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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Toxicity to reproduction, screening (OECD 422, rat, m/f): NOAEL reproductive = 1000 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
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:
other: 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 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.
Details on mating procedure:
- M/F ratio per cage: 1:1 from within the same treatment groups.
- Length of cohabitation: Up to 15 days. All females were mated successfully within 7 days.
- Proof of pregnancy: Mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal washing. Upon the confirmation of mating, vaginal lavage was discontinued, and the male was removed from the cage. The day on which mating was confirmed was designated GD 0.
- After successful mating each pregnant female was caged individually.
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
Details on study schedule:
not applicable for an OECD 422 study
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.
Parental animals: Observations and examinations:
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
Oestrous cyclicity (parental animals):
Daily vaginal lavage (washings) was conducted for all females for 14 consecutive days prior to dosing, from the start of dosing until the confirmation of mating, and on the morning of lactation Day 14, prior to necropsy.
Sperm parameters (parental animals):
Organ weights were collected for epididymides, prostate, seminal vesicles with coagulating glands and testes. Sections of testes and epididymides were collected, stained with Periodic Acid-Schiff (PAS) and qualitatively assessed for stages of spermatogenesis and interstitial testicular cell structure.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes, litters were culled to 10 pups/litter, with 5 pups/sex, where possible. Pups were selected for cull randomly, with no bias as to size or clinical status. Culled pups had sex confirmation at necropsy and were discarded following completion of blood sampling. Surplus pups were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose).

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups (daily), stillbirths, live births, postnatal mortality, clinical observations (daily), body weights (on post natal Day (PND) 1, 4, 7 and 13), presence of gross anomalies, anogenital distance (AGD, on PND 4), presence of nipples/areolae in male pups (PND 13). Particular attention was be paid to the external reproductive genitals which were examined for signs of altered development; gross evaluation of external genitalia.


GROSS EXAMINATION OF DEAD PUPS:
Yes, where possible a macroscopic examination (external) with an assessment of stomach for milk content was performed for prematurely dead pups.

THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood: On PND 4, blood from surplus pups was collected by decapitation. On PND 13, blood was collected by cardiac puncture.
- Anaesthetic used for blood collection: No anaesthesia was used.
- Animals fasted: No
- How many animals:
On PND 4: from at least 2 pups/ litter when available
On PND 13: from 2 male and 2 female pups/ litter
- Parameters examined: thyroxine (T4) and thyroid stimulating hormone (TSH).

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
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.
Postmortem examinations (offspring):
SACRIFICE
- Surplus pups culled on post natal Day 4 (PND 4) and pups sent to necropsy on PND 13 were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable plane of anaesthesia was established, PND 4 pups were decapitated and PND 13 pups were exsanguinated by severing a major blood vessel.

GROSS NECROPSY
- Full macroscopic examinations were conducted for all decedents and one pup/sex/litter sacrificed on PND 13. External examinations for macroscopic abnormalities, with particular attention to the external reproductive genitals, were conducted for all remaining pups sent to necropsy on PND 13.

HISTOPATHOLOGY / ORGAN WEIGTHS: Thyroid glands of pups culled on PND 13 (1 pup/sex/litter across group) were weighed approximately 24 h post fixation and preserved in 10% neutral-buffered formalin. Preserved pup thyroid glands from the control and high dose group were embedded in paraffin wax (block stage), sectioned at 5 µm and stained with hematoxylin and eosin for microscopical examination.
Statistics:
Please refer to the attached document "Statistical analysis_OECD 422" under "Attached background material".
Reproductive indices:
Female mating index (%) = (Mated females / Females cohabitated excluding females sacrificed during cohabitation) x 100
Male mating index (%) = (Number of males mated with at least 1 female / Number of males cohabitated with at least 1 female) x 100
Female fecundity index (%) = (Pregnant females / Mated females excluding females with an undetermined pregnancy status) x 100
Male fecundity index (%) = (Number of males impregnating at least 1 female / Number of males mating with at least 1 female) x 100
Female fertility index (%) = (Pregnant females / Females cohabitated excluding females sacrificed during cohabitation or with an undetermined pregnancy status) x 100
Male fertility index (%) = (Number of males impregnating at least 1 female / Number of males cohabitated with at least 1 female) x 100
Gestation index (%) = (Number of females with live born pups / Number of pregnant females) x 100

In addition, the following parameters were recorded: pre-coital time in males, duration of gestation, number of delivering females, number of females with liveborn and stillborn pups and number of females with no liveborn pups.
Offspring viability indices:
Post-implantation loss (%) = ((Number of implantations – Number of live born pups)/ Number of implantations) x 100
Livebirth index (%) = (Number of live born pups / Number of pups born) x 100
Day 4 viability index (%) = (Number of pups alive on PND 4 pre-cull / Number of pups born alive) x 100

In addition, the following parameters were recorded: number of pups delivered, number of stillborn pups, number of females delivering live pups, number of females died or killed during lactation, number of females removed due to total litter loss, number of females surviving to scheduled sacrifice, number of dead pups, number of pups missing/ presumably cannabalised, number of pups surviving at 13 days, sex ratio (number and mean males % per litter on Day 1, Day 4 pre-cull and Day 13) and number of live pups per litter with live pups (on Day 1, Day 4 pre- and post-cull, Day 7 and Day 13).
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”.
Dermal irritation (if dermal study):
not examined
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 reduced in the first study week (-8%) 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
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".
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
A slight increase in the mean length of oestrous cycles was observed prior to pairing for females administered 1000 mg/kg bw/day when compared with control animals. This increase was due to 3/10 females administered 1000 mg/kg bw/day which were observed with 5 day cycles. Rat oestrous cycles are generally 4 - 5 days in length and at least one 5-day cycle was also recorded for these females prior to the start of dosing; therefore, the observation was not attributed to treatment.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Males:
Pre-coital time: There was a slight shift in increased pre-coital interval observed for males in the 300 and 1000 mg/kg bw/day group when compared to control animals (refer to Table 16 under “Any other information on results incl. tables”). As no treatment-related effect was noted o the oestrus cycle, the slight increase in pre-coital interval was considered to be a secondary effect due to the compromised physical health of the animals, which resulted in a reduced initial desire for males to mate. In addition, all males mated within 7 days of pairing, which is in line with the historical control range of the testing laboratory. Thus, the shift was considered not to represent an adverse or test item-related effect.

Male mating index: There was no effect on the male mating index. Mating indices were 100% for all control and test item-treated groups.

Male fecundity index: There was no effect on male fecundity index. Fecudinty indices were 100%, 90%, 90% and 100% at 0, 100, 300 and 1000 mg/kg bw/day.

Male fertility index: There was no effect on male fertility index. Fertility indices were 100%, 90%, 90% and 100% at 0, 100, 300 and 1000 mg/kg bw/day.

Females:
Female mating index: 2/10 females at 300 mg/kg bw/day and 4/10 females at 1000 mg/kg bw/day all came into oestrus but failed to mate until they were in oestrus for a second time. There was no effect on the female mating index. Mating indices were 100% for all control and test item-treated groups.

Female fecundity index: There was no effect on female fecundity index. Fecudinty indices were 100%, 90%, 90% and 100% at 0, 100, 300 and 1000 mg/kg bw/day.

Female fertility index: There was no effect on female fertility index. Fertility indices were 100%, 90%, 90% and 100% at 0, 100, 300 and 1000 mg/kg bw/day.

Gestation length: At 1000 mg/kg bw/day, gestation length was marginally increased when compared with control animals, with gestation lengths observed to be either 23 or 24 days for the high dose group, versus 22 - 24 days for the control group. Although the values fell within the range of the laboratory’s historical control data (22.7 - 24.2 days), gestation lengths of 22 days were recorded for one or two females from the remaining dose groups, including controls, which are lower than the historical control data range. Based on the findings of this study, the marginal shift to a higher mean gestation length cannot be disregarded and was considered test article-related.
One female administered 1000 mg/kg bw/day (Animal R0709) had 2 stillborn pups and a total litter size of 11 pups; the gestation length for this animal was 23 days. Another female administered 1000 mg/kg bw/day (Animal R0701) had one stillborn pup and a gestation length of 24 days, although this animal only produced a small litter of 6 pups in total. Another female administered 1000 mg/kg bw/day (Animal R0710) had 4 stillborn pups, and a gestation length of 23 days, with an average litter size of 8 pups. It is not unusual to observe stillborn pups following the birth of a large litter or prolonged gestation. The gestation length for Animal R0704 of the 1000 mg/kg bw/day group, which also possibly had a stillborn pup (not confirmed by a lung float test), was also 24 days. The treatment-related finding on gestation length was considered non adverse as the finding was within the laboratory’s historical control data. For details, please refer to Table 17 under “Any other information on results incl. tables”.


Gestation index: At 1000 mg/kg bw/day, there was a slight decrease in gestation index (-10%) when compared to control animals. Gestation indices were 100%, 100%, 100% and 90% at 0, 100, 300 and 1000 mg/kg bw/day. The finding was attributed to 1/10 females with signs of dystocia and considered treatment-related.
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:
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
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
1 000 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
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no effects observed
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, pups of a single litter were noticed to have thin fur (dorsal) on post natal Day 13. The finding was isolated to the one litter and therefore considered to have arisen incidentally.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
There was an increased incidence (statistically not significant) of post-implantation loss noted for the females of the 100 mg/kg bw/day (10.70%) and the1000 mg/kg bw/day dose group (17.00%) when compared to control animals (5.02%). The values of control animals fell within the range of historical control data.
In addition, at 1000 mg/kg bw/day, the number of stillborn pups was increased and outside the laboratory’s historical control range (7 pups at 1000 mg/kg bw/day versus 0-6 pups in historica control range), whereas no stillborn pups were found in the remaining dose groups. Further, 2 dead pups were observed for litters of the high dose group on PND 0 and 1, whereas no dead pups were observed in the remaining dose groups. The finding remained within historical control data of the testing facility (0-5 dead pups on PND 0-4).
There was further a slight increase in the incidence of missing/ presumably cannibalised pups in the animals of this group (0, 2, 1 and 4 pups at 0, 100, 300 and 1000 mg/kg bw/day). The incidences of missing/ presumably cannibalised pups at the low and intermediate dose level was not dose-dependent and considered to have arisen incidentally. The incidences of missing/ presumably cannibalised pups at the high dose level fell within the range of the testing facility's historical control data (0-7 pups).
The observations in the high dose group resulted in a higher number of pup mortality between PND 0 – 4 for litters when compared to control animals. At 1000 mg/kg bw/day, the livebirth index and the Day 4 viability index were slightly reduced (-9% and -7%) when compared to control animals. However, the number of pups in the high dose group litters were within the historical control data range and there was no positive correlation between a larger litter size and stillborn pups.
The observations made in the high dose group were considered treatment-related but non-adverse, due to the lack of statistical significance. Any abnormalities observed were attributed to adverse effects in the dams rather than to represent a reproductive toxicity effect. For details, please refer to Table 18 under “Any other information on results incl. tables”.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no statistically significant changes on pup body weight noted for male and female offspring. At 1000 mg/kg bw/day, pup weight adjusted for litter size on PND 1 was 6% lower for males and 10% lower for females when compared to control animals. By PND 13, mean body weights were 6% or 5% lower than control for pups from litters from males or females administered 1000 mg/kg bw/day, which showed improvement for the females, compared with the PND 1 body weights. Body weight gain was comparable for all groups, hence the marginal changes in body weight were considered non-adverse.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
There were no effects on sexual maturation noted for any sex at any dose level.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There were no changes in ano-genital distance observed. Mean ano-genital distances for pups from litters from females administered 300 or 1000 mg/kg bw/day were longer than those of pups from control animals, but statistical significance was not gained and the values fell within those of historical control data (1.7 - 2.44 mm).
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipple retention was evident for male pups from litters from test article-treated or control females.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There was no effect on thyroid weight or thyroid : body weight noted for pups from any test item or control group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, each one male and one female pup from a single litter were found to have no milk in the stomach. As this finding was confined to a single litter, the observation was not attributed to treatment.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related histopathological findings observed in the thyroids of pups.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis: There were no statistically significant changes in T4 and TSH levels observed. The marginal increase in TSH levels in pups from dams exposed to 300 and 1000 mg/kg bw/day were statistically not significant and remained within those of historical controls (0.12 - 0.58 µIU/mL).
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
1 000 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
Key result
Reproductive 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 levels in adult animals

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

Table 16: Pre-coital intervals noted for adult males

Dose group (mg/kg bw/day) Males
0 100 300 1000
Pre-coital interval (Days) - Number of females mated  1 3/10 1/10 1/10 3/10
2 2/10 3/10 1/10 1/10
3 4/10 5/10 2/10 2/10
4 1/10 0/10 3/10 0/10
5 0/10 0/10 1/10 0/10
6 0/10 1/10 1/10 1/10
7 0/10 0/10 1/10 3/10
Historical control range 1 - 7 days

Table 17: Gestation length in adult females

Dose group (mg/kg bw/day) Females
0 100 300 1000
Duration of gestation (days) 22.9 ± 0.6 22.8 ± 0.4 23.0 ± 0.5 23.2 ± 0.4
Historical control range 22.8 - 24.0 days

Table 18: Parturition and litter data

Dose group (mg/kg bw/day) Females
0 100 300 1000
Number of pups/litter  9.90 ± 3.11 10.78 ± 1.79 10.67 ± 1.58 10.33 ± 2.50
HCD number of pups/litter 4.7 - 15.0
Females with stillborn pups (total) 0 0 0 3
Females with stillborn pups (%) 0 0 0 33.33
Stillborn pups (total) 0 0 0 7
Stillborn pups (mean ± SD) 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.78 ± 1.39
HCD stillborn pups 0 - 6
Dead pups (total) 0 0 0 2
Missing/ presumed cannabalised pups (total) 0 2 1 4
Dead pups, missing or cannibalised Days 0-4 (total) 0 2 1 6
Dead pups, missing or cannibalised Days 5-13 (total) 0 0 0 0
Post-implantation loss (mean ± SD) 0.60 ± 0.84 1.56 ± 2.74 0.44 ± 0.53 1.67 ± 1.87
Post-implantation loss (%) 5.02 10.70 3.99 17.00
Livebirth index (mean %) 100 100 100 91
Day 4 viability index (mean %) 100 98 99 93
HCD: Historical control data of the testing laboratoy from 2013 - 2020; SD: standard deviation
Conclusions:
Under the conditions of the study, the NOAEL for systemic toxicity was 300 mg/kg bw/day for male and female rats, 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.
There was a marginal shift towards a longer gestation length in dams administered 1000 mg/kg bw/day and a marginally lower gestation index in females of the high dose group, which was attributed to a single dam with signs of dystocia in this group. In addition, a slight increase in post-implantation loss was noted at 1000 mg/kg bw/day and the number of stillburn pups was slightly outside the historical control range. Based on these effects, a NOEL of 300 mg/kg bw/day for reprodcutive toxicity was derived in the study report.
The changes in gestation length at 1000 mg/kg bw/day were within those of historical control animals and only a single dam with dystocia was observed in the high dose group. Post-implantation loss observed for the high dose group animals remained within those of historical control data and the number of stillborn pups was only slightly outside those of historical control data. Therefore, the NOAEL for reproductive toxicity was established at 1000 mg/kg bw/day. As no adverse effects on development were observed, the NOAEL for developmental toxicity was 1000 mg/kg bw/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, of Regulation (EC) No. 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction

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.

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 (Covance, study no. 8422999). The following parameters of systemic toxicity were evaluated in the study: 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.

In addition, the following parameters on reproduction were examined: Oestrous cycle, mating and fertility indices (mating index, fecundity index and fertility index for male and female animals), pre-coital intervals, duration of gestation and gestation index.

For details on parental systemic toxicity (mortality, clinical signs, functional observations, haematology, clinical chemistry, organ weights, gross pathology and histopathological findings), please refer to Section 7.5 “Repeated dose toxicity”.

All females were successfully mated. Except for 1/10 females at 100 mg/kg bw/day and 1/10 females at 300 mg/kg bw/day, all females were pregnant and produced a litter. 2/10 females at 300 mg/kg bw/day and 4/10 females at 1000 mg/kg bw/day all came into oestrus but failed to mate until they were in oestrus for a second time. Mating, fecundity and fertility indices in male and female animals were unaffected by treatment.

In males of the 300 and 1000 mg/kg bw/day dose groups, there was a slight shift in increased pre-coital interval observed when compared to control animals. As no treatment-related effect was noted in the oestrus cycle, the slight increase in pre-coital interval was considered to be a secondary effect due to the compromised physical health of the animals, which resulted in a reduced initial desire for males to mate. In addition, all males mated within 7 days of pairing, which is in line with the historical control range of the testing laboratory. Females of the high dose group had a marginally increased gestation length when compared to control animals, with gestation lengths observed to be either 23 or 24 days for the high dose group, versus 22 - 24 days for the control group. Although the values fell within the range of the laboratory’s historical control data, gestation lengths of 22 days were recorded for one or two females from the remaining dose groups, including controls, which were lower than the historical control data range. Based on the findings of this study, the marginal shift to a higher mean gestation length was considered treatment-related. As the finding was within the historical control range, the shift in gestation length was considered non-adverse. The slight decrease in gestation index (-10%) in the animals of the high dose group was attributed to 1/10 females with dystocia.

Post-implantation losses were increased at 100 and 1000 mg/kg bw/day, but the findings gained no statistical significance, remained within those of historical control data and no dose-response relationship was evident.

Based on the reproductive findings observed, the authors concluded a NOEL for reproductive toxicity of 300 mg/kg bw/day for males and female rats.

However, the changes in gestation length at 1000 mg/kg bw/day were within those of historical control animals and only a single dam with dystocia was observed in the high dose group. Post-implantation loss observed for the high dose group animals remained within those of historical control data and the number of stillborn pups was only slightly outside those of historical control data. In conclusion, there was no evidence for adversity in any of the parameters investigated. Therefore, under the conditions of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the NOAEL for reproductive toxicity was established at 1000 mg/kg bw/day for male and female rats.

 

Effects on developmental toxicity

Description of key information

Toxicity to reproduction/development, screening (OECD 422, rat, m/f): NOAEL developmental = 1000 mg/kg bw/day

Developmental toxicity (OECD 414, rat, m/f): NOAEL developmental = 1000 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Jul 2020 - 17 Aug 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 2018
Deviations:
yes
Remarks:
criteria used for categorising fetal external, soft tissue and skeletal examination not specified, only a limit set of historical control data provided, short acclimatisation period as time-mated females were purchased for the study
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Margate, United Kingdom
- Age at the time of mating: 9 – 11 weeks
- Weight at dosing: 180.6 – 258.2 g
- Housing: Individually 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: VRF1 diet (Special Diets Services Ltd., Witham, United Kingdom), ad libitum
- Water: Main tap supply water, ad libitum
- Acclimation period: The animals were delivered to the testing laboratory by gestation Day (GD) 3. Acclimatisation was limited by mated status, but all animals were given a clinical inspection for ill health upon arrival and before the start of dosing to ensure suitability for the study.

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: 24 Jul 2020 To: 17 Aug 2020
Route of administration:
oral: gavage
Vehicle:
water
Details on 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 whilst stirring for approximately 10 minutes. Formulations were prepared weekly and stored at room temperature (15 - 25°C) in a sealed container.
VEHICLE
- Concentration in vehicle: 10, 30 and 100 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 in a previous study and found to be stable over a period of 14 days for formulations of 1 and 250 mg/mL at 15 - 25°C (Covance study no. 8422999). In the present study, formulations prepared for use at the beginning (gestation Day (GD) 6 and end of dosing (GD 20) were analysed to determine the achieved concentration. Triplicate samples were taken from the middle of the test article formulations and were analysed.
The mean achieved concentrations were 110% (GD 6) and 100 - 104% (GD 20) of the nominal concentration with relative standard deviations ranging from 0.33 – 0.53% (GD 6) and 0.28 – 0.60% (GD 20). 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.
Details on mating procedure:
The females were mated prior to arrival in the testing facility.
Duration of treatment / exposure:
Days 6 - 21 of gestation.
Frequency of treatment:
daily, 7 days/week
Duration of test:
Day 21 of gestation
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:
20 time-mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels for the main developmental toxicity study were selected based on the results of a preliminary dose-finding study (Covance study no. 8417090). Pregnant rats were administered dose levels of 100, 300 and 1000 mg/kg bw/day. The test article was well tolerated at all dose levels, with only transiently lower food consumption observed following administration of 1000 mg/kg bw/day during early pregnancy. No fetal variations or malformations were noted; as such, the limit dose of 1000 mg/kg bw/day was considered suitable as the high-dose level for use in the present study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (at the beginning and end of each working day)
- Cage side observations included: mortality, clinical signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination on gestation Days (GD) 3, 6, 7, 8, 9, 12, 15, 17, 19, 20, and 21.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GD 6, 7, 8, 9, 12, 15, 17,19, 20, and 21. An additional, unscheduled body weight was recorded on GD 20 for 1/20 females at 100 mg/kg bw/day.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The amount of food consumed was determined from GD 6 - 7, 7 - 8, 8 - 9, 9 - 12, 12 - 15, 15 - 17, 17 - 19, 19 - 20, and 20 – 21.

WATER CONSUMPTION: No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 21
- Organs examined: Ovaries and uteri

OTHER:
Thyroid Hormone analysis:
- Time schedule for collection of blood: Blood samples were collected from the abdominal aorta at scheduled necropsy on gestation Day 21.
- Anaesthetic used for blood collection: Yes (isoflurane)
- How many animals: all animals in all groups
- Parameters examined: triiodothyronine (T3), thyroxin (T4) and thyroid stimulating hormone (TSH)

Thyroid weights were recorded from the parental females of all groups at scheduled caesarian section.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Pregnancy status, terminal body weight and number of dead fetuses.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
- Other: Anogenital distance and sex ratio
Statistics:
For details on statistical analysis please refer to the document "Statistical analysis_OECD 414" under "Attached background material".
Indices:
Maternal parameters:
Pre-implantation loss (%) = ((Number of corpora lutea – Number of implantations)/Number of corpora lutea) x 100
Post-implantation loss (%) = ((Number of implantations – Number of live embryos)/Number of implantations) x 100
Corrected body weight (Carcasse weight) = Terminal body weight – uterine weight
Corrected weight change = Carcasse weight – gestation Day 6 body weight
Total weight change = Gestation Day 21 body weight – gestation Day 6 body weight

Litter data:
Male fetuses (%) = (Number of male fetuses/Number of fetuses of determined sex) x 100
Historical control data:
Historical data was provided for fetal skeletal variations only to allow comparison with concurrent controls.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Excessive salivation was noted in the animals at 100 mg/kg bw/day and above (in 4/20, 6/20 and 10/20 at 100, 300 and 1000 mg/kg bw/day). In addition, mouth rubbing was observed in animals of the mid (7/20) and high dose groups (20/20). The observations were made immediately after dosing, but were also observed in some animals at the end of the day observations of gestation Day (GD) 20. Isolated incidences of paddling behaviour was noted for 1/20 and 2/20 animals on GD 13 or 15 at 300 and 1000 mg/kg bw/day. The findings were attributed to unpalatability or the unpleasant taste of the test article and therefore considered non-adverse. For details please refer to Table 1 under “Any other information on results incl. tables”.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day there was a statistically significant reduction in body weight gain observed for gestation Days (GD) 9 – 12, 20 – 21 and over the entire treatment period (GD 6 – 21). Initial body weight losses were also observed for the animals of this group, but did not reach statistical significance. The findings were consistent with a reduced food consumption in the animals of this group and attributed to treatment. However, in absence of a detrimental effect on pregnancy or the developing fetus, these findings were considered non-adverse.
No effect on body weight or body weight change was noted following administration of 100 or 300 mg/kg bw/day. For details please refer to Table 2 under “Any other information on results incl. tables”.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day there was a statistically significant reduction in food consumption observed from the start of dosing up to gestation Day (GD) 15 and on the last day of dosing (GD 21). Overall mean food consumption was -15% when compared to control animals. The findings were in line with a reduced body weight development in these animals and related to treatment. However, in the absence of a detrimental effect on pregnancy or the developing fetus, the findings were considered not to represent an adverse effect of the test article.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not specified
Description (incidence and severity):
The following ED-related parameters were investigated in the study: T4 and TSH level, anogenital distance, genital abnormalities, thyroid weight and histopathology, gravid uterus weight, foetal development, litter size, litter/pup weight, number of implantations/ corpora lutea, number of embryonic or foetal deaths and viable foetuses, post-implantation losses, pre-implantation losses, presence of foetal anomalies (external, visceral and skeletal) and sex ratio.
For details, please refer to the respective result fields and the endpoint summary.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test article-related macroscopic findings were recorded for the thyroid of animals administered 100, 300, or 1000 mg/kg bw/day. All tissues were macroscopically unremarkable, or the findings observed were generally consistent with the usual pattern of findings in rat of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test article-related microscopic findings were recorded for the thyroid of animals administered 100, 300, or 1000 mg/kg bw/day. All tissues were microscopically unremarkable, or the findings observed were generally consistent with the usual pattern of findings in rat of this strain and age.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyriod hormone levels were not affected by treatment.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
A total in utero litter loss was noted for 1/20 females at 100 mg/kg bw/day and for 1/20 females at 1000 mg/kg bw/day. The observations were within the laboratories historical control range, occurred without any dose response relationship and, in the absence of any pre- or post-implantation losses, was considered unrelated to treatment.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
1/20 females administered 100 mg/kg bw/day and 1/20 females administered 1000 mg/kg bw/day started parturition prior to the terminal sacrifice. An increased incidence of early parturition was recently observed in this strain of animal from the animal supplier; therefore, this finding was unrelated to the test article.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
1/20 females of the control group and 1/20 females administered 100 mg/kg bw/day were not pregnant. These animals were supplied non-pregnant, and, as such, the lack of pregnancy for these animals was unrelated to the test article.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were observed in 2 fetuses of the control group and in 1 fetus of the 1000 mg/kg bw/day group. Abnormal findings in the control group comprised a shortened 13th rib on the right in a female fetus and a male fetus showing a malrotated hindlimb on the right ankle joint, a malpositioned talus of the right hindlimb and hyperextention of the hindlimb on the right ankle joint. At 1000 mg/kg bw/day, a malrotated hindlimb of the ankle joint was observed bilaterally in 1 male fetus. The findings were considered to be incidental.
At 1000 mg/kg bw/day, reduced incidences of incomplete interparietal ossification of the skull was noted in the fetuses (1.93% of the fetuses) when compared to control animals (16.4% of the fetuses). In addition, incomplete metatarsal ossification of the hindlimbs was occasionally observed in animals of the control and test item-treated groups. The findings were within the range of the laboratories historical control data and thus considered to have arisen incidentally. For details please refer to Table 4 under “Any other information on results incl. tables”.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 100 mg/kg bw/day, diaphragmatic hernia in the muscular region, protruding from the liver was noted in 1 male fetus. At 1000 mg/kg bw/day, 1 male fetus showed edema. The latter finding was considered a congenital abnormality. The fetus was also recorded with adrenal variations, abnormal blood vessels, and dilated brain ventricles, which was associated with the severe edema. The weight of this fetus was also greater than other fetuses maternally exposed at this dose (7.28 g), and the higher fetal weight was most likely due to increased fluid retention. This is an unusual finding; however, as this was a single finding, it was considered congenital and unrelated to the test article.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
1 000 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
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: Summary of clinical signs of toxicity

Dose group (mg/kg bw/day) 0 100 300 1000
Mouth rubbing 0/20 0/20 7/20 20/20
Paddling 0/20 0/20 1/20 2/20
Excessive salivation, mild, clear 0/20 1/20 3/20 4/20
Excessive salivation, mild, colorless 0/20 0/20 3/20 4/20
Excessive salivation, moderate, clear 0/20 0/20 0/20 1/20
Excessive salivation, moderate, colorless 0/20 1/20 0/20 1/20

Table 2: Body weight and body weight gain

Dose group (mg/kg bw/day) 0 100 300 1000
Body weight (g)
BW GD 6 214.5 ± 19.66 211.4 ± 14.10 211.7 ± 14.17 211.4 ± 10.84
% control 100 99 99 99
BW GD 12 235.0 ± 23.15 234.8 ± 15.16 234.3 ± 14.23 226.8 ± 11.08
% control 100 100 100 97
BW GD 21 314.8 ± 32.94 313.0 ± 25.63 312.0 ± 23.41 295.9 ± 19.63
% control 100 99 99 94
Body weight gain (g)
GD 6-7 2.9 ± 4.27 2.9 ± 3.39 2.7 ± 4.49 1.4 ± 3.68
% control 100 100 93 48
GD 9-12 14.4 ± 4.15 16.0 ± 2.93 13.9 ± 4.30 10.6 ± 3.42**
% control 100 111 97 74
GD 17-19 22.9 ± 5.32 22.8 ± 6.00 21.8 ± 5.34 19.6 ± 6.74
% control 100 100 95 86
GD 20-21 15.8 ± 4.42 14.3 ± 4.20 15.0 ± 4.47 7.0 ± 11.93**
% control 100 91 95 44
GD 6-21 100.3 ± 16.89 101.2 ± 14.57 100.3 ± 19.81 83.5 ± 12.74**
% control 100 101 100 83
**: statistically significant at p < 0.01; BW: body weight; GD: gestation day

Table 3: Food consumption

Dose group (mg/kg bw/day) 0 100 300 1000
GD 6-7 20.3 ± 3.65 19.2 ± 3.15 19.4 ± 3.17 15.6 ± 3.14***
% control 100 94 96 77
GD 9-12 19.1 ± 2.60 19.9 ± 2.23 19.4 ± 2.41 14.9 ± 2.13***
% control 100 104 102 78
GD 15-17 21.5 ± 3.24 22.3 ± 2.39 22.2 ± 2.51 20.1 ± 2.12
% control 100 104 103 79
GD 20-21 20.6 ± 3.94 19.2 ± 3.17 18.8 ± 3.31 14.2 ± 5.98***
% control 100 93 91 69
GD 6-21 20.8 ± 2.33 21.0 ± 1.91 20.6 ± 1.78 17.6 ± 1.63***
% control 100 101 99 85
*, ** and ***: statistically significant at p < 0.05, p < 0.01 and p < 0.001; GD: gestation day

Table 4: Skeletal malformations in fetuses

Dose group (mg/kg bw/day) 0 100 300 1000 HCD
Skull
Interparietal: incomplete ossification
Litter (%) 47 39 47 11 5 - 75
Fetal (%) 16.4 12.22 13.73 1.93** 0.76 - 49.17
Hindlimb
metatarsal: incomplete ossification Litter (%) 21 0 11 26 0 - 43
Fetal (%) 5.44 0 1.93 10.33 0 - 14.74
**: statistically significant at p< 0.01; HCD: historical control data generated in the testing facility (time frame for data generation not specified)
Conclusions:
The test item had no effect on intrauterine development. The NOAEL for maternal and developmental toxicity were both 1000 mg/kg bw/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance, and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.6, of Regulation (EC) No. 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity

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 (Covance study no. 8421688), as well as on a second preliminary range-finding study performed in pregnant rats (Covance study no. 8417090).

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 a 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.

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.

The following parameters of systemic toxicity were evaluated in the study: 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.

In addition, oestrous cycle and reproductive parameters were monitored. The following parameters on developmental toxicity were examined: Litter size, viability indices (post-implantation loss, livebirth index and Day 4 viability index), number of dead, stillborn and missing/ presumably cannibalised pups, sex ratio, clinical signs of toxicityand early post-natal development (body weights, ano-genital distance, areola/nipple retention in male pups, thyroid hormone TSH and T4 levels (post natal Day (PND) 4 and PND 13), macroscopic analysis and histopathology of the thyroid glands).

For details on parental systemic toxicity (mortality, clinical signs, functional observations, haematology, clinical chemistry, organ weights, gross pathology and histopathological findings), please refer to Section 7.5 “Repeated dose toxicity”. For details on reproductive toxicity, please refer to “Effects on fertility” described above.

There was an increased incidence (statistically not significant) of post-implantation loss noted for the females of the 100 mg/kg bw/day (10.70%) and the1000 mg/kg bw/day dose group (17.00%) when compared to control animals (5.02%). In addition, at 1000 mg/kg bw/day, the number of stillborn pups was increased and outside the laboratory’s historical control range, whereas no stillborn pups were found in the remaining dose groups. Further, 2 dead pups were observed for litters of the high dose group on PND 0 and 1, whereas no dead pups were observed in the remaining dose groups. The incidence of dead pups was, however, within the range of the laboratory's historical control data. There was also a slight increase in the incidence of missing/ presumably cannibalised pups in the animals of this group (0, 2, 1 and 4 pups at 0, 100, 300 and 1000 mg/kg bw/day). The incidences of missing/ presumably cannibalised pups at the low and intermediate dose level was not dose-dependent, fell within the range of the laboratoy's historical control data and was considered to have arisen incidentally.

The observations in the high dose group resulted in a higher incidence of pup mortality between PND 0 – 4 for litters when compared to control animals. At 1000 mg/kg bw/day, the livebirth index and the Day 4 viability index were slightly reduced (-9% and -7%) when compared to control animals. However, the number of pups in the high dose group litters were within the historical control data range and there was no positive correlation between a larger litter size and stillborn pups.

The observations made in the high dose group were considered treatment-related but non-adverse, due to the lack of statistical significance. Any abnormalities observed were attributed to adverse effects in the dams rather than to represent a reproductive toxicity effect.

The surviving offspring showed no treatment-related signs of systemic or local toxicity and body weight development was comparable to those of control animals. In addition, there were no statistically significant changes in T4 and TSH levels observed following treatment with the test item. Sexual maturation, ano-genital distance and nipple retention in male pups were not affected. At macroscopical examination, one male and one female pup both from one high dose group litter were found to have no milk in the stomach. As this finding was confined to a single litter, the observation was not related to treatment. Thyroid weight was unaffected in the pups and there were no histopathological findings in the thyroids.

In conclusion, all adverse findings observed for pup viability and survival were attributed to adverse effects in the dams and there were no adverse effects on pup development. Under the conditions of the study, the NOAEL for developmental toxicity was 1000 mg/kg bw/day. 

 

 

 

The test item was further investigated for its teratogenic potential in a prenatal developmental toxicity study in rats according to OECD guideline 414 and in compliance with GLP (Covance, 2021). The test substance was dissolved in purified water and administered by oral gavage to groups of 20 pregnant rats from gestation Days (GD) 6 to 21. Based on the results of a preliminary toxicity study in pregnant rats of this strain, test item dose levels of 100, 300 and 1000 mg/kg bw/day were chosen. A similar constituted group received the vehicle and served as control.

The animals were observed for mortality and clinical signs of toxicity and individual body weights, as well as food consumption were recorded at regular intervals during gestation. On Day 21 of pregnancy, all dams were sacrificed and maternal and fetal animals were examined. Blood was collected from all females prior to Caesarian section for analysis of thyroid hormone levels. The uteri and ovaries were dissected and evaluated for gravid uterus weight, the number of corpora lutea, implantations, early and late resportions and live and dead fetuses. In addition, pre- and post-implantation loss indices were determined. Fetuses were evaluated for sex ratio and anogenital distance and examined for external, visceral and skeletal abnormalities.

Homogeneity and stability of the test item formulated in vehicle were confirmed by analytical methods. There was no mortality observed during the whole study period. Test item-related maternal systemic toxicity was not observed. The animals of all dose groups showed excessive salivation, with increasing severity at higher concentrations. Mouth rubbing was noted in animals of the mid and high dose groups and isolated incidences of paddling was noted in the animals of the two dose groups. However, the findings were attributed to unpalatability or the unpleasant taste of the test article and therefore considered non-adverse.

Animals of the 1000 mg/kg bw/day group showed a reduction in body weight and statistically significant reductions in body weight gain (-17%) and food consumption (-15%) over the entire treatment period. However, in the absence of a detrimental effect on pregnancy or the developing fetus, the findings were considered not to represent an adverse effect of the test article. There were no findings in thyroid weight and hormone levels and no macroscopic or microscopic findings in the animals of any dose groups that were related to treatment with the test item.

No findings were observed in Caesarian section data with respect to the number of corpora lutea, implantation sites, pre- and post-implantation loss, early and late resorptions, total resorptions, dead and live fetuses. In addition, gravid uterine weight, corrected body weight (carcasse weight), corrected and total weight change from GD 6 – 21 of maternal animals were not affected.

There was no effect on the number of females with live fetuses, the sex ratio, mean fetal weights or anogenital distance data.

There were no test item-related external, skeletal or visceral malformations observed when compared to the vehicle control animals. Incidences of skeletal malformations were observed in a few animals of the control and test-item treated groups, but occurred without dose response relationship and remained within the ranges of historical control data and were therefore not attributed to treatment.

Based on these results and under the conditions of this study, a NOAEL for maternal toxicity and fetal developmental toxicity was ≥ 1000 mg test item/kg bw/day in Crl:WI(Han) rats.

Justification for classification or non-classification

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

Additional information

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