Tetrasodium {5-[(4-chloro-6-{[4-(alkenylsulfonyl)phenyl]amino}-heteromonocycl-2-yl)amino]-4-(hydroxy-κO)-3-{[2-(hydroxy-κO)phenyl]diazenyl-κN}naphthalene-disulfonato(4-)}[3-(hydroxy-κO)-4-{[2-(hydroxy-κO)naphthalen-1-yl]diazenyl-κN}naphthalene-sulfonato(3-)]chromate(4-)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study was conducted between the 09 June 2014 and 10th July 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:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: FAT 20341/A TE
Batch: BOP 02-12 (Navy PLK 241, BS)
Purity: 80.1% w/w
Physical state / Appearance: black powder, solid at 20 °C
Expiry date: 21 November 2017
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:

Three 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 2.46 to 3.27 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: 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 material was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball.

- Concentration (if solution):
Undiluted and used as supplied

VEHICLE
Not applicable - test material was used as supplied

Duration of treatment / exposure:

72 hours

Observation period (in vivo):

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

Number of animals or in vitro replicates:

3 animals were tested in total. (After consideration of the ocular responses produced in the first treated animal, two additional animals were treated).

Details on study design:

PROCEDURE:
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 86 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.

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, two additional animals were similarly treated.

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. For more details the draize scale can be found in the any other information on results section.

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: 74365 Female

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: 74431 female

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks:

No effects observed

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal: 74467 female

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Results 4

Irritation parameter:

iris score

Basis:

animal: 74365 female

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 24 hours

Results 5

Irritation parameter:

iris score

Basis:

animal: 74431 female

Time point:

other: Mean 24, 48 and 72 hours

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Results 6

Irritation parameter:

iris score

Basis:

animal: 74467 female

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 24 hours

Results 7

Irritation parameter:

other: redness

Basis:

animal: 74365 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Results 8

Irritation parameter:

other: redness

Basis:

animal: 74431 female

Time point:

other: Mean 24, 48 and 72 hours

Score:

1.66

Max. score:

3

Reversibility:

fully reversible within: 7 days

Results 9

Irritation parameter:

other: redness

Basis:

animal: 74467 female

Time point:

other: Mean 24, 48 and 72 hours

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Results 10

Irritation parameter:

chemosis score

Basis:

animal: 74365 female

Time point:

other: Mean 24, 48 and 72

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Results 11

Irritation parameter:

chemosis score

Basis:

animal: 74431 female

Time point:

other: Mean 24, 48 and 72 hours

Score:

1.66

Max. score:

4

Reversibility:

fully reversible within: 7 days

Results 12

Irritation parameter:

chemosis score

Basis:

animal: 74467 female

Time point:

other: 24, 48 and 72 hours

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritant / corrosive response data:

Ocular Reactions:
Individual and group mean scores for ocular irritation are given in Table 1 and Table 2*.

Black staining of the cornea, iris and conjunctival membranes was noted in all treated eyes 1 hour after treatment and prevented accurate evaluation of corneal, iridial and conjunctival redness effects.

Black staining of the fur was noted around the treated eye of two animals at all observations.

No corneal effects were noted at the 24-Hour observation and at subsequent observations.

Iridial inflammation was noted in one treated eye at the 24, 48 and 72 Hour observations.

Moderate conjunctival irritation was noted in all treated eyes at the 1 and 24 Hour observations. Moderate conjunctival irritation was noted in one treated eye with minimal conjunctival irritation noted in two treated eyes at the 48 Hour observation. Minimal conjunctival irritation was noted in one treated eye at the 72 Hour observation.

Two treated eyes appeared normal at the 72 Hour observation and one treated eye appeared normal at the 7 Day observation.

* All tables can be found in the any other information on results section.

Other effects:

All animals showed expected gain in body weight during the study.

Any other information on results incl. tables

TABLES:

Table 1     Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	74365Female				74431Female					74467Female
	IPR= 0				IPR = 0					IPR = 0
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	?s	0	0	0	?s	0	0	0	0	?s	0	0	0
F = Area of Cornea Involved	?s	0	0	0	?s	0	0	0	0	?s	0	0	0
Score (E x F) x 5	0-80	0	0	0	0-80	0	0	0	0	0-80	0	0	0
IRIS
D	?s	0	0	0	?s	1	1	1	0	?s	0	0	0
Score (D x 5)	0-10	0	0	0	0-10	5	5	5	0	0-10	0	0	0
CONJUNCTIVAE
A = Redness	?s	1	1	0	?s	2	2	1	0	?s	2	1	0
B = Chemosis	2	1	1	0	2	2	2	1	0	2	2	1	0
C = Discharge	2Sf	2Sf	1Sf	0Sf	2Sf	1Sf	1Sf	1Sf	0Sf	2	1	1	0
Score (A + B + C) x 2	8-14	8	6	0	8-14	10	10	6	0	8-14	10	6	0
Total Score	8-104	8	6	0	8-104	15	15	11	0	8-104	10	6	0

IPR=Initial pain reaction

Sf =        Black staining of fur around treated eye

?s =        Degree of ocular staining prevented accurate evaluation of reactions

- =         Observation not required - considered to be zero for calculation of Group Mean Score

 

Table 2     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		7 Days
74365Female		8-104		8		6		0		-
74431Female		8-104		15		15		11		0
74467Female		8-104		10		6		0		-
Group Total		24-312		33		27		11		0
Group Mean Score		8-104		11.0		9.0		3.7		0.0

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item produced a group mean score in the range of 8.0 to 104.0 and was considered to be at least 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 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 three rabbits produced iridial inflammation and moderate conjunctival irritation. Thedegree of ocular staining prevented accurate the evaluation of the cornea, iris and conjunctival redness 1 hour after treatment. Two treated eyes appeared normal at the 72‑Hour observation and one treated eye appeared normal at the 7‑Day observation.

 

Conclusion:

The test item produced a group mean score in the range of 8.0 to 104.0 and was considered to be at least 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 Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.