Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 02 March 2000 to 27 September 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline Study performed according to GLP

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
The testes/epididymides for the male animal #3055 were fixed in 10% buffered formalin and not in Bouin's solution as required by protocol. 2. The Week 3 high spiked control sample for the diet analysis was not within +15% of the low spiked control sample.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): ST 08 C 99
- Substance type: pure active substance
- Physical state: clear liquid
- Analytical purity: 98.5%
- isomers composition: ca. 10-12% cis- and 88-90 % trans- isomers
- Lot/batch No.: F 84912-08D
- Expiration date of the lot/batch: 2000-07-01
- Storage condition of test material: 2 - 30° C, in closed containers protected from light.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Kingston, New York 12484- USA
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males: 180-231g, females: 140-207g
- Housing: Animals were doubly housed in elevated, stainless steel, wire mesh cages during the first week of the acclimation period and individually housed thereafter
- Diet (e.g. ad libitum): ad libitum (Certified Rodent Diet, No. 5002; (Meal) (pMI Nutrition International, St. Louis, Missouri))
- Water (e.g. ad libitum): ad libitum (regularly tested)
- Acclimation period: 16 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 40-74 (excursions outside the specified range (30-70%) were not considered to have affected the integrity of the study)
- Air changes (per hr): not mentionned in study report
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 09 March 2000 To: 01 June 2000

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: food
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): every 14 days
- Mixing appropriate amounts with (Type of food): poured with the Certified Rodent Diet N. 5002
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All dietary levels were assayed weekly for the first month (2 samples per concentration). Subsequent assays were performed at monthly intervals for the remainder of the study.
The analytical method involved methanol extraction of ST 08 C99 from rodent diet, followed by quantification using a gas chromatograph and flame ionization detection (GC/FID).
The variance between nominal concentration and measured concentration in rodent diet was acceptable as measured concentrations in rodents diets were within ±15% of nominal concentrations (87.1 – 115.0 %).
Homogeneity analysis & Stability analysis: dietary mixes were found to be homogenous as analysis of sample originating from the bottle, the middle and the top of the dietary mixes were less than 5 % divergent. Samples were furthermore found to be stable when stored at room temperature for 14 days.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily, 7 days each week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 50 & 100 mg/kg bw/day (male/female)
Basis:
nominal in diet
No. of animals per sex per dose:
20 rats (10 males and 10 females) per dose. (See Table 1 "experiment outline" in "Any information on material & methods incl. Tables")
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Doses were selected by the Sponsor based on a 2-week preliminary range-finding study in rats (HLS Study No. 99-2620).
Higher doses than 100 mg/kg bw/day could not be used due to palatability problems.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once in the morning and once in the afternoon) for mortality and general condition. Daily (concurrent with one viability
check) for any signs of poor health or toxic or pharmacologic effects (e.g., abnonnalities in general condition, appearance, activity, behavior, respiration, etc.). Unusual signs were also recorded.
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily (concurrent with one viability check)


BODY WEIGHT: Yes
- Time schedule for examinations: Animals were removed from their cages and weighed twice pretest, weekly during treatment and at termination. Fasted body weights were obtained just prior to necropsy.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study) :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, calculated from food consumption data and based on nominal dietary concentrations.


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 data


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretest: Study Day -10, Test: Study day 90 (during last week of the test, i.e. week 14).
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at Termination (Week 14).
- Anaesthetic used for blood collection: Yes (light CO2/O2 anesthesia)
- Animals fasted: Yes (overnight)
- How many animals: up to 10 animals/sex/group
- Parameters checked in table [YES] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination on Week 14.
- Animals fasted: Yes
- How many animals: up to 10 animals/sex/group
- Parameters checked in table [YES] were examined.


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Pretest and during Week 13
- Dose groups that were examined: all (blind observations, i.e., the observer did not know the identity of the animal's dose group)
- Battery of functions tested: grip strenght / motor activity / other:
Locomotor activity (modified version of Schulze's procedures) using an automated Photobeam Activity System (San Diego instruments, Inc) (60 min sessions divided into 12 5-min intervals)
Functional observational battery (Moser, 1989); the following test were performed as part of tthe functional observational battery:
Home Cage Evaluations (posture, vocalization and palpebral closure);
Handling Evaluations (reactivity to general stimuli (handling); assessment of signs of autonomic function: lacrimation, salivation, altered fur appearance, or red/crusty deposits around eyes);
Open Field Evaluation (arousal level and gait; count of urination and defecation; convulsions, tremors, abnormal movements or behaviors, excessive or repetitive actions; piloerection and exophthalmos);
Reflex Assessments (response to visual (approach response) and auditory (finger snap) stimuli; response to a tail pinch; pupillary function);
Grip Strength (Meyer et al., 1979): Grip strength was measured using a Grip Strength Meter (Columbus Instruments International
Corporation, Columbus, Ohio);
Landing Foot Splay (a small dot of paint was applied to each animal's hindpaws. Each animal was then dropped onto a flat surface from a height of one foot. The distance between the marks left by the hindpaws was measured in centimeters);
Hindlimb Extensor Strength (Animals were held in a vertical position facing the observer with a finn grasp around the thorax. The observer placed one finger against the bottom of each hindpaw and pressed towards the animal. Muscular resistance and pressure exerted by the animals were scored);
Air Righting Ability (Animals were held upside down and dropped from a height of two feet into a container of bedding. The landing position of each animal was recorded);
Body Weight: Animals were removed from their cages and weighed using a Mettler Balance, Model PE4000 (Mettler Instrument Corporation, Hightstown, New Jersey).


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A complete macroscopic examination was performed on all animals, including animal humane euthanized; all abnormal observations were recorded. The necropsy consisted of an external examination, including identification of all clinically recorded lesions, as well as a detailed internal examination. Animals were fasted prior to scheduled sacrifices. Organs (adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate, spleen, testes, thymus, thyroid/parathyroids, uterus (with cervix)) were weighed for all animals at the scheduled sacrifice interval. Prior to weighing, the organs were carefully dissected and properly trimmed to remove adipose and other contiguous tissues in a uniform manner. Organs were weighed as soon as possible after dissection in order to avoid drying. Paired organs were weighed together.
HISTOPATHOLOGY: Yes
The tissues (see list below) were obtained at termination and preserved for all animals. In addition, slides of the indicated tissues were prepared and examined microscopically for all animals in the 0 and 100 mg/kg/day groups. Any abnormalities not noted during macroscopic examinations that were seen during histology processing were recorded. Tissues preserved and examinated: Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (medulla, pons, cerebrum and cerebellum), epididimydes, oesophagus,eyes (with optic nerve), Heart, Kidneys, Lachrymal gland/Harderian glands, large intestine (cecum, colon, rectum), larynx, Liver, Lungs (with bronchi), Lymph nodes (mediastinal and mesenteric), Mammary gland, Muscle (biceps femoris), Nerve, Ovaries, Pancreas, Pharynx, Pituitary, Prostate, Salivary glands (submandibular), Seminal vesicles, Skin, Small intestine (duodenum, ileum, jejunum), Spinal cord (thoracic, lumbar and cervical), Spleen, Stomach, Testes (with epididymides), Thymus, Thyroid/parathyroid, Trachea, Urinary bladder, Uterus, Vagina, Zymbal’s gland, Gross lesions.
Other examinations:
None
Statistics:
The following parameters were analyzed statistically: mean body weight values, mean food consumption values, mean hematology values, mean coagulation values, mean clinical chemistry values, mean terminal organ weights, organ/body and organ/brain weight ratios, mean motor activity counts, forelimb and hindlimb grip strength measurements, landing foot splay measurements
Mean values of all dose groups were compared to the mean value for the control group at each time interval.
Evaluation of equality of group means was made by the appropriate statistical method, followed by a multiple comparison test if needed. Bartlett's test was performed to determine if groups had equal variances. For all parameters except organ weights, if the variances were equal, parametric procedures were used; if not, nonparametric procedures were used. Organ weight data was analyzed only by parametric methods. The parametric method was the standard one-way analysis of variance (ANOVA) using the F ratio to assess significance (Annitage, 1971; Dunlap and Duffy, 1975). If significant differences among the means were indicated, additional tests were used to determine which means were significantly different from the control: Dunnett's, Williams, or Cochran and Cox's modified t-test. The nonparametric method was the Kruskal-Wallis test and if differences were indicated, Shirley's test, Dunn's test, or Pairwise Comparison with Bonferroni Correction (Games and Howell, 1976) were used to determine which means differed from control. Bartlett's test for equality of variance was conducted at the 1% significance level; all other statistical tests were conducted at the 5% and 1% significance levels.

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:
no effects observed
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:
not examined
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No test article-related mortality was observed during the study. One female (animal No. 1554) administered 0 mg/kg/day of ST 08 C99
was sacrificed for humane reasons, due to an injury, during Month 2 of test article administration.

BODY WEIGHT AND WEIGHT GAIN
Body weights and bodyweight gains were comparable between control and test article-treated groups throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption values of test article treated groups were comparable to control values during the study.

FOOD EFFICIENCY
Test article intake, calculated from food consumption data and based on nominal dietary concentrations, confirmed that animals received the desired levels.

OPHTHALMOSCOPIC EXAMINATION
Administration of ST 08 C99 did not result in any test article-related ocular findings.

HAEMATOLOGY
No test article-related effects were seen in the hematology parameters examined. Statistically significant differences from
controls in mean hematocrit values of treated males were attributed to normal biological variability and fell within historical control
data ranges.

CLINICAL CHEMISTRY
No test article-related effects were seen in the clinical chemistry parameters examined. Statistically significant differences from control in mean potassium values of all treated males and a statistically significant decrease in mean globulin value of males fed 100 mg/kg day were considered to be due to normal variability since they occurred in only one sex and fell within historical control data ranges.

NEUROBEHAVIOUR
Motor Activity: No test article-related effects were evident in the motor activity for either sex in any of the treatment groups.
Functional Observation Battery: Test article administration did not affect the neurological condition of the animals as measured by a functional observational battery of assessments. Landing foot splay and grip strength for all test article treated groups were comparable to control values or within
the range of normal variation. No abnormal movements were observed.

ORGAN WEIGHTS
Organ weight data of rats fed ST 08 C99 were considered comparable to control weights or were within normal variability at termination of the study. The statistically significant difference in mean organ to bodyweight ratio of kidneys of males administered 100 mg/kg/day was not considered test article-related since it fell within historical control data ranges and there were no associated microscopic findings in the kidney.

GROSS PATHOLOGY
There were no test article-related macroscopic findings. Findings observed occurred with comparable incidence and severity in the control and test article treated groups or they occurred sporadically. These incidental findings have been seen in rats of this strain and age used in other studies conducted in this facility.

HISTOPATHOLOGY
There were no test article-related microscopic findings.
Microscopic findings observed occurred with comparable incidence and severity in the 0 and 100 mg/kg/day groups or they occurred sporadically. These incidental findings have been seen in rats of this strain and age used in similar studies conducted in this facility.

OTHER FINDINGS

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day (nominal)
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:
Administration of dietary concentrations of 10, 50 and 100 mg/kg/day of ST 08 C99 to Sprague-Dawley rats for up to 90 days did not result in any adverse, toxicological effects. The NOEL (No Observable Effect Level) was 100 mg/kg/day.
Executive summary:

Introduction. This study complies with the recommendations of the OECD Guidelines for Testing of Chemicals n° 408. It wa

s designed to investigate the systemic toxicity of the test material when administered orally, via dietary admixture, to Sprague-Dawley CD® rats (10/sex/group) at dose levels of 10, 50, and 100 mg/kg bw/ day for a period of 90 days. Control animals (10/sex) received untreated standard laboratory diet.

 

Results and discussion. Clinical observations and body weight measurements were performed on all animals pretest and weekly during the study period. Ophthalmoscopic examinations were conducted on all animals pretest and at termination of the dosing period. Food consumption was evaluated weekly beginning one week prior to initiation of dosing. Test substance intake measurements were performed weekly beginning one week after initiation of dosing. Motor activity, functional observational battery and ophthalmology observations were performed pretest and during the twelfth week of dosing.

 

After 3 months of treatment, hematology, coagulation and clinical chemistry evaluations were performed, surviving animals were euthanatized, selected organs were weighed and organ/body weight and organ/brain weight ratios calculated. Complete macroscopic postmortem examinations were performed on all animals, and histopathological evaluation of all tissues was conducted for animals in the 0 and 100 mg/kg/day groups.

 

Administration of dietary concentrations of 10, 50 and 100 mg/kg/day of ST 08 C99 to Sprague-Dawley rats for up to 90 days did not result in any adverse toxicological effects. Therefore, the NOEL (No-Observable-Effect-Level) was 100 mg/kg/day.