Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 May to 24 Jun 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study run to a method comparable with current guidelines and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Batch No.: CPo84700201-022901
Purity: 98%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 6 weeks.
- Weight at study initiation:
- Fasting period before study:
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material and paper as cage-enrichment. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cageenrichment or bedding material.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet. During motor activity measurements, animals had no access to food.
- Water (e.g. ad libitum): Free access to tap water except during motor activity measurements.
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 40-70%
- Air changes (per hr): 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity of the test substance.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Stability for at least 6 hours at room temperature is confirmed over the concentration range 0.6 to 15 mg/mL.
- Concentration in vehicle: 0.6, 3, 15 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
Duration of treatment / exposure:
At least 28 days. Animals were dosed up to the day prior to necropsy.
Frequency of treatment:
Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrations
Remarks:
Doses / Concentrations:
3, 15, 75 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for selecting satellite groups: Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).

Examinations

Observations and examinations performed and frequency:
Mortality / Viability: At least twice daily.

Clinical signs: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately after dosing. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded.

Functional Observations: During Week 4 of treatment, the following tests were performed at no specific time point, but within a similar time period after dosing for the respective animals(abbreviations mentioned in the respective tables indicated between brackets):
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent): in Group 1 and 4.
- fore- and hind-limb grip strength were recorded as the mean of three measurements (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands): in Group 1 and 4.
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA): in all animals. Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
Since the hearing ability, pupillary reflex, static righting reflex and/or grip strength measurements did not reveal treatment-related effects in Group 4 animals, these respective tests were not performed for Group 2 and 3 animals at the end of the treatment period.

Body weights: Weekly.

Food consumption: Weekly.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Clinical laboratory investigations: Blood samples were collected under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m. on the day of necropsy at the end of the treatment phase. Animals were deprived of food overnight (for a maximum of 24 hours), but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids. The following parameters were determined:
Haematology: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets;
Clotting Potential: Prothrombin time, Activated Partial thromboplastin time;
Clinical Biochemistry: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.
Sacrifice and pathology:
GROSS PATHOLOGY: All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.
HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all Group 1 and 4 animals,
- liver (males and females) and thyroid (females) of all animals of groups 2 and 3, based on (possible)
treatment-related changes in these organs in group 4,
- all gross lesions
Other examinations:
Organ weights: The following organ weights and terminal body weight were recorded from the animals on the scheduled day of necropsy: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid.
Statistics:
If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
The Steel-test was applied if the data could not be assumed to follow a normal distribution.
The Fisher Exact-test was applied to frequency data.
The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
Mortality
No mortality occurred during the study period.

Clinical signs
No clinical signs of toxicity or abnormalities during weekly arena observations were noted during the observation period.
Salivation seen after dosing among animals at 15 and 75 mg/kg on Days 27 and 28 was considered to be a physiological response rather than a sign of systemic toxicity, considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test substance.

Functional observations
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals.
Motor activity was considered similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. The apparent higher motor activity of males at 3, 15 and 75 mg/kg was ascribed to control values being lower than expected based on background control values. Since the mean motor activity of treated males showed no dose-related increase and remained with the background range, motor activity of these males was considered to have been unaffected by treatment.

Body weights
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

Food consumption
Food consumption before or after correction for body weight remained similar to the control level over the study period.

Haematology
No toxicologically relevant changes occurred in haematological parameters of treated rats.
The (statistically significant) lower values of prothrombin time and activated partial thromboplastin time of females at 15 and/or 75 mg/kg were considered to be of no toxicological significance. A lower prothrombin time and activated partial thromboplastin time has no toxicological relevance since an opposite effect is expected in case of target organ toxicity and these lower values occurred in the absence of a dose-related trend and/or remained within the range considered normal for rats of this age and strain.

Clinical biochemistry
The following changes in clinical biochemistry parameters at 75 mg/kg were considered to be related to treatment:
- Lower albumin level in males (and a similar trend in females, not statistically significant),
- Lower bilirubin level in males,
- Higher alanine aminotransferase activity (ALAT) in females,
- Higher aspartate aminotransferase activity (ASAT) in females,
- Lower cholesterol level in males (not statistically significant),
- Higher cholesterol level in females (not statistically significant),
- Lower glucose level in males and females (not statistically significant),
- Higher bile acid level in females (not statistically significant),
Any other statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological significance as these occurred in the absence of a dose-related trend and/or remained within the range considered normal for rats of this age and strain.

Macroscopic examination
Macroscopic examination revealed an enlarged and/or pale discolored liver of males (4/5) and females (5/5) at 75 mg/kg. In addition, an accentuated lobular pattern of the liver was observed for females (4/5) at 75 mg/kg.
The incidence of other necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.

Organ weights
Statistically significantly higher absolute and relative liver weights were present in males and females at 75 mg/kg (relative weights were increased with approximately 51% and 96% for males and females, respectively).
Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

Microscopic examination
Microscopic examination revealed hepatocellular vacuolation (mainly macrovesicular, periportal) in one male and two females treated at 15 mg/kg at minimal degree and in all males and females treated at 75 mg/kg up to marked degree. In addition hepatocellular hypertrophy (mainly centrilobular) was present in all males and females treated at 75 mg/kg at slight degree.
All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Males and females at 75 mg/kg had marked increase in liver weight with hepatocellular vacuolation up to marked degree and slight hypertrophy.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the marked increase in liver weight with hepatocellular vacuolation up to marked degree and slight hypertrophy in males and females at 75 mg/kg, a No Observed Adverse Effect Level (NOAEL) for the test substance of 15 mg/kg was established.