Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC, Part B-26 (2001)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF (2000)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): BIOBANTM CS-1246
- Molecular formula (if other than submission substance):C7H13NO2
- Molecular weight (if other than submission substance): 143.09
- Physical state: Pale yellow liquid
- Analytical purity: 98.55% pure by gas chromatography-Flame Ionization Detection (GC-FID) and Fourier Transform Infared (FT-IR) spectroscopy
- Lot/batch No.TC1031LAH3
- Stability under test conditions: A previously conducted toxicity study has shown the test material to be stable for at least 25 days in corn oil at dose levels ranging from 0.025 to 25%. The established concentration range and duration will span those used in this study

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, INC. (Raleigh, North Carolina, USA)
- Age at study initiation: Approximately 7 weeks of age at study start.
- Weight at study initiation: For males Approx. 230-262 gms and for females 170-175 gms
- Housing: Rats were housed one per cage after randomization in stainless steel cages. Cages had wire mesh floors and were suspended above absorbent paper.Cages contained a feed crock and a pressure activated lixit valve-type watering system
- Diet (ad libitum): Animals were provided LabDiet Certified Rodent Diet #5002 in meal form
- Water: Animals were provided ad libitum muncipal water adlibitum.
- Acclimation period: At least one week prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): As per standards
- Humidity (%): As per standards
- Air changes (per hr): As per standards
- Photoperiod (hrs dark / hrs light): As per standards


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
All dosing solutions were prepared by mixing the test material in corn oil at concentrations of 2.5, 12.5, or 62.5 mg/ml and administered at a dose volume of 4 ml/kg bw to achieve the targeted dose levels. Dose solutions were not adjusted for purity. Dose volumes were adjusted weekly based on individual body weights. The control rats were dosed with corn oil at 4 ml/kg bw.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose confirmation analyses of all dose solutions, plus control were determined pre-exposure, near the middle and end of the study. The homogeneity of the low-dose and the high-dose test solutions was determined concurrent with dose confirmation. The method used for analyzing the test material in corn oil was gas chromatography-mass spectrometry (GC/MS) with internal and external standards. A previously conducted toxicity study showed CS-1246 to be stable for at least 25 days in corn oil at dose levels ranging from 0.025 to 25%. The established concentration range and duration spanned those used in this study. Therefore, additional stability analyses were not conducted.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
10 mg/kg/day
Basis:
other: Nominal in corn oil
Remarks:
Doses / Concentrations:
50 mg/kg/day
Basis:
other: Nominal in corn oil
Remarks:
Doses / Concentrations:
250 mg/kg/day
Basis:
other: Nominal in corn oil
No. of animals per sex per dose:
10 / sex / dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels selected were based on preliminary results following completion of the 28-day study. The high dose was expected to produce perioral soiling, body weight depression, hyperplasia of the stomach limiting ridge, and multifocal or diffuse hyperplasia and hypertrophy of mucous cells in the glandular mucosa of the stomach. The mid- and low-dose levels were expected to provide dose response data for
any treatment-related effects observed in the high-dose group. The low-dose was expected to be a no-observed-effect level (NOEL).
- Rationale for animal assignment: Random
- Rationale for selecting satellite groups: Groups of ten male and ten female CD rats were administered the control, mid- and high-dose for 90 days and were then given control feed for an additional 28 days to evaluate the potential reversibility of any effects induced during the 90-day exposure phase of the study
- Post-exposure recovery period in satellite groups: 28 days
Groups of ten male and ten female Crl:CD(SD) rats were administered CS-1246 in corn oil by gavage at dose levels of 0, 10, 50, or 250 mg/kg body weight/day (mg/kg/day, mkd) for at least 90 days to evaluate the potential for systemic toxicity. Groups of ten male and ten female CD rats were administered the control, mid- and high-dose for 90 days and were then given control feed for an additional 28 days to evaluate the potential reversibility of any effects induced during the 90-day exposure phase of the study.

Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Groups of ten male and ten female CD rats were administered the control, mid- and high-dose for 90 days and were then given control feed for an additional 28 days to evaluate the potential reversibility of any effects induced during the 90-day exposure phase of the study
BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed pre-exposure and weekly throughout the dosing period.
FOOD CONSUMPTION: Yes
Feed consumed was determined pre-exposure and at least weekly for all animals by weighing feed containers at the start and end of a measurement cycle.
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all animals were examined by a veterinarian pre-exposure and prior to the scheduled necropsy using indirect ophthalmoscopy
- Dose groups that were examined: All dose groups
HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necrpsy
- Anaesthetic used for blood collection: Yes CO2
- Animals fasted: Yes
- How many animals: All dose groups
- Parameters checked: Hematologic parameters were assayed using an Advia 120 Hematology Analyzer, and included the following parameters: Hematocrit (HCT), Hemoglobin (HGB) concentration, Red blood cell (RBC) count, Total white blood cell (WBC) count, Differential WBC count, Platelet (PLAT) count, Reticulocyte (RET) count, Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin Concentration (MCHC), and Prothrombin time (PT).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to necropsy
- Animals fasted: Yes
- How many animals: All dose groups
- Parameters checked: Clinical Chemistry parameters were measured using a Hitachi 912 Clinical Chemistry Analyzer (Roche Diagnostics, Indianapolis, Indiana), and included: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (GGT), Albumin (ALB), Albumin/Globulin Ratio (A/G Ratio), Cholesterol (CHOL), Creatinine (CREA), Electrolytes (NA, K, PHOS, CL and CA), Globulin (GLOB), Glucose (GLU), Total bilirubin (TBIL), Total protein (TP), Triglycerides (TRIG), and Urea nitrogen (UN).

URINALYSIS: Yes
- Time schedule for collection of urine: week prior to the scheduled necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: Urine was obtained from all animals the week prior to the scheduled necropsy and analyzed for color, appearance, specific gravity (refractometer) and urine volume, pH, Bilirubin, Glucose, Protein, Ketones, Blood, Urobilinogen, and microscopic Examination
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: conducted pre-exposure and during the last week of the treatment period for the main study animals only.
- Dose groups that were examined: All dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity

OTHER: Rectal temperature also recorded.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes The necropsy of all animals included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised.
HISTOPATHOLOGY: Yes All tissues were processed by standard histologic procedures from control and high-dose group animals and all animals that died or were sacrificed in a moribund condition. Relevant gross lesions, and all target tissues were microscopically examined from all animals in the low- and intermediate dose groups. Selected histopathologic findings were graded to reflect the severity of specific lesions.
Similar necropsy procedures were followed for animals found dead or moribund, except that body weights, organ weights, and blood samples were not obtained.
Other examinations:
Organ weights: The brain, liver, kidneys, heart, adrenals, testes, epididymides, uterus, ovaries, thymus, and spleen were trimmed and weighed immediately.
Statistics:
See the attachment-1

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
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:
no effects observed
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:
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: The only treatment-related clinical observation was clear perioral soiling (salivation), noted in males (7/20) and females (5/20) given 250 mg/kg/day. All other clinical observations appeared to be incidental and had no relationship to treatment.
There were no animals with the observation of clear perioral soiling during the 28-day recovery period. All rats survived the 90-day dosing phase and the 28-day recovery period.
Sensory Evaluation :Examinations performed on males and females at baseline and prior to termination revealed no treatment-related findings.
Rectal Temperature:There were no treatment-related effects seen in rectal temperature.
Grip Performance:There were no treatment-related effects seen in grip performance.
Motor Activity:There were no treatment-related effects seen in motor activity counts at either time interval.
BODY WEIGHT AND WEIGHT GAIN: There were no treatment-related alterations in mean body weights and body weight gains of males or females at any dose level. Males given 10 or 250 mg/kg/day had slightly lower mean body weights (not statistically identified) and body weight gains throughout the 90-day dosing phase. At study termination the mean body weight gains of males given 10 or 250 mg/kg/day were 9.3% and 8.0% lower than controls, respectively. These lower body weight gains were interpreted to be unrelated to treatment because of the lack of a dose response. Males given 50 mg/kg/day and females given 10, 50, or 250 mg/kg/day had mean body weights and body weight gains that were comparable to, or higher than, controls.
The body weights and body weight gains of males given 250 mg/kg/day gradually increased relative to controls during the 28-day recovery phase. By the end of the 28-day recovery period, the body weights and body weight gains of males given 250 mg/kg/day were comparable to controls. The body weights and body weight gains of males given 50 mg/kg/day, and females given 50 or 250 mg/kg/day were comparable to or higher than controls during the 28-day recovery phase

FOOD CONSUMPTION:There were no treatment-related effects on the feed consumption of males or females at any dose level throughout the 90-day dosing period.The feed consumption of females given 250 mg/kg/day was slightly higher than controls throughout the 28-day recovery period. The feed consumption of males given 50 or 250, and females given 50 mg/kg/day was comparable to controls during the 28-day recovery phase.

OPHTHALMOSCOPIC EXAMINATION: Pre-exposure examination on all rats placed on study indicated all rats were suitable for use in the study. A pale fundus was noted in one male given 250 mg/kg/day and one female given 50 mg/kg/day prior to study termination. These observations were interpreted to be unrelated to treatment due to their low incidence and lack of a dose response.

HAEMATOLOGY: Males and females given 250 mg/kg/day had statistically identified higher mean reticulocyte counts that were interpreted to be treatment related. However, based on the lack of any additional treatment-related hematologic effects and the normal microscopic appearance of the bone marrow and spleen in all treated animals, the higher reticulocyte counts were considered to be of minimal toxicologic significance. In addition, males and females given 250 mg/kg/day had lower mean hematocrits and hemoglobin concentrations that were not statistically identified. These alterations were interpreted to be unrelated to treatment because the values were within or near the historical control mean ranges of 90-day Crl:CD(SD) rat studies recently conducted at this laboratory, and the lack of a dose response in the mid- and low-dose groups.
There were no treatment-related alterations in the prothrombin times of male and female rats.
Following the 28-day recovery period, the mean hemoglobin concentration and hematocrits of males and females given 50 or 250 mg/kg/day were comparable to controls. The mean reticulocyte count of rats given 250 mg/kg/day was statistically identified as lower than Table A6.4.1/01-1, Table A6.4.1/01-2

Males and females given 250 mg/kg/day had statistically identified higher mean reticulocyte counts that were interpreted to be treatment related. However, based on the lack of any additional treatment-related hematologic effects and the normal microscopic appearance of the bone marrow and spleen in all treated animals, the higher reticulocyte counts were considered to be of minimal toxicologic significance. In addition, males and females given 250 mg/kg/day had lower mean hematocrits and hemoglobin concentrations that were not statistically identified. These alterations were interpreted to be unrelated to treatment because the values were within or near the historical control mean ranges of 90-day Crl:CD(SD) rat studies recently conducted at this laboratory, and the lack of a dose response in the mid- and low-dose groups.

There were no treatment-related alterations in the prothrombin times of male and female rats.

Following the 28-day recovery period, the mean hemoglobin concentration and hematocrits of males and females given 50 or 250 mg/kg/day were comparable to controls. The mean reticulocyte count of rats given 250 mg/kg/day was statistically identified as lower than controls for males and comparable to control for females following the 28-day recovery period. Therefore, there was recovery of the higher reticulocyte counts noted in both sexes following the 90-day portion of the study.
CLINICAL CHEMISTRY: There were no treatment-related alterations in any of the clinical chemistry parameters for male and female rats. There were statistically identified lower mean albumin (250 mg/kg/day), cholesterol (250 mg/kg/day), and potassium concentrations (10 mg/kg/day) for males. These alterations were interpreted to be unrelated to treatment because the albumin and cholesterol concentrations were within or near the historical control mean ranges of 90-day Crl:CD(SD) rat studies recently conducted at this laboratory, and because of the lack of a dose response for the potassium concentration values.

URINALYSIS: Females given 250 mg/kg/day had an increase in the incidence and severity of blood in the urine, relative to controls. This alteration was interpreted to be treatment related; however, there were no histopathologic alterations in the urinary tract (kidneys and urinary bladder examined microscopically) that corresponded to blood in the urine for females at any dose level. Females given 250 mg/kg/day also had a lower mean urine volume and a higher mean urine specific gravity, which were not statistically identified. These alterations were interpreted to be unrelated to treatment because the specific gravity was within the historical control mean ranges of 90-day Crl:CD(SD) rat studies recently conducted at this laboratory, and because of the lack of a dose response for the lower urine volume. There were no treatment-related alterations in the urinalysis parameters for male rats at any dose level.
Following the 28-day recovery period, the level of blood in the urine of females given 50 or 250 mg/kg/day was comparable to controls.

ORGAN WEIGHTS: There were no treatment-related alterations in mean final body weights or organ weights of male and female rats at any dose. Males given 50 mg/kg/day had a statistically identified higher absolute brain weight. This alteration was interpreted to be unrelated to treatment because of the lack of a dose response.

GROSS AND HISTOPATHOLOGY PATHOLOGY: All males and 8/10 females given 50 mg/kg/day, and all males and females given 250 mg/kg/day had treatment related thickening of the limiting ridge of the stomach. In addition, one male given 250 mg/kg/day had an ulcer of the glandular mucosa of the stomach that was interpreted to be treatment related. There were no treatment-related gross observations in males or females given 10 mg/kg/day. All males and females given 50 or 250 mg/kg/day had grossly normal stomachs following the 28-day recovery period.

The primary organ affected by the gavage administration of CS-1246 was the stomach. All males and 6/10 females given 50 mg/kg/day, and all males and females given 250 mg/kg/day had treatment related slight hyperplasia of the squamous epithelium lining the limiting ridge of the stomach. Treatment-related very slight or slight hypertrophy of the surface epithelial cells lining the glandular mucosa of the stomach was present in 2/10 males given 50 mg/kg/day, and in all males and females given 250 mg/kg/day. Treatment-related subacute to chronic inflammation of the glandular mucosa and submucosa occurred in a few males and females given 50 mg/kg/day and in the majority of males and females given 250 mg/kg/day. Five males given
250 mg/kg/day had increased numbers of mitotic figures in the mucous cells of the glandular mucosa. Additional stomach effects consisted of multifocal erosions in the glandular mucosa of one male given 250 mg/kg/day, a focal ulcer in the glandular mucosa of another male given 250 mg/kg/day, and focal necrosis of the glandular mucosa of one female given 250 mg/kg/day. All of the above effects were interpreted to be the result of direct irritation to the gastric mucosa by CS-1246. There were no treatment-related effects in the stomachs of males and females given 10 mg/kg/day.

A treatment-related histopathologic effect was also present in the liver of males given 250 mg/kg/day. This alteration consisted of an increase in the incidence of very slight altered tinctorial properties (increased eosinophilia) of centrilobular hepatocytes. Based on the lack of corresponding clinical chemistry or liver weight changes, the alteration of centrilobular hepatocytes was interpreted to be a non-adverse effect with minimal toxicologic significance.

Following the 28-day recovery period, treatment-related chronic inflammation of the glandular mucosa of the stomach still was present in a few males and females given 50 mg/kg/day, and in the majority of males and females given 250 mg/kg/day. The other stomach alterations (hyperplasia of the limiting ridge, hypertrophy of the glandular mucosal epithelium, and erosions/ulcers) were completely resolved following the 28-day recovery period. The livers of males given 50 or 250 mg/kg/day were comparable to controls following the recovery period


Effect levels

Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The no-observed-effect level (NOEL), based on localized gastric irritation, for Crl:CD(SD) rats of either sex was 10 mg/kg/day CS-1246. The NOEL for systemic toxicity was 50 mg/kg/day for both sexes.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
The no-observed-effect level (NOEL), based on localized gastric irritation, for Crl:CD(SD) rats of either sex was 10 mg/kg/day CS-1246. The NOEL for systemic toxicity was 50 mg/kg/day for both sexes.
Executive summary:

Ten male and ten female Crl:CD(SD) rats per group were administered CS-1246 in corn oil by gavage at dose levels of 0, 10, 50, or 250 milligrams per kilogram body weight per day (mg/kg/day) for 90 days to evaluate the potential for systemic toxicity. Parameters evaluated during the 90-day dosing phase were daily cage-side observations, daily clinical observations, weekly detailed clinical

observations, functional tests, ophthalmic examinations, body weights, feed consumption, hematology, prothrombin time, urinalysis, clinical chemistry or selected organ weights, and gross and histopathologic examinations. An additional ten male and ten female rats from the control, midand high-dose groups were held untreated for 28 days following the dosing period to assess recovery from treatment-related effects.

90-Day Dosing Phase : There were no treatment-related effects in functional tests, body weights, feed consumption, ophthalmic observations, prothrombin time, clinical chemistry parameters, or organ weights. Males and females given 250 mg/kg/day had a treatment-related increase in the incidence of clear perioral soiling (salivation).

Males and females given 250 mg/kg/day had statistically identified higher mean reticulocyte counts that were interpreted to be treatment related. However, based on the lack of any additional treatment-related hematologic effects and the normal microscopic appearance of the bone marrow in all treated animals, the higher reticulocyte counts were considered to be of minimal toxicologic

significance.

Females given 250 mg/kg/day had an increase in the incidence and severity of blood in the urine, relative to controls. This alteration was interpreted to be treatment related; however, there were no histopathologic alterations in the urinary tract that corresponded to blood in the urine.

The primary organ affected by the gavage administration of CS-1246 was the stomach. All males and 8/10 females given 50 mg/kg/day, and all males and females given 250 mg/kg/day had treatment-related gross pathologic observations of a thickened limiting ridge of the stomach. In addition, one male given 250 mg/kg/day had a treatment-related ulcer of the glandular mucosa of the

stomach. There were no treatment-related gross observations in males or females given 10 mg/kg/day. Microscopically, all males and 6/10 females given 50 mg/kg/day, and all males and females given 250 mg/kg/day had slight hyperplasia of the squamous epithelium lining the limiting ridge of the stomach. Very slight or slight hypertrophy of the surface epithelial cells lining the glandular mucosa of the stomach was present in 2/10 males given 50 mg/kg/day, and in all males and females given 250 mg/kg/day. Subacute to chronic inflammation of the glandular mucosa and submucosa occurred in a few males and females given 50 mg/kg/day and in the majority of males and females given 250 mg/kg/day. Five males given 250 mg/kg/day had increased numbers of mitotic figures in the mucous cells of the glandular mucosa. Additional stomach effects consisted of multifocal erosions in the glandular mucosa of one male given 250 mg/kg/day, a focal ulcer in the glandular mucosa of another male given 250 mg/kg/day, and focal necrosis of the glandular mucosa of one female given 250 mg/kg/day. All of the above effects were interpreted to be the result of direct irritation to the gastric mucosa by CS-1246. There were no treatment-related effects in the stomachs of males and females given 10 mg/kg/day.

Males given 250 mg/kg/day had a treatment-related increase in the incidence of very slight altered tinctorial properties (increased eosinophilia) of the centrilobular hepatocytes. Based on the lack of corresponding clinical chemistry or liver weight changes, the alteration of centrilobular hepatocytes was interpreted to be a non-adverse effect with minimal toxicologic significance. 28-Day Recovery There was partial recovery of the inflammation of the stomach, with treatmentrelated chronic inflammation of the glandular submucosa still present in a few males and females given 50 mg/kg/day, and in the majority of males and females given 250 mg/kg/day, following the 28-day recovery period. There was complete recovery of all other treatment-related effects.

The no-observed-effect level (NOEL), based on localized gastric irritation, for Crl:CD(SD) rats of either sex was 10 mg/kg/day CS-1246. The NOEL for systemic toxicity was 50 mg/kg/day for both

sexes.