Direct Yellow 169

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline- and GLP-compliant 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))

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): FAT 40'403/A
- Physical state: yellowish solid

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., Wolferstrasse 4, CH-4414 Fuellinsdorf
- Age at study initiation: Males : 6 weeks; Females: 6 weeks
- Weight at study initiation: Males: 165 - 178 g; Females 160 - 178 g
- Fasting period before study: no data
- Housing: The rats were housed individually in Makrolon type-3 cages with standard softwood bedding, ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum): The animals were provided with pelleted standard Kliba 343, rat maintenance diet. Batch 70/90 ('Kliba*, Klingentalmuehle AG, CH-4303 Kaiseraugst) ad libitum.
- Water (e.g. ad libitum):Community tap water from Itingen was available, ad libitum.
- Acclimation period:7 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:

PREPARATION OF DOSING SOLUTIONS:
The test article was weighed into a glass beaker on a tared Mettler PE 360 balance and the vehicle, bidistilled water, was added. The mixture was
prepared using a homogenizer. Homogeneity of the vehicle was maintained during treatment using a magnetic stirrer.
Frequency of preparation: Daily prior to each application

VEHICLE
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): 10 ml/kg body weight and treatment day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test article/vehicle mixture were determined during acclimatization. Further samples for analysis were taken during week 3 of the test and subsequently analyzed. The analyses were performed in the Analytical Laboratory of RCC Umweltchemie AG,
according to a method which was supplied by the sponsor.
Acclimatization/Pretest
At 3 weeks of the test August 28, 1990 September 18, 1990
The supply of the analytical method by the sponsor
was delayed. Therefore the samples were deep frozen
directly after preparation and analyzed at September
11 and 21, 1990, respectively.

Duration of treatment / exposure:

28 days
plus 14 days recovery

Frequency of treatment:

once daily
plus 14 days recovery

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10 males, 10 females (groups 1 and 4)
5 males, 5 females (groups 2 and 3)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based upon data received from acute studies, especially an acute oral toxicity study (RCC 273881) and a 5-day range-finding study (RCC 273947) in which FAT 40'403/A was administered orally by gavage to rats.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded on the same days as the food
consumption using the same recording system


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



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


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Dates: at 4 weeks; at 6 weeks
- Dose groups that were examined: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).
Pictures were taken of all abnormal findings.


HAEMATOLOGY: Yes
Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted for 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 bioloqical variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus.


CLINICAL CHEMISTRY: Yes
Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted for 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 bioloqical variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus.


URINALYSIS: Yes
Urine was collected over an 18-hour 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. Blood and urine sampling: at 4 weeks; at 6 weeks
NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
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
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. Necropsies were performed by experienced prosectors. All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination, after 4 weeks
after 6 weeks; Samples of the following tissues and organs were collected from all animals at necropsy and fixed in phosphate buffered neutral 4 % formaldehyde solution: Adrenals; Aorta; Bone with 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; Nasopharynx; 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.
Slides of Adrenals, Heart, Kidneys, Liver, Spleen and Stomach collected at terminal sacrifice from the animals of the control and high-dose groups w re 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 p erformed. 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.
The Fisher's exact test for 2x2 tables was applied to the overall spontaneous mortality data and for the overall ophthalmoscopy data.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were round-off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded-off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no treatment-related clinical signs. The administration of FAT 40'403/A was not associated with any treatment related deaths.

BODY WEIGHT AND WEIGHT GAIN
There were no treatment-related effects on body weight gain.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no treatment related effects on food consumption or relative food consumption.


OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related effects on ophthalmoscopy parameters.

HAEMATOLOGY
Hematology
There were no treatment-related effects on hematology parameters

CLINICAL CHEMISTRY
Clinical Biochemistry
There were no treatment-related effects on clinical biochemistry parameters
at up to 1000 mg/kg FAT 40'403/A in females and up to 200 mg/kg in males.
In males receiving 1000 mg/kg FAT 40'403/A uric acid, bilirubin and LDH levels
were higher and sodium and chloride levels lower than in control males after
4 weeks of treatment. LDH was also slightly higher after the 2 week treatment free
recovery period.

URINALYSIS
There were no treatment-related effects on urinalysis parameters at 50 or
200 mg/kg FAT 40'403/A.
At 1000 mg/kg there was a treatment-related increase in low-grade bilirubinuria
in both sexes.
In 7/10 males at 1000 mg/kg the urine was dark yellow, but this is attributed
to coloration by the test article which is yellow in the solid state.


ORGAN WEIGHTS
There were no treatment-related effects on organ-weights.

GROSS PATHOLOGY
There were no treatment-related changes in the macroscopic appearance of any
organ or tissue, except for yellow discoloration of the gastointestinal mucosa
in 4/10 rats at 1000 mg/kg after 4 weeks of treatment. This is attributed to
the presence of FAT 40'403/A which is yellow in the solid state.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related changes in the microscopic appearance of any
organ or tissue.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: Based upon the results obtained in this study, the "no-adverse-effect-level" of FAT 40'403/A is 200 mg/kg body weight for male and female rats when administered orally by gavage for a period of 28 days.

Target system / organ toxicity

Critical effects observed:

not specified