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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2015- December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD guideline and in accordance with GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): EXP1503090
- Physical state: Brown, clear liquid
- Storage condition of test material: at room temperature (18°C to 24°C), under a nitrogen blanket

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, USA
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 145 g to 195 g (males), 120 g to 154 g (females)
- Fasting period before study: no
- Housing: 2 to 3 per cage by sex in solid bottom cages containing ground corncob bedding material
- Diet: PMI Nutrition International, LLC, Certifi ed Rodent, LabDiet® 5002 (meal), ad libitum
- Water: Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 to 22.5
- Humidity (%): 41.5 to 55.9
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 August 2015 To: 18 December 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Mineral oil, USP and Corn oil, NF
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The vehicle control mixture was prepared approximately weekly for administration to the control group. Aliquots were prepared for daily dispensation to the control group and stored at room temperature (18°C to 24°C), protected from light. The vehicle control was mixed throughout the preparation, sampling, and dose administration procedures. The test substance formulations were prepared daily or up to every 5 days as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18°C to 24°C), protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

The control substance was mineral oil, USP, and corn oil, NF, mixture. The concentration of mineral oil in the control was 32 mg/mL, based on the concentration of mineral oil in the high-dose formulation: (30 mg/mL x 2.85 correction factor) x 37% mineral oil content.

The vehicle and test substance formulations were administered orally by gavage via an appropriately sized flexible Teflon®-shafted, stainless steel ball-tipped dosing cannula. The dose volume for all groups was 10 mL/kg bw. Individual doses were based on the most recently recorded body weights to provide the correct mg/kg bw/day dosage. Adjusted doses became effective the day of collection of the weekly body weights.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In a validation study, test substance homogeneity and stability following at least 5 days of room temperature storage was established for test substance concentrations in the diluent at 1 and 100 mg/mL. Therefore, stability was not conducted as part of the current study.
Samples for concentration and homogeneity determination were collected from the top, middle, and bottom strata of the 3 and 30 mg/mL dosing formulations and from the middle of the control and 10 mg/mL dosing formulations prepared for use on study day 0. In addition, samples for homogeneity determination were collected from the top, middle and bottom strata of the 3 and 30 mg/mL dosing formulations prepared on study day 15. Samples for resuspension homogeneity were collected from the top and bottom strata of these same 3 and 30 mg/mL dosing formulations following room temperature (18°C to 24°C) storage for at least 5 days. In addition, samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared for use during study weeks 3, 7, and 12. One duplicate set was analyzed and the remaining duplicate set was stored at room temperature (18°C to 24°C) and retained as backup samples. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated gas chromatography method using flame ionization detection.
Duration of treatment / exposure:
90 or 91 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 100, 300 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
15 (control group and 300 mg/kg bw/day group)
10 (30 mg/kg bw/day group and 100 mg kg bw/day group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosage levels were determined from results of a previous range-finding study in rats (WIL-537040). In that study, male and female Sprague Dawley rats were administered for 14 consecutive days at dosage levels of 0, 10, 30, 100, 300, and 450 mg/kg bw/day. In the females, there were no significant clinical signs and no effects on mean body weights, body weight gains, or food consumption at any dose level tested. Significant increases in ALP and ALT were observed in the 300 and 450 mg/kg bw/day groups when serum chemistry was evaluated at the time of necropsy. Additional support comes from two other studies for the Calcium Salicylate (CAS 114959-46-5) which is an appropriate read across for the Magnesium Salicylate. A 14-day range-finding study performed was conducted at doses of 0, 50, 125, 250, 500, and 1000 mg/kg bw/day to both male and female Sprague Dawley rats. Test substance was dosed, uncorrected, from material supplied at 43% w/v Active Ingredient (AI). Minor effects were observed with body weight or body weight changes only at the highest dose. Serum chemistry (primarily the liver) was affected in both sexes at doses of 125, 250, 500, and 1000 mg/kg bw/day. In a 28-day, repeat oral dose study, doses were set at 0, 50, 150, and 500 mg/kg bw/day uncorrected, from material supplied at 43% w/v AI. Slight changes were observed with slightly lower body weight gains, longer prothrombin tim es and several elevated liver enzymes all in 500 mg/kg bw/day male animals. In the 500 mg/kg bw/day group, there were also increased liver weights (in both males and females) and increased thyroid weights in males. All findings resolved during the 14-day recovery period.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily during recovery period)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: within 4 days of receipt, on the day of randomization, weekly (± 2 days) during the study period, and on the days of the scheduled necropsies.

BODY WEIGHT: Yes
- Time schedule for examinations: on study day 0 (prior to dosing), weekly (± 2 days) during the dosing and recovery periods, and on the day prior to the first day of the scheduled necropsies (nonfasted). Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the day of the scheduled necropsies.

FOOD CONSUMPTION:
- 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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimation (all animals) and near the end of the dosing period (study week 12, control group and 300 mg/kg bw/day group).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at scheduled necropsy (study weeks 13 and 17)
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Total leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Prothrombin time, Activated partial thromboplastin time, Reticulocyte count Percent, Absolute, Mean platelet volume, Differential leukocyte count (percent and absolute neutrophil, lymphocyte, monocyte, eosinophil, basophil, large unstained cell), Red cell distribution width, Hemoglobin distribution width, Platelet estimate, Red cell morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at scheduled necropsy (study weeks 13 and 17)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Albumin, Total protein, Globulin (by calculation), Albumin/globulin ratio (by calculation), Total bilirubin, Urea nitrogen, Creatinine, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Glucose, Total cholesterol, Calcium, Chloride, Phosphorus, Potassium, Sodium, Sorbitol dehydrogenase, Triglycerides, Magnesium, Appearance.

URINALYSIS: Yes
- Time schedule for collection of urine: at scheduled necropsies (study weeks 13 and 17)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Specific gravity, pH, Urobilinogen, Total volume, Color, Clarity, Protein, Glucose, Ketones, Bilirubin, Occult blood, Leukocytes, Nitrites, Microscopy of sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during study week 12/13
- Dose groups that were examined: all
- Battery of functions tested: FOB, home cage observations / handling observations / open field observations / sensory observations / neuromuscular observations / physiological observations /motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thora cic, abdominal, and pelvic cavities, including viscera.
ORGAN WEIGHTS: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries with oviducts, Pituitary, Prostate with seminal vesicles, Spleen, Testes, Thymus, Thyroid with parathyroids, Uterus.
HISTOPATHOLOGY: Yes. The following tissues and organs were collected: Adrenals, Aorta, Bone with marrow, Femur, Sternum, Bone marrow smear (from femur), Brain (7 levels), Cervix, Epididymides, Eyes with optic nerve, Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys, Larynx, Liver (sections of 2 lobes), Lungs (including bronchi), Lymph nodes (Axillary, Mandibular, Mesenteric), Ovaries with oviducts, Pancreas, Peripheral nerve (sciatic), Peyer’s patches, Pharynx, Pituitary, Prostate, Salivary glands (mandibular), Seminal vesicles, Skeletal muscle (rectus femoris), Skin with mammary gland, Spinal cord (cervical, thoracic, lumbar), Spleen, Testes, Thymus, Thyroid (with parathyroids), Tongue, Trachea, Urinary bladder, Uterus, Vagina, Gross lesions (when possible).
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests 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 to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Functional observational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test.
All repeated measures analysis of variance (RANOVA) statistical analyses during study week 12/13 were conducted using SAS® version 9.2 software. 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. 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 was evaluated and, if significant at the 0.05 level, trend tests on treatment means were performed at the 0.05 level for each time interval. 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. Pairwise comparisons were made for each individual test substance-treated group with the control group through linear contrasts (for each time interval or across the pooled time intervals for the entire session), conducted at the 0.01 significance level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
clinical signs at 100 and 300 mg/kg bw/day
Mortality:
mortality observed, treatment-related
Description (incidence):
clinical signs at 100 and 300 mg/kg bw/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decreased in 300 mg/kg bw/day males
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:
effects observed, treatment-related
Description (incidence and severity):
at 300 mg/kg bw/day
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):
statistically significant higher weights of liver (m/f), adrenal glands (m/f), spleen (m) and kidneys (f), non-statistically significant increased weight of thyroid/parathyroid (f)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
liver and adrenal cortex at the primary necropsy
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no test substance-related deaths. Three males were found dead prior to the primary necropsy, with on study day 49 one control male and one one high dose group male, and on study day 34 one low dose male. The cause of death in all three animals was undetermined. There were no macroscopic or microscopic findings in each of the 3 unscheduled deaths that were considered to be related to administration of the test substance.
Test substance-related clinical observations noted for the 100 and 300 mg/kg bw/day group males and females included the following: rales, red nasal discharge, salivation, and/or red, yellow, and/or clear material on various body surfaces. These findings were noted in a dose-related manner at the detailed physical examinations, at the time of dose administration, and/or 1-2 hours following dose administration generally throughout the dosing period. No remarkable clinical findings were observed during the recovery period. All other clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.

BODY WEIGHT AND WEIGHT GAIN: Test substance-related effects on body weights were noted in the 300 mg/kg bw/day group males. Lower mean body weight gains and mean cumulative body weight gains were noted throughout the dosing period, and these differences were primarily statistically significant. As a result, mean body weights for the 300 mg/kg bw/day group males were 13.9% lower (statistically significant) than the control group at the end of the dosing period (study week 13). Mean body weight gains for the 300 mg/kg bw/day group males were similar to the control group during the recovery period. However, mean body weights were statistically significantly lower ( 14.8% to 17.2%) than the control group during the recovery period. As the body weights were as much as 13.9% lower during the dosing period and did not recover, the effects on body weights in the 300 mg/kg bw/day group males were considered to be adverse.
There were no other test substance-related effects on body weight. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. As these differences did not occur in a dose-related manner or were transient in nature, they are considered not toxicologically relevant.

FOOD CONSUMPTION: Test substance-related effects on food consumption were noted for the 300 mg/kg bw/day group males. Slightly lower mean food consumption values were noted in the 300 mg/kg bw/day group males generally throughout the dosing period. The differences were occasionally statistically significant and correlated with the effects on body weights noted for these males. Mean food consumption for the 300 mg/kg bw/day group males was similar to the control group during the recovery period. There were no other test substance-related effects on food consumption. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. These differences were not considered to be test substance-related as they were transient in nature and/or the direction of change was not toxicologically relevant.

OPHTHALMOSCOPIC EXAMINATION: No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups.

HAEMATOLOGY: Test substance-related, non-statistically significant, higher total white blood cell and absolute lymphocyte values were noted in the 300 mg/kg bw/day group males and females at the primary necropsy. Six individual 300 mg/kg bw/day group males had values outside the range of the control group males and two individual 300 mg/kg bw/day group females had values outside the range of control group females. There were no histologic correlates, and the magnitude difference was considered to be biologically irrelevant. There were no other test substance-related alterations in hematology or coagulation parameters at the scheduled necropsies.

CLINICAL CHEMISTRY: Test substance-related and statistically significantly higher serum liver enzymes were noted in the 300 mg/kg bw/day group males (alkaline phosphatase (+579%), alanine aminotransferase (+185%), and aspartate aminotransferase (+33%)) and females (alkaline phosphatase (+706%) and alanine aminotransferase(180%) at the primary necropsy. Additionally, higher serum alkaline phosphatase values were noted in the 100 mg/kg bw/day group males. Serum liver enzymes were similar to control groups at the recovery necropsy. There were no other statistically significant differences in serum chemistry parameters; in high dose males non-significant increases in cholesterol and triglyceride levels were observed at the primary necropsy. At the end of the recovery period levels of cholesterol and glucose were non-significantly decreased. These observations were not considered toxicologically relevant. In high dose females an increased blood triglyceride level was observed.

URINALYSIS: Statistically significantly lower urine specific gravity and higher total urine volume values were noted in the 300 mg/kg bw/day group males when compared with the control group at the primary necropsy. There were no microscopic correlates and no dose response relationships. Values were within the WIL Research historical control database range with the exception of 1 individual male with minimally lower urine specific gravity. While several control group males had individual specific gravity and total urine volume values outside the historical control database range, the differences were attributed to an aberration in the concurrent control group and not related to test substance administration. There were no statistically significant differences in urinalysis parameters at the recovery necropsy.

NEUROBEHAVIOUR: Home cage observations were unaffected by test substance administration. The slightly lower number of males and females noted to be sitting or standing normally was not considered test substance-related. Handling observations, open field observations, sensory observations, neuromuscular observations and motor activity were unaffected by test substance administration. Test substance-related, statistically significantly lower mean body weights were noted in the 300 mg/kg bw/day group males at the study week 13 evaluation. The differences were consistent with effects noted in the weekly body weights.

ORGAN WEIGHTS: Test substance-related statistically significant higher liver weights were noted in 300 mg/kg bw/day (32% and 30% relative increase in males and females, respectively). In addition in females a 10% increase in liver weight was observed at 100 mg/kg bw/day. Statistically significant higher relative adrenal gland weights (56% and 35% in males and females, respectively) were noted in the 300 mg/kg bw/day groups. In high dose males a statistically significant higher prostate/seminal vesicles weight (16%) and spleen weight (27%) was observed. In high dose females statistically significant increased relative weights of kidneys (8%) and a non-statistically significant increase in relative thyroid/parathyroid weight (16%) were observed.
Observed non-statistically significant increased relative weights of testes and thymus (males), are considered to be related to the decreased body weight in high dose males compared to controls and not toxicologically relevant.
Organ weights with test substance-related differences at the primary necropsy were similar to the control groups at the recovery necropsy. Statistically significant differences in organ weights noted at the recovery necropsy (higher brain, seminal vesicle/prostate, and testes weights relative to body weight and lower absolute liver weight in the 300 mg/kg bw/day group males) were considered to be a result of effects on final body weight and not test substance-related.

GROSS PATHOLOGY: Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance.

HISTOPATHOLOGY, NON-NEOPLASTIC: Test substance-related microscopic findings were noted in the liver (100 (f) and 300 (m/f) mg/kg bw/day) and adrenal cortex (female, 300 mg/kg bw/day) at the primary necropsy. Minimal hepatocellular hypertrophy was noted at 100 (1 female) and 300 mg/kg bw/day (6 females, 2 males). The finding on liver was observed together with higher liver weights and higher serum liver enzyme values. Adrenal cortical cell hypertrophy was noted in the 300 mg/kg bw/day group females (4 females), together with higher adrenal gland weights. There were no test substance-related microscopic findings noted at the recovery necropsy. There were no other test substance-related histologic changes.

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: body weight (gain), liver enzymes, organ weights and histopathology in liver and adrenals

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The analyzed dosing formulations contained 90.2% to 110% of the test substance which was within the WIL Research SOP range of target concentrations for suspensions (85% to 115%), and were homogeneous. The test substance was not detected in the analyzed vehicle formulation that was administered to the control group.

Incidence of Selected Histopathologic Findings, Study Week 13 Primary Necropsy

 

Males

Females

Dosage (mg/kg/day):

0

30

100

300

0

30

100

300

 

 

 

 

 

 

 

 

 

Liver

10

9

10

10

10

10

10

10

  Hypertrophy, hepatocellular

0

0

0

2

0

0

1

6

      Minimal

-

-

-

2

-

-

1

6

 

 

 

 

 

 

 

 

 

Adrenal cortex

10

0

1

10

10

10

10

10

  Hypertrophy, cortical cell

0

NA

0

0

0

0

0

4

      Minimal

-

NA

-

-

-

-

-

4

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, oral administration of EXP1503090 to Crl:CD(SD) rats at dosage levels of 30, 100, and
300 mg/kg/day for a minimum of 90 consecutive days resulted in adverse, lower body weights (up to 13.9% lower) with
corresponding lower food consumption in the 300 mg/kg/day group males. Test article-related, nonadverse microscopic
findings of hepatocellular hypertrophy was noted for the 300 mg/kg/day group males and 100 and 300 mg/kg/day group
females and adrenal cortical cell hypertrophy was noted for the 300 mg/kg/day group females. These findings correlated
with effects on organ weights and/or higher serum liver enzyme values but were considered to be associated with P450
enzyme induction. Therefore, due to the adverse lower body weights in the 300 mg/kg/day group males, the no observed
adverse-effect level (NOAEL) was 100 mg/kg/day for males and 300 mg/kg/day for females.
Executive summary:

Due to the adverse lower body weights in the 300 mg/kg/day group males, the no observed adverse-effect level (NOAEL) was 100 mg/kg/day for males and 300 mg/kg/day for females.