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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Oct 2012 - 24 Feb 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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 October 2008)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
(adopted 30 May 2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Erlangen, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain specification: Crl: WI(Han)
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 152 - 172 g (males), 130-150 g (females)
- Housing: individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 190612)
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 1039), ad libitum
- Water: tap water (sulphur acidified to a pH of approximately 2.8), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- 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
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulation was prepared freshly on each administration day before the administration procedure. The time of preparation was recorded for all dosing formulations. The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration. Before the first administration, the pH value of the formulations was measured.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.
- Concentration in vehicle: 15, 50 and 120 mg/mL (low-, mid- and high dose)
- Amount of vehicle: 5 mL/kg bw
- Manufacturer: Fresenius Kabi (Ampuwa)
- Lot/batch no.: 19F630WA
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For determination of the concentration of test item in dosing formulations, samples of at least 25 mL were retained from all groups once weekly during the treatment period and stored between -15 and -35 °C. Stability of the dosing formulations was tested once at the beginning of the treatment period. From the low and high dose groups samples of dosing formulations were frozen at 0 hours and 6 hours after the preparation and stored at -15 and -35 °C. In the 1st and 4th week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared high and low dose formulations and stored between -15 and -35 °C.
The determination of test item concentration in the dosing formulations was performed by Eurofins Agrosciences Services EcoChem GmbH. The analytical method met the requirements of guideline SANCO/3029/99. Formulation analytics were performed as stand-alone study. Analytical results were provided to the study director for calculation of the actual test item concentrations.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
- 10 per sex in the control and high dose groups
- 5 per sex in the low and mid dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the previous 14-day range-finding study (section 7.5.1: supporting, Allingham, 2013, 14-day range finder, rat, RL2)
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day
- Cage side observations included: health condition (once daily), morbidity and mortality (twice daily, except on weekends and public holidays when observations were made once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first administration and at least once a week thereafter
- Detailed clinical observations included observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size

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

FOOD CONSUMPTION:
- Time schedule for examinations: weekly during the treatment and recovery period

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: 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
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery period prior to or as part of the sacrifice
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), prothrombin time (PT), activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery period prior to or as part of the sacrifice
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K)

URINALYSIS: Yes
- Time schedule for collection of urine: prior to or as part of the sacrifice of the animals
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes (overnight)
- Parameters examined: colour/ appearance , specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- 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 dose groups
- Battery of functions tested: multiple detailed behavioural observations
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals)
ORGAN WEIGHTS: Yes (all surviving animals: liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/ parathyroid glands, testes, spleen, epididymides, brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries, heart; paired organs were weighed separately)
HISTOPATHOLOGY: Yes (all animals of the control and high dose groups: brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), eye, uterus with cervix (females), liver, vagina (females), kidneys, testes (males), adrenal glands, epididymides (males), small and large intestines (including Peyer´s patches), prostate and seminal vesicles with coagulating glands as a whole (males), stomach, urinary bladder, thymus, lymphnodes (mesentric and axillary), thyroid glands, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, spleen, sternum with bone marrow, lung and trachea, pituitary gland, mammary glands, oesophagus, skin, gross lesions; kidneys, urinary bladder and stomach (glandular and non-glandular) and gross lesions were also examined in all animals of the low and mid dose groups)
Statistics:
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 of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. Statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test. These statistics were performed with GraphPad Prism V.5.01 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
dose-dependent clinical signs associated with local irritation of the test item
Mortality:
mortality observed, treatment-related
Description (incidence):
dose-dependent clinical signs associated with local irritation of the test item
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
significantly higher kidney weights (absolute weight 16%, relative weight 15% above controls) in high dose group males (600 mg/kg bw/day)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg bw/day: multifocal tubular degeneration/regeneration, tubular dilation + crystalline deposits in altered tubules, focally extensive transitional cell hyperplasia in renal pelvis, focally extensive transitional cell hyperplasia of urinary bladder
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
One low dose group female (75 mg/kg bw/day) was found dead on Study day 24. In view of its isolated occurrence and lack of dose relationship this death was not considered to be test item-related.
Male and female animals of the high dose group (600 mg/kg bw/day) moved the bedding immediately after administration. This was mostly associated with slight or moderate piloerection and later also slight, moderate or – in few animals severe salivation at the time of administration. These observations can be ascribed to a local irritant effect of the test item. Moving the bedding and slight piloerection were also noted after administration occasionally in mid dose group animals (250 mg/kg bw/day), and slight signs of irritation (moving the bedding and salivation) were also observed in a single female animal of the low dose group (75 mg/kg bw/day) on a single day. Signs of local irritation were not observed during the recovery period. Slight piloerection was only seen in few animals on the first days of the recovery period.
Observations related to a lesion during gavaging (emesis, severe piloerection and half eye-lid closure, slightly increased spontaneous activity and abnormal breathing, vocalization, severe salivation and hypothermia) were noted in 3 animals of the high dose group and were thus concluded to be not test substance-related.
Occasional isolated findings, like red nasal discharge or eschar were also observed in control animals and are not assumed to be related to the test item.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

BODY WEIGHT AND WEIGHT GAIN
Throughout the treatment and recovery period, body weights were within the normal range of variation for this strain. Body weight gain was not considerably different between the groups and neither for male nor for female animals were there any significant differences in body weight between the dose groups and the control group of this study.

FOOD CONSUMPTION
No significant difference in food consumption was found in the treatment or recovery period between the dose groups and the control group of both genders.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopic findings in any of the animals of this study.

HAEMATOLOGY
The test item had no effect on haematological parameters and coagulation parameters analysed at the end of the treatment and recovery period. There were no toxicologically relevant differences in any of the parameters between dose groups and control group. Slightly – but statistically significantly higher platelet count and prolonged activated partial thromboplastin time (aPTT) in male animals of the high dose group (600 mg/kg bw/day) at the end of the recovery period and of large unstained cells of female animals of the high dose group (600 mg/kg bw/day) at the end of the treatment period were in the normal range of historical data and are not assumed to be biologically relevant.

CLINICAL CHEMISTRY
The test item had no effect on clinical biochemistry markers analysed at the end of the treatment and recovery period. There were no toxicologically relevant differences in any of the parameters between dose groups and control group. Slightly – but statistically significantly lower albumin levels in male animals of all dose groups observed at the end of the treatment period – but not in high dose group animals (600 mg/kg bw/day) at the end of the recovery period are not assumed to be toxicologically relevant. Statistically significantly lower total bilirubin levels in female – but not male animals of the high dose group (600 mg/kg bw/day) at the end of the recovery period are not assumed to be toxicologically relevant.

URINALYSIS
The test item had no effect on urinary parameters analysed at the end of the treatment and recovery period. A high count of red blood cells was found in the urine of a single female each of the mid dose group (250 mg/kg bw/day) at the end of the treatment period and of the high dose group (600 mg/kg bw/day) at the end of the recovery period.

NEUROBEHAVIOUR
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

ORGAN WEIGHTS
At the end of the treatment period a slight and statistically significantly higher kidney weight (absolute weight 16%, relative weight 15% above controls) was found in male animals of the high dose group (600 mg/kg bw/day) and was associated with histopathological findings in the kidney of these animals.
A slightly – but not statistically significantly lower thymus weight (21% below controls) in female – but not male animals of the high dose group (600 mg(kg bw/day) is not considered to be the result of a primary toxicological effect of the test item. When compared to controls, absolute thyroid weight of female animals of the mid dose group (250 mg/kg bw/day; 22% below controls) and high dose group (600 mg/kg bw/day; 27% below controls and statistically significant) was slightly lower than in controls. As this is in contrast to thyroid weight of male animals, where slightly higher thyroid weight was found in mid dose group (250 mg/kg bw/day; 27% above controls) and high dose group (600 mg/kg bw/day; 19% above controls), this is not assumed to be a test item related effect. Slight but statistically significant differences in absolute brain weight of male animals of the low dose (75 mg/kg bw/day) and high dose (600 mg/kg bw/day) groups are not assumed to be toxicologically relevant as values were within the normal range of historical data. At the end of the recovery period, relative kidney weight (to brain weight) of female animals of the high dose group (600 mg/kg bw/day) was slightly (16% above controls) and statistically significantly higher than in controls. Besides, there were no considerably altered organ weights in male and female animals. Statistically significantly higher heart weight of animals of the high dose group (600 mg/kg bw/day) and pituitary gland weight in male high dose group animals (600 mg/kg bw/day) is not considered toxicologically relevant as the values were within the normal range of historical data.

GROSS PATHOLOGY
Only single specific gross pathological changes were recorded for male and female animals at the end of the treatment period and are not considered to be treatment-related. These single findings were a yellow spot on one epididymis and a fluid-filled uterus in a control and high dose (600 mg/kg bw/day) animal, respectively, discoloured red axillary lymph nodes and thymus in one female animal of the low dose group (75 mg/kg bw/day), dark discoloured lungs in another low dose animal (75 mg/kg bw/day), one whitish focus on one adrenal gland and kidney of an mid dose animal (250 mg/kg bw/day) and an uterus distended with fluid. A fluid-filled uterus was also found in one control animal at the end of the recovery period. According to the authors, the above-mentioned alterations were incidental findings and were not assumed to be related to the test item.

HISTOPATHOLOGY: NON-NEOPLASTIC
No histopathological findings were noted that could be associated with the death of the low dose female (75 mg/kg bw/day) found dead. In view of its isolated occurrence and lack of dose relationship this death was not considered to be test item-related.
Test item-related histopathological findings considered to be toxicologically relevant were seen in the kidney and urinary bladder. In the kidney, minimal to moderate multifocal tubular degeneration/regeneration was observed in all high dose group males and females (600 mg/kg bw/day), in all males accompanied by minimal tubular dilation and in most animals showing crystalline deposits within altered tubules. In one single female of this dose group, a minimal focally extensive transitional cell hyperplasia was seen in the renal pelvis. In the urinary bladder focally extensive transitional cell hyperplasia was also noted in two animals of the high dose group (600 mg/kg bw/day). Renal and urinary bladder changes noted in high dose animals (600 mg/kg bw/day) were not reversible after the 14-day recovery period and were considered to be adverse. Minimal multifocal tubular degeneration/ regeneration, noted in 2/5 mid dose group males (250 mg/kg bw/day) at terminal sacrifice, was not considered adverse as it was not accompanied by any changes in clinical pathology or organ weight parameters.
Furthermore, at terminal sacrifice there was indication of a local irritant effect of the test item formulation to the gastric epithelium, predominantly comprising diffuse hyperplasia of the nonglandular epithelium and/or epithelial hyperplasia of the limiting ridge in both sexes of the high dose group (600 mg/kg bw/day) and in females of the mid dose group (250 mg/kg bw/day). These lesions did not resolve during the recovery period. In the glandular part of the stomach, an increased incidence and severity of submucosal mixed cell infiltrate(s) and some minor (multi)focal mucosal congestion/haemorrhage were noted in the high dose group (600 mg/kg bw/day), but were no longer seen after the recovery period. As gastric lesions were related to the specific administration route used in this study, they were considered to be irrelevant for risk assessment in humans. Likewise, in the lung, multifocal minimal or mild chronic (peri)bronchitis was noted in two high dose males (600 mg/kg bw/day), at terminal sacrifice, and was considered to indicate a local irritant effect of the test item formulation, when accidentally partially instilled into the lung.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion