Registration Dossier

Diss Factsheets

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

Constituent 1
Chemical structure
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

Resultsopen allclose all
Irritation parameter:
other: %viability
Value:
149.334
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
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.