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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:
November 2012-February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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
Specific details on test material used for the study:
EXP1200078
Lot no. E00275-350
Exp. date: End-2013
Dark brown, opaque, viscous liquid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 51 days
- Weight at study initiation: male 147-168 grams, female 127-152 grams
- Fasting period before study: prior to clinical pathology blood collection
- Housing: Upon arrival animals were housed 2 to 3 per cage by sex for approximately 3 days. Thereafter, all animals were housed individually in stainless steel, wire-mesh cages.
- Diet: ad libitum PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal)
- Water: ad lbitum Reverse osmosis-treated (on-site) drinking water
- Acclimation period: 22 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0-22.1
- Humidity (%): 32.9-54.9
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 December 2012 To: 02 January 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: mineral oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The vehicle suspension was dispensed approximately weekly for administration to the control group and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature. The vehicle was mixed throughout the dispensation, sampling, and dose administration procedures.The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: (0, 20, 60 and 200 mg/mL)
- Amount of vehicle (if gavage): 5 mL/kg
- Lot no.: 1AL0557
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test substance formulations at concentrations that bracket those used on the present study was confirmed in a previous study (Coffee, 2013, WIL 537029) following 5- and 12-day storage at room temperature. Therefore, additional assessments of formulation stability were not conducted.
Quadruplicate samples for homogeneity determination were collected from the top, middle, and bottom strata of the first preparation of the 20 and 200 mg/mL dosing formulations. The last aliquot used for dosing from the first formulation preparation at these concentrations was also sampled for resuspension homogeneity (quadruplicate samples collected from the top and bottom strata). Quadruplicate samples for concentration analysis were collected from the middle stratum of the dosing formulations prepared for use during study weeks 0 and 3 (including the control group). One duplicate set was analyzed and the remaining duplicate set was stored at room temperature and retained as backup samples. All analyses were conducted by the WIL Research using a validated high performance liquid chromatography method using fluorescence detection.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Mineral oil vehicle
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 14-day oral range-finding toxicity study (Haas, 2013, WIL-537025) in which there were non-adverse findings of wet clear material around the mouth at 1 to 2 hours post-dosing in the 1000 mg/kg/day group males.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: within 3 days of receipt, 1 week (± 2 days) prior to randomization, on the day of randomization, weekly (± 2 days) during the study, and on the day of the scheduled necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: within 3 days of receipt, 1 week prior to randomization, on the day of randomization, prior to dosing on study day 0, weekly during the study, and on the day prior to the scheduled necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

FOOD EFFICIENCY:
- 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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: study day 28
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters according to guideline were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: study day 28
- Animals fasted: Yes
- How many animals: all animals
- Parameters according to guideline were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: study day 28
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters according to guideline were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during pretest and during study week 3 (prior to dose administration)
- Dose groups that were examined: all groups
- Battery of functions tested: FOB: sensory activity / grip strength / motor activity / handling observations / open field observations / neuromuscular observations / physiological observations

OTHER: organ weight
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. A complete necropsy was conducted on all animals. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The following tissues and organs were collected and placed in 10% neutral-buffered formalin:
Adrenals (2)
Aorta
Bone with marrow
Femur
Sternum
Bone marrow smear (from femur) a
Brain
Cervix
Epididymides (2) b
Eyes with optic nerve (2) c
Gastrointestinal tract
Esophagus
Stomach
Duodenum
Jejunum
Ileum
Cecum
Colon
Rectum
Heart
Kidneys (2)
Larynx
Liver (sections of 2 lobes)
Lungs (including bronchi, fixed by inflation with fixative)
Lymph nodes
Axillary (2)
Mandibular (2)
Mesenteric Ovaries with oviducts (2) d
Pancreas
Peripheral nerve (sciatic)
Peyer’s patches
Pharynx
Pituitary
Prostate
Salivary glands (mandibular [2])
Seminal vesicles (2)
Skeletal muscle (rectus femoris)
Skin (with mammary gland) e
Spinal cord (cervical, lumbar, thoracic)
Spleen
Testes (2) b
Thymus
Thyroid (with parathyroids, if
present [2]) d
Tongue
Trachea
Urinary bladder
Uterus
Vagina
Gross lesions (when possible)

a = Bone marrow smears were obtained at scheduled necropsy, but not placed in formalin; slides were not examined.
b = Fixed in modified Davidson’s solution.
c = Fixed in Davidson’s solution.
d = Parathyroids and oviducts were examined if in the plane of section and in all cases where a gross lesion of the organ was present.
e = For females; a corresponding section of skin was taken from the same anatomic area for males.


HISTOPATHOLOGY: Yes. After fixation, protocol-specified tissues were trimmed and processed into paraffin blocks, sectioned at 4 to 5 microns, mounted on glass microscope slides, and stained with hematoxylin and eosin.
Microscopic examination was performed on all tissues mentioned by gross pathology from the animal found dead and from all animals in the control and 1000 mg/kg bw/day groups at the scheduled necropsy. In addition, gross lesions were examined microscopically from all animals in the 100 and 300 mg/kg bw/day groups.
Statistics:
Conducted by WIL Research: All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Body weight, body weight change, food consumption, continuous FOB, clinical pathology, and organ weight data were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. FOB parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test (Steel and Torrie, 1980).
Conducted by BioSTAT Consultants, Inc: All statistical analyses were conducted using SAS® version 9.2 (SAS Institute, Inc., 2002-2008), or higher. The SAS® procedure PROC MIXED was used for analysis with the random effect of animal included as the repeated measurement. The covariance structure across time was selected by comparing Akaike’s Information Criterion (AIC).
The monotonic dose-response relationship was evaluated using sequential linear trend tests based on ordinal spacing of dose levels. The linear dose by time interaction (LinTrt*Time) was evaluated. If the linear dose by time interaction was not significant, the trend test was conducted across the pooled time intervals for the entire session only.
Nonmonotonic dose responses were evaluated whenever no significant linear trends were detected but TRT and/or TRT*TIME interaction was significant at the 0.01 level. Within the framework of the RANOVA, pairwise comparisons were made for each individual test substance-treated group with the control group through linear contrasts. These nonmonotonic dose-response comparisons were conducted at the 0.01 significance level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
test substance-related clear material around the mouth
Mortality:
mortality observed, treatment-related
Description (incidence):
test substance-related clear material around the mouth
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:
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):
Test substance-related higher mean liver weights were noted in the 1000 mg/kg/day group males and females and a lower mean seminal vesicles weight was noted in the 1000 mg/kg/day group males.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no test substance-related deaths. At a low incidence, test substance-related clear material around the mouth was noted in the 1000 mg/kg/day group males (only noted 1 to 2 hours after dose administration beginning on study day 11) and females (at the time of dosing and 1 to 2 hours post-dosing beginning on study day 8).

HAEMATOLOGY: Lower mean absolute neutrophil counts were noted in the 100 and 300 mg/kg/day group males; however, these changes did not show a clear dose-related response. Slightly higher, not statistically significant, mean white blood cell counts and mean absolute lymphocyte counts were noted in the 1000 mg/kg/day group males and females. However, the group means and all individual animal values for the 1000 mg/kg/day group males and females were within the WIL Research historical control database ranges. These observed changes were due to individual animal biological variation and were not considered to be test substance related.

ORGAN WEIGHTS: Higher mean liver weights (absolute, relative to final body weight, relative to brain weight) were noted in the 1000 mg/kg/day group males (12.6%, 10.5%, and 14.8%, respectively) and females (11.5%, 7.0%, and 10.3%, respectively); differences were not statistically significant in the females. However, all individual animal absolute liver weight values were within the WIL Research historical control database range of ± 2 standard deviations from the mean with the exception of 1 individual animal value in the 1000 mg/kg/day group females. There were no clinical pathology and/or histologic changes correlating to the higher liver weights.
A lower mean seminal vesicles weight relative to final body weight (17.7%) was noted in the 1000 mg/kg/day group males. However, the lower mean absolute seminal vesicles weight and mean seminal vesicles weight relative to brain weight differences were not statistically significant (16.2% and 14.9%, respectively), all individual animal absolute seminal vesicle weight values were within the WIL Research historical control database reference range, and there were no histologic changes correlating to the lower seminal vesicles weight. This change was considered to be toxicologically unimportant.


Effect levels

Key result
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: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The analyzed dosing formulations were within WIL Research’s SOP range for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the vehicle formulation that was administered to the control group.

Applicant's summary and conclusion

Executive summary:

EXP1200078 was administered by daily oral gavage during 28 days to Crl:CD rats (5/sex) at dose levels of 0, 100, 300 and 1000 mg/kg/day, according to OECD guideline 407.

There were no test substance-related deaths. Body weight, food consumption, functional observational battery, locomotor activity, hematology, coagulation, serum chemistry, and urinalysis parameters were unaffected by test substance administration. There were no test substance-related gross necropsy observations or histologic changes.

Test substance-related clinical observations of clear material around the mouth were noted in the 1000 mg/kg/day group males and females.

Test substance-related higher liver weights were noted in the 1000 mg/kg/day group males and females and a lower seminal vesicles weight was noted in the 1000 mg/kg/day group males. These observations were not accompanied by histopathological changes, and considered to be not relevant/adverse.

Based on the results of this study, the NOAEL was considered to be 1000 mg/kg/day, the highest dosage level administered.