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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:
from 04-DEC-1996 to 01-APR-1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guidance test with GLP compliance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Directive 92/69/EEC, B. 7. "Subacute Toxicity Oral", July 31, 1992
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
other: Japanese Guidelines for Screening Toxicity Testing of Chemicals: Testing Methods for New Substances, enacted July 13, 1974, amended December 5, 1986
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Code number: FAT45'176/A
Batch No.: WP2/96
Purity: ca. 60 %
Appearance: orange-brown powder
Storage: room temperature
Expiration date: 11/2004

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4 4414 Fuellinsdorf/Switzerland
Age at delivery: 6 weeks
Body weight and range at acclimatization/pretest: Males: 126 - 172 g (mean 152 grams), Females: 99 - 145 g (mean 131 grams)
Acclimatization: 7 days under test conditions following a health examination. Only animals without any visible signs of illness were used for the study
Accommodation: Groups of five in Makrol on type-4 cages with standard softwood bedding ('Lignocel' Schill AG, 4132 Muttenz / Switzerland).
Diet: Pelleted standard Kliba no. 343 rat maintenance diet (KLIBA Mühlen AG, 4303 Kaiseraugst/Switzerland) was available ad libitum (Batch no. 81/96). The feed batch was analyzed for contaminants.
Water: Tap water was available ad libitum in water bottles.

Conditions
Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with a target range for room temperature of 2 2±3 degrees centigrade and for relative humidity between 40-70 %. 12 hours fluorescent light/ 12 hours dark (light period between 6.00 a.m. and 6.00 p.m.), music during the light period.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Dose volume: 10 ml/kg body weight per treatment day,
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean concentrations of the homogeneity samples were found to be 101.0 %, 96.4 %, and 100.0 % of the nominal concentrations of dose group 2 (5 mg/ml), dose group 3 (20 mg/ml), and dose group 4 (100 mg/ml), respectively. The individual concentrations varied in the range from -4 % to +6 % of the mean concentrations.
FAT 45'176/A was found to be stable in bi-distilled water at room temperature for at least two hours.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days per week for a total of 28 days.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200 1000 mg/kg per treatment day
Basis:
nominal in diet
No. of animals per sex per dose:
Groups 1 and 4: 10 males; 10 females
Groups 2 and 3: 5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
TEST ARTICLE PREPARATION
FAT 45'176/A was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using an magnetic stirrer. Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer.
Frequency of preparation: Daily, prior to each application
Positive control:
no data

Examinations

Observations and examinations performed and frequency:
MORTALITY / VIABILITY AND CLINICAL SIGNS: Observations for mortality/viability were recorded twice daily and clinical signs of toxicity were recorded at least once daily.
FOOD CONSUMPTION: The food consumption was recorded once during the pretest period and weekly thereafter using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.
BODY WEIGHTS: Body weight was recorded once weekly during pretest, treatment and recovery, and twice before necropsies; using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.
OPHTHALMOSCOPIC EXAMINATIONS: At 4 weeks and at 6 weeks
Sacrifice and pathology:
NECROPSY: At 4 weeks and at 6 weeks
All animals were weighed and necropsied and descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by an experienced veterinary pathologist. At the end of the treatment or recovery period the animals designated according to the necropsy schedule were anesthetized by intraperitoneal injection of sodium pentobarbitone and sacrificed by exsanguination.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS: The following organ weights were recorded on the scheduled dates of necropsy: Adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thyroid including parathyroid gland. The organ to terminal body weight ratios as well as the organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.

HISTOTECHNIQUE: All organ and tissue samples, as defined under Histopathology were processed, embedded, cut at an approximate thickness of 4 micrometers, and stained with hematoxylin and eosin.

HISTOPATHOLOGY: Slides of adrenals, heart, kidneys, liver, lungs, spleen, stomach and testes collected at terminal sacrifice from the animals of group 1 (control) and group 4 (high-dose) as well as all gross lesions from rats of all groups were examined by a pathologist.
Other examinations:
Clinical Laboratory Investigations
Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia.The animals were fasted in metabolic cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected between the hours of 06.30 and 07.35 to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
Urine was collected during the 18-hour fasting period into a specimen vial.
Statistics:
When the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was
applied for the comparison between the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
The Fisher's exact test was applied to the ophthalmoscopy data and macroscopical findings.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All animals survived the scheduled treatment period. One group 4 (1000 mg/kg) female (no. 53) died during blood sampling on the day of the scheduled necropsy.
Mortality:
mortality observed, treatment-related
Description (incidence):
All animals survived the scheduled treatment period. One group 4 (1000 mg/kg) female (no. 53) died during blood sampling on the day of the scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect on body weight.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption values were similar for treated groups and controls.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes on the ophtha!mological findings.
Haematological findings:
no effects observed
Description (incidence and severity):
no remarkable findings were noted.
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights, organ to body weight and organ to brain weight ratios were unaffected by treatment with test article.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All gross and microscopic lesions recorded in this study were considered to be spontaneous in origin. Their incidence and severity was similar in control and treated rats at both sacrifices.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All gross and microscopic lesions recorded in this study were considered to be spontaneous in origin. Their incidence and severity was similar in control and treated rats at both sacrifices.
Histopathological findings: neoplastic:
not specified
Details on results:
Clinical Signs: There were no clinical signs noted in groups 2 (50 mg/kg) and 3 (200 mg/kg). Animals of group 4 showed dark feces from treatment day 13 throughout the treatment period and during the first day of the treatment-free recovery period.

Hematology / Clinical Biochemistry / Urinalysis
The results of the hematology, clinical biochemistry and urinalysis data indicated no changes of toxi cological significance at termination of the treatment nor at the end of the treatment-free recovery period. However, the following changes recorded between the controls and treated rats of group 4
(1000mg/kg) at termination of the treatment were considered to be test articlerelated:
Clinical Biochemistry: slightly increased uric acid level in females (p<0.01), moderately increased total bilirubin level in both males and females (p<0.01), slightly increased total cholesterol and phospholipid levels in males (p<0.01), slightly increased triglyceride level in females (p<0.01), slightly lower potassium level and slightly increased total protein and globulin levels in males (p<0.05). In addition, orange plasma discoloration was observed in all animals of group 4.
Urinalysis: apart from a higher incidence in the score for urine bilirubin (statistically non-significant) and urine discoloration in both sexes of group
4, no remarkable findings were noted. Urine discoloration as observed in both sexes of group 4 ranged from a deep-yellow (2 males, 6 females) to light-orange (4 males, 2 females) to orange (2 males) to deep-orange (2 males). Moreover, a deep-yellow urine discoloration was also noted in 4 out of 5 males and 2 out of 5 females of group 3 (200 mg/kg), and in 3 out of 5 males and 1 out of 5 females of group 2 (50 mg/kg).
Recovery period: with the exception of a slightly lower potassium level in males (p<0.05), all of the findings recorded during the treatment period indicated reversibility and/or were similar to those of the controls, and no longer statistically significant. Subsequently, no further plasma or urine discoloration was observed.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, 200 mg/kg FAT 45'176/A was established as the no-observed-effect level (NOEL) - except for urine discoloration - and 1000 mg/kg as the no-observed-adverse-effect level (NOAEL).
Executive summary:

In this subacute toxicity study, FAT 45'176/A was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days.

The study comprised 5 animals per group and sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, food consumption and body weights were recorded periodically during the treatment and recovery periods. Ophthalmoscopic examinations were performed at pretest, the end of the treatment and recovery periods.

The oral administration of FAT 45'176/A to Wistar rats at doses of 50, 200 and 1000 mg/kg body weight/day, for 28 days, resulted in no effects on mortality, food consumption, body weight, Ophthalmoscopical findings, organ weights, macroscopic and microscopic findings.

Animals of group 4 (1000 mg/kg) showed dark feces from teatment day 13 throughout the treatment period and during the first day of the treatment-free recovery period.

In group 4 (1000 mg/kg) slightly increased uric acid level in females, moderately increased total bilirubin level in both males and females, slightly increased total cholesterol and phospholipid levels in males, slightly increased triglyceride level in females, slightly lower potassium level and slightly increased total protein and globulin levels in males, higher incidence in the score for urine bilirubin were considered to be related to treatment with test article. Plasma discoloration was observed in all animals of group 4 (1000 mg/kg) and urine discoloration in most of the animals of group 4 (1000 mg/kg) and in some animals of groups 3 (200 mg/Kg) and 2 (50 mg/kg). Urine discoloration was reversible after the treatment-free recovery period.

Based on the results of this study, 200 mg/kg FAT 45'176/A was established as the no-observed-effect level (NOEL) - except for urine discoloration - and 1000 mg/kg as the no-observed-adverse-effect level (NOAEL).