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Administrative data

short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 February 1996 to 17 June 1996
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
according to guideline
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
according to guideline
other: Revised Japanese Chemical Substance Law (1987) according to the notification of Dec. 9, 1986 by EA, Environmental Agency (No. 700); MHW, Ministry of Health and Welfare (No. 1039) and MITI, Ministry of International Trade and Industry (No. 1014).
GLP compliance:

Test material

Constituent 1
Reference substance name:
FAT 40'549/A
FAT 40'549/A
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
Identity: FAT 40'549/A
Batch No.: TV 1
Expiration date: January 01, 2001
Stability of Test Article: Stable under storage conditions
Stability of Test Article in Vehicle: Stable in bi-distilled water for 48 hours
Description: yellow-brown powder
Storage conditions: In the original container at room temperature, away from direct sunlight.
Safety Precautions: Routine hygienic procedures were used to ensure the health and safety of the personnel
Specific details on test material used for the study:
Identity: FAT 40'549/A
Batch No: TV1
Expiration date: 01 Jan 2001
Stability in water: for atleast 48 hours
Solubility in water: approximately 90 mg/L at 20 degree celcius.
Aggregate state under storage conditions: solid (powder)
Colour: yellow brown.
Storage conditions: at room temperature
Safety precautions: Routine hygenic procudures are sufficient to assure personnel health and safety.

Test animals

Details on test animals or test system and environmental conditions:
Test animal: Rat, HanIbm: WIST (SPF)
Total number of animals per group: Groups 1 and 4: 10 males; 10 females; groups 2 and 3: 5 males and 5 females
Total number of animals: 30 males and 30 females
Age at delivery: 6 weeks
Body weight and range at acclimatization/pretest: Males: 149 - 173 g (mean 161 g), Females: 120 - 148 g (mean 135 g)
Identification: Cage card and individual ear tattoo.
Randomization: Computer-generated random algorithm.
Acclimatization: 7 days under test conditions following a health examination. Only animals without any visible signs of illness were used for the study.
Standard Laboratory Conditions. Air-conditioned with 10 - 15 air changes per hour, and continuously monitored environment with a target range for temperature of 22 ± 3 degrees centigrade and for relative humidity between 40 - 70 %. 12 hours fluorescent light/ 12 hours dark, and at least 8 hrs music/light period.
Accommodation: Groups of five in Makrolon type-4 cages with standard softwood bedding
Diet: Pelleted standard Kliba no. 343 rat maintenance diet (KLIBA Mühlen AG, 4303 Kaiseraugst/Switzerland) was available ad libitum (Batch no. 72/96). The feed batch was analyzed for contaminants.
Water: Tap water was available ad libitum in water bottles.
Duration of acclimatization: 7 days
Duration of treatment: 28 days
Duration of recovery: 14 days.

Administration / exposure

Route of administration:
oral: gavage
Details on oral exposure:
Method: Oral by gavage.
Frequency: Once daily, 7 days per week for a total of 28 days.
Dose volume: 10 ml/kg body weight per treatment day,
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability of the test article/vehicle mixtures were determined in samples taken during acclimatization and during
week 3 of the treatment. The analyses were performed in the Analytical Laboratories of RCC Umweltchemie AG according to a method supplied by
the sponsor.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Group 1: Control group
Dose / conc.:
50 mg/kg bw/day (nominal)
Group 2: Low dose group
Dose / conc.:
200 mg/kg bw/day (nominal)
Group 3: Mid dose group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Group 4: High dose group
No. of animals per sex per dose:
5 males and 5 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment: Randomsed.
- Fasting period before blood sampling for clinical biochemistry: yes 18 hours before blood sampling.
- Post-exposure recovery period in satellite groups: 2 weeks


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 once daily.
FOOD CONSUMPTION: The food consumption was recorded once during the acclimatization period and weekly thereafter using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

BODY WEIGHTS: Body weights were recorded weekly and before necropsy

OPHTHALMOSCOPIC EXAMINATIONS: Dates: At 4 weeks: 28-MAR-1996, At 6 weeks (i.e. 2 weeks recovery): ll-APR-1996. The cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes of all animals were examined after the application of a mydriatic solution (Ciba Vision AG, CH-3172 Niederwangen) using a Miroflex 2 Ophthalmoscope (Eisenhut Vet. AG, CH-4123 Allschwil). A description of any abnormality was recorded.
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 08.00 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. Blood and urine sampling: After 4 weeks and after 6 weeks.
Sacrifice and pathology:
After 4 weeks and after 6 weeks.
All animals were weighea and necropsied and descriptions of all macroscopic abnormalities were recorded. Prior to necropsy, the animals were fasted , but free access to water was provided. 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 (Narcoren, Rhône Merieux GmbH, Laupheim) at a dose of 2.0 ml/kg body weight (equivalent to 320 mg sodium pentobarbitone/kg body weight), weighed and sacrificed by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in phosphate buffered neutral 4 % formaldehyde solution.

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 using an on-line electronic recording system consisting of a Mettler balance connected to a computer system.

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.

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) were examined by a pathologist. Kidneys and all gross lesions from rats of all groups were also examined.
The following data were recorded on-line: food consumption, body weights, organ weights, clinical signs, mortality, macroscopic findings at necropsy and histopathology.
Clinical Laboratory data were recorded on-line (Sysmex TOA E-4000 Multiparameter Automated Hematology Analyzer, Sysmex TOA R-1000 Automated Reticulocyte Analyzer, Instrumentation Laboratory ACL 300 Coagulation System, Eris Selective Multi-Test Analyzer 6170, Boehringer Miditron M Semi-Automated Urine Chemistry Analyser) or on data sheet and then transferred into the computer system.
Ophthalmoscopic data were recorded on data sheets and transcribed for compilation and analysis into the computer system.
The computer-generated values which appear in the tables represent the roundedoff results of the raw data values or of calculations which used the exact raw data values.
Group means were calculated according to the definition of any mean value using the individual values per animal and the number of animals.

The following statistical methods were used to analyze the body weights, food consumption, organ weights and all ratios and clinical laboratory data:
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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
All animals survived the scheduled study period. No clinical signs were noted in any group.
no mortality observed
Description (incidence):
All animals survived the scheduled study period. No clinical signs were noted in any group.
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):
There were no effects on food consumption or relative food consumption.
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 ophthalmoscopic findings noted for any group.
Haematological findings:
no effects observed
Description (incidence and severity):
The assessment of hematology, indicated no changes of toxicological significance at termination of the treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Iincrease in the total bilirubin concentration (p<0.01) was observed.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urine discoloration was observed in both sexes of group 4 (1000 mg/kg). For the latter, the discoloration ranged from a deep yellow to light orange to orange. These findings are considered to be related to the pigmentation of the test article, FAT 40'549/A.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No statistically significant differences of organ weights, organ to body weight and organ to brain weight ratios in relation to treatment with FAT 40'549/A were recorded.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Morphologic findings related to treatment were seen in the kidneys and consisted of an increased incidence and severity of lipofuscin pigment in the proximal tubules in all rats at 1000 mg/kg at both sacrifices
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not specified

Effect levels

open allclose all
Dose descriptor:
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
other: No toxic effects observed upto 1000 mg/kg
Dose descriptor:
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical biochemistry

Target system / organ toxicity

Critical effects observed:

Applicant's summary and conclusion

Based on the findings of this study, the no-observed-effect level (NOEL) and no-observed-adverseeffect level (NOAEL) were found to be 200 mg/kg bw and 1000 mg/kg bw respectively.
Executive summary:

A sub-acute repeat dose study was conducted to assess the repeat dose toxicity of FAT 40549/A. This study was conducted in accordance with OECD test guideline 407 and Directive 92/69/EEC, B.7 in a GLP certified laboratory.

The oral administration of FAT 40549/A to Wistar rats at doses of 50, 200 and 1000 mg/kg/day, for 28 days, resulted in animals of both sexes from the high dose group in increased total bilirubin concentration, urine discoloration, and increased incidence and severity of lipofuscin pigment in the epithelium of the proximal tubules.

After the recovery period the higher bilirubin concentration and urine discoloration were found to be reversed and similar to those of the control, whereas the lipofuscin pigment was noted at the end of treatment and also following the recovery phase. At the end of the recovery period slightly higher score for blood in urine of high dose males was recorded.

Higher bilirubin concentration and a more intense yellow coloration of the plasma, as well as the urine discoloration were considered to be related to the yellow brown color of the test article and in the absence of relating morphological changes of no toxicological relevance. The increase in the incidence and severity of lipofuscin pigment in the proximal tubular renal epithelium in all animals of group 4 is derived from oxidation of tissue lipids and probably represents an increased metabolic turnover in the affected cells.

Based on the results of this study, 1000 mg/kg FAT 40549/A/A was established as the no-observed-adverse-effectllevel (NOAEL) and the NOEL, the no-observed-effect-level as 200 mg/kg.