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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 20-Oct-2010 Experimental Completion Date: 29-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:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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
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 / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Test Animals:
Animals: Young Adult New Zealand White Rabbit, SPF
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories U.K. Ltd., Hillcrest, Dodgeford Lane, Belton, Loughborough, Leics, LE12 9TE / UK
Number of Animals per Test: 3 males
Age (when treated): 16 weeks
Body Weight Range (when treated): 3041 - 3243 g
Identification: By unique cage number and corresponding ear number.
Randomization: Selected by hand at time of delivery. No computer generated randomization program.
Acclimatization: Under laboratory conditions after health examination. Only animals without any visual 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 17-23 °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: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls.
Diet: Pelleted standard Teklad Global High Fiber Rabbit Diet 2031C (batch no. 25/10, Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad
libitum. Results of analyses for contaminants are archived at Harlan Laboratories Ltd. A piece of wood (batch no. 102240, imported by Indulab AG, Gams / Switzerland from ABEDD® - LAB & VET GmbH, 1160 Vienna / Austria) and a haystick 4642 (batch no. 80/09, Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) were also provided for environmental enrichment.
Water: Community tap water from Füllinsdorf ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at Harlan Laboratories Ltd.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

The test item was applied as a weight of 0.1 g/animal, the dose specified in the test guidelines for
solid test items.

Duration of treatment / exposure:

single exposure

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 males

Details on study design:

Purpose:
The purpose of this primary eye irritation study was to assess the irritation potential of a single dose of Acid Yellow RN 2903 when placed in the conjunctival sac of rabbit eyes. This study should provide a rational basis for risk assessment in man as ocular contact is one of the possible routes of human exposure.

Preparation of Dose Formulations:
The test item was used as delivered by the Sponsor. The pH of the test item was specified by the Sponsor as 7.5 to 8.5 at 10 g/L. According to Commission Regulation (EC) No 440/2008, B.5. and OECD Guideline 405, a test item does not need to be tested if the pH-value is less than 2 or greater than 11.5, owing to its predictable corrosive properties.

Test Item Administration:
The test item was applied as a weight of 0.1 g/animal, the dose specified in the test guidelines for solid test items. The eyes of the animals were examined one day prior to test item administration. On the day of treatment, the test item was applied with an eye glass to the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of test item. The right eye remained untreated and served as the reference control. A single animal was treated first. As neither a corrosive effect nor a severe irritant effect was observed after the 1- and 24-hour examinations, the test was completed using the two remaining animals.
Rationale: The application form and dose were used to detect an irritating potential of the test item applied.

Observations
Viability / Mortality: Daily from acclimatization of the animals to termination of the test.
Clinical Signs (systemic): Daily from acclimatization of the animals to termination of the test.
Body Weights: At start of acclimatization, on the day of application and at termination of observation.

Pathology:
Necropsy: No necropsy was performed on the animals sacrificed at termination of observation. The animals were sacrificed by intravenous injection of 1.0 mL/kg body weight of a solution of 162 mg/mL sodium pentobarbitone into the ear vein and discarded.

Statistical Analysis:
No statistical analysis was performed.

Data Compilation:
Viability/mortality, clinical signs and eye reactions were recorded on data sheets and transcribed for compilation and analysis.
Body weights were recorded on-line.
The RCC Tox Computer System (RCC-Tox-Lims) had been validated with respect to data collection, storage and retrievability.

Determination of Eye Irritating Potential:

Observation and Scoring: (see Appendix 1 and 2 in attached section for scoring system)

The eye reactions were assessed according to the numerical scoring system listed in the Commission Regulation (EC) No 440/2008, B.5, at approximately 1, 24, 48 and 72 hours after administration. Scleral reddening and ocular discharge were also assessed. Eye examinations were made with a Varta Cliptrix diagnostic-lamp (Roth AG, 4153 Reinach / Switzerland).
Data was summarized in tabular form, showing for each animal the irritation scores for the designated observation time, a description of the degree and nature of irritation, the presence of serious lesions and non-ocular effects. The scores of each animal at the following reading times (24, 48, 72 hours) were used in calculating the respective mean values (with the exception of the sclerae) for each type of lesion.
The test item was classified according to the referred classification criteria.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Results 2

Irritation parameter:

iris score

Basis:

mean

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Results 3

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: 42 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.33

Reversibility:

fully reversible within: 48 hours

Results 4

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: 43 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.67

Reversibility:

fully reversible within: 72 hours

Results 5

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: 44 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.33

Reversibility:

fully reversible within: 72 hours

Results 6

Irritation parameter:

chemosis score

Basis:

animal: 42 and 44 Males

Time point:

other: Mean 24, 48 and 72

Score:

0

Results 7

Irritation parameter:

chemosis score

Basis:

animal: 43 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.33

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

The mean score was calculated separately for each animal across three scoring times (24, 48 and 72 hours after instillation) for corneal opacity, iris light reflex, redness and chemosis of the conjunctivae, respectively. The individual mean scores for corneal opacity and iris light reflex were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.33, 0.67 and 0.33 for reddening and 0.00, 0.33 and 0.00 for chemosis.

Moderate reddening of the conjunctivae was noted in all animals at the 1-hour observation and persisted as slight reddening until the 24-hour reading in two animals (Nos. 42 and 44) and up to the 48-hour reading in one animal (No. 43). Slight to obvious swelling (chemosis) of the conjunctivae and slight to moderate reddening of the sclerae were observed in all animals at the 1-hour reading. The conjunctival swelling and the reddening of the sclerae persisted as slight in one animal (No. 43) up to the 24-hour reading. Slight to moderate ocular discharge was present in all animals at the 1-hour observations. No abnormal findings were observed in the treated eyes of any animals 48 hours (Nos. 42 and 44) or 72 hours (No. 43) after treatment, the end of the observation period for all animals.

Coloration:
Slight yellow staining of the treated eye produced by the test item was noted in all animals at the 1-hour reading.

Test Item Remnants:
Yellow test item remnants were observed in the treated eyes of all animals at the 1-hour reading.

Corrosion:
No corrosion of the cornea was observed at any of the reading times.

Other effects:

Viability / Mortality:
No intercurrent deaths occurred during the course of the study.

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

Body Weights:
One animal (No. 43) slightly lost body weight during acclimatization. This loss was therefore not induced by the treatment and the animal regained weight during the treatment. Otherwise, the body weight of the animals was within the range commonly recorded for this strain and age.

Applicant's summary and conclusion

Conclusions:

Based upon the referred classification criteria (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008), Acid Yellow RN 2903 does not have to be classified with respect to eye irritation in rabbits.

Executive summary:

The primary eye irritation potential of Acid Yellow RN 2903 was investigated according to OECD test guideline no. 405.

The test item was applied by instillation of 0.1 g into the left eye of each of three young adult New Zealand White rabbits.

Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test item instillation.

The mean score was calculated separately for each animal across three scoring times (24, 48 and 72 hours after instillation) for corneal opacity, iris light reflex, redness and chemosis of the conjunctivae, respectively. The individual mean scores for corneal opacity and iris light reflex were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.33, 0.67 and 0.33 for reddening and 0.00, 0.33 and 0.00 for chemosis.

The instillation of Acid Yellow RN 2903 into the eye resulted in mild to moderate, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, ocular discharge and chemosis. These effects were reversible and were no longer evident 72 hours after treatment, the end of the observation period for all animals. No abnormal findings were observed in the cornea or for the iris light reflex of any animals at any of the examinations. No corrosion was observed at any of the measuring intervals. Slight yellow staining produced by the test item was noted in the treated eyes of all animals at the 1-hour reading. No clinical signs were observed.

Thus, the test item did not induce significant or irreversible damage to the rabbit eye.

Based upon the referred classification criteria (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008), Acid Yellow RN 2903 does not have to be classified with respect to eye irritation in rabbits.