and neurobehavioral examinations were performed during study week 12. At scheduled necropsy (after 91 - 93 days of treatment), blood was collected from all animals for analysis of haematology, clinical chemistry and thyroid hormone analysis. All animals were subjected to a full macroscopic examination. For animals of the control and high dose groups, histopathological examination was performed.

Homogeneity and stability of the test item formulated in vehicle was confirmed by analytical methods. There was no mortality observed during the whole study period.

Post-dosing, mouth rubbing, excessive salivation and incidences of jaw chomping were noted in males and females of all test-item treated groups and attributed to the palatability of the test item. Excessive salivation was observed for the high dose group animals of both sexes (8/10 males and 4/10 females at 300 mg/kg bw/day, respectively) during the entire study period. Increased incidences of mild or moderate vocalization were observed predominantly in females of all treatment groups, but occurred also in females of the control group. Further observations included decreased activity, red discharge from the nose, piloerection, hunched posture and audible respiration in animals among all groups. The findings were not severe, occurred sporadically in a small number of individuals and were mostly observed on one occasion only. Without a dose-response relationship, the findings were considered to be incidental and non-adverse.

The clinical observational measurement and motor activity changes were mainly noted at early intervals, soon after handling, and, therefore, could be attributed to handling. Clinical observations noted in the functional observatory battery (FOB) were also sporadic in nature, with eye closure the only observation noted for all test article-treated groups, but this was limited to one animal at each dose level. Changes noted in the FOB appeared to be sporadic in nature or were not dose-related.

Body weight, body weight development and food consumption were unaffected by treatment. Fluctuations were occasionally observed during the course of the study, but small in magnitude, transient in nature and/or without statistical significance. There were no remarkable observations on ophthalmoscopy noted in either sex.

Haematological findings comprised a statistically significant increase in white blood cell count (+35%) and in lymphocytes (+45%) in males at 300 mg/kg bw/day. In females of the high dose level, there was a statistically significant increase in platelet count (+17%) when compared to control animals and slight increases in lymphocytes (+18%) and reticulocytes (+12%), both statistically not significant. The changes were considered indicative of an infection response, but as no organ weight or macroscopic or microscopic changes occurred, these changes were, therefore, considered non adverse.

Findings in clinical chemistry were limited to a statistically significant increase in inorganic phosphate in males at 300 mg/kg bw/day (+31%). In the absence of any macroscopic or microscopic findings, the observation was considered to be non-adverse. At urinalysis, an increased urine volume was noted in males (+38%, statistically not significant) and females (+76%, statistically significant) administered 300 mg/kg/day. In the absence of any findings in the kidneys the increase was considered non-adverse.

Thyroid hormone analysis revealed no statistically significant findings. An increase in T4 levels in both sexes at 100 mg/kg bw/day (+20% in males and +14% in females) was statistically not significant. In addition, increased TSH levels in males at 10, 100 and 300 mg/kg bw/day (+75, +144 and +69%) were statistically not significant. In the absence of a dose-response relationship and any histopathological findings, the changes in thyorid hormone levels were considered non-adverse.

Organ weight measurements revealed a statistically significant increase in relative liver to brain weight (+13%) in males of the high dose group when compared to control animals and a statistically significant increase in relative adrenals to body weight (+19%) in females of the high dose group when compared to control animals. The findings were attributed to normal biological variation, of small magnitude, inconsistent between both sexes and, in the absence of any correlating histopathological findings, not related to treatment with the test item.

There were no macroscopical and no histopathological findings that were related to treatment. All abnormal findings were generally consistent with the usual background pattern of findings in rats of this strain and age and therefore considered to be incidental.

Based on the experimental findings, the NOAEL for systemic toxicity was 300 mg/kg bw/day in male and female rats.

Justification for classification or non-classification

The available information on oral repeated dose toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.