Disodium {sulfonylbis[4,1-phenylenediazene-2,1-diyl(1-ethyl-6-hydroxy-4-methyl-2-oxo-1,2-dihydropyridine-5,3-diyl)]}dimethanesulfonate

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 14-Sep-2010 Experimental Completion Date: 05-Oct-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Acid Yellow RN 2903

IUPAC Name:

Acid Yellow RN 2903

Details on test material:

Identification: Acid Yellow RN 2903
Description: Yellow to orange powder
Batch Number: Vers. Kilo 6, 26.05.2009
Purity: 65.7 %
Correction Factor for the Purity: No
Stability of Test Item: Stable under storage conditions
Stability of Test Item Dilution: Stable in water for 4 days
Expiry Date: 01-Aug-2015
Storage Conditions: At room temperature (range of 20 ± 5 °C, provided by Harlan Laboratories Ltd.), light protected.
Safety Precautions: Routine hygienic procedures were used to ensure the health and safety of the personnel.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animals:
Animals: Rat, RccHan: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM / The Netherlands
Number of Animals per Group: 5 males and 5 females
Total Number of Animals: 5 males and 5 females
Age (when treated): Males: 9 weeks, Females: 11 weeks
Body Weight Range (when treated): 214.0 g – 244.8 g (males), 186.8 g – 207.0 g (females)
Identification: Unique cage number and corresponding color-coded spots on the tail. The animals were marked on the last day of acclimatization.
Randomization: Selected by hand at time of delivery. No computer generated randomization program.
Acclimatization: One week under laboratory conditions, after health examination. Only animals without any visible signs of illness were used for the study.

Environmental Conditions:
Conditions: Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with a room temperature of 22 ± 3 °C and a relative humidity between 30-70%, automatically controlled light cycle of 12 hours light and 12 hours dark and music played during the daytime light period.
Accommodation: During acclimatization in groups of five per sex in Makrolon type-4 cages with standard softwood bedding. Individually in Makrolon type-3 cages with standard softwood bedding (‘Lignocel’ J. Rettenmaier&Söhne GmbH&CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) during treatment and observation. Paper enrichment, Reference no. 207057, batch no. 67, (Enviro-dri from Lillico, Biotechnology, Surrey / UK) was included.
Diet: Pelleted standard Teklad Rat-Mouse Diet 2914C, irradiated, batch no. 30/10 (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) ad libitum. Results of analyses for contaminants are archived at Harlan Laboratories Ltd.
Water: Community tap water from Füllinsdorf ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at Harlan Laboratories Ltd.

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

water

Details on dermal exposure:

One day before treatment, the backs of the animals were clipped with an electric clipper, exposing an area of approximately 10% of the total body surface. Only those animals without injury or irritation on the skin were used in the test. On test day 1, the test item was applied evenly on the intact skin with a syringe and covered with a surgical gauze pad (ca. 5 x 5 cm) held in contact with the skin by means of an adhesive hypoallergenic aerated semi-occlusive dressing and an elastic adhesive restrainer bandage wrapped around the abdomen.

The application dose was 2000 mg/kg body weight. The application volume was 6 mL/kg.

Twenty-four hours after the application the dressing was removed and the skin was flushed with lukewarm tap water and drapped off with disposable paper towels. Thereafter, the reaction sites were assessed. All animals were re-shaved on test day 11 to facilitate the reading of the local reactions.
Rationale: Dermal administration was used as this is one possible route of human exposure during manufacture, handling and use of the test item.

Duration of exposure:

24 hours

Doses:

2000 mg /kg body weight

No. of animals per sex per dose:

5

Control animals:

not required

Details on study design:

Purpose:
The purpose of this study was to assess the acute dermal toxicity of Acid Yellow RN 2903 when administered to rats by single semi-occlusive dermal application, followed by an observation period of 14 days. This study should provide a rational basis for risk assessment.

Vehicle:
The vehicle was chosen after a non-GLP solubility trial which was performed before the study initiation date. Therefore, solubility testing will be excluded from the statement of compliance. The test item was prepared at 33% (w/w) in purified water. A concentration of 33 % was soluble after treatment with an Ultra Turrax. Therefore, this concentration was considered dermally applicable.
The following information was provided by Harlan Laboratories Ltd.:
Purified water was prepared at Harlan Laboratories Ltd. (deionised water which was processed and treated by the PURELAB Option-R unit, linking four purification technologies: reverse osmosis, adsorption, ion-exchange and photo oxidation).

Preparation of Dose Formulations:
Dose levels are in terms of the test item as supplied by the Sponsor. The test item was weighed into a tared glass beaker on a suitable precision balance and the vehicle added (weight:volume). The formulation was prepared shortly before the application using a magnetic stirrer, a spatula and an
Ultra-Turrax as homogenizers. Homogeneity of the test item in the vehicle was maintained during administration using a magnetic stirrer.

Observations:
Viability / Mortality: Daily during the acclimatization period. Once before treatment, within the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2-15.
Clinical Signs: Daily during the acclimatization period. Once before treatment, within the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1. Once daily during days 2-15. All abnormalities will be recorded.
Local Dermal Signs: Once daily during days 2 (following dressing removal) through day 15 using the numerical scoring system described in Appendix I (see section any other information on materials and methods).

Body Weights: On test days 1 (prior to administration), 8 and 15.

Pathology:
Necropsy: All animals were sacrificed at the end of the observation period by carbon dioxide asphyxiation and discarded after macroscopic examinations were performed. An external examination and opening of the abdominal and thoracic cavities for examinations of major organs was performed.
The appearance of any macroscopic abnormalities was recorded. No organs or tissues were retained.

Determination and Scoring of Clinical Signs and Local Dermal Signs:
Data was summarized in tabular form, showing for each animal the clinical and local signs at each measurement interval. All findings were described.
The skin reaction was assessed according to the numerical scoring system listed in the Commission Regulation (EC) No 440/2008 B.4.

Data Compilation
Viability/mortality was recorded on data sheets.
Body weights were recorded on-line with the ToxControl Computer System.
Clinical signs, local dermal signs, mortality data and macroscopic findings were compiled into the ToxControl Computer System during recording.
The ToxControl Computer System had been licenced for Harlan Laboratories Ltd. and validated with respect to data collection, storage and retrievability.

Statistics:

No statistical analysis was performed.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No intercurrent deaths occurred during the course of the study.

Clinical signs:

other: No clinical signs were recorded throughout the entire observation period.

Gross pathology:

No macroscopic findings were recorded at necropsy.

Other findings:

Local Dermal Signs:
Very slight to well-defined erythema were observed in all animals from test day 2 (after removing the dressing) to day 4. Slight crusts of a maculated shape were present in two males and one female from test day 3 to 12 (1 male, 1 female) or 15 (1 male). Slight yellow staining of the skin was caused by the test item in all animals and detected from test day 2 to test day 5 or 6.

Any other information on results incl. tables

Due to the nature and quantity of the tables, all tables have been attached in the attached section.

Applicant's summary and conclusion

Conclusions:

The median lethal dose of Acid Yellow RN 2903 after single dermal administration to rats of both sexes, observed over a period of 14 days, is:
LD50 (rat): greater than 2000 mg/kg body weight

Executive summary:

Five male and five female RccHan:WIST (SPF) rats were treated with Acid Yellow RN 2903 at 2000 mg/kg by dermal application. The test item was formulated in purified water at a concentration of 0.333 g/mL and administered at a volume dosage of 6 mL/kg. The application period was 24 hours. The animals were examined daily during the acclimatization period and mortality, viability and clinical signs were recorded. All animals were examined for clinical signs before treatment, within the first 30 minutes and at approximately 1, 2, 3 and 5 hours after treatment on day 1 and once daily during test days 2-15. Local signs were noted once daily from test day 2 to 15. Mortality/viability was recorded before treatment, within the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2-15. Body weights were recorded on day 1 (prior to administration) and on days 8 and 15. All animals were necropsied and examined macroscopically. No intercurrent deaths occurred during the course of the study. No clinical signs were recorded throughout the entire observation period. Local dermal signs were observed among all animals, including very slight to well-defined erythema, slight crusts of a maculated shape and slight yellow staining produced by the test item. These dermal signs were mostly reversible. Only one out of ten animals still showed skin reactions after 15 days, the end of the observation period. One female animal lost body weight (-2.2%) during the first week after treatment. However, the animal regained weight until the end of the observation period. Otherwise, the body weight of the animals was within the range commonly recorded for animals of this strain and age. No macroscopic findings were observed at necropsy. The median lethal dose of Acid Yellow RN 2903 after single dermal administration to rats of both sexes, observed over a period of 14 days, is: LD50 (rat): greater than 2000 mg/kg body weight