C.I. Acid Yellow 246

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 June 1985 to 16 October 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

not specified

GLP compliance:

yes

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 20306/B
Description: powder
Batch No.: HT 2025/50
Purity: 97 % (guaranteed by the sponsor)
Stability of test article stable; expiration: 2014
Stability of test article in vehicle stable for at least 2 hours
Instruction for test article storage at room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST SYSTEM
Species: Wistar Rat, KFM-Han., outbred, SPFquality
Rationale: Recognised by the international guidelines as the recommended test system.
Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf / Switzerland
Total number of animals: 20 males, 20 females
Age at start of treatment: males: 9 weeks, females: 11 weeks
Body weight range at start of treatment: males: 238 - 283 g, females: 208 - 252 g
Identification: By unique cage number and corresponding ear tag.
Randomization: Computer-generated random algorithm
Acclimation: One week under laboratory conditions, after veterinary examination
- Diet: standard Kliba 343, Batch 22/85 rat maintenance diet ("KLIBA"-Futter, Klingentalmuehle AG, Switzerland) ad libitum.
-Water: Community tap water from Itingen

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2
- Humidity (%): 55 ±10
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 hours of artificial fluorescent light each day. Music was played during light period.

Administration / exposure

Type of coverage:

occlusive

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

4 % dilution of CMC

Details on exposure:

Twenty-eight (28) dermal applications were administered to the shaved skin of the back. The skin area involved in the dermal application was equivalent to about 10 % of the total body surface. The hair was clipped from the dorsal surface once weekly throughout the study. The test article was applied evenly on the shaved skin and covered with an occlusive dressing. The dressing was wrapped around the abdomen and fixed with an elastic adhesive bandage, for 6 hours per day, 7 days per week for a total of 28 application days. The application volume was 4 ml/kg body weight. The animals of the control group were treated with the vehicle alone in the same way (4 ml/kg body weight). Test article remainders were washed off with luke-warm tap water after termination of the daily treatment in animals of groups 2 to 4. They were dried with disposable paper towels.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

According to sponsor the stability and homogeneity of the test article/vehicle mixture has found to be stable if the mixture is prepared daily.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours per day, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Middle dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Test Article Preparation: FAT 20306/B was weighed into a glass beaker on a tared Mettler PK 4800 balance and the vehicle added. The mixture was prepared using a homogenizer.
Frequency of Preparation: Daily prior to application.

Examinations

Observations and examinations performed and frequency:

Symptoms (systemic): Twice daily
Symptoms (local): Daily prior to the following application according to Draize score.
Viability / Mortality: Twice daily
Food Consumption: Weekly
Body Weights: Twice weekly
Ophthalmoscopic Examinations: Observation by ophthalmoscope (Heine- Bifocal Ophthalmoscope, miroflex type) at termination of the application period

Sacrifice and pathology:

ORGAN WEIGHTS: The following organ weights were recorded on the scheduled date of necropsy: adrenal glands, ovaries, kidneys, testes, liver
NECROPSY AND HIST0PATH0L0GY: All animals mere necropsied and descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by a pathologist. All animals were killed by intraperitoneal injection of sodium pentobarbital. Organs and tissues were fixed in 4 % neutral phosphate buffered formalin and embedded in Paraplast. Samples of the following tissues were collected from all animals a necropsy.

Other examinations:

CLINICAL Laboratory Investigations:
General:
Blood, samples for haematology 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 7.30 and 9.00 a.m. to reduce biologic variation caused by circadian rhythm. Blood samples were drawn from the retro-orbital plexus. Urine was collected into a specimen vial using a metabolism cage.
Blood and urine sampling: after 4 weeks. The assays of blood parameters were performed under quality control conditions to assure reliable test results.

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 distri-bution, 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) in the summary tables.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Description (incidence and severity):

Only one animal (female no. 39) of group 4, 1000 mg/kg showed slight erythema between day 4 and 8 and slight edema between day 4 and 8 of test on the treated skin.

Mortality:

no mortality observed

Description (incidence):

One female animal from group 04 died incidentally after blood sampling just before the regular autopsy. No treatment- related findings could be detected in this animal/ nor was an evident cause of death detectable.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

However, reduced body weight gain was noted in females of groups 3 and 4 during the last two weeks of treatment. These findings were related to the initial differences between the animals of the test article and control groups and not to test article treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

However, reduced food consumption values were noted in the female animals of groups 3 and 4. These findings were related to the initial differences betweeen the treated groups and the controls and not to test article treatment.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

no changes of toxicological significance after 4 weeks of treatment.

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The observed differences in female animals of groups 2, 3 and 4 were related to the significantly reduced final body weights, which were not related to test article treatment.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Effect levels

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

haematology

mortality

ophthalmological examination

Remarks on result:

other: Only local findings, described as a slight edema and erythema, were observed in one animal of this qroup.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The "no-toxic effect level" (NOEL) of FAT 20306/B for 28 day dermal toxicity study in male and female rats is considered to be 1000 mg/kg bw/day.

Executive summary:

In a GLP-compliant dermal toxicity study with FAT 20306/B was conducted in Wistar rats for 6 hours/day (7 days/meek) for a total of 28 d applications according to OECD guideline 410 and EU method B.9. The study was comprised of 4 groups (0, 40, 200, 1000 mg/kg), for a total of 40 rats. No treatment-related effects were noted on most parameters observed during the study (mortality, food consumption, body weight, clinical signs, ophthalmoscopic examination, haematology, clinical chemistry, urine analysis, organ weight etc.). Only one animal (female no. 39) of group 4, 1000 mg/kg showed slight erythema between day 4 and 8 and slight edema between day 4 and 8 of test on the treated skin. No other local or systemic symptoms were observed in the animals of the test article-treated or control group. There were no adverse effects on gross and microscopic pathology. Based on the findings observed during this study the "no-toxic effect level" (NOEL) of FAT 20306/B for 28 day dermal toxicity study in male and female rats is considered to be 1000 mg/kg bw/day.