Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and 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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
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

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 10-12 weeks.
- Weight at study initiation: All animals were within +/- 20% of the sex mean.
- Housing: Males and females were housed in groups of 5 in macrolon cages. During mating, males and females were caged together in macrolon cages. During lactation, pups were kept with the dams until termination.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet, except during motor activity assessments for the selected males and females, and for 24 hours prior to blood chemistry sampling.
- Water (e.g. ad libitum): Free access to tap water, except during motor activity assessments for the selected males and females.
- Acclimation period: At least 5 days prior to the start of treatment

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance. Test formulations were stored at room temperature protected from light until use. Thevehicle was chosen based on the results of trial formulations performed prior to treatment.

VEHICLE
- Concentration in vehicle: 0, 2.5, 5 and 10 mg/mL
- Amount of vehicle (if gavage): Dose volume of 5 mL/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was conducted at a single sample point during the study according to a validated method. Samples of formulation were analysed for homogeniety (highest and lowest concentration) and accuracy of preparation (all concentrations). The stability in the vehicle over 6 hours at room temperature under protection from light was also determined (highest and lowest concentration). The accuracy, homogeniety and stability of test samples as appropriate were reported to be in the satisfactory ranges, ensuring the acceptability of dosing formulations.
Duration of treatment / exposure:
Males were exposed for 29 days (i.e. 2 weeks prior to mating, during mating and up to the day prior to scheduled necropsy). Females were exposed from 42-55 days (i.e. during 2 weeks prior to mating, during mating, during post-coitum (gestation) and during at least 4 days of lactation up to the day prior to scheduled necropsy). The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.
Frequency of treatment:
Parental males and females daily via oral gavage
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 12.5, 25 and 50 mg/kg/day
Basis:
other: Nominal concentration
No. of animals per sex per dose:
- A total of 80 rats were used on study (40 males and 40 females).
- Each test group consisted of 10 males and 10 females (including 10 males and females in the control groups).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based on the reults of a 10-day dose range finding study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards and up until the day prior to necropsy. Detailed observations were made in all animals at least 1 hour (+/- 30 minutes after dosing). The schedule was selected based on the peak period of anticipated effects from the dose range finding study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the start of treatment at at weekly intervals during the treatment period this was performed outside the home cage and in a standard arena. Any signs observed were graded for severity.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum (gestation) and during lactation on days 1 and 4.

FOOD CONSUMPTION: Yes
- Food cunsumption was measured weekly except for males and females that were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum (gestation) and during lactation on days 1 and 4.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy for the selected 5 animals/sex/group.
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes (a maximum of 24 hours prior to sampling)
- How many animals: 5 animals/sex/group
- Parameters examined: Parameters examined: Leukocyte, RBC, HGB, RDW, Haematocrit, MCV, MCH, MCHC, reticulocyte ratio, differential leukocyte count/ratio, platelets, PT and APTT.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy for the selected 5 animals/sex/group.
- Animals fasted: Yes (a maximum of 24 hours prior to sampling)
- How many animals: 5 animals/sex/group
- Parameters examined: ALAT, ASAT, ALP, Total protein, Albumin, Total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The tests were performed on 5 animals/sex/group. The selected males were tested during week 4 of treatment and the females were tested towards the end of the scheduled lactation period (at least 1 hour after dosing).
- Dose groups that were examined: All
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and locomotor activity.
Sacrifice and pathology:
All animals surviving until the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated and subject to a full post mortem examination (with special attention paid to reproductive organs).

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).

Selected 5 animals/sex/group and animal number 35 that dies spontaneously.
Adrenal glands, Aorta, Brain (cerebellum, midbrain, cortex (7 levels)), caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, eyes (with optic nerve and haderian gland), female mammary gland area, femur including joint, heart, Ileum, Jujunum, kidneys, Lacrimal gland (exorbital), Larynx, liver, lung (infused with formalin), Lymph nodes (mandibular and mesenteric), Nasopharynx, esophagus, ovaries, pancreas, peyer's patch, pituitary gland, preputial gland, prostate gland, rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, midthoracic and lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid, tongue, trachea, urinary bladder, uterus, vagina and all gross lesions.

All remaining animals and females that failed to deliver:
cervix, clitoral gland, coagulation gland, epididymides, mammary gland area, ovaries, preputial gland, prostate, seminal vesicles, testes, uterus, vagina and all gross lesions.

The epididymides, eyes and testes were fixed in modified Davidson's solution and water, and transferred to formalin after fixation for at least 24 hours.

Histopathology
Tissues from 5 high dose and control animals were processed and stained with haematoxylin and eosin prior to microscopic examination. The additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males suspected to be infertile or which died before mating to examine stages of spermatogenesis. All gross lesions were also examined and the reproductive organs of all males that failed to sire and all females that failer to deliver healthy pups.

A peer review of the histopathology data was performed by a second pathologist.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-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 (many-to-one rank 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. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

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 different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY:
One male at 50 mg/kg was found dead on Day 2 of treatment at approximately 1 hour after dosing. No clinical signs were observed prior to death, and no macro/microscopic abnormalities were observed. As no futher mortality was observed in the treatment period, and this death happened after a short time, it is not considered to be related to treatment.

In all test groups, flat posture, calm behaviour, ventro-lateral recumbency, ptosis and/or animals lying dispersed in cages were shown by all males onDays 28 and/or 29 of study. Flat posture was also noted among all females in all test groups during Week 5 onwards. In addition, all females at 25 and 50 mg/kg showed ptosis, with lethargy, abnormal posture, and ventro-lateral recumbency (at lower frequencies) across all treated groups during Week 5 onwards.

It could not be excluded that these signs were also present earlier during the treatment period, and were first noticed at a later time point when the scheduled functional observation tests were conducted (just after conduct of the clinical signs at ca. 1 hour after dosing). Additional observations observed on subsequent days showed that these signs were intermittent in nature and showed reversibility by approximately 3-7 hours after dosing.

BODY WEIGHT AND WEIGHT GAIN:
Males in all treatment groups had a slightly lower bodyweight gain during the mating period, achieving statistical significance on most occassions. At the end of the study the total mean bodyweight gain of males was slightly lover than controls, although no dose response was noted for this affect. Mean bodyweight gain for females was lower during the lactation period in females receiving 50 mg/kg with no statistical significance. This was attributed to a slight weight loss for two females in this group.

FOOD CONSUMPTION:
For females a level of statistical significance was attained on all occassions at 50 mg/kg, and on several occassions at 12.5 and 25 mg/kg. During lactation lower food consumption was recorded for females at all dose groups, attaining statistical significance at 12.5 and 50 mg/kg. These changes showed no dose-related trend. No statistically significant effects were observed in males.

HAEMATOLOGY:
Haematology parameters were considered to have been unaffected by treatment.

CLINICAL CHEMISTRY:
Clinical chemistry parameters were considered to have been unaffectd by treatment, however females receiving 50 mg/kg showed lowered creatinine levels. This effect showed no significant morphological correlate.

NEUROBEHAVIOUR: Females at 25 and 50 mg/kg showed a lower motor activity for both total movements and ambulations, being statistically significant only for total movements. Motor activity (total movements) of males at 12.5, 25 and 50 mg/kg also appeared lower than the control mean (not statistically significant). Since the number of ambulations of these males was similar to control levels, this indicated that the number of fine body movements of these males was lower than controls. No clear dose-related response was apparent for any of these changes. In contrast to males, there were no apparent differences in motor activity habituation profile between female treated and control groups. Hearing ability, pupillary reflex and static righting reflex were normal in all selected males and females of all groups. Grip strength was similar across the dose groups.

ORGAN WEIGHTS:
Organ weights and organ to body weight ratios of treated animals were considered to be unaffected by treatment.

GROSS PATHOLOGY:
There were no test substance related macroscopic abnormalities.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no test substance related morphological alterations or abnormalities up to and including 50 mg/kg.

Effect levels

Dose descriptor:
NOAEL
Effect level:
12.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results observed on study, it was concluded that a NOAEL value of 12.5 mg/kg was appropriate and a conservative value based on the effects observed.








Executive summary:

The experimental laboratory concluded in the report that no NOAEL for parental effects could not be derived based on the clinical signs observed in combination with lower motor activity, and changes in bodyweight and food intake observed. On review of the data, it was considered that the effects observed at 12.5 mg/kg were not considered adverse in nature and as such the parental NOAEL was set as 12.5 mg/kg. The following justifications are provided showing a NOAEL value of 12.5 mg/kg as appropriate:

No adverse effects were observed in the majority of experimental endpoints including clinical pathology, macro and microscopic pathology, FOB parameters (excluding locomotor activity) and organ weights in any dose group along with no observed treatment related deaths at any dose.

The clinical signs observed were shown intermittently and showed full reversibility at approximately 3-7 hours post dose in both sexes. The signs observed were less apparent in animals that received 12.5 mg/kg and all signs observed were graded as slight or moderate rather than severe in the laboratories grading system.

With regards to the locomotor activity, females that received 25 or 50 mg/kg showed reduced motor activity scores for total movements and ambulations with statistical significance only for total movements, with no statistically significant effects observed in animals that received 12.5 mg/kg. In males, minor effects were observed although no statistical significance was obtained in any dose and no clear dose response was apparent.

With regards to bodyweight gain, males in all dose groups had a slightly lower bodyweight gain during the mating period and by the end of the study bodyweight gain in treated males was 4-5% lower than controls. As no dose response was indicated for this change it was not be considered adverse, and also the possible bodyweight effects in females were considered as not significant (a result of slight weight loss in two animals).

With regards to food consumption, no statistically significant effects were observed in males for food consumption and in females the effects observed during lactation showed no dose related change and as such are considered non-adverse.

Based on this information it could be concluded that no adverse effects were observed at any dose, although based on the effects, it was considered that a NOAEL value of 12.5 mg/kg was conservative and as such appropriate.