Reaction mass of Tetrasodium 3-{[5-({4-[alkyl(3-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)amino]-6-fluoro- heteromonocycle-2-yl}amino)-2-sulfonatophenyl]diazenyl}-4-hydroxy-5-(alkanoylamino)naphthalene-2,7-disulfonate and Trisodium 3-[{5-[(4-{[3-(ethenylsulfonyl)phenyl](alkyl)amino}-6-fluoro-heteromonocycle-2-yl)amino]-2-sulfonatophenyl}diazenyl]-4-hydroxy-5-(alkanoylamino)naphthalene-2,7-disulfonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 10 November 2015 and 19 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Animal Information
Two male New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 2.93 or 3.08 kg and were 12 to 20 weeks old. After an acclimatization period of at least 5 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.

Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, 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 12 hours continuous light and 12 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.

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

Test item was used as supplied.

Controls:

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

Amount / concentration applied:

A volume of 0.1 mL of the test item, which was found to weigh approximately 65 mg (as measured by gently compacting the required volume into an adapted syringe).

Duration of treatment / exposure:

The test item was not removed.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

Two males

Details on study design:

Experimental Design and Study Conduct
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.
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.

A volume of 0.1 mL of the test item, which was found to weigh approximately 65 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. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to a six point scale.

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.

Serial Observations
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to a numerical evaluation (Draize, J.H, 1977).
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

75272 Male

Basis:

mean

Time point:

other: 24,48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Results 2

Irritation parameter:

cornea opacity score

Remarks:

75263 Male

Basis:

mean

Time point:

other: 24,48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Results 3

Irritation parameter:

iris score

Remarks:

75272 Male

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: No effects observed

Results 4

Irritation parameter:

iris score

Remarks:

75263 Male

Basis:

mean

Time point:

other: 24,48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: No effects observed

Results 5

Irritation parameter:

conjunctivae score

Remarks:

75272 Male

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0.666

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Results 6

Irritation parameter:

conjunctivae score

Remarks:

75263 Male

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Results 7

Irritation parameter:

chemosis score

Remarks:

75272 Male

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0.666

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Results 8

Irritation parameter:

chemosis score

Remarks:

75263 Male

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0.666

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritant / corrosive response data:

Ocular Reactions
There was no initial pain reaction, in either animal, following instillation of the test item.
Red 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 Hour observation. Minimal conjunctival irritation was noted in both treated eyes at the 48 Hour observation.
Both treated eyes appeared normal at the 72 Hour observation.

Other effects:

Body Weight
One animal showed no gain in body weight and the other animal showed expected gain in body weight during the study.

Any other information on results incl. tables

Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	75272Male				75263Male
	IPR= 0				IPR = 0
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0
IRIS
D	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	2	1	1	0	2	2	1	0
B = Chemosis	1	1	1	0	2	1	1	0
C = Discharge	1Sf	1Sf	1Sf	0Sf	1Sf	1Sf	0Sf	0Sf
Score (A + B + C) x 2	8	6	6	0	10	8	4	0
Total Score	8	6	6	0	10	8	4	0

IPR=Initial pain reaction

Sf = Red colored staining of fur around treated eye

Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number and Sex	Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours
75272Male	8	6	6	0
75263Male	10	8	4	0
Group Total	18	14	10	0
Group Mean Score	9.0	7.0	5.0	0.0

Individual Body Weights and Body Weight Change

Rabbit Number and Sex	Individual Body Weight (kg)		Body Weight Change (kg)
	Day 0	Day 3
75272Male	2.93	2.99	0.06
75263Male	3.08	3.08	0.00

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item produced a maximum group mean score of 9.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals.

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 male rabbits produced moderate conjunctival irritation. Both treated eyes appeared normal at the 72‑Hour observation.

Conclusion

The test item produced a maximum group mean score of 9.0 and was classified as amildirritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item does not meet the criteria for classification according to the Globally Harmonized Systemof Classification and Labelling of Chemicals.