Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 23, 2015 - Mar 27, 2017
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted October 03, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: Chinese Guidelines for testing of Chemicals (HJ/T153-2004), Appendix A, State Environmental Protection Administration, 2004-06-01.
Deviations:
no
Qualifier:
according to
Guideline:
other: Chinese Guidance for New Chemical Substance Notification (first version), Nr. 7.1, Chemical Registration Center of SEPA, October 2004.
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species: Rat
Strain: Crl: WI (Han)
Breeder: Charles River, Germany
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 8w
- Weight at study initiation: males: 233 g (mean), 212-249 g (range), females: 171 g (mean), 158-189 g (range)
- Fasting period before study: no
- Housing: grouped
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 23.7°C
- Humidity (%): 31.5 - 74.2 %
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Administration was done orally by gavage, once daily, 7 times a week for 4 weeks. The control rats received the vehicle, 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium), at the same frequency as the animals treated with the test material. The volume of administration was 10 mL/kg body weight, the volume of administration per animal was calculated by means of the LIM-System.
Vehicle:
other: 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)
Details on oral exposure:
Administration was done orally by gavage, once daily, 7 times a week for 4 weeks. The control rats received the vehicle, 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium), at the same frequency as the animals treated with the test material. The volume of administration was 10 mL/kg body weight, the volume of administration per animal was calculated by means of the LIM-System.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity data of the test material in 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium) were evaluated in parallel to the current study. According to the stability and homogeneity data, the formulations were prepared for up to 7 study days and before the first use the formulations were stirred overnight. Formulations were stored under room conditions. Exposure to light was kept to a minimum. Determination of test item concentration was performed for 2 preparations: each time at the beginning of a preparation period and at the end of a preparation period. Formulation analysis was performed using an HPLC method. The preparations were stored under normal room conditions. Exposure to light was kept to a minimum. Analytical results are given in mg/mL.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
once daily, 7 times a week for 4 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
0 mf/kg: 20 (10 m / 10 f)
100 mg/kg: 10 (5 m / 5 f)
300 mg/kg: 10 (5 m / 5 f)
1000 mg/kg: 20 (10 m / 10 f)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected on the basis of results of a 7-day oral pilot toxicity study in rats with the test material, a single dose and two 4-week repeat-dose toxicity studies in rats with two structurally similar compounds.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: recovery
- Post-exposure recovery period in satellite groups: 2 weeks
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Clinical Signs
Mortality, the behavior and appearance of each animal were checked daily. Parameter and symptoms were recorded with the LIM-System.

Body Weight
Body weight was recorded before treatment and thereafter weekly with the LIM-System.

Food consumption
Food consumption was determined once a week by weighing the food per cage which had not been consumed. The parameter was recorded with the LIM-System.

Water consumption
Water consumption was determined twice a week by measuring the water per cage which had not been consumed. The parameter was recorded with the LIM-System.

Laboratory tests
In week 5 and 7 approximately 2.5 mL blood was taken sublingually before necropsy under inhalation anesthesia. The blood samples were divided for haematological and clinico-chemical examinations. Before blood sampling the animals were kept in metabolism cages for the collection of urine for approximately 18 hours without food.

Gross Pathology
At the day of necropsy, all rats were anesthetized by a carbon dioxide air mixture and exsanguinated by opening the abdominal vessels. Then, the animals were necropsied, examined for gross pathological alterations, the weights of selected organs were recorded and histotechnical procedures and histopathological examinations were performed.

Body and Organ Weights
The body and organs weights were recorded by the LIM-system. Based on the absolute organ weights the relative organ weights (related to 100 g body weight) were calculated. The following weights were determined:
Terminal body weight (after exsanguination)
Heart
Liver
Kidneys (together)
Spleen
Thymus
Testes (together)
Prostate
Uterus
Ovaries (together)
Adrenals (together after fixation)
Thyroids with Parathyroids (together after fixation)
Brain (after fixation)
Epididymides
Seminal vesicle

Histopathology
The organs and tissues of main kill animals were fixed, histotechnically processed and examined as listed below:
Adrenal (2)
Aorta
Bone (femur)
Bone marrow (sternum, femur)
Brain (cerebrum, cerebellum, brain stem)
Esophagus
Eye (2)
Heart
Intestine, large
Cecum
Colon
Rectum
Intestine, small
Duodenum
Jejunum
Ileum
Kidney (2)
Knne joint
Larynx
Liver (left lateral and right medial lobe)
Lung (with mainstem bronchi)
Lymph nodes
mandibular (2)
mesenteric
Mammary gland (inguinal)
Micro transponder
Muscle, skeletal (thigh)
Nasal turbinates
Nerve, optic (2)
Nerve, sciatic
Pancreas
Parathyroid (2)
Peyer’s Patches
Pituitary
Reproductive organs, female
Ovary (2)
Oviduct (2)
Uterus (cornu/corpus/cervix)
Vagina
Reproductive organs, male
Epididymis (2)
Prostate
Seminal vesicle
Testis (2)
Salivary gland (2) (submandibular, parotid, sublingual)
Skin (inguinal)
Spinal cord (cervical, thoracal, lumbal)
Spleen
Stomach (proventricular, fundic, pyloric)
Thymus
Thyroid (2)
Tongue
Trachea
Ureter (2)
Urinary bladder
Zymbal's gland (2)
All tissues showing abnormalities

In addition, mesenteric lymph nodes, thymus and liver were also evaluated in all group 2 and 3 main and in all recovery kill animals. All histopathology findings were recorded with the LIM-System.
Sacrifice and pathology:
At the day of necropsy, all rats were anesthetized by a carbon dioxide air mixture and exsanguinated by opening the abdominal vessels. Then, the animals were necropsied, examined for gross pathological alterations, the weights of selected organs were recorded and histotechnical procedures and histopathological examinations were performed.
Statistics:
To compare the treatment groups with the control group, the following statistical procedures were applied separately for each sex and each measuring point. To take the number of dose groups into account, all the test procedures used maintain a multiple significance level of alpha = 0.05. For absolute body weight, body weight gain (differences to baseline values on day 1), body temperature, food and water consumption, organ weights (relative and absolute), clinical pathology parameters (hematology, clinical chemistry serum parameters, specific gravity and urine weight), dose groups were compared with those of the control, using the multiple two-sided Dunnett-Test (Dunnett 1955; 1964). For all parameters (for urinalysis only specific gravity and urine weight) mean values, standard deviation, and number of animals (N) were calculated. Software: Body weight, body temperature, food and water consumption, organ weights, hematological parameters, clinico-chemical parameters (for urinalysis only specific gravity and urine weight), were evaluated within the LIM-System. The urinalysis examinations were recorded with the LIMSystem.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One 100 mg/kg/d male showed hair loss. Hair loss is a common background finding in this rat strain and was also observed in one male of the control group. Therefore, this finding is considered incidental. The control male animal that showed hair loss also showed skin wounds at the foreleg and the back.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight gain was statistically significantly increased in 300 mg/kg/d females on day 7. However, this finding was without a dose relationship and not accompanied by increased food consumption during week 1. Therefore, this findings is considered incidental. Body weight and body weight gain did not show any dose-dependent changes at the end of the treatment and recovery period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was dose-dependently and statistically significantly decreased in week 2 (day 7-14) in 300 and 1000 mg/kg/d males and in week 3 and 4 (day 14-28) in all male dose groups. These slight effects are considered treatment-related, but not adverse.
In females food consumption was statistically significantly increased in week 1 (day 1-7) at 1000 mg/kg/d, in week 2 (day 7-14) at 1000 and 100 mg/kg/d and in week 3 (day 14-21) at 100 mg/kg/d. However at 300 mg/kg/d food consumption was statistically significantly decreased from week 2 to 4 in females. These in- and decreases in the female animals are not considered treatment-related, because they were not dose dependent and did no show a correlation to effects on body weight/body weight gain.
During the recovery period food consumption was statistically significantly decreased in 1000 mg/kg/d animals of both sexes from day 28 to 35 and in 1000 mg/kg/d females from day 35 to 42. These slight effects are considered treatment-related, but not adverse.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water consumption was statistically significantly decreased in 1000 mg/kg/d males from day 1 to 7 and statistically significantly increased from day 28 to 31 and 35 to 38. In females, water consumption was statistically significantly increased at 1000 mg/kg/d from day 7 to 21 and day 24 to 28 and at 100 mg/kg/d from day 10 to 21 and day 24 to 28. The effects on water consumption were not dose-dependent and no correlation with changes in urinalysis, serum clinical chemistry parameters, or histopathological changes in the urinary tract were observed. Therefore, a treatment-relationship is questionable.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A decrease of white blood cell count (up to 0.4-fold) and lymphocytes (absolute up to 0.3-fold; relative, up to 0.7-fold) and increase of relative neutrophilic granulocytes (up to 2.0-fold) were detected in both sexes at 1000 mg/kg. A decrease of white blood cell count (0.6-fold) and lymphocytes (absolute, 0.5-fold; relative, up to 0.8-fold) and increase of relative neutrophilic granulocytes (1.7-fold) were detected in both sexes at 300 mg/kg. At 100 mg/kg, decreases of white blood cell count (0.6-fold) and absolute lymphocytes (up to 0.5-fold) were detected in both sexes and in males, relative numbers of lymphocytes were decreased (0.8-fold) and neutrophilic granulocytes increased (1.6-fold). At the end of the recovery period, these changes normalized but not completely in the high dose group (white blood cell count, 0.8-fold; lymphocytes, absolute, up to 0.6-fold; lymphocytes, relative, up to 0.8-fold; neutrophilic granulocytes, relative, up to 1.5-fold).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Alterations seen in clinical chemistry were slight in degree, not dose-dependent and mostly within internal reference limits. They are considered incidental and not treatment-related.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis did not reveal any pathological alterations in the treated animals.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In the functional observational battery (FOB) no treatment-related changes in autonomous, neuromuscular, sensomotoric and central nervous system were noted. The motor activity investigations revealed a dose dependent increase of total counts on day 28 at all dose levels in both sexes, however, statistical significance was reached in 1000 mg/kg/d males only. Corresponding effects were observed on the derived motor activity parameters: counts in 5-minute segments (in most cases), total distance, on-time, rearing time and rearing number. The increases of motor activity parameters were slight in degree and no further central nervous system effects were observed in the FOB. Therefore the changes in motor activity parameters were not considered adverse.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Organ weight changes were noticed in females. The absolute and relative spleen weights were statistically significant decreased to a minor degree in females at 1000 mg/kg/day. As this finding was without a histological correlate it is considered to be of questionable toxicological relevance. All other organ weight changes were regarded as incidental as they showed no dose-dependency or histological correlate. After a 2-week treatment-free period, no spleen weight changes were noticed in females. In
males, a minor statistic significant decrease of relative spleen weights was seen. This finding is regarded as incidental as there were no spleen weight changes and no histological findings in main kill males.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross pathology revealed no treatment-related findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related and dose-dependent histopathological findings in the mesenteric lymph node, thymus and liver (females only) of mid and high dose animals.
In the mesenteric lymph node, a slight sinus histiocytosis and minimal to moderate multifocal macrophage aggregates were seen. These findings are considered to be related to a resorption of the test item. The slight multifocal lymphoid necrosis and occasionally lymphoid depletion noticed in the paracortex of females are regarded as a secondary phenomenon most likely related to the resorption of the test item. After a 2-week treatment-free period, the sinus histocytosis and the lymphoid depletion were completely reversible. The lymphoid necrosis showed a clear trend to recovery. However, macrophage aggregates were still diagnosed with a slightly higher incidence or degree of severity compared with rats of the main kill group.
The findings in the thymus consisted of a slight decrease of the cortex/medulla ratio in rats of both genders and might be stress-related. The findings were completely reversible in females after the 2-week treatment-free period and showed a trend to reversibility in males.
In the liver, a minimal diffuse hepatocellular vacuolation was noticed in females. This finding showed complete reversibility after the recovery period of 2 weeks.
A unilateral nephroblastoma was diagnosed in the kidney of one low dose female (No. 30) treated with 100 mg/kg/day. This findings is considered to be incidental and spontaneous in nature.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Findings were considered non-adverse as only low in severity and/or reversibility or at least a trend to reversibility could be shown and no indicators of decreased organ/tissue functionality were visible.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Daily oral treatment with the test material at doses of 100, 300 and 1000 mg/kg was tolerated over 4 weeks and revealed treatment-related and dose-dependent histopathological findings in the liver (only females), mesenteric lymph node and thymus at 1000 and 300 mg/kg/d. After a 2-week treatment-free period, a complete reversibility or a clear trend to recovery was seen for nearly all findings in the mesenteric lymph node. Macrophage aggregates in the mesenteric lymph node showed no recovery. The findings in the thymus were completely reversible in females and showed a trend to recovery in males. The findings in the liver were completely reversible.
The findings in the mesenteric lymph node (sinus histiocytosis, macrophage aggregates, lymphoid necrosis and depletion), thymus (decreased cortex/medulla ratio) and in the liver (discrete hepatocellular vacuolation) are considered non-adverse as only low in severity and/or reversibility or at least a trend to reversibility could be shown and no indicators of decreased organ/tissue functionality were visible.
The hematological effects (decrease of white blood cells and absolute lymphocytes) also showed a clear trend towards recovery and were not considered adverse.
The effects on food/water consumption and motor activity were only slight in degree and not considered adverse.
The No Observed Adverse Effect Level (NOAEL) in Wistar (Han) rats was established at 1000 mg/kg/d.
Executive summary:

The informtion for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 407.

Daily oral treatment with the test material at doses of 100, 300 and 1000 mg/kg was tolerated over 4 weeks and revealed treatment-related and dose-dependent histopathological findings in the liver (only females), mesenteric lymph node and thymus at 1000 and 300 mg/kg/d. After a 2-week treatment-free period, a complete reversibility or a clear trend to recovery was seen for nearly all findings in the mesenteric lymph node. Macrophage aggregates in the mesenteric lymph node showed no recovery. The findings in the thymus were completely reversible in females and showed a trend to recovery in males. The findings in the liver were completely reversible.

The findings in the mesenteric lymph node (sinus histiocytosis, macrophage aggregates, lymphoid necrosis and depletion), thymus (decreased cortex/medulla ratio) and in the liver (discrete hepatocellular vacuolation) are considered non-adverse as only low in severity and/or reversibility or at least a trend to reversibility could be shown and no indicators of decreased organ/tissue functionality were visible.

The hematological effects (decrease of white blood cells and absolute lymphocytes) also showed a clear trend towards recovery and were not considered adverse.

The effects on food/water consumption and motor activity were only slight in degree and not considered adverse.

The No Observed Adverse Effect Level (NOAEL) in Wistar (Han) rats was established at 1000 mg/kg/d.