Registration Dossier

Administrative data

Description of key information

Oral (OECD 422), rat: NOAEL systemic ≥ 400 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Oct 2018 – 09 Jan 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 29 Jul 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Slovak National Accreditation Service, Bratislava, Slovak Republic
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
outbreed
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: The Lab Animals Breeding Center “Pushchino”, Pushchino, Russia
- Age at study initiation: approximately 11 weeks
- Weight at study initiation: 348 ± 27 g (males) and 213 ± 14 g (females)
- Housing: Animals were housed in groups of 2/sex in polycarbonate cages type IV (595 x 380 x 200 mm) until mating. During the mating period, males were housed alone and cohabited with females in the home cage of the male. After mating, females were housed individually in these cages. Males and females of the satellite group (not mated) were housed 2-3 animals per cage. Commercial autoclaved woodchip bedding was used.
- Diet: Laboratory Rodent Diet (SSNIFF V1534-300, Spezialdiaten GmbH, Soest, Germany), ad libitum
- Water: filtered tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 30 - 63
- Air changes (per hr): at least 12 times
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Aug 2018 To: 09 Jan 2019
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Required volumes of the test item (taking into account the purity and the density of the liquid) were mixed with the calculated volume of vehicle (water) and stirred to create a homogeneous water emulsion. Dosing formulations were prepared every three days, aliquoted to the required volumes of days of administration and stored at room temperature in tightly closed glass jars in the fume hood. On the day of dosing, the aliquot of each formulation was mixed thoroughly and stirred continuously during dosing.

VEHICLE
- Concentration in vehicle: 5, 20 and 80 mg/mL
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing formulations were analysed for homogeneity, stability and concentration. Stability of the test item in the vehicle prepared at concentrations of 5, 20 and 80 mg/mL was confirmed following 3 days of storage at room temperature during method validation study. Additional data on the stability of dose formulations were obtained by analyzing the medium strata of all dose formulations after their 2-day storage and resuspension at the beginning of in-life phase of the study. Analysis of formulations for homogeneity and concentration was conducted in the test facility using a LC/MS validated method at the beginning, in the middle and at the end of in-life phase. In addition, homogeneity analysis was performed at the beginning of the study after 2 days of dose storage after resuspension.
For stability, the mean concentration of formulation samples of 5 and 80 mg/mL after 3 days of storage and resuspension should be within the acceptable range of the target concentration (85 – 115% with RSD < 10%), and within the range 85-115% of the time zero point.
Stability of the test substance formulations was confirmed after three days of storage at room temperature and after 2-day storage incl. resuspension, except for dose group 2 (20 mg/mL) which exceeded the acceptable range (120.5%) presumably due to problems of homogenization and sample preparing.
For dose formulations 5, 20 and 80 mg/mL all analysed concentrations were within the acceptable range of 85 – 115% of the target concentration for all dates of analysis, excluding formulation 20 mg/mL on date of 27 Nov 2018, for which the actual concentration was 81.0% of the target level.
The samples were considered homogeneous since the relative standard deviation (RSD) for the mean concentration was ≤ 10% at a concentration within the acceptable limits (85 – 115% of target concentration) with the following exceptions: The RSD value exceeded 10% on date of 27 Nov 2018 for 20 mg/mL (10.2%) and 80 mg/mL (12%) with a mean concentration for 20 mg/mL formulation less than the acceptable limit (79.6%). The homogeneity data of the formulations after 2-day storage and re-mixing were outside the acceptable range for the formulation of 5 mg/mL (RSD 10.9%) and 20 mg/mL (RSD 10.2% and mean concentration 117.8% of target). However, the described outputs from the acceptable range were slight, and the deviations are considered to be mild. In general, it can be concluded that dose formulations homogeneity was acceptable, not affecting the interpretation of calculated dosages administered to animals.
Duration of treatment / exposure:
Males: Treatment for 28 days, beginning 14 days prior to mating, throughout the mating period until necropsy
Females: Females which delivered were treated for 49 – 56 days, beginning 14 days prior to mating until lactation day 13. Females which failed to deliver/ with no evidence of mating were treated for 55 days.
Animals of the recovery group were not mated and dosed for 28 days (males) or 55 days (females) with following two weeks recovery period.
Frequency of treatment:
daily, 7 days/week
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 (main group)
5 (recovery group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Available toxicity data of structurally similar analogue substances revealed that doses of the test item used in the study on repeated dose toxicity can be in the range of 25 to 400 mg/kg bw/day. A pilot dose-range finding study with the test item was performed and confirmed that a dose of 400 mg/kg bw/day will be tolerated without causing death or obvious suffering. Test item doses of 25 and 400 mg/kg bw/day were administered daily for 21 days to each three male and three female Sprague Dawley rats. No mortalities and no adverse clinical signs were observed, except for excessive salivation in one female treated at 400 mg/kg bw/day. No effects on food consumption and body weight gain was noted and there were no gross macroscopic findings at necropsy. Increased liver weights were observed in males at 25 and 400 mg/kg bw/day and in females at 400 mg/kg bw/day and assumed to be caused by hepatocellular hypertrophy. Based on these results and based on the published data of analogues toxicity, doses of 25, 100 and 400 mg/kg bw/day were chosen for the current study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily mortality/morbidity
- Cage side observations included: All animals were observed for general condition and clinical signs of toxicity 5 - 20 min following dose administration. Females expected to deliver were observed twice daily for dystocia and other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of treatment and on a weekly basis throughout the study
- Observations included: chromodacryorrhea, palpebral aperture, hunching, piloerection, respiration, alertness, reactivity to handling, vocalization, mucous membranes, locomotion/hypokinesia, gait and stereotypes/bizzares

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on Day 1 prior to dosing and weekly thereafter. During pregnancy, mated females were weighed on Days 0, 7, 14, 20 and within 24 h of parturition and on post-natal days (PND) 2, 4 and 13.

FOOD CONSUMPTION: Yes
- Food consumption was measured prior to dosing and weekly until cohabition. Food consumption was not recorded during mating. After mating, food consumption of females was measured on gestation Days 0-1, 6-7, 13-14 and 19-20 and on lactation Days 1-2, 4-5 and 12-13.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once prior to start of treatment and on a weekly basis throughout the study.
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the scheduled necropsy from the inferior vena cava; for satellite animals following two weeks post-treatment as part of euthanasia
- Anaesthetic used for blood collection: Yes (Zoletil® and Xyla® (tiletamine+zolazepam+xylazine))
- Animals fasted: Yes
- How many animals: 6/sex randomly selected of each main group; all satellite animals
- Parameters checked in Table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the scheduled necropsy from the inferior vena cava; for satellite animals following two weeks post-treatment as part of euthanasia. Tail-tip amputation was performed for assessing the level of glucose.
- Anaesthetic used for blood collection: Yes (Zoletil® and Xyla® (tiletamine+zolazepam+xylazine))
- Animals fasted: Yes
- How many animals: 6/sex randomly selected of each main group; all satellite animals
- Parameters checked in Table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- The following parameters were examined: glucose, protein, bilirubin, urobilinogen, pH value, specific gravity, blood cells, ketones, nitrites, leukocytes, creatinine

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on Day 28 of dose administration (males) and on lactation Day 13 (females)
- How many animals: 6/sex randomly selected
- Dose groups that were examined: all dose groups
- Battery of functions tested: home cage / handling / open field / sensory activity / grip strength / locomotor activity

IMMUNOLOGY: No

OTHER:
THYROID HORMONE ANALYSIS
- Time schedule for collection of blood: on the day of scheduled necropsy from the inferior vena cava; for satellite animals following two weeks post-treatment as part of euthanasia
- Anaesthetic used for blood collection: Yes (Zoletil® and Xyla® (tiletamine+zolazepam+xylazine))
- Animals fasted: Yes
- How many animals: 6/sex randomly selected of each main group; all recovery animals
- Parameters checked: thyroxine (T4)
Sacrifice and pathology:
SACRIFICE
- Male animals: All surviving animals were euthanized by anesthesia (Zoletil® / Xyla®) following completion of the mating period and 28 days of dose administration.
- Female animals: All surviving dams were euthanized on lactation Day 14 after litters have been euthanized on post-natal Day (PND) 13. Females which have not delivered were euthanized after 55 days of dosing. All females were euthanized by anesthesia (Zoletil® / Xyla®).
- Satellite animals: All satellite animals were subjected to euthanasia at the end of two weeks post-treatment period. All animals were euthanized by anesthesia (Zoletil® / Xyla®).

GROSS NECROPSY
- Gross necropsy included examination of the external surface of the body, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal and pelvic cavities including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: adrenals, brain, cowper’s glands, epididymides, heart, glans penis, kidneys, levator ani and bulbocavernosus muscles complex, liver, ovaries, pituitary, prostate, seminal vesicles and coagulating glands, spleen, testes, thymus, thyroid and uterus. The tissues indicated in Table No. 3 were prepared for light microscopic examination from six randomly selected adult males and females in the control and high-dose groups. Liver, kidneys, spleen, stomach, testes, epididymides, and seminal vesicles with coagulating glands were examined in the low- and mid dose groups as well as in satellite subgroup animals. Microscopic examination of thyroid glands was done for all adult males and femlaes in all groups including satellite subgroups, and one male and female of day 13 pup from each litter in all groups.
Statistics:
All statistical tests were performed separately for each sex and compared the treated groups to the control group.
- Multi-factor analysis of variance ANOVA-2, followed by the Duncan test: mean body weights, body weight gain and food consumption data
- Parametric one-way analysis of variance (ANOVA), followed by Dunnett's test in case of significance (p < 0.05): clinical pathology values and functional observational battery (FOB) data, organ weights (absolute and relative to body weights and relative to brain weights)
- T-test: clinical pathology values and FOB values of satellite animals, comparison of organ weights
- Fisher’s exact test: FOB parameters which yield scalar or descriptive data, histopathological findings
- Kruskal-Wallis nonparametric ANOVA test with Dunn’s test: gamma-glutamyltransferase data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
25 mg/kg bw/day: piloerection, reactivity to handling and contraction of whisker pad (1/12 males); nasal red-brown discharge/crust and rales (1/12 females): non-adverse
100 mg/kg bw/day: nasal red-brown discharge/crust (1/12 males), transient effect; piloerection (1/12 males): non-adverse
400 mg/kg bw/day: excessive salivation 15 min after administration (4/17 males, 13/17 females); chromodacryorrhea (3/17 females); piloerection and reactivity to handling (2/17 females); nasal red-brown discharge/crust (1/17 males, 2/17 females): non-adverse
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
25 mg/kg bw/day: significant monocytosis in males; decrease in segmented neutrophils and increase in lymphocyte relative level (not-significant) in females: non-adverse
100 mg/kg bw/day: significant monocytosis in males; decrease in segmented neutrophils and increase in lymphocyte relative level (significant) in females: non-adverse
400 mg/kg bw/day: increased monocyte level in males (not significant); decrease in segmented neutrophils and increase in lymphocyte relative level (not significant) in females; increased granulocytes count and decreased lymphocyte level in females of the recovery group: non-adverse
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
25 mg/kg bw/day: lower calcium concentration in males (significant): non-adverse
100 mg/kg bw/day: lower calcium concentration in males (significant): non-adverse
400 mg/kg bw/day: lower calcium concentration in males (not significant); decreased chloride concentration in females (significant); decreased inorganic phosphates concentration and decreased total creatinine kinase after 2 weeks of recovery in females; increased triglycerides in males and females (not significant); decrease in glutamate dehydrogenase (not significant): non-adverse
Urinalysis findings:
effects observed, non-treatment-related
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
400 mg/kg bw/day:
- increased liver weight in males and females compared to control animals, associated with microscopic findings
- slightly increased testis weight, non-significant, unclear relationship with test item administration
- decreased testicular and seminal vesicle weight in males of the recovery group, accompanied by an increase in the glans penis weight
- slight decrease in the relative weight of the spleen in males of the recovery group
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
100 mg/kg bw/day:
- one large incapsulated kidney in 1/12 females (microscopic analysis revealed aseptic hydronephrosis), incidential, non-adverse;
- deformed spleen in 1/12 males, associated with a slightly increased spleen weight, lack of correlation to microscopic findings, unclear relationship with test item administration
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
25 mg/kg bw/day:
- Hepatocellular hypertrophy, glycogen accumulation in females: adaptive changes, non-adverse
- Epithelial hypertrophy in follicles of the thyroid gland (not significant), test item related but species specific, non-adverse
- Mineralization of kidney tubules in 2/6 females (also found in 1/6 control females), uncertain relationship to test item
- Neutrophilic or macrophage infiltration of deep mucosa and/or submucosa in the forestomach of single males, slight and incidental, uncertain relationship to test item

100 mg/kg bw/day:
- Multifocal hepatocellular hypertrophy in 2/6 males
- Hepatocellular hypertrophy, glycogen accumulation in females: adaptive changes, non-adverse
- Epithelial hypertrophy in follicles of the thyroid gland (not significant), test item related but species specific, non-adverse
- Mineralization of kidney tubules in 3/6 females (also found in 1/6 control females), one female had severe unilateral hydronephrosis, uncertain relationship to test item
- Neutrophilic or macrophage infiltration of deep mucosa and/or submucosa in the forestomach of single males, slight and incidental, uncertain relationship to test item

400 mg/kg bw/day:
- Generalized hepatocellular hypertrophy (slight grade) in 4/6 males, statistically significant
- Hepatocellular hypertrophy, higher liver weight and glycogen accumulation in females, adaptive changes, non-adverse
- Epithelial hypertrophy in follicles of the thyroid gland (statistically significant in males), test item related but species specific, non-adverse;
- Mineralization of kidney tubules in 1/6 males and 2/6 famles, uncertain relationship to test item
- Atrophy and fibrosis of red pulp in the spleen in 2/6 males, not correlating to hematological changes and changes in spleen weight
- Neutrophilic or macrophage infiltration of deep mucosa and/or submucosa in the forestomach of single males, focal degeneration of epithelium in non-glandular stomach in 1/6 females, uncertain relationship to test item
- Significantly higher relative count in granulocytes (p < 0.05) in bone marrow smear in females (satellite group), due to the increase in the level of segmented neutrophils, findings correlated to the granulocyte absolute and relative count in peripheric blood after recovery period, non-adverse
- Slightly reduced erythrocariocytes in bone marrow smear of females (satellite group), leading to an increased myeloid-erythroid ratio, non-adverse
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
MORBIDITY AND MORTALITY
There was no morbidity and mortality of animals caused by the test item administration.

CLINICAL SIGNS:
Excessive salivation was observed 15 min after administration in 4/17 males and 13/17 females treated at 400 mg/kg bw/day, starting from the third week of dosing. Transient salivation following dosing is considered to be a sign of an adverse test item taste or a locally irritating effect. It is not considered toxicologically relevant.
In addition, 3 females of the high dose group had chromodacryrrhea starting from the 7th week of administration with an associated unilateral palpebral closure noted for one of them. Despite its low frequency, chromodacryorrhea is considered to be related to the test item administration.
Transient nasal red-brown discharge/crust was observed in one female treated at 25 mg/kg bw/day and one male and two females in the 400 mg/kg bw/day dose group. In one female nasal discharge was associated with the short-term rales. In addition, rales were noted for one female in the 25 mg/kg bw/day dose group. Incidents of nasal discharge and rales can be caused by aspiration of formulation, were not correlated with test item related microscopic findings and not considered to be toxicologically relevant. One male treated at 25 mg/kg bw/day had clinical signs of slight distress including piloerection, reactivity to handling and contraction of whisker pad starting , starting from the end of the second treatment week. No apparent cause of the poor clinical condition was determined. Clinical observations were not associated with any gross findings and significant microscopic changes in selected organs.
In the 400 mg/kg bw/day dose, the test item related piloerection and reactivity to handling were observed in individual females (No. 138 and No. 98, respectively) by the end of the administration period. Besides, piloerection was noted for a single male (No.6) in the 100 mg/kg bw/day dose group.

BODY WEIGHT AND WEIGHT CHANGES:
Mean body weights and body weight gains were unaffected by the test item for all study periods. .

FOOD CONSUMPTION:
Mean food consumption of animals of all treatment groups was similar to that in the vehicle control group for any treatment periods.

FUNCTIONAL OBSERVATION BATTERY:
Home cage, handling, open field, sensory, neuromuscular and physiological parameters evaluated during Functional Observation Battery testing as well as locomotor activity patterns were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the vehicle control group on study Day 27 (males), on PND 13 (females) or after a two-week recovery period.

HAEMATOLOGICAL FINDINGS:
The test item significantly increased the level of monocytes in males of dosage group 25 and 100 mg/kg bw/day and showed a similar trend (not significant) in animals treated at 400 mg/kg bw/day. The effect was considered treatment related, but non-adverse, as a dose-relationship was lacking and no changes in bone marrow were observed. After two weeks of recovery, hematological parameters in the 400 mg/kg bw/day male group were comparable with values in the control group. In blood smear of females, a statistically significant test-item related decrease in segmented neutrophils and an increase in relative lymphocyte relative level (p < 0.05) was observed in the 100 mg/kg bw/day dose group. The same trend was observed for females treated at 25 and 400 mg/kg bw/day, but without statistical significance.
Females of the 400 mg/kg bw/day dose recovery group exhibited, on the contrary, an increase in granulocytes count with a decrease in lymphocytes level, due to the recovery of the described above changes. Changes in the blood of the 400 mg/kg bw/day recovery females were correlated to the findings in the bone marrow smear. Thus, the hematological changes in females were slight, reversible and considered as non-adverse.

CLINICAL BIOCHEMISTRY:
A statistically significant decrease in calcium concentration was observed in males treated at 25 and 100 mg/kg bw/day (by 4.1%, p < 0.01) and a non-significant decrease in males of the 400 mg/kg bw/day dose group (by 1.7%). However, after the 2-week post-treatment period in this group, calcium concentration was increased compared with the control group, probably due to a recovery rebound effect. In addition, a slight (2.6%) but statistically significantly reduced chloride concentration (p < 0.05) was observed in females treated at 400 mg/kg bw/day. The effect did not correlate to the microscopic changes in kidneys and was thus considered as non-adverse.
Decreased inorganic phosphate and decreased total creatinine kinase activity was observed in females of the 400 mg/kg bw/day post-treatment subgroup and attributed to an impaired phosphate exchange.
In addition, increased triglycerides values were observed in males and females of the high dose group and may be associated with microscopic findings in the liver. The findings were slight, statistically non-significant and not considered adverse.
Slightly decreased glutamate dehydrogenase activity in animals treated at 400 mg/kg bw/day were not considered toxicologically relevant, as there was no statistical significance and other liver parameters were not found to be altered.

URINALYSIS:
A slightly higher mean diuresis value (non-significant) was observed in animals treated at 400 mg/kg bw/day. The effect was considered not test item-related.
There were no statistically significant differences for urinalysis parameters in F0 males at the end of the 28-day treatment period when the vehicle control and test item-treated groups were compared. After the 2-week post-treatment period, urinalysis values in 400 mg/kg bw/day dose group were also similar with values in a vehicle control group.
Analysis of creatinine in urine did not reveal significant changes in creatinine concentration in all dose treated males a the and of 28-day administration period as well as after 2-week post-treatment period.

ORGAN WEIGHT FINDINGS:
Increased absolute organ weights were found only in animals of the high dose group (400 mg/kg bw/day). An increased liver weight was observed in males (14.6%, p < 0.05) and females (12.1%, p < 0.05) when compared to control animals. The effect was associated with microscopic findings and considered test-item related. After the 400 mg/kg bw/day dose post treatment period, the absolute and relative liver weight in males and females did not significantly differ from the values in the control group.
In 400 mg/kg bw/day recovery subgroup males, a decrease in absolute and relative weights of testes was observed (p < 0.05). Furthermore, there was a tendency to a reduction in the relative weight of the seminal vesicles. The decrease in the testicular and seminal vesicles weight in recovery males was accompanied by an increase in the weight of the glans penis, which was statistically significant as compared with the control group for the absolute value (by 11.7%, p < 0.05). In contrast, during the administration period, there was a slight increase in the mean value of the testes weight noticeable for the right of the pair (by 12.3 % for absolute value, and by 11.7 % relative to body weight value). However, change in the weight of testes in the treatment period was not significant, was not associated with the microscopic findings for both treatment and post-treatment period, and therefore is regarded as change with an unclear relationship with the test item administration. In addition, the slight decrease in the relative weight of spleen was observed in the 400 mg/kg bw/day post-treatment males. This observation with an unclear relationship with the test item administration did not correlate to the microscopic findings in the spleen for post-treatment males. At the same time, in males of the 400 mg/kg bw/day main subgroup, incidents of atrophy and fibrosis were found in the spleen without any notable changes in the organ weight.

GROSS PATHOLOGICAL FINDINGS:
One female in the 100 mg/kg bw/day dose group had one large incapsulated kidney, that was associated with the microscopic findings (aseptic hydronephrosis). This finding was considered incidental and most likely not related to the test item administration.
One male in the 100 mg/kg bw/day dose group had a visually deformed spleen. This gross observation was associated with a slight increase in individual spleen weight, was not correlated to the microscopic findings in the spleen of males necropsied after 28 days of test item treatment. The spleen is regarded as an organ with an unclear relationship with the test item administration.
Macroscopic findings in thymus, lymph nodes, thyroids, adrenals, and pituitary concerned visual changes in organs size, occurred at similar frequencies in the test item and the vehicle control groups, did not correlate with a statistical change in the weights, and were associated with individual variability in organs size or weights.
Gross finding in urinary bladder (opaque, dense area) was noted in some males and females including the control group, was not correlated to the changes in organ weight or any microscopic observations, and thus considered not to be test item-related.

HISTOPATHOLOGICAL FINDINGS, NON-NEOPLASTIC:
Test item-related findings were observed in the liver, the thyroid glands and in the bone marrow. Findings with unclear relation to the administration of the test item were observed in kidneys, spleen and stomach.
Multifocal hepatocellular hypertrophy was noted in two of six examined males at 100 mg/kg bw/day and generalized hepatocellular hypertrophy of slight grade was observed in four of six males at 400 mg/kg bw/day. In the high dose group, the incidence of observation was statistically higher compared to the control vehicle group (p < 0.05).
In females, hepatocellular hypertrophy was found in all groups, including the vehicle control group, but was not observed in females of the control satellite subgroup and can be due to the physiological state of pregnancy and lactation. However, in all examined females at 25, 100 and 400 mg/kg bw/day hepatocellular hypertrophy was of more severe grade compared to the vehicle control group. Hepatocellular hypertrophy correlated to the increased absolute and relative liver weights and clinical pathology changes, and can be associated with microsomal enzyme induction. Besides, cytoplasmic alteration (glycogen accumulation) of slight to marked grade was found in a half of examined females of all dose treated group. Hepatocellular hypertrophy and higher liver weights as well as glycogen accumulation in the cytoplasm of hepatocytes were reversible, considered adaptive changes and hence non-adverse.
Epithelial hypertrophy in follicles of the thyroid gland was observed in some males and females in all test item-treated groups. In males at 400 mg/kg bw/day, an increase in finding frequency was statistically significant (p < 0.05). Follicular epithelial hypertrophy can occur with enzyme induction and increased hepatic clearance of thyroxin, was reversible and is considered to be species-specific and non-adverse. In parental animals, microscopic findings in thyroid gland were not correlated to changes in the thyroxin level as well as to the changes in organ weight. There were no test item-related findings in thyroid gland in 400 mg/kg bw/day post-treatment animals. At the same time, a lower level of thyroxin associated with increased thyroids weight was revealed in F1 offspring. So, the changes in the thyroid gland of parental rats are considered as test item-related; however, are considered to be species-specific and non-adverse.
Findings of uncertain relationship to the test item included mineralization of the tubules in the kidneys. Mineralization of tubules in papilla or inner stripe of the outer medulla was found in 1 of 6 females in the control group, 2 of 6 females in the 25 mg/kg bw/day group, 3 of 6 females in the 100 mg/kg bw/day group and 2 of 6 females in the 400 mg/kg bw/day group, and in single male in the 400 mg/kg bw/day group. Mineralization in the corticomedullary junction is a common finding in rats. There was no statistically significant increase in the frequency of mineralization. However, in one female in the 100 mg/kg bw/day group the severe unilateral hydronephrosis was revealed, which possibly could have been caused by mineralization and obstruction of the urinary tract. The signs of chronic progressive nephropathy were observed in approximately similar frequency in males and less often in females of all groups including the control group.
In two males from the 400 mg/kg bw/day group, atrophy, and fibrosis of red pulp in the spleen with or without atrophy of white pulp was observed. These findings did not correlate to the hematological changes and changes in spleen weight. The slight decrease in the mean relative weight of spleen was noted for males in the 400 mg/kg bw/day post-treatment group; however, this change was not statistically significant compared to the control group.
In the forestomach of single males from of each dose group, the neutrophilic or macrophage infiltration of deep mucosa and/or submucosa of the minimum and moderate grade was found with concomitant focal degeneration of epithelium in one male. Besides, the focal degeneration of epithelium in nonglandular stomach was observed in one female in the 400 mg/kg bw/day group. Observations in the forestomach can potentially indicate a local effect of the test item administered by gavage. However, these findings were incidental and slight, and the relationship with the test item is considered unclear.
No test item-related histological findings in organs of the male and female reproductive system were revealed. The decrease in absolute and relative weights of testes and a tendency to a reduction in the relative weight of the seminal vesicles in the 400 mg/kg bw/day recovery males did not correlate to any microscopic changes in these organs in the test item treated males euthanized on day 29 or after 2-weeks post-treatment period.
During qualitative examination of the testes with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure, no test item related spermatogenic disturbance, tubular vacuolation, contraction, dilatation, necrosis, dilated rete, nor Leydig cell atrophy, hypertrophy, hyperplasia, adenoma were revealed. In the 400 mg/kg bw/day dose group, inflammation in the tunica albuginea of one testis was observed in single male. In another male, chronic inflammation in the ventral lobe of the prostate was found. These findings were of minimal and slight grade and considered to be not test item-related.
In ovaries and oviducts of the females, there were no significant microscopic changes that could be associated with the test item. Findings in the uterus and vagina, observed with a similar frequency in the control group and 400 mg/kg bw/day group, are assumed to be related to parturition.
There were no significant abnormalities in the ovaries, oviducts, and uterus of non-gravid females No.103 (group 3) and No.138 (group 4), which could have affected the ability of these females for pregnancy. However, in the female No.138, findings in liver were observed, which are supposed to be associated with the test item administration (glucogen accumulation of minimal grade and pigment in Kupffer cells of moderate grade). Microscopic alterations in bone marrow smear were observed only in 400 mg/kg bw/day dose post-treatment females and comprised a significantly higherrelative count in granulocytes (p < 0.05) due to the increase in the level of segmented neutrophils. The findings correlated to the granulocyte absolute and relative count in peripheric blood after the recovery period and were considered as non-adverse. In addition, in this female dose group the percentage of erythrocariocytes was slightly reduced leading to an increased myeloid-erythroid ratio (non-adverse).
Dose descriptor:
NOAEL
Effect level:
>= 400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at highest dose
Critical effects observed:
no
Conclusions:
Based on the results of this study, the no observed adverse effect level (NOAEL) for systemic toxicity was ≥ 400 mg/kg bw/day.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information on repeated dose toxicity comprises an adequate and reliable study (Klimisch score 1), and are thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test substance 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 (Biological Testing Laboratory, 2019). Based on the results of a preliminary range-finding study, the dose levels for the study were selected. Groups of 12 male and 12 female Sprague Dawley rats were treated once daily by oral gavage of the test item at doses of 25, 100 and 400 mg/kg bw/day. A similar constituted group of male and female rats received the vehicle (water) and served as control. For recovery, 5 additional animals per sex were added to each of the control and the 400 mg/kg bw/day dose group.

Males were treated for 28 days, beginning 14 days prior to mating, during mating and up to scheduled necropsy. Females that delivered were treated for 49 – 56 days, beginning 14 days prior to mating until lactation Day 13. Females which failed to deliver were treated for 55 days. Animals of the recovery group were not mated and dosed for 28 days (males) or 55 days (females), followed by a 2-week recovery period without treatment.

The following parameters were evaluated in the study: Mortality/morbidity, clinical sings, functional observations, body weight, food consumption, hematological findings, clinical chemistry parameters, thyroid hormone levels, urinalysis, organ weights, gross necropsy findings and histopathological findings.

There was no mortality or morbidity caused by the test substance administration. The main clinical sign related to test substance administration was excessive salivation in the 400 mg/kg bw/day group (4/17 males and 13/17 females). Transient salivation following dosing is considered to be a sign of an adverse test item taste or a locally irritating effect. It is not considered toxicologically relevant. Three females of the high dose group had chromodacryorrhea starting from the 7th week of administration with an associated unilateral palpebral closure noted for one of them. Further clinical signs in individual animals among all dose groups included piloerection, reactivity to handling and nasal red-brown discharge/crust in single animals and were considered non-adverse. Mean body weights and body weight gain in all dose groups were similar to the vehicle control group during the pre-mating and overall treatment periods in males, as well as during the pre-mating, gestation and lactation periods in females. Food consumption remained unaffected by treatment at any dosage level. No effects on Functional Observation Battery parameters (home cage, handling, open field, sensory, neuromuscular and physiological) and no effects on locomotor activity were noted at any dose level.

Hematological analysis revealed a statistically significant, treatment-related monocytosis in males at 25 and 100 mg/kg bw/day compared to the vehicle control. At 400 mg/kg bw/day, the increase in monocytes level was non-significant. A decrease in segmented neutrophils and an increased lymphocyte relative count in females treated at 100 mg/kg bw/day was observed, and the same tendency was noted for doses of 25 and 400 mg/kg bw/day. Blood hematology changes during the treatment period in males and females did not correlate to any changes in the bone marrow, were reversible with rebound post-treatment changes in the blood and bone marrow noted only for females, and considered as non-adverse.

There were no findings in urinalysis for the test item-treated groups when compared to the vehicle control group.

Regarding clinical chemistry parameters, in males, the concentration of calcium decreased in the 25 and 100 mg/kg bw/day groups. Also at 400 mg/kg bw/day, a minimal but not statistically significant decrease in calcium concentration was noted in males. In the 400 mg/kg bw/day recovery subgroup, calcium level was increased compared with the control group, probably due to a recovery rebound effect. In females, the slight decrease in chloride ions concentration was observed in the 400 mg/kg bw/day group. The change did not correlate to the microscopic findings in kidneys and is considered to be non-adverse. There were no significant changes in calcium and inorganic phosphate level in females revealed at the end of the dose treatment period. However, in the 400 mg/kg bw/day post-treatment subgroup, the decreased concentration of inorganic phosphates and decreased activity of total creatine kinase were noted, which can be associated with the impaired phosphate exchange. An increase in the mean value of triglycerides (notable in males and females in the 400 mg/kg bw/day group) may be associated with microscopic findings in the liver. However, this change was statistically non-significant, and not considered adverse.

There were no significant test item-related gross observations revealed during necropsy. A test item-related increase in organ weight was recorded for the liver. The changes in weight of testes, seminal vesicles, and spleen of unclear relationship with the test item administration were found in recovery males. Test item-related microscopic findings were observed in the liver and thyroid glands; the findings with unclear relation to the administration of the test item were observed in kidneys, spleen, and stomach.

Hepatocellular hypertrophy (males and females) and increased glycogen accumulation in hepatocytes (females) were noted at 400 mg/kg bw/day and correlated to the increased absolute and relative liver weights and a tendency to an increase in serum triglycerides (males and females). Hepatocellular hypertrophy and higher liver weights as well as glycogen accumulation in the cytoplasm of hepatocytes were reversible, considered adaptive changes and hence non-adverse.

In thyroid follicles, epithelial hypertrophy was recorded for some males and females in all test item-treated groups. In males at 400 mg/kg bw/day, an increase in finding frequency was statistically significant. Follicular epithelial hypertrophy can occur with enzyme induction and increased hepatic clearance of thyroxin, was reversible and is considered to be species-specific and non-adverse. In parental animals, microscopic findings in thyroid gland were not correlated to changes in the thyroxin level as well as to the changes in organ weight.

Based on these findings, the no observed adverse effect level (NOAEL) for systemic toxicity was derived to be ≥ 400 mg/kg bw/day.

Justification for classification or non-classification

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