Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Feb - 18 Apr 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

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 03 Oct 2008
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
adopted 31 May 2008
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050 (28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted July 2000
GLP compliance:
yes (incl. certificate)
Remarks:
OGYEI National Institute of Pharmacy and Nutrition, Budapest, Hungary
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han:WIST
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Toxi-Coop Zrt., Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: less than 9 weeks
- Weight at study initiation: 233 - 252 g (males) and 164 - 190 g (females)
- Housing: Up to 3 animals of the same sex in type III polypropylene/polycarbonate cages embedded with certified laboratory wood bedding
- Diet: ssniff® SM R/M-Z+H complete diet (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: The food was considered not to contain any contaminants at levels that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Water quality control analysis and microbiological assessment are performed once in every six months by National Public Health and Medical Officer Service. The water was considered not to contain any contaminants at levels that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 8-12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 Feb To: 27 Mar 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: The test item was formulated in vehicle at concentrations of 10, 30 and 100 mg/mL. Formulations were prepared beforehand for not longer than 3 days and were stored in a refrigerator (5 ± 3 °C) until use.

- VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Lot no.: 17/154012

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing formulations were analysed for homogeneity and concentration of the test item twice during the study. The analyses of the formulations on the first and last week of treatment demonstrated homogeneity of the dose formulations with achieved concentrations in the range of 94 % to 108 % of nominal concentrations.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 7 days/week
Doses / concentrationsopen allclose all
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:
5 (main groups)
5 (recovery period: controls and high-dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: The dose levels were chosen on the basis of the results of a 14-day dose range finding study in rats (Toxi-Coop study no. 673-400-4231). The test item was formulated in polyethylene glycol and administered at doses of 100, 300 and 1000 mg/kg bw/day by oral gavage to five rats per group and sex for a period of 14 days. There was no mortality and no clinical signs of toxicity noted up to the highest dose tested. Body weight development, food consumption, haematology and clinical chemistry were as well unaffected and gross necropsy revealed no abnormalities.
- Rationale for selecting satellite groups: Assessment of reversibility, persistence or delayed occurrence of potential toxicological effects
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (at the beginning and end of each working day)
- Parameters examined: clinical signs, morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
- Parameters examined on general appearance: Skin, fur, eyes and mucous membranes; special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.
- Parameters examined on autonomic activity: lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions
- Parameters on battery of functions: circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 (prior to first treatment), once weekly during the treatment and recovery periods and immediately prior to sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, food consumption was evaluated and reported by weekly interval for each group.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the acclimatisation period and prior to test termination
- Dose groups that were examined: control and the high dose groups (1000 mg/kg bw/day)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last treatment and at the end of the recovery period
- Anaesthetic used for blood collection: Yes (Isoflurane CP)
- Animals fasted: Yes
- How many animals: all animals of all dose groups
- Parameters examined for haematology: white blood cell count (WBC), red blood cell count (RBC), haemoglobin concentration (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration MCHC), platelet count (PLT), mean platelet volume (MPV) and reticulocytes (RET)
- Parameters examined for blood coagulation: activated partial thromboplastin time (APTT) and prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last treatment and at the end of the recovery period
- Anaesthetic used for blood collection: Yes (Isoflurane CP)
- Animals fasted: Yes
- How many animals: all animals of all dose groups
- Parameters examined: alanine aminotransferase activity (ALT), aspartate aminotransferase activity (AST), gamma glutamyltransferase activity (GGT), alkaline phosphatase activity (ALP), total bilirubin concentration (TBIL), creatinine concentration (CREA), urea concentration, glucose concentration (GLUC), cholesterol concentration (CHOL), inorganic phosphate concentration (Pi), calcium concentration (Ca++), sodium concentration (Na+), potassium concentration (K+), chloride concentration (Cl-), albumin concentration (ALB), total protein concentration (TPROT) and albumin/globulin ratio (A/G ratio)

URINALYSIS: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals underwent gross pathological examination at termination of the treatment treatment, on Day 28 (main groups) and at the end of the recovery period on Day 42 (recovery groups). The external appearance (surface of the body, all orifices) was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically for each animal. All observations were recorded with details of the location, color, shape and size.
The following organs were weighed: Liver, kidneys, testes, epididymides, prostate and seminal vesicles with coagulating glands as a whole, uterus and fallopian tubes, thymus, spleen, brain and heart, adrenal glands and ovaries. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
The following organs/tissues were preserved for histopathological examination: adrenal glands, aorta (thoracic and abdominal), bone with joint and marrow (femur), brain (cerebrum, cerebellum, pons and medulla oblongata), esophagus, eyes (lachrymal gland with Harderian glands), gonads (testes with epididymides, ovaries, uterus with vagina, fallopian tubes), gross lesions, heart, kidneys, large intestine (caecum, colon, rectum, including Peyer’s patches), liver, lungs (with main stem bronchi), lymph nodes (submandibular and mesenteric), mammary gland, muscle (quadriceps), nasal turbinates, pancreas, pituitary, prostate, salivary glands (submandibular), sciatic nerve, seminal vesicles with coagulating gland, skin, small intestine (duodenum, ileum jejunum), spinal cord (at cervical, mid-thoraic and lumbar level), spleen, sternum, stomach, thymus, thyroids and parathyroid, trachea, urinary bladder
Histopathological examination was performed on the preserved organs and tissues of the control animals and the animals from the high dose group including recovery animals.
Statistics:
Statistical analysis was performed on body weight, food consumption, haematology, blood coagulation, clinical chemistry and organ weight. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.
For evaluation of data obtained during the recovery period, the homogeneity of variance between groups was checked by F-test. Depending on the result, pooled or separate variance estimate of the Two-Sample t-test was performed.
Frequency of clinical signs, pathological and histopathological findings by sex and dose was calculated.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Soft faeces were noted among all groups from study Day 9 until the end of the treatment period, and within the first two days of the recovery period (study Days 28-29). The faeces of the animals were normal from the third day of the recovery period. The findings were attributed to the vehicle and no related to the test item.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males there was a trend toward reduced body weight and body weight gain at all doses. However, statistically significant changes were only noted in the high dose during the first week of treatment (30.3 g versus 37.3 g in the control in males). At the end of the treatment period mean body weights were reduced by 1.1, 3.5 and 4.2% at 100, 300 and 1000 mg/kg bw/day dose levels, respectively. Although this reduction in final body weights was dose-related it was not considered of toxicological relevance due to the slight degree of the effect.
In females of the high dose group, the mean body weight on study Day 7 was significantly reduced when compared to controls (188.6 g versus 195.0 g in the control). Similar to the observation in males, the mean body weight gain in females during the first week was significantly reduced (9.4 g versus 16.3 g in the control). These slight changes had no influence on the overall body weight gain of these animals therefore it was judged to be without any toxicological relevance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
During the treatment period, a statistically significant decrease of the mean food consumption was observed in male animals at 100 and 1000 mg/kg bw/day during the first week, in male animals at 300 and 1000 mg/kg bw/day during the third week and in male animals at 1000 mg/kg bw/day during the last week of the treatment period. In females, a statistically significant decrease of the mean food consumption was observed at 1000 mg/kg bw/day during the first and last week of the treatment period. These occasionally fluctuations were considered to be without toxicological meaning.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related adverse changes in the examined haematological parameters at any dose group. At the end of the treatment period, a number of adverse effects was observed in males:
100 mg/kg bw/day: statistically significant decrease in the mean HCT volume and statistically significant increases in the MCHC and in MPV
300 mg/kg bw/day: biologically significant decrease of HGB, HCT volume and statistically significant decrease in the mean RBC and statistically significant increase in the MCHC
1000 mg/kg bw/day: biologically significant decrease in the mean RBC, HGB concentration, HCT volume and biologically significant increase in the MCHC
Although the mentioned differences in RBC, HGB, HCT and MCHC between control and treated groups reached statistical significance, they were of minor degree and mostly remained within the historical control ranges (refer to Table No. 1 under “Any other information in results incl. tables”). The statistically significant increase in MPV at the lowest dose group was considered to be without a toxicological meaning. All alterations in haematology parameters in males were fully reversible and recovered to those of control levels during the recovery period.
At the end of the recovery period, there was a statistically significant increase in the mean percentage of reticulocytes at 1000 mg/kg bw/day. The finding was judged to be toxicologically not relevant.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
A number of statistically significant alterations was noted in males and females among all dose groups, but the changes were considered not test item-related and not toxicologically relevant:
100 mg/kg bw/day: slightly lower mean K+ levels in males and decreased AST activity in females after 28 days of treatment
300 mg/kg bw/day: decreased CHOL in females after 28 days of treatment
1000 mg/kg bw/day: increased mean A/G ratios in males and decreased CHOL in females after 28 days of treatment; decreased CREA after 14 days of recovery in females
Urinalysis findings:
not examined
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):
After 28 days of treatment, the absolute mean heart weight was statistically significantly reduced in males at 300 mg/kg bw/day (-13%) and 1000 mg/kg bw/day (-11%) when compared to control animals. There were no significant alterations of the relative heart weight. At 300 and 1000 mg/kg bw/day, mean relative testes weights (relative to body and/or brain weights) were significantly increased. Absolute testes weight was similar in all groups. The alterations were considered as significant differences without biological meaning in the absence of any histopathological findings.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
After 28 days of treatment, bilateral pyelectasia was observed in 2/5 males of the 1000 mg/kg bw/day group and in 1/5, 2/5 and 1/5 females of the control, 100 and 300 mg/kg bw/day groups, respectively. There were no inflammatory or other pathological signs related to this finding. Pyelectasia is a common finding in rats of this strain, thus the findings were not attributed to treatment.
After the recovery period, 2/5 females of the control group and 2/5 females of the high dose group showed slight or moderate hydrometra. Hydrometra is a frequent observation in experimental rats. Due to the lack of inflammatory or other pathological signs and the absence of a dose-response relationship the finding was judged to be toxicologically not relevant and not related to the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related microscopical lesions observed. A number of histopathological abnormalities was occasionally observed in control animals and animals of the high dose group:
Alveolar emphysema was noted in the lungs of 1/5 recovery control males, in 1/5 recovery control females, in 1/5 males and 1/5 females at 1000 mg/kg bw/day and in 1/5 recovery 1000 mg/kg bw/day females.
Hyperplasia of bronchus associated lymphoid tissue (without inflammatory lesions) was noted in 1/5 control males, 1/5 recovery control males and in 1/5 males at 1000 mg/kg bw/day. The finding represents a physiological immunomorphological phenomenon without pathological significance.
Uni- or bilateral pyelectasia was noted in1/5 females and 2/5 males at 1000 mg/kg bw/day. The finding was without degenerative, inflammatory or other histopathological lesions and is commonly found in experimental rats, thus the observation was considered without toxicological significance.
Dilatation of the uterus horns was observed in 1/5 control females, 2/5 recovery control females and in 2/5 females of the recovery 1000 mg/kg bw/day group. The finding was related to the prooestrus phase of the inner genital organs and not related to treatment.
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)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1: Body weight gain in male and female rats

Dose Group
[mg/kg bw/day]
Body weight gain (g, mean ± SD) between days
Treatment period Recovery period
0-7 7-14 14-21 21-27 0-27 27-34 34-41 27-41
MALES
Control 37.3 ± 6.3 36.1 ± 7.4 25.5 ± 6.4 11.3 ± 5.7 110.2 ± 16.1 15.8 ± 6.2 15.2 ± 9.9 31.0 ± 11.5
100 36.0 ± 6.0 32.6 ± 5.2 24.2 ± 6.6 15.4 ± 4.5 108.2 ± 15.9 nd nd nd
300 34.6 ± 4.2 33.4 ± 5.5 18.8 ± 4.0 12.2 ± 7.5 99.0 ± 9.0 nd nd nd
1000 30.3 ± 4.8* 37.4 ± 7.1 19.1 ± 8.4 8.3 ± 5.9 95.1 ± 16.4 19.2 ± 4.7 13.8 ± 6.8 33.0 ± 6.6
FEMALES
Control 16.3 ± 4.6 14.5 ± 4.8 3.1 ± 8.6 9.3 ± 9.0 43.2 ± 12.7 -0.2 ± 7.4 5.0 ± 3.2 5.2 ± 8.8
100 16.8 ± 5.2 11.4 ± 6.9 7.0 ± 7.5 5.2 ± 7.2 40.4 ± 11.1 nd nd nd
300 14.0 ± 3.8 12.2 ± 3.3 6.2 ± 10.6 6.2 ± 8.8 38.6 ± 9.4 nd nd nd
1000 9.4 ± 7.3* 16.2 ± 6.8 4.5 ± 7.3 6.9 ± 7.2 37.0 ± 11.0 2.0 ± 4.0 6.2 ± 4.5 6.0 ± 3.4
± %: Percent deviation versus control; * = p < 0.05; ** p < 0.01; nd: no data
Number of animals: control animals and 1000 mg/kg bw/day group: 10 male + 10 female during treatment, 5 male + 5 female during recovery; 100 and 300 mg/kg bw/day: 5 male + 5 female during treatment, no recovery animals 

Table 2: Haematology parameters

Group
(mg/kg bw/day)
RBC (1012/L) HCD range
(1012/L)
HGB (g/L) HCD range
(g/L)
HCT (L/L) HCD range
(L/L)
MCHC (g/L) HCD range
(g/L)
Control 8.67 7.4 - 9.9 163.4 142 - 184 0.50 0.39 - 0.52 328.20 350 - 375
100 8.31 159.2 0.47 340.40
300 8.17 156.6 0.46 342.00
1000 7.99 154.0 0.44 346.60

RBC: red blood cells; HCD: historical control data; HGB: haemoglobin; HCT: hematocrit; MCHC: mean corpuscular haemoglobin concentration

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the NOAEL for systemic toxicity was 1000 mg/kg bw/day, which corresponds to the highest dose tested.