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Repeated dose toxicity: oral

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short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-02-05 to 2016-03-14
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 03 October, 2008
according to
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
May 30, 2008
GLP compliance:
yes (incl. certificate)
Limit test:

Test material

Test material form:
solid: particulate/powder

Test animals

Details on test animals and environmental conditions:
- Age at study initiation: 7-8 weeks
- Weight at study initiation: males: 162 – 211 g, females: 133 – 182 g
- Housing: individually, IVC cages (type III H, polysulphone cages) on Altromin saw fibre bedding
- Diet (e.g. ad libitum): ad libitum, Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)

- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
polyethylene glycol
PEG 400
Details on oral exposure:
The test item formulation was prepared freshly on each day of administration.

- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on the test item’s characteristics and on results obtained from another study.
- Concentration in vehicle: 0, 2, 6, 20 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): S7106885
Analytical verification of doses or concentrations:
The HPLC-UV method was used for determination of the test material
Details on analytical verification of doses or concentrations:
Concentration analysis of formulation samples was determined at three concentrations, 2.0 mg/mL, 6.0 mg/mL and 20.0 mg/mL in study weeks 1, 2, 3 and 4. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 104.1%, 96.5% and 100.9% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups in study week 1 and 2 as measured concentrations were within acceptance criterion of 15%.In study week 3 however, the single samples no. 22 (LD), 23 (MD) and 24 (HD) did not meet the acceptance criteria as recovery was higher than 15% of nominal concentration. These results were verified by reanalysis. In study week 4 measured concentrations for LD and HD were within acceptance criterion of 15% but MD sample 27 showed a recovery lower than 15% of nominal value.
Reanalysis of 27 (MD) in triplicate proved the concentration to be within the acceptance criterion.
Homogeneity of formulation samples was determined at two concentrations, 2.0 mg/mL and 20.0 mg/mL, in study weeks 1 and 4. The mean recoveries observed for the LD dose group was 98.9% and 90.0% of the nominal value and 99.8% and 88.4% of the nominal value for HD dose group.
The coefficients of variation (COV) of the different sampling locations (top, middle, bottom) were 0.8% and 0.4% in LD dose group, 1.4% and 0.5% in HD dose group. All samples were homogenous, as COV was below or equal 15%.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
According to the results of a previous dose range finding study (dose levels: 100, 300, 1000 mg/kg bw) and in consultation with the sponsor the above listed doses were applied.
- Rationale for animal assignment (if not random):
The animals were weighed and adjacent assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively.
- Rationale for selecting satellite groups:
In order to detect any possible delayed occurrence or persistence of or recovery from toxic effects
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
not applicable


Observations and examinations performed and frequency:
- twice daily

- Time schedule: Detailed cage side observations were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.

- Time schedule: Clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing.
- parameters: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size.

- Time schedule for examinations: Once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment and the recovery period

- Time schedule: weekly

- 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


OPHTHALMOSCOPIC EXAMINATION: Yes, before the first administration and in the last week of the treatment period as well as at the end of the recovery period in the recovery animals.

- Time schedule for collection of blood: At the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
- Anaesthetic used for blood collection: Yes: Xylazin/ketamin
- Animals fasted: Yes, over night
- How many animals: all animals
- Parameters: haematocrit, haemoglobin, red blood cell count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocytes, platelet count, white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells, blood coagulation, i.e. prothrombin time and activated partial thromboplastin time

- Time schedule for collection of blood: At the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
- Animals fasted: Yes, over night
- How many animals: all animals
- Parameters: alanine aminotransferase, aspartate-aminotransferase, alkaline phosphatase, creatinine, total protein, albumin, urea, total bilirubin, total bile acids, total cholesterol, glucose, sodium, potassium

- Time schedule for collection of urine: A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes, over night
- Parameters: specific gravity, nitrite, pH, protein, glucose, ketone bodies, urobilinogen, bilirubin, erythroctes, leukocytes

- Time schedule for examinations:
Once before the first exposure and once in the fourth week of exposure as well as in the last week of the recovery period
- Dose groups that were examined:
all animals
- Battery of functions tested:
grip strength / sleeping / moving in cage / piloerection / vocalization / grooming / salivation / lacrimation / changes in skin / cyanosis / exophthalmos / eyes opening / respiration / response to handling / arousal / fear / finger approach / head touch / body position / spontaneous activity / ataxic gait / hypotonic gait / twitches / tremors / seizures / unusual behavior / stereotypie / rearing supported / rearing not supported / urination / defecation / feaces consistency / abnormal vocalization / aggressiveness / startle response / equilibrium reflex / positional passivity / visual placing / pinching the tail / toe pinch reflex / limb tone / hind limb reflex / righting reflex ground / air righting reflex / pupil response / body temperature / anterior chamber of the eye / fundus of the eye
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table1)

HISTOPATHOLOGY: Yes (see table1)
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Furthermore, statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test or Mann-Whitney U-Test when appropriate. These statistics were performed with Ascentos 1.1.3 software or GraphPad Prism V.6.01 software (p < 0.05 is considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Moving the bedding was observed in in all groups, immediately after administration. This transient clinical finding is closely related to the dosing procedure and, with a lower duration and incidence, was also observed in the negative control. Also abnormal breathing and nasal discharge were recorded in the HD group with an incidence of 6/10 males and 3/10 females for a period of 1 to 5 days. Those findings are considered to be related to events during the dosing procedure and to be the reasons for morbidity of 5 animals. An effect of the test item is not assumed. Other clinical findings were observed on few or single animals for a very short period and are therefore considered isolated incidental findings which are toxicologically not relevant.
mortality observed, non-treatment-related
Description (incidence):
Animals no. 27 (recovery HD male) and 48 (HD female) were found dead, both on study day 3. In addition, animals no 28, 30 (recovery HD males) and 60 (HD female) were euthanised for ethical reasons on study day 9, 17 and 28, respectively. Before euthanasia or day before they were found dead, all animals showed clinical findings in the respiratory tract such as abnormal breathing or in addition nasal discharge. For all animals, but animal no 60, histopathological findings in the respiratory tract were reported. Histopathologically for those animals the cause of morbidity is deemed to be incidental or accidental events related to the dosing procedure, but not directly related to the test item. In histology animal no 60 showed forestomach hyperkeratosis, squamous cell hyperplasia and submucosal edema in the forestomach, moderate thymic atrophy as well as lymphoid depletion in the spleen (marked), axillary lymph node (minimal) and mesenteric lymph node (minimal), and slight adrenocortical diffuse hypertrophy. Considering the lack of systemic toxicity in the survivors, the reason for death is assumed to be related to stress.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
A slightly statistically significantly higher body weight increase was recorded from day 22 to 28 in the LD males. However, this isolated finding is considered toxicologically not relevant. No considerable or statistically significant differences in mean body weight were recorded between any of the dose groups and the corresponding controls at the end of the treatment and of the recovery phase.
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:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
At the end of the treatment period, statistically significantly higher mean values of WBC were observed in HD males (7.7 E9/mL) compared to the controls (4.7 E9/mL). However this effect was not recorded for females and was absent in males at the end of the recovery phase. In LD females, at the end of the treatment phase, the level of MCH was slightly but statistically significantly lower (18.1 pg) compared to the corresponding control group (19.02 pg). This effect was neither observed in higher dosed females, nor in any of the males groups. At the end of the recovery phase there was no considerable and no statistically significant difference in MCH in the HD female animals compared to the control. Moreover, the higher levels of WBC in HD males and the lower levels of MCH in females are within the normal range of variation of historical control data for this strain. All other hematology parameters were within the normal range of variation of historical control data for this strain and differences between dose and control groups are not assumed to be biologically relevant. Blood coagulation was not affected by the test item.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At the end of the treatment and of the recovery phase, all parameters of clinical chemistry in all groups were within the normal range of variation of historical control data for this strain.
Urinalysis findings:
no effects observed
Description (incidence and severity):
All parameters of urinalysis of the respective treatment groups at the end of the treatment and recovery period were not considerably different to the corresponding control and were within the normal range of variation of historical data for this strain.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
In the recovery HD group, statistically significantly higher body temperature pre-values were recorded (recovery HD males 38.60°C, females 38.48°C) compared to the C group (C males 37.96°, females 37.94°C). However, the difference is only very slight and within the expected range of variation for this parameter. At the end of the study, i.e. after 4 weeks of treatment, in the HD males the value for supported rearing was statistically significantly lower compared to the corresponding control with values of 1.2 (HD males) vs 5.0 (C group). Due to the fact that at 4 weeks only 2 males remained in the recovery HD no statistical test was possible. Nonetheless, with a mean value of 1.0 the recovery HD males showed a similar effect at 4 week time point. At the end of the recovery phase the mean value for this supported rearing was similar in both the control and the recovery HD males, which suggest a recovery. Moreover, this effect was not observed in females. Therefore the effect of the test item on supported rearing is considered to be restricted to male animals and to be not adverse.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
At the end of the treatment period, in LD males for 1/5 animals a higher weight of epididymides was recorded with a value of 3.0 g versus 1.1 g in mean of the control males. This is assumed to be an isolated finding which is not toxicologically relevant. In females no. 38 (LD) and 45 (MD) very high weights of pituitary gland were assessed with values of 0.1620 g (animal no. 38) and 0.1368 g (animal no. 45) compared to the mean of 0.0119 g in female controls. The initially reported high values could not be confirmed. Therefore, values recorded for weight of pituitary glands of animals 38 and 45 were considered to be invalid and not test item related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In MD male no 13 and in HD males no 18 and 19 enlarged axillary lymph nodes on one side were recorded which correlated in histology with sinus dilatation or bilateral activation.
In control male no 3 yellow epididymides were observed which correlated in histopathology with bilateral sperm granuloma.
In HD male no 26 at the end of the recovery phase small testes were found. In histopathology this finding correlated with tubular atrophy.
Female of the LD group, no 39, showed a cyst at corpus luteum of the ovaries. In that organ no abnormalities were detected in histopathology. In the LD group the animal no 36 showed a fluid filled uterus which was correlated with cyclic change. Those pathological changes recorded at the end of the treatment and recovery phase were either isolated incidental findings, were observed in control animals or were related to normal female cyclic changes and are therefore considered to be normal background alterations.
In HD animal no 28, euthanized on study day 9, a dark lung was found which correlated with aspiration pneumonia in histopathology.
In HD female no 48 a lung with bloody appearance was observed. There was no corresponding finding in histopathology.
In female no 60 (HD recovery group), which was euthanized on day 28, on the right side an enlarged adrenal gland with dark discoloration were recorded. The findings correlated in histopathology with diffuse hypertrophy and hemorrhage.
A dark and marbled thymus was found in HD male 28 which was euthanised on study day 9. There was no correlation found in histology.
Gas filled organs, i.e. stomach, duodenum and ileum were partially found in HD recovery males 27 (found dead on study day 3), 28 (euthanized on study day 9), 30 (euthanized on study day 17) and in the HD recovery female no 60 (euthanized on study day 28). No corresponding abnormalities were found for these organs in histopathology. Gaseous distention in the stomach and/or small intestine was histologically deemed to be a non-specific, postmortem change which is frequently recorded in dead animals.
Several autolytic organs were recorded in HD female no 48 which was found dead on study day 3.
As outlined above the death of all that animals is considered to relate to events during the dosing procedure.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Under the conditions of this study, treatment-related histomorphologic changes were observed in the stomach of Group 4 (100 mg/kg bw/day). They consisted of diffuse hyperkeratosis as well as squamous cell hyperplasia in the forestomach, forestomach submucosal edema, and increased basophilia of foveolar cells, increase in globular leukocytes and increased mucous neck cells in the glandular stomach. Among these findings, changes recorded in the forestomach were treatment-related local lesions by the direct contact of the test item to mucosa, but those were deemed not to be relevant to humans. Histomorphologic changes recorded in the glandular stomach were deemed to be an adaptive response to irritative stimuli by the direct contact of the test item to the mucosal surface, and were considered not to be adverse, since there was no indicator of cellular and tissue injuries. Findings of forestomach and glandular stomach described above were no longer present in any animals that survived until the end of recovery period. There is a possibility that the daily administration of the highest dose level, 100 mg/kg bw/day, gives local irritative stress to animals, and this dose level might be near the maximum tolerated dose in the rat model with the regimen of repeated dosing for approx. 4 weeks. The remainder of findings recorded in the present study was within the range of normal background lesions which may be recorded in animals of this strain and age, or was incidental lesions that were not related to treatment with the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no treatment-related effects observed up to 100 mg/kg bw

Target system / organ toxicity

Key result
Critical effects observed:

Applicant's summary and conclusion

On the basis of the present study, the 28-Day Repeated Dose Oral Toxicity study with test substance in male and female Wistar rats, with dose levels of 10, 30, and 100 mg/kg body weight day the following conclusions can be made:
From the result of histopathology, the histomorphological no-observed-adverse effect level (NOAEL) for systemic toxicity could be established at 100 mg/kg bw/day. It was interpreted that the treatment-related findings recorded in the forestomach are not relevant to humans. The treatment-related findings recorded in the glandular stomach are deemed to be an adaptive response and not adverse under the condition of this study, although the test item may give local irritative effects when the large amount of it was exposed to mucosal tissues regardless of animal species. The local findings in stomach correlated with administration related clinical findings and findings in FOB. The clinical findings represent a reaction to local toxicological effects and the findings in FOB were reversible and are considered not adverse.
Therefore, the overall NOAEL of the test item for systemic toxicity in this study is considered to be at 100 mg/kg/day.