C.I. Acid Yellow 246

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:

20 July 1987 to 06 April 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

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

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: FAT 20 306/B
Description: powder
Batch number: HT 2025/50
Purity: 96 %
Stability of test article: stable; expiration: year 2014
Stability of test article dilution: stable for at least 2 hours
Instructions for test article storage: room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Strain: Wistar rat, KFM-Han., outbred, SPF quality
- Source: Kleintierfarm Madoerin AG CH 4414 Fuellinsdorf/Switzerland
- Age at pretest: 7 - 8 weeks
- Weight at pretest: males: 153 - 174 g; females: 150 - 184 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding
- Diet: Pelleted standard Kliba no. 343, Batch 73/87 and 77/87 rat maintenance diet ('Kliba', Klingentalmuehle AG, 4303 Kaiseraugst, Switzerland) ad libitum
- Water: Community tap water from Itingen was available ad libitum.
- Acclimation period: Seven days under laboratory conditions, after veterinary examination

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

4% (CMC)

Details on oral exposure:

TEST ARTICLE PREPARATION: The test article was weighed into a glass beaker on a tared Mettler PK 300 balance and the vehicle, carboxymethyl cellulose 4 % (CMC) in distilled water, was added. The mixture was prepared dally prior to administration using a homogenizer and kept stable during application with a magnetic stirrer.
Method: Oral by gavage, once daily. The control animals received the vehicle without the test article.
Frequency: Once daily, 7 days per week
Dose volume: 10 ml/kg body weight

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 was determined during pretest. Intercurrent sampling for analyses was additionally performed during week 3 of test. Analyses were performed in the RCC Analytical Chemistry Laboratories, according to a method supplied by the sponsor.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 days per week

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:

5/sex for test concentrations of 50 mg/kg and 200 mg/kg
10/sex for test concentrations of 0 mg/kg and 1000 mg/kg

Control animals:

yes, concurrent vehicle

Details on study design:

TEST ARTICLE PREPARATION
The test article was weighed into a glass beaker on a tared Mettler PK 300 balance and the vehicle, carboxymethyl cellulose 4 % (CMC) in distilled water, was added. The mixture was prepared dally prior to administration using a homogenizer and kept stable during application with a magnetic stirrer.

TEST METHOD
In this subacute toxicity study, test article was administered daily by 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. After termination of the treatment period animals were observed for a further 14 -day treatment-free recovery period.

Positive control:

no data

Examinations

Observations and examinations performed and frequency:

VIABILITY / MORTALITY: 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 acclimation period and weekly thereafter using an on-line electronic recording system consisting of a Mettler PK 4800 balance connected to the RCC computer.
BODY WEIGHTS: The body weight of each animal was recorded weekly during the acclimation and treatment period using an on-line electronic recording system consisting of a Mettler PK 4800 balance connected to the RCC computer.
OPHTHALMOSCOPIC EXAMINATIONS: at 4 weeks and at 6 weeks. Ophthalmoscopic examinations were performed on all animals. A description of any abnormality was recorded. Examinations were performed at termination of treatment and recovery period.

Sacrifice and pathology:

ORGAN WEIGHTS: The following organ weights were taken from all animals necropsied after 4 and 6 weeks respectively: Adrenals, Kidneys, Liver, Testes.
NECROPSY: after 4 weeks and after 6 weeks. All animals were necropsied, and descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by a pathologist. 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.
HISTOTECHNOLOGY: All organ and tissue samples, as defined under Histopathology (following) were processed, embedded and cut at a thickness of 2 - 4 micrometers and stained with hematoxylin and eosin.
HISTOPATHOLOGY: Tissue samples of Adrenals, Heart, Kidneys, Liver, Spleen and Gross lesions collected at the end of the assigned study periods from the animals of the control and high-dose 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
Blood and urine sampling: at 4 weeks and at 6 weeks

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.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) 1n the summary tables.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Group 2 - 5 0 mg/kg:
One male animal (no. 12) showed slight sedation (days 17-18) and slight ruffled fur (days 17-29) and one female animal (no. 43) showed slight ruffled fur (days 17-29) during the treatment period. As shown above the animals had recovered from clinical signs at day 2 of the recovery period.
Group 3 - 200 mg/kg:
Four males (nos. 16, 18, 19 and 20) and two females (nos. 47 and 49) showed slight sedation, dyspnea, ruffled fur and alopecia at different days during weeks 2 to 4 and at day 29 (week 5, first day of recovery). The symptoms described were not all observed in the same animals as well as not all during the same observation period.
Group 4 - 1000 mg/kg:
Nine males (nos. 21-29) and ten females (nos. 51-60) showed slight respectively slight to moderate sedation, dyspnea, rales, ruffled fur, hunched posture, loss of weight and stiff gait. These symptoms were observed at different days during weeks 2 to 4. The intensity and duration varied among the animals. The animals which were kept for an additional recovery period of 14 days were not 1n all cases free of symptoms at termination of recovery. Nevertheless, most symptoms had recovered between days 2 to 10 of the treatment-free period.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related findings were noted at termination of treatment and/or recovery.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The absolute kidney weights of the male rats of group 3 as well as kidney to body weight ratios of the male rats of group 4 were increased at termination of treatment period.
In addition, the liver weight ratios of the female rats of group 4 were reduced at termination of the recovery period.
These statistical differences were considered to be incidental and of no toxicological or biological relevance.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Effect levels

Key result

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: Based on study findings

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The "no observed adverse effect level" (NOAEL) and "no-toxic-effect-level" (NOEL) of FAT 20306/B is 1000 and 50 mg/kg bw respectively for male and females rats when administered by oral gavage.

Executive summary:

In a GLP-compliant subacute 28-day toxicity study, FAT 20306/B was administered daily by gavage to Wistar rats according OECD guideline 407. The study was comprised of four groups (0, 50, 200 and 1000 mg/kg body weight) with a 14-day recovery period. Starting with day 17 of treatment one male and one female rat of the low-dose group showed slight sedation and ruffled fur. In addition, four males and two females of the mid-dose as well as nine males and ten females of the high-dose group showed dyspnea, ruffled fur, alopecia, sedation, rales, hunched posture, stiff gait and loss of weight. These symptoms were classified as slight to moderate. They were different in duration and intensity among the animals. A clear dose-relation was observed. However, 2 days following exposure these symptoms disappeared (reversible).

No treatment-related effects were noted on all the parameters observed during the study such as mortality, food consumption, body weight, clinical signs, ophthalmoscopic examination, haematology, clinical chemistry, urine analysis, organ weight, gross and histo pathology.

Based upon the results the "no observed adverse effect level" (NOAEL) and  "no-toxic-effect-level"  (NOEL) of FAT 20306/B is 1000 and 50 mg/kg bw respectively for male and females rats when administered by oral gavage.