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Toxicological information

Repeated dose toxicity: oral

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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
Study period:
From 06 December 2011 to 02 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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 (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Clear colourless to slightly yellowish viscous liquid
- Purity: UVCB
- Storage condition of test material: At room temperature in the dark

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: Age at start F0-treatment: Approximately 10 weeks.
- Weight at study initiation: Males: Mean: 284-286 g. Females: Mean: 196-197 g
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: Acclimatization F0: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 ºC
- Humidity (%): 40 to 70%
- Air changes (per hr): approximately 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 06 December 2011 To: 02 April 2012

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. Adjustment was made for density (1.084 g/cm3) of the test substance.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations analysed in the formulations at dose concentrations of 10, 30, 100 and 200 mg/ml were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

The formulations at the entire range were homogeneous (i.e. coefficient of variation ≤ 10%).

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.
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 for 41-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Frequency of treatment:
Once daily for 7 days per week.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on the results of a 10-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg bw/day.

Examinations

Observations and examinations performed and frequency:
MORTALITY / VIABILITY: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

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 and during lactation on Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Weekly, except for males and females which 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 postcoitum and on Days 1 and 4 of lactation.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: Blood samples were collected from the selected 5 animals/sex/group.
- Parameters:
White blood cells (WBC)
Differential leucocyte count:
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
Red blood cells
Reticulocytes
Red blood cell distribution width (RDW)
Haemoglobin
Haematocrit
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
Platelets
Prothrombin Time (PT)
Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment
- Animals fasted: Yes
- How many animals: Blood samples were collected from the selected 5 animals/sex/group.
- Parameters:
Alanine aminotransferase (ALAT)
Aspartate aminotransferase (ASAT)
Alkaline Phosphatase (ALP)
Total protein
Albumin
Total Bilirubin
Bile acids
Urea
Creatinine
Glucose
Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic Phosphate (Inorg. Phos)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (from lactation Day 4 onwards). These tests were conducted after dosing (all before blood sampling) at no specific time point, but within a similar time period after dosing for the respective animals.
- Dose groups that were examined: 5 animals/sex/group
- Battery of functions tested:
- hearing ability
- pupillary reflex
- static righting reflex
- grip strength
- locomotor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy was conducted on the following days:
Females which delivered: Lactation Days 5-7.
Females which failed to deliver: Post-coitum Days 26-27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
Males: Following completion of the mating period (a minimum of 28 days of dose administration).

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY: Yes
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin.

Selected 5 animals/sex/group:
Identification marks: not processed
Ovaries
Adrenal glands
(Pancreas)
(Aorta)
Peyer's patches [jejunum, ileum] if detectable
Brain - cerebellum, mid-brain, cortex
Pituitary gland
Caecum
Preputial gland
Cervix
Prostate gland
Clitoral gland
Rectum
Colon
(Salivary glands - mandibular, sublingual)
Coagulation gland
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides
Skeletal muscle
Eyes (with optic nerve (if detectable) and Harderian gland)
(Skin)
Spinal cord -cervical, midthoracic, lumbar
Female mammary gland area
Spleen
Femur including joint
Sternum with bone marrow
Heart
Stomach
Ileum
Testes
Jejunum
Thymus
Kidneys
Thyroid including parathyroid if detectable
(Lacrimal gland, exorbital)
(Tongue)
(Larynx)
Trachea
Liver
Urinary bladder
Lung, infused with formalin
Uterus
Lymph nodes - mandibular, mesenteric
Vagina
(Nasopharynx)
All gross lesions
(Esophagus)

All remaining animals, females which failed to deliver:
Cervix
Preputial gland
Clitoral gland
Prostate gland
Coagulation gland
Seminal vesicles
Epididymides
Testes
Mammary gland area
Uterus
Ovaries
Vagina
Identification marks: not processed
All gross lesions

The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.
Other examinations:
ORGAN WEIGHTS:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group:
Adrenal glands
Spleen
Brain
Testes
Epididymides
Thymus
Heart
Uterus (including cervix)
Kidneys
Prostate
Liver
Seminal vesicles including coagulating glands
Ovaries
Thyroid including parathyroid

All remaining males:
Epididymides
Testes
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 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 following additional methods of statistical analysis were used:
Locomotor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test 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 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
Food efficiency:
not examined
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):
no effects observed
Description (incidence and severity):
(A statistically significantly higher locomotor activity noted for females at 1000 mg/kg bw/day as compared to controls was not considered adverse)
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period.

No clinical signs of toxicity were noted during the observation period. Incidental findings that were noted for control and/or treated animals included alopecia and scabbing on different parts of the body. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment.

The higher reticulocyte count with concurrent increased red blood cell distribution width noted for one female at 300 mg/kg bw/day was considered as a chance finding, since it was an isolated finding in the mid dose group only. Moreover, values remained within the normal range of biological variation.

CLINICAL CHEMISTRY
Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment.

Significantly lower total bilirubin was recorded for males at 300 mg/kg bw/day. In the absence of a treatment-related distribution, this finding was not considered as a sign of toxicity. At the individual level, higher alanine aminotransferase and aspartate aminotransferase values were recorded for one male at 1000 mg/kg bw/day. Both values were above the normal range of biological variation. However, since for this male no adverse effects were seen on clinical appearance, body weights, liver organ weight, macroscopy and microscopy, these isolated findings were not considered treatment-related.

NEUROBEHAVIOUR
Females at 1000 mg/kg showed a statistically significant higher locomotor activity (total movements and ambulations) as compared to controls. The habituation profile was comparable to that of the control, 100 and 300 mg/kg bw/day groups, with very high activity in the first interval that decreased over the duration of the test period. However, for females at 1000 mg/kg bw/day, locomotor activity started higher and remained higher throughout the observation period. No treatment-related effects on locomotor activity were seen for females at 100 and 300 mg/kg bw/day and males up to 1000 mg/kg bw/day. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.

ORGAN WEIGHTS
There were no toxicologically relevant changes in organ weights or organ to body weight ratios seen with treatment up to 1000 mg/kg.

Statistically significant changes included lower seminal vesicle organ weights (absolute and relative to body weight) for males at 100, 300 and 1000 mg/kg bw/day, higher relative brain weights for males at 300 mg/kg bw/day and higher relative adrenal weights for males at 1000 mg/kg bw/day. These findings were considered not to be toxicologically relevant as changes were very slight, a treatment-related trend was absent, no corroborative histopathological changes were evident and/or the reproductive ability of the males was not affected.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

Incidental findings seen for control and/or treated animals included pelvic dilation of the kidneys (unilateral), greenish, soft nodule on the tail of the epididymides (both unilateral and bilateral), redbrown discoloration of the thymus, enlarged mandibular lymph nodes (unilateral), tan discoloration of the clitoral glands, and alopecia on different sides of the body. The incidence of these macroscopic findings 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. They were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic observations attributed to the test article.

No significant abnormalities were seen in the reproductive organs of the paired animals that failed to conceive.

Staging of spermatogenesis did not provide any evidence of test article related impairment to the spermatogenetic cycle

Effect levels

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 at the highest dose level.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on these results, a No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.
Executive summary:

The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300, or 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41- 54 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

No parental toxicity was evident up to the highest dose level tested (1000 mg/kg bw/day). The statistically significantly higher locomotor activity (total movements and ambulations) noted for females at 1000 mg/kg bw/day as compared to controls was not considered adverse, since changes were relatively slight, and occurred in the absence of concurrent clinical signs or changes in other

functional observation tests.

No treatment-related effects were noted on any of the remaining parental parameters investigated in this study (i.e. clinical appearance, hearing ability, pupillary reflex, static righting reflex and grip strength, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

Based on these results, a No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.