JAUNE REACTIF 181

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July 1990 to 4 September 1990

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

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 40'400/A
- Lot/batch No.: BG 3247/TV 6
- Expiration date of the lot/batch: March 1995
- Stability: Stable
- Stability of test article dilution: at least 2 hours

Specific details on test material used for the study:

Test substance: FAT-40400/A
Expiry Date: 3/95
Stability of test article: Stable
Stability of test article in vehicle: stable for at least 2 hours
Storage: in the original container, at room temperature, protected from light

Test animals

Species:

rat

Strain:

Wistar

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 weeks
- Weight at acclimatization: males: 159 - 177 g, females: 161 - 175 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz)
- Diet: Pelleted standard Kliba 343, Batches 70/90, 71/90, 73/90 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

Vehicle:

water

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 mixtures were determined during acclimatization. Further samples for analysis were taken during
acclimatization and during week 3 of the test, than kept deep frozen until later determination of analysis.

Duration of treatment / exposure:

up to 28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Middle dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

The number of rats assigned to toxicity and recovery testing per group were:
- 5 per sex for 50 and 200 mg/kg bw for the toxicity test
- 5 per sex for 0 and 1000 mg/kg bw for the recovery test

Control animals:

yes, concurrent vehicle

Details on study design:

FAT 40400/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 was based upon data received from acute studies and a 5-days 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 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. Blood samples were collected from each animal between the hours of 7.00 and 9.30 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.
- Urinalysis: Urine was collected during the 18-hour fasting period into a specimen vial using a metabolism cage, during which time the animals were deprived of food but allowed access to water ad libitum. Parameters being measured: volume (18 hour), specific gravity, pH, color, 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. 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 or had to be killed in extremis. Upon detection of treatment-related morphologic changes in the organs of any high-dose animal, histologic evaluation of the same organs in all dose groups were performed. All abnormalities were described and included in the report.

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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The lower values seen in males receiving 50 mg/kg FAT 40400/A are considered not to be due to treatment because of the lack of a dose-relationship.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The lower values seen in males receiving 50 mg/kg FAT 40'400/A are considered not to be due to treatment because of the lack of a dose-relationship.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

However, in the urinalysis 5 out of 10 male rats of group 4 (1000 mg/kg) indicated a deep yellow urine discoloration at termination of the treatment. This observation was attributed to the high concentration and yellowish nature of the test article and is not an indication of renal insufficiency. At termination of the treatment-free recovery period this finding was no longer observed.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The following conclusions are based on examination of absolute organ weights, organ/body weight ratios and organ/brain weight ratios. In males, although there were no statistically significant differences in absolute organ weight between treated and control group means, there was evidence from organ/body and organ/brain weight ratios of a slight increase in liver and heart weights at 1000 mg/kg FAT 40400/A after 4 weeks of treatment. After the 14-day treatment-free recovery period there was no evidence of a treatment-related effect. In females, heart weight in rats receiving FAT 40'400/A was lower than in controls after 4 weeks of treatment but the fact that the differences were greater in the groups receiving 50 or 200 mg/kg than in the high dose group receiving 1000 mg/kg FAT 40400/A suggests that this was not due to treatment. Similarly, although liver weight in high dose females after the 14-day treatment-free recovery period was higher than in control females, the absence of any treatment-related difference at the end of the dosing period suggest that this was not due to the administration of FAT 40400/A.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effect level based on clinical symptoms, body weight, food consumption, ophthalmoscopic examination, clinical laboratory investigations, and pathology

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) of FAT 40400/A was determined to be 1000 mg/kg/bw.

Executive summary:

In a GLP-compliant repeated dose toxicity study, performed according to OECD guideline 407 and EU method B.7. Wistar rats were treated with the test substance (50, 200, and 1000 mg/kg bw) 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. 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. No treatment-related effects were observed on mortality, clinical signs, food consumption, body weight, ophthalmoscopic examinations, clinical laboratory investigations, macroscopic and microscopic findings. The lower values of food consumption seen in males receiving 50 mg/kg FAT 40400/A are considered not to be due to treatment because of the lack of a dose-relationship. However, in the urinalysis 5 out of 10 male rats of group 4 (1000 mg/kg) indicated a deep yellow urine discoloration at termination of the treatment. This observation was attributed to the high concentration and yellowish nature of the test article and is not an indication of renal insufficiency. At termination of the treatment-free recovery period this finding was no longer observed. There was evidence that treatment of male rats with 1000 mg/kg of the test substance for 4 weeks was associated with a slight increase in liver weight which regressed during the treatment-free recovery period. There were no other differences in organ weight which could be attributed to treatment with the test substance. So, based on the study results, the No Observed Adverse Effect Level (NOAEL) of FAT 40400/A was determined to be 1000 mg/kg/bw.