Trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January 2017 (histopathology 5 June 2017 to 16 June 2017)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Principles of method if other than guideline:

High opacity scores were obtained in the test and the corneas were stained dark purple by the substance, and thus, the opacity scores may have been due to the staining. Therefore conform the guidance additional histopathology was performed

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate

EC Number:

228-327-6

EC Name:

Trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate

Cas Number:

6227-20-9

Molecular formula:

C34H27N5O11S3.3Na

IUPAC Name:

trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate

Test material form:

solid: flakes

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir
- Number of animals: not indicated
- Storage, temperature and transport conditions of ocular tissue: placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL), transported over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
- Time interval prior to initiating testing: not indicated
- indication of any existing defects or lesions in ocular tissue samples: checked and only undamaged tissue used

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL: 20% w/v solution in sodium chloride 0.9% w/v.

Duration of treatment / exposure:

240 minutes at 32 ± 1 ºC

Duration of post- treatment incubation (in vitro):

post treatment opacity measurement immediately
permeality measurement after exposure to fluorescein for 90 min at 32 ± 1 ºC

Details on study design:

QUALITY CHECK OF THE ISOLATED CORNEAS:based on visual examination and pre-treatment opacity check

NUMBER OF REPLICATES: 2/treatment

TREATMENT METHOD:closed chamber

REMOVAL OF TEST SUBSTANCE: 3 rinses with EMEM containing phenol red

MEASURED ENDPOINTS: Corneal opacity and Corneal permeability

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS Classification
≤ 3 No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55 No prediction of eye irritation can be made
> 55 Category 1. UN GHS or EU CLP Causes serious eye damage

Histologically process and microscopically evaluation of the isolated bovine eye corneas was performed after in vitro testing.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Value:

138.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

It can not be excluded that staining of the cornea could have influenced the opacity readings

Results 2

Irritation parameter:

histopathological observations

Value:

>= 1 - <= 2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

indication of irritation is not sufficient to allow the conclusion severely damaging to the eyes

Other effects / acceptance of results:

The positive control was slightly out of range. This was evaluated and considered not to have affected the study validity.

The purple staining of cornea by the test substance may have influenced the opacity reading. This was checked by histologically processing and microscopically evaluation of the isolated bovine eye corneas after in vitro testing (see below)

The histopathology findings were only low grade and restricted to the upper layers of the epithelium, so were not typical of the histopathological findings that would be expected following exposure of the cornea to substances having the potential to cause serious eye damage associated with dense corneal opacity (i.e. not consistent with the corneal effects commonly induced by Category 1 substances). So, it is very likely that the high scores for opacity in the BCOP test were due primarily to staining of the cornea by the test item.

Any other information on results incl. tables

	Opacity		Permeability
Cornea	Post-Treatment- Pre-Treatment	Corrected Value	OD	Corrected Value	In Vitro Irritancy Score
Negative control	0		0.029
	1		0.022
	1		0.054
Mean	0.7*		0.035		1.2
Positive Control	83	82.3	1.239	1.204
	84	83.3	1.609	1.574
	82	81.3	1.482	1.447
Mean		82.3		1.408	103.5
Test Item	182	181.3	0.230	0.195
	135	134.3	0.043	0.008
	94	93.3	0.243	0.208
Mean		136.3		0.137	138.4

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the results of the test the substance is considered not severely damaging to the eyes

Executive summary:

In a BCOP assay bovine corneas were exposed to the substance and assessed for opacity and permeability. Opacity was strongly increased, while permeability was slightly increased compared to the negative controls. The calculation of the In Vitro Irritancy Score showed that the substance is severely damaging to the eyes. As it could not be excluded that staining of the cornea could have influenced the opacity readings, semi-quantitative histopathological evaluation of the corneas from eyes exposed to the substance was performed. This investigation gave scores below the threshold for classification of this material as Eye Category 1. Therefore the conclusion of the study is that the substance is not considered severely damaging to the eyes.