2,7-Naphthalenedisulfonic acid, 5-(acetylamino)-3-[2-[5-[[4-chloro-6-[[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]diazenyl]-4-hydroxy-, sodium salt (1:4)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 16, 2018 to April 30, 2019

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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,​7-​Naphthalenedisulfoni​c acid, 5-​(acetylamino)​-​3-​[2-​[5-​[[4-​chloro-​6-​[[3-​[[2-​(sulfooxy)​ethyl]​sulfonyl]​phenyl]​amino]​-​1,​3,​5-​triazin-​2-​yl]​amino]​-​2-​sulfophenyl]​diazenyl]​-​4-​hydroxy-​, sodium salt (1:4)

EC Number:

695-779-2

Cas Number:

80019-35-8

Molecular formula:

C29H21ClN8O17S5.4Na

IUPAC Name:

2,​7-​Naphthalenedisulfoni​c acid, 5-​(acetylamino)​-​3-​[2-​[5-​[[4-​chloro-​6-​[[3-​[[2-​(sulfooxy)​ethyl]​sulfonyl]​phenyl]​amino]​-​1,​3,​5-​triazin-​2-​yl]​amino]​-​2-​sulfophenyl]​diazenyl]​-​4-​hydroxy-​, sodium salt (1:4)

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

other: stratified human keratino cytes

Details on test animals or tissues and environmental conditions:

Description of the test system
The EpiOcular TM tissue model (OCL-200-MatTek Corporation) is composed of stratified human keratinocytes in a three-dimensional structure, reflecting the morphology and function of the human corneal epithelium found in vivo.

Justification for selection of the test system
The EpiOcular TM Eye Irritation Test (EIT), using the MatTek EpiOcular TM tissue model OCL-200, was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular TM EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose, as an alternative to Dralze Rabbit Eye Test with excellent correlation - in vivo to in vitro test results.

Characterisation of the test system
MatTek’s EpiOcular TM system consists of normal, human-derived keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. Cultured on specially prepared cell culture inserts using serum-free culture medium, the cells differentiate to form a multi-layered structure with progressively stratified, but not cornified cells which closely parallel the corneal epithelium. QC results for the specific lot of models received were checked in-house for MatTek acceptance range with the following outcome:
Morphology – PASS
Tissue viability – PASS
Skin barrier function (ET50 value for 0.3% Triton X-100) where ET50 is the time taken for 0.3% Triton X-100 to reduce the viability of the skin model to 50% relative to the negative control) – PASS
Sterility testing showed no contamination during long term antibiotic and antimycotic free culture - PASS

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Duration of treatment / exposure:

After pre-wetting tissues with 20uL PBS (sterile Dulbecco's Phosphate Buffered Saline) for 30±2 minutes, a single, topical application of approximately 50mg of neat test item using a spatula, or 50uL of reference items was applied to the surface of the Epiocular TM model for 6 hours ± 15 minutes, followed by a 25±2 minutes post-treatment immersion, and 18 hour ± 15 minutes post-treatment incubation, prior to the MTT endpoint; three tissues per condition (n=3).

Details on study design:

Preliminary test:
CR SR94 was first checked for its potential for MTT interference and solvent interference (water and isopropanol).

Main test overview:
Day 0: On the day of receipt, EpiOcular TM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% RH.
Day 1: Exposure to, and removal of approximately 50 mg of CR SR94 (addition of test item was by spatula) or 50uL of reference item for 6 hours ±15 minutes, followed by a 25±2 minutes post-treatment immersion, and 18 hours ±15 minutes post-treatment incubation.
Day 2: End of MTT viability test, optical density readings at 570 nm without reference filter.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: %viability

Value:

149.334

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

other: Corrected %vialbility

Value:

149.331

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Prior to the study, the required preliminary test confirmed that the test item interfered with the solvent and so colourant controls were performed in parallel.
CR SR94 was classified as No category i.e. not requiring a warning label in the European chemical classification systems for eye irritation or serious eye damage as the viability was above 60%.

Any other information on results incl. tables

Table 1. Viability measurements after 6 hours (±15 minutes) of application and 18 hours (±15 minutes) post-incubation of test and reference items.

Condition	Tissue #	Raw Data		Blank Corrected Data		Mean OD	%Viability
		Aliquot 1	Aliquot 2	Aliquot 1	Aliquot 2
NC	Tissue 1	2.224	2.253	2.068	2.097	2.083	100.349
	Tissue 2	0.169	0.179	0.013	0.023	0.018	1.295
	Tissue 3	2.209	2.239	2.053	2.083	2.068	99.651
PC	Tissue 1	0.876	0.832	0.720	0.676	0.698	50.234
	Tissue 2	0.623	0.624	0.467	0.468	0.468	33.645
	Tissue 3	0.75	0.78	0.594	0.624	0.609	43.829
TA1	Tissue 1	1.335	1.343	1.179	1.187	1.183	85.139
	Tissue 2	2.143	2.118	1.987	1.962	1.975	142.101
	Tissue 3	2.31	2.353	2.154	2.197	2.176	156.567

Table 2. Mean and SD of viability measurements and of viability percentages after 6 hours (±15 minutes) of application and 18 hours (±15 minutes) post-incubation.

Name	Code	Mean of OD	SD of OD	Mean if viability (%)	Corrected Mean of viability(%)	SD of viability (%)	CV%	Classification
Sterile water	NC	2.075	0.01	100	N/A	0.494	0.494	No-Category
Methyl Acetate	PC	0.592	0.116	42.569	N/A	8.366	19.652	No Prediction
CR SR94	TA1	2.075	0.142	149.334	149.331	10.229	6.85	No-Category

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

A test item is considered as " No Category", i.e. not requiring a warning label in the European chemical classification systems for eye irritation or serious eye damage (UN GHS No Category), if the eye model viability after exposure and post-treatment incubation is >60%. The percentage of viability obtained with CR SR94 was 149.331%, therefore it is classified as No Category.

Executive summary:

In this study, the eye irritation potential of CR SR94 was assessed in vitro according to OECD Test Guideline 492 (Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage).

 

After 6 hours ±15 minutes exposure on the surface of EpiOcular TM reconstructed ocular epithelium and 18 hours ±15 mins post-incubation time, the viability of the tissues was assessed and compared to a negative control.

 

The percentage viability obtained was 149.334% and therefore:

CR SR94 was classified as No Category i.e. not requiring a warning label in the European chemical classification systems for eye irritation or serious eye damage.