Reaction products of 6-hydroxy-5-nitrosonaphthalene-2-sulfonic acid, chelated with iron (3+), 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:

April 20 to 21, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Version / remarks:

adopted 28 July 2015

Deviations:

yes

Remarks:

length of incubation during the Direct MTT reduction test was 1 instead of 3 hours. This did not have any impact on the outcome of the study.

Principles of method if other than guideline:

SOP: EpiOcular EIT for the prediction of acute occular irritation of chemicals, Version 9, April 16, 2015, MatTek corp.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Acid Green 001

IUPAC Name:

Acid Green 001

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: the reconstructed human cornea-like epithelial model EpiOcular™ (OCL-200 ver. 2.0) is supplied from MatTek, Bratislava, SK. The RhCE tissues are reconstructed from primary human cells, which have been cultured for several days to form a stratified, highly differentiated squamous epithelium, morphologically similar to that found in the human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo. Lot No. of tissues used for this test: 23776 kit A.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg.

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

two (test substance), three (negative and positive control)

Details on study design:

DIRECT MTT REDUCTION: the test substance was added to 1 ml of 1 mg/ml MTT solution and incubated at standard culture conditions (37 ± 1 °C, in a humidified atmosphere of 5 ± 1 % CO2) for 1 hour. At the end of the exposure time, the presence and intensity of the staining was observed. 50 μl of sterile deionized water in MTT solution was used as negative control.

COLOUR INTERFERENCE: the test substance was applied on two viable tissue replicates, which underwent the entire testing procedure but were incubated with medium instead of MTT solution during the MTT incubation step to generate a non-specific colour (NSCliving) control.

TISSUES PRETREATMENT: on the day of receipt, EpiOcularTM tissues were conditioned to release transport stress related compounds and debris by incubation in assay medium delivered by MatTek for test performance for 1 hour at standard culture conditions and, after media replacement, overnight (following 16-24 hours) also standard at culture conditions. After pre-incubations, tissues were wetted with 20 μl of DPBS (Dulbecco's Phosphate Buffered Saline (Ca2+/Mg2+-free DPBS) spread across entire tissue surface.

APPPLICATION OF TEST SUBSTANCE: after 30 minutes incubation (in the dark at standard culture conditions) the tissues were topically exposed to the test chemical (50 mg of substance/surface ratio 39.7 mg/cm2 per tissue) for 6 ± 0.25 hours at standard culture conditions. At the end of the 6 ± 0.25 hours treatment time, the test substance was removed by extensively rinsing the tissues with PBS brought to room temperature. After rinsing, tissues were immediately transferred to and immersed in 5 ml of previously warmed assay medium (room temperature) in a pre-labelled 12-well plate for a 25 ± 2 minute immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test substance absorbed into the tissue. At the end of the post-soak immersion, each insert was removed from the assay medium, the medium is decanted off the tissue, and the insert was blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 ml of (room temperatured) assay medium. The tissues were incubated for 18 ± 0.25 hours at standard culture conditions (post-treatment incubation). At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material.

TISSUE VIABILITY MEASUREMENTS: two tissues were placed into the 24-well plate containing 0.3 ml of MTT solution at 1 mg/ml and incubated for 180 ± 10 minutes at standard culture conditions. In the end of staining (incubation with MTT solution) the bottom of all inserts was blotted on absorbent material and inserts were then transferred to a pre-labelled 6-well plate containing 2 ml of isopropyl alcohol in each well so that no isopropyl alcohol was flowing into the insert. The plates were sealed with parafilm, and were extracted the same day. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2 hours and 11 minutes at room temperature. After extraction, extracts were collected, mixed and two 200 μl aliquots from every well were transferred to the appropriate wells of a pre-labelled 96-well plate for OD570 reading. Concurrent negative (ultra pure water) and positive (methyl acetate) controls were included in each run.

OD570 MEASUREMENTS: OD570 was measured on a plate reader Biotek Epoch. Isopropyl alcohol serves as a blank. No external filter was used.














- Justification for the use of a different negative control than ultrapure H2O (if applicable)
- Justification for the use of a different positive control than neat methyl acetate (if applicable)
- Description of any modifications to the test procedure
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
- Description of the method used to quantify MTT formazan
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable)
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
- Complete supporting information for the specific RhCE tissue construct used
- Reference to historical data of the RhCE tissue construct
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
- Positive and negative control means and acceptance ranges based on historical data
- Acceptable variability between tissue replicates for positive and negative controls
- Acceptable variability between tissue replicates for the test chemical

Results and discussion

In vitro

Results

Irritation parameter:

other: Tissue viability (%)

Value:

58.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DATA CORRECTION: average viability of the treated tissues was 59.1 % of the negative control viability. This value is close to cut off value. Negligible OD570 was observed in colorant control tissues (colour intereference test) though correction could be done. The true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the interfering test chemical and incubated with MTT solution minus the percent non-specific colour obtained with living tissues exposed to the interfering test chemical and incubated with medium without MTT solution, run concurrently to the test being corrected (%NSCliving).
Data are then corrected as follows: True viability = % Viability of treated tissue –% NSCliving = 59.1-0.2 = 58.9 %
No Direct MTT reduction is observed. The MTT remained coloured green for all the time of incubation without any change towards blue.

OTHER EFFECTS:
- Visible damage on test system: after treatment medium was coloured green and enhanced level of liquid occurred in inserts which is evidence of permeability of tissues. After washing of the test substance off tissues, tissues remained coloured green. Tissues remained coloured green even after extraction with isopropyl alcohol

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the average negative control OD570 was 1.447 which is > 0.8 and < 2.5. This criterion was fulfilled.
- Acceptance criteria met for positive control: the mean relative viability of the positive control was 32.6 % which is below 50 % of negative control viability. This criterion was fulfilled.
- Other: the difference of viability between the three relating tissues of the negative control was 9.2 %. The difference of viability between the two relating tissues of the test substance was 7.6 % and for colorant control 7.7 % therefore less than < 20 %. This criterion was fulfilled. The difference of viability between the positive control tissues was > 20 %. This criterion was not fulfilled; this deviation had no impact on the outcome of study.

DEVIATION: length of incubation at direct MTT reduction was one hour which is differencet from the OECD TG 492 as well as from MatTek’s Protocol where 3 hours are recommended. One hour was the time recommended in former MatTek’s Protocol. According to the laboratory, one hour is sufficient time for demonstration of the direct reduction. This deviation had no impact on the outcome of study.

Any other information on results incl. tables

The OD570 values obtained at the MTT test, their averages, standard deviations and relative viabilites are presented in the table below. The data are corrected by subtraction of OD₅₇₀isopropyl alcohol itself (blank, 0.039).

Table: results

Treatment	Tissue 1	Tissue 2	Tissue 3	avg (%)	SD (%)	Average viability (%NC)
water	1.522	1.261	1.558	1.447	0.132	100.0
% NC	105.21	87.17	107.7	100	9.2
Test substance	0.92	0.79	-	0.855	0.065	59.1
% NC	63.59	54.61	-	59.1	7.6
NSCliving	0.004	0.003	-	0.003	0.000	0.2
% NC	0.24	0.21	-	0.2	7.7
Positive control	0.858	0.309	0.247	0.471	0.274	32.6
% NC	59.31	21.36	17.07	32.6	58.2

NC = Negative control

Applicant's summary and conclusion

Interpretation of results:

other: the substance potentially requires classification for eye irritation or eye damage (Cat.2 or Cat.1) according to the CLP Regulation (EC) No.1272/2008

Conclusions:

The test substance is identified as a substance potentially requiring classification for eye irritation or eye corossion.

Executive summary:

The potential of the susbtance to cause eye irritation or corrosion was evaluated in the in-vitro system, reconstructed human cornea-like epithelial model EpiOcular™ (OCL-200 ver. 2.0) according to the OECD Guideline 492. The EpiOcular™ RhCE tissues are reconstructed from primary human cells and consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo. The test substance was applied in on the tissue surface, and the after 6 hrs of exposure at standard culture conditions, the substance was removed and the tissues were incubated with medium for 18 hours (post-treatment incubation). At the end of the post-treatment incubation the tissues were incubated with the MTT solution for 180 minutes. In the end of the staining, the plates were placed on an orbital plate shaker and shaken for 2 hours and 11 minutes at room temperature for the extraction of MTT formazan. The extracted MTT formazan was quantified using either a standard absorbance (Optical Density (OD)) measurement. Concurrent negative (sterile water) and positive (methyl acetate) controls run in parallel. Direct MTT reduction and colour interference controls were also performed; no direct MTT reduction or colour intereference were observed.

Tissue viability = 58.9 % of negative control viability.

The test substance is identified as a substance potentially requiring classification for eye irritation or eye corossion. Further investigation is required for the classification of the substance.