Reaction mass of Potassium sodium 3-{[4-({3-(substitutedamino)-4-[(2-sulfonato-4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]phenyl}amino)-6-chloro-heteromonocyl-2-yl]amino}-2-substituted-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate (1:3:1) and Potassium sodium 3-[(4-{[3-(subsitutedamino)-4-{[4-(ethenylsulfonyl)-2-sulfonatophenyl]diazenyl}phenyl]amino}-6-chloro-heteromonocy-2-yl)amino]-2-substituted-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate (3:9:3)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study conducted between 9th September 2014 and 25th September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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

Test material form:

other: red solid

Details on test material:

Identification: FAT 40863/A TE
Sponsor description: yellowish to brown solid
Harlan description: red solid
Batch: BOP 02-13 (BS-ROE1670)
Purity: 82.1% (w/w)
Expiry Date: 05 June 2018
Storage Conditions: room temperature in the dark

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.96 or 3.23 kg and were twelve to twenty weeks old. After an acclimatization period of at least five days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Justification
The rabbit is the preferred species of choice as historically used for irritation studies and is specified in the appropriate test guidelines. The number of animals used was the minimum required to achieve the objectives of the study. Testing was conducted in two animals and the response in those animals was such that exposure of a third animal would not affect classification of the test item, therefore, no further testing was needed.

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

For the purpose of the study the test item was used as supplied.

Controls:

other: EXAMPLE: The left eye remained untreated and was used for control purposes.

Amount / concentration applied:

TEST MATERIAL

- Amount(s) applied (volume or weight with unit):
A volume of 0.1 mL of the test item, which was found to weigh approximately 96 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes

VEHICLE
Not applicable, for the purpose of the study the test item was used as supplied.

Duration of treatment / exposure:

72 hours

Observation period (in vivo):

Approximately 1 hour, 24, 48 and 72 hours and 7 days following treatment.

Number of animals or in vitro replicates:

2 males

Details on study design:

PRE STUDY:
Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

TESTING PROCEDURE:
Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre dose anesthesia of ocular anesthetic (two drops of 0.5% tetracaine hydrochloride) was applied to each eye.

Eight hours after test item application, a subcutaneous injection of post dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.

After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.

Observations were made, 1, 24, 48 and 72 hours. Any clinical signs of toxicity, if present, were also recorded and bodyweights were recorded too. An additional observation was made in one treated eye on Day 7 to assess the reversibility of the ocular effects.

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
Not applicable
- Time after start of exposure:
Not applicable

SCORING SYSTEM:
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the draize scale which can be found from (Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.48 to 49). The Draize scoring system has been included in the any other information on methods section (including tables).

TOOL USED TO ASSESS SCORE:
Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal: 74708 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal: 74726 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Results 3

Irritation parameter:

iris score

Basis:

animal: 74708 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: no effects observed

Results 4

Irritation parameter:

iris score

Basis:

animal: 74726 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: no effects observed

Results 5

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: 74708 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

1.66

Max. score:

3

Reversibility:

fully reversible within: 7 days

Results 6

Irritation parameter:

other: redness

Basis:

animal: 74726 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Results 7

Irritation parameter:

chemosis score

Basis:

animal: 74708 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 7 days

Results 8

Irritation parameter:

chemosis score

Basis:

animal: 74726 Male

Time point:

other: mean 24, 48 and 72 hours

Score:

0.661

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritant / corrosive response data:

Individual and group mean scores for ocular irritation are given in Table 1*

Orange colored staining of the fur around the treated eye was noted in both animals at all observations.

No corneal or iridial effects were noted during the study.

Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment. Moderate conjunctival irritation was noted in one treated eye with minimal conjunctival irritation noted in the other treated eye at the 24 and 48 Hour observations. Minimal conjunctival irritation was noted in one treated eye at the 72 Hour observation.

One treated eye appeared normal at the 72 Hour observation and the other treated eye appeared normal at the 7 Day observation.

* Table 1 can be found in the any other information on results (including tables section of the robust study summary).

Other effects:

Body weights:
Both animals showed expected gain in body weight during the study.

Any other information on results incl. tables

Table 1     Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	74708Male					74726Male
	IPR= 0					IPR = 0
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0	0
IRIS
D	0	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	2	2	2	1	0	2	1	1	0
B = Chemosis	2	2	1	1	0	1	1	1	0
C = Discharge	2Sf	2Sf	1Sf	1Sf	0Sf	1Sf	1Sf	0Sf	0Sf
Score (A + B + C) x 2	12	12	8	6	0	8	6	4	0
Total Score	12	12	8	6	0	8	6	4	0

IPR=Initial pain reaction

Sf =        Orange colored staining of the fur around the treated eye

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

Introduction:

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.

 

Results:

A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. One treated eye appeared normal at the 72‑Hour observation and the other treated eye appeared normal at the 7‑Day observation.

 

Conclusion: 

The test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.