Reaction products of diazotised 2-amino-5-{[2-(sulfooxy)ethyl]sulfonyl}benzenesulfonic acid coupled with 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid under acidic conditions, further coupled with diazotised reaction products of 2,4,6-trifluoro-1,3,5-triazine with 2-[(2-anilinoethyl)sulfonyl]ethyl hydrogen sulfate and 2,4-diaminobenzenesulfonic acid (1:1:1) under alkaline conditions, potassium sodium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Experimental start date 26 November 2018 Experimental completion date 26 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of diazotised 2-amino-5-{[2-(sulfooxy)ethyl]sulfonyl}benzenesulfonic acid coupled with 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid under acidic conditions, further coupled with diazotised reaction products of 2,4,6-trifluoro-1,3,5-triazine with 2-[(2-anilinoethyl)sulfonyl]ethyl hydrogen sulfate and 2,4-diaminobenzenesulfonic acid (1:1:1) under alkaline conditions, potassium sodium salts

EC Number:

948-562-4

Molecular formula:

UVCB

IUPAC Name:

Reaction products of diazotised 2-amino-5-{[2-(sulfooxy)ethyl]sulfonyl}benzenesulfonic acid coupled with 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid under acidic conditions, further coupled with diazotised reaction products of 2,4,6-trifluoro-1,3,5-triazine with 2-[(2-anilinoethyl)sulfonyl]ethyl hydrogen sulfate and 2,4-diaminobenzenesulfonic acid (1:1:1) under alkaline conditions, potassium sodium salts

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Collection and Transport of Eyes to the Laboratory
Multiple chicken heads (Spring chickens (Gallus Gallus e.g. Ross 308 Broiler)), were supplied by Baileys Turkeys Ltd., Cheshire, UK. The chickens weighed approximately 3 kg and were approximately 56 days old prior to being humanely killed for human consumption.

Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline.

Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.03g of test item

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.

Number of animals or in vitro replicates:

The test item and positive control item groups consisted of three eyes and the negative control item group consisted of two eyes.

Details on study design:

Eye Preparation
Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.

Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are ≤ 0.5.

Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.

Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ±1.5 °C.

Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit lamp microscope at the center of each cornea.

Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.

After the approval process the eyes were incubated for approximately 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

Application of Test Item, Negative and Positive Control Items
The test item and positive control item groups consisted of three eyes and the negative control item group consisted of two eyes.

Immediately following the zero reference measurements, each test eye (including clamp) was removed from the superfusion apparatus and placed horizontally (cornea facing upwards) into a petri dish. 0.03 g of test item was applied to the cornea. The entire surface of the cornea was evenly covered. The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline. The treated eye (including clamp) was subsequently returned to the superfusion apparatus in the original upright position.

0.03 g of the positive control item, Imidazole, was similarly applied to the cornea of each positive control eye and 0.03 mL of the negative control item was applied to the cornea of each negative control eye.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Test item

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

Test item

Value:

0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Results 3

Irritation parameter:

other: Corneal swelling compared to time zero

Run / experiment:

Test item

Value:

5.07

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Other effects / acceptance of results:

Corneal Opacity Scores
Very faint opacity was noted in the negative control treated eyes.

‘Complete corneal opacity; iris invisible’ was noted in all positive control treated eyes.

Very faint opacity was noted in one test item treated eye.

‘Scattered or diffused areas; details of the iris are clearly visible’ of opacity were noted in two test item treated eyes.

No morphological effects were noted in the test item, positive or negative control item treated eyes.

Fluorescein Retention Scores
No fluorescein retention was noted in one negative control treated eye during the study period.

Very minor single cell staining was noted in one negative control treated eye.

‘Confluent large areas of the cornea retaining fluorescein’ was noted in all positive control treated eyes.

Very minor single cell staining was noted in two test item treated eyes.

‘Single cell staining scattered throughout the treated area of the cornea’ was noted in one test item treated eye.

Any other information on results incl. tables

  Ocular Reactions

The ocular reactions observed in treated eyes were as follows:

Test Item
Maximal mean score for corneal opacity	:	0.8	ICE Class II
Mean score of Fluorescein Retention	:	0.7	ICE Class II
Maximal mean corneal swelling compared to time zero	:	5.07%	ICE Class II
Positive Control Item
Maximal mean score for corneal opacity	:	4.0	ICE Class IV
Mean score of Fluorescein Retention	:	3.0	ICE Class IV
Maximal mean corneal swelling compared to time zero	:	26.36%	ICE Class III
Negative Control Item
Maximal mean score for corneal opacity	:	0.5	ICE Class I
Mean score of Fluorescein Retention	:	0.3	ICE Class I
Maximal mean corneal swelling compared to time zero	:	-3.42%	ICE Class I

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No prediction can be made following assessment of the data for all endpoints.

Executive summary:

Introduction

The study was performed to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye.

Method

0.03 g of thetest item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes were treated with negative control item. 

Results

Maximal ocular irritation observations recorded for the test item treated eyes were as follows:

Mean Corneal Opacity (ICE class)	Mean Fluorescein Retention (ICE class)	Mean Corneal Thickness compared to time zero % (ICE class)					Combination of the 3 Endpoints
		30 mins	75 mins	120 mins	180 mins	240 mins
0.8 (II)	0.7 (II)	1.38 (I)	-1.38 (I)	1.38 (I)	5.07 (II)	2.76 (I)
		(II)					3 x II
Classification:		No prediction can be made

 

Conclusion

No prediction can be madefollowing assessment of the data for all endpoints.

 

mins=Minutes following treatment