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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:
14 Nov 1990 to 30 Aug 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Remarks:
Swiss GLP
Limit test:
yes

Test material

Constituent 1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): FAT 40'406/A
- Lot/batch No.: Vers. Nr. SR 6947/14
- Expiration date of the lot/batch: July 1995
- Stability: Stable
- Stability of test article dilution: Stable for at least 48 hours
- Storage: At room temperature, protected from light
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: SR 6947/14
- Expiration date of the lot/batch: July, 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (approx. 20 °C) in the dark
- Stability under test conditions: Stable for >48 h

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Hanlbm: WIST (SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., Wölferstrasse 4, CH-4414 Füllinsdorf
- Age at acclimation: 6-7 weeks
- Weight at acclimatization: males: 160 - 175 g, females: 144 - 155 g
- Housing: Individually in Makrolon type-3 cages (size: 22 x 37.5 x 15 cm) with standard softwood bedding ('Lignocel', Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kliba 343, Batches 77/90 and 78/90 rat maintenance diet ("Kliba" Klingentalmuehle AG, CH-4303 Kaiseraugst,) available ad libitum
- Water: Community water from Itingen, available ad libitum
- Acclimation period: Seven days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral by gavage, once daily. Rationale for oral route is accidental oral ingestion is a possible route of human exposure.
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on oral exposure:
MAXIMUM DOSE VOLUME APPLIED: 10mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability of the test article/vehicle mixtures were determined during acclimatization. Further samples for analysis were taken during week 3 of the test and subsequently analyzed.
Duration of treatment / exposure:
up to 28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1 - Control
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Group 2: Low dose
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Group 3: Mid Dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4: High Dose
No. of animals per sex per dose:
- 5 per sex/dose (Group 2 and 3)
Additionally, 5/sex for control and high dose recovery group (Group 1 & 4)
Control animals:
yes, concurrent vehicle
Details on study design:
FAT 40406/A was administered to SPF-bred Wistar rats by repeated oral gavage, for a period of 28 days. The study was comprised of four groups, each containing five male and five female rats. The test article/vehicle mixture was administered on a mg/kg bw basis by oral gavage. The dose selection was based upon data received from acute studies and a 5-day oral (range-finding) gavage study. The animals of the control group were treated similarly with the vehicle alone. For the post-exposure recovery period two satellite groups exposed to 0 and 1000 mg/kg bw for 28 days consisting of five male and five female rats each were monitored for an additional 14 days after the end of the 28-days treatment period.

Examinations

Observations and examinations performed and frequency:
- Mortality/viability: Observations for viability and mortality were recorded once daily.
- Clinical signs: Signs of toxicity were assessed once daily. Descriptions of all abnormalities were recorded and the subsequent progress was monitored.
- Food consumption: The food consumption was recorded once during the acclimatization period and weekly thereafter.
- Body weights: The body weight of each animal was recorded on the same days as the food consumption.

- Ophthalmoscopic examination: Ophthalmoscopic examinations were performed on all animals. A description of any abnormality was recorded. Examinations were performed at termination of treatment and a second time on the recovery individuals of groups 1 and 4 at termination of the recovery period. Ten minutes after the application of a mydriatic solution (Dispersa AG, CH-8400 Winterthur) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine Miroflex 2 Ophthalmoscope (Eisenhut Vet. AG, CH-4123 Allschwil).

- Haematology/clinical chemistry: Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia at termination of treatment. The animals were fasted for approximately 18 hours before blood sampling but water was provided ad libitum. Blood samples were collected from each animal between the hours of 6.05 and 8.15 a.m. to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus. Parameters being measured: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, nucleated erythrocytes normoblasts, heinz bodies, methemoglobin, total leukocyte count, differential leukocyte count, red cell morphology, thromboplastin time, activated partial thromboplastin time, glucose, urea, creatinine, uric acid, bilirubin, cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase, alkaline phosphatase, gamma-glutamyl-transferase, calcium, phosphorus, sodium, potassium, chloride, albumin, protein total, globulin, albumin/globulin ratio.

- Urinalysis: Urine was collected during the 18-hour fasting period into a specimen vial using a metabolism cage. Parameters being measured: volume (18 hour), specific gravity, pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, urine sediment.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were necropsied and descriptions of all macroscopic abnormalities were recorded. Prior to necropsy, the animals were fasted for approximately 18 hours, but water was provided. The following organ weights were taken from all animals necropsied at termination of treatment or recovery: Adrenals, aorta, bone (sternum, femur), bone marrow (sternum, femur), brain, cecum, colon, duodenum, epididymides, esophagus, eyes with optic nerve and harderian gland, female mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, larynx, lacrimal gland (extra-orbital), liver, lung infused with formalin, lymph nodes (mandibular, mesenteric), nasal cavities, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary gland (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder infused with formalin, uterus, vagina, gross lesions.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were taken from all animals necropsied at termination of treatment or recovery: adrenals; brain; heart; kidneys; liver; ovaries; pituitary gland; spleen; testes; thyroid gland.

HISTOPATHOLOGY: Yes
- Slides of adrenals, heart, kidneys, liver, spleen and stomach collected at terminal sacrifice from the animals of the control and high-dose groups were examined by a pathologist. The same applied to all gross lesions and to all animals which died spontaneously. Upon detection of treatment related morphologic changes in the kidneys of any high-dose animal, histologic evaluation of this organs in all dose groups were performed.
Statistics:
The following statistical methods were used to analyze the body weights, food consumption, organ weights and clinical laboratory data: Univariate one-way analysis of variance was used to assess the significance of intergroup differences. If 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 for 2x2 tables was applied to the overall spontaneous mortality data and for the overall ophthalmoscopy data.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
During most of treatment period, in all treated animals of any groups slight diarrhea was observed. Those findings were considered to be an effect of the vehicle (PEG 400) used which empirically induce diarrhea. Male and female rats of groups 3 and 4 (200 mg/kg and 1000 mg/kg, respectively) showed red discoloration of the feaces from treatment start until the end of the 28-day study period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female control" (group 1, no. 33) animal died during blood sampling immediately prior to schedule necropsy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weights of male rats of group 3 (200 mg/kg) were statistically significantly higher than their corresponding controls on treatment days 22 to 28, this was coherent to their food consumption rates and considered to be toxicologically not significant. The body weights of any other animal of the treated groups were not affected by the treatment with FAT 40'406/A.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Male rats of group 3 (200 mg/kg) consumed statistically significantly more food than the respective controls on treatment days 15 to 28. Female rats of group 4 (1000 mg/kg) consumed less food during the treatment-free recovery period, without reading any statistically significance. The relative food consumption of group 3 (200 mg/kg) males did not differ significantly from controls nor did the absolute or relative food consumption rates of any other animal of the treated groups. The observed differences were considered to be incidental and of normal pattern for rats of this strain and age.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
However, a corneal ulcer was found in the right eye of the high-dose female no. 60 towards to the end of the recovery period. This effect was considered to be spontaneous and not related to test article treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The assessment of hematological data indicated the following effects at termination of the treatment for female rats of group 4 (1000 mg/kg), when compared to the controls:
- Slight decrease of the erythrocyte count, hemoglobin concentration and hematocrit
- slight increase of 'the mean corpuscular volume and mean corpuscular hemoglobin indices;
- slight increase of the total leukocyte count;
- slight increase in the absolute proportion of lymphocytes of the differential leukocyte count.
The findings observed reflect slight anemia for the females of group 4. However, the findings were not considered to be of toxicological significance since the findings were not supported by any other hematological or histopathological changes which would indicate increased erythropoietic activity. In addition, the findings were within the normal range of the historical control data. At termination of the treatment-free recovery period these findings were found to be reversed and comparable to those of the controls. All other statistical differences in the results of the hematology parameters were considered to be incidental and of normal biological variation for rats of this 'strain and age (see also historical reference values for untreated Wistar Han. rats, pp. 175-182).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
For biochemical data the following effects were noted at termination of the treatment for rats of group 4 (1000 mg/kg), when compared to the controls:
- Slight decrease of the glucose and urea concentrations for females;
- slight increase of the total bilirubin concentration for both sexes;
- slight decrease of the total cholesterol and phospholipid concentrations for females;
- slight decrease of the calcium concentration for both sexes;
- slight decrease of the phosphorus concentration for females;
- slight decrease of the albumin, total protein and globulin concentrations for females.
The changes noted, with the exception of total bilirubin, were considered to be of metabolic nature with no toxicological relevance. As for total bilirubin, there was no direct indication of increased hemolysis or increased erythropoietic activity nor evidence of biliary obstruction or hepatocellular damage which would suggest an increase in the plasma bilirubin concentration. In the assay method used, bilirubin reacts with 2.5-dichlorophenyldiazonium salt to give a red azo dye. Since the test article was a dark "reactive red" dye, one can expect a direct effect on the final concentration measurement. It is therefore only likely, that the higher bilirubin concentration is related to interference with the test article FAT 40406/A. At termination of the treatment-free recovery period these findings were found to be reversed and comparable to those of the controls. All other statistical differences in the results of the clinical biochemical parameters were considered to be incidental and of normal biological variation for rats of this strain and age (see also historical reference values for untreated Wistar Han. rats, pp. 183-190).
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinalysis data indicated a slightly lower overnight urinary output (volume/18h) and a correspondingly lower specific gravity for males of group 4 (1000 mg/kg). A lower specific gravity was also observed for females of this group. In addition, both sexes of group 4 indicated an orange urine discoloration. At termination of the treatment-free recovery period the lower overnight urinary output for males and the orange urine discoloration for both sexes were still to be noted, whereas the lower specific gravity findings were comparable to those of the controls. The findings were considered an effect of the treatment. All other differences in the results of the urinalysis parameters were considered to be incidental and of normal biological variation for rats of this strain and age (see also historical reference values for untreated Wistar Han. rats, pp. 191-198).
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
After termination of the 28-day treatment period male rats of group 3 (200 mg/kg) showed heart to body weight ratios statistically significantly lower than the corresponding controls. Females of group 3 (200 mg/kg) showed absolute heart weights and the ratios to body- and brain weight statistically significantly lower than the corresponding controls. Females of the high-dose (group 4, 1000 mg/kg) showed statistically significantly higher spleen weights than those of the controls. After the treatment-free recovery period female rats of the high-dose group 4 (1000 mg/kg showed statistically significantly increased absolute and relative kidney weights compared to those of control.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Dark red discoloration of the kidneys was seen in group 4 animals, both main test and recovery.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Renal tubular vacuolation was seen histologically in group 4 (main test and recovery) animals. The vacuoles appeared to contain pigment which is thought to be the test article. The degree of vacuolation or amount of pigment did not reduce after 15 days without treatment. There was an increase in renal tubular basophilia, mainly in male recovery animals, which suggests that the presence of the pigment for a prolonged period may cause some degeneration. The kidneys of group 2 and 3 (50 and 200 mg/kg, respectively) animals were indistinguishable from those of controls. There were no other treatment related findings identified.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
No observed adverse effect level (NOAEL) of FAT 40406/A was determined to be 200 mg/kg/bw.
Executive summary:

In a GLP-compliant repeated toxicity study, performed according to OECD guideline 407, Wistar rats were treated with the test substance (0, 50, 200, and 1000 mg/kg bw/day) by repeated oral gavage, for a period of 28 days. The study comprised of four groups, each containing five male and five female rats. For the post-exposure recovery period two satellite groups exposed to 0 and 1000 mg/kg bw/day for 28 days consisting of five male and five female rats each were monitored for an additional 14 days after the end of the 28 days treatment period. No treatment related effects were observed on mortality, clinical signs, food consumption, body weight, organ weight and ophthalmoscopic examinations. Haematological investigations indicated a slight anemia for the females in the high dose group, but the findings were not supported by any other hematological or histopathological changes, were reversible and were within the normal range of the historical control data. In the clinical biochemistry investigations, the effects observed were considered to be of metabolic nature with no toxicological relevance. Urinalysis data indicated no changes of toxicological significance at termination of the treatment. The kidneys of group 4 (1000 mg/kg bw/day, main test and recovery) animals were macroscopically discolored dark red. This was considered an effect of the dark red color of the test article itself. Renal tubular vacuolation was seen histologically in group 4 (main test and recovery) animals. The vacuoles appeared to contain pigment which is thought to be the test article. The degree of vacuolation or amount of pigment did not reduce after 15 days without treatment. There was an increase in renal tubular basophilia, mainly in male recovery animals, which suggests that the presence of the pigment for a prolonged period may cause some degeneration. The kidneys of group 2 and 3 (50 and 200 mg/kg bw/day, respectively) animals were on the other hand indistinguishable from those of controls. Therefore, no observed adverse effect level (NOAEL) of FAT 40406/A was determined to be 200 mg/kg bw/day based on the effects observed in the kidney.