Disodium 7-amino-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

Endpoint summary

Description of key information

To assess the skin irritation potential of the substance, an in vitro skin irritation test (EPISKIN(TM) reconstructed human epidermis model) has been conducted.

There are no studies available for respiratory irritation.

To assess the eye irritation potential of the substance, an in vitro eye irrtitation test (Human Cornea Model test) has been conducted.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September to November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 28 July 2015

Deviations:

yes

Remarks:

3 deviations from the Study Plan occurred but were considered to have not affected the integrity or validity of the study. See detailed explanations in the section "Overall remarks, attachments"

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

yes

Remarks:

3 deviations from the Study Plan occurred but were considered to have not affected the integrity or validity of the study. See detailed explanations in the section "Overall remarks, attachments"

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test item was used as supplied.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM reconstructed human epidermis model Kit: SkinEthic Laboratories, Lyon, France (27-09-2016),
- Tissue batch number(s): EpiSkin Tissues (0.38 cm2) lot number 16-EKIN-039; Maintenance Medium lot number: 16-MIAN3-066; Assay Medium lot number: 16-ESSC-042
- Production date: nd
- Shipping date: nd
- Delivery date: 27 september 2016
- Date of initiation of testing:

Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C / room temperature
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 μL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface.

- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2 in air for 42 hours
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium
- Incubation time: The tissues were incubated for 3 hours at 37 °C, 5%
- Spectrophotometer:Anthos 2001 microplate reader
- Wavelength:562 nm
- Filter: without reference filter
- Filter bandwidth: nd
- Linear OD range of spectrophotometer: nd

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Water-killed tissues
- Procedure used to prepare the killed tissues (if applicable): Water-killed tissues were prepared prior to the study by placing untreated EPISKINTM tissues in a 12-well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5% CO2 in air for 48 ± 1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (−14 to −30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.
- N. of replicates : the MTT reducing test item was applied to three water-killed tissues. In addition, three water-killed tissues remained untreated.
- Method of calculation used:
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
Approximately 10 mg of test item was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.
The test item was also found to have the potential to produce color interference with the MTT endpoint if residual test item remained on the tissues after rinsing.
Therefore a parallel test was performed using living tissues that was identical to the main test with the exception of the MTT loading step being replaced with a 3 hour incubation in assay medium. Three living tissues were treated with the test item and three tissues remained untreated.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: pre-test followed by main test

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean tissue viability following the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period is equal or less than 50%.
The test substance is considered to be non-irritant to skin if the mean tissue viability following the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period is higher than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (26.3 mg/cm2)
- Concentration (if solution): n.a.

VEHICLE
- Amount(s) applied (volume or weight with unit): 5 microL
- Concentration (if solution): n.a.
- Lot/batch no. (if required): n.a.
- Purity: n.a.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 microL
- Concentration (if solution): 100%

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 microL
- Concentration (if solution): 5% w/w

The negative control item, DPBS, was used as supplied.
The positive control item, SDS, was prepared as a 5% w/v aqueous solution.
A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.

Duration of treatment / exposure:

15 min (room temperature)

Duration of post-treatment incubation (if applicable):

Incubation at 37ºC, 5% CO2 in air for 42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of 3 experiments

Value:

110.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative mean viability of the test item treated tissues was 110.6% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.
Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

- OTHER EFFECTS:
- Visible damage on test system:
- Direct-MTT reduction: The test item was found to be a possible direct MTT reducer. The MTT solution did not turn blue or purple but became a dark red color; therefore, as a precaution the test item was treated as if it was a direct MTT reducer and an additional procedure using water-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.
- Colour interference with MTT: The solution containing the test item was a dark red color. Therefore, an additional procedure using living tissues with no MTT loading step was performed. However, the results obtained showed that negligible color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The relative mean viability of the test item treated tissues was 110.6% after a 15-Minute exposure period and 42-Hour post-exposure incubation period.
It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 0.874 and the standard deviation value of the viability was 5.7%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 5.5% relative to the negative control treated tissues and the standard deviation value of the viability was 1.4%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 5.5%. The test item acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline:

Deviations from the study plan:

Deviation 1 An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

Deviation 2 An assessment found the test item had the potential to cause color interference with the MTT endpoint. Therefore, an additional procedure using living tissues with no MTT loading step was performed. However, the results obtained showed that negligible color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

Deviation 3 To clarify Deviation 1. The test item was found to be a possible direct MTT reducer.

Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item:

OD = Optical Density

SD = Standard deviation

∗ = The mean viability of the negative control tissues is set at 100%

Item	OD562 of tissues	Mean OD562 of triplicate tissues	+-SD of OD562	relative individual tissue viability (%)	relative mean viability (%)	+-SD of relative mean viability (%)
negative control item	0.927 0.828 0.867	0.874	0.050	106.1 94.7 99.2	100*	5.7
positive control item	0.058 0.034 0.051	0.048	0.012	6.6 3.9 5.8	5.5	1.4
test item	0.991 0.912 0.998	0.967	0.048	113.4 104.3 114.2	110.6	5.5

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant. The following classification criteria apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was found to be a possible direct MTT reducer and therefore additional non-viable tissues were incorporated into the testing for correction purposes. The test item was also found to have the potential to produce color interference with the MTT endpoint and therefore additional living tissues were incorporated into the testing for correction purposes.

At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 562 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June to July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

July 2015

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcularTM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation's Reconstructed Human EpiOcularTM Model; 29 June 2015

GLP compliance:

yes (incl. QA statement)

Species:

other: Human Cornea Model

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability:
1. Assessment of Direct Test Item Reduction by MTT
Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test item prior to conducting any assays with viable tissues. For this purpose approximately 50 mg of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a 6-well plate and the mixture was incubated in the dark at 37+-1.5ºC in a humidified atmosphere of 5+-0.5% CO2 in air for three hours. A control (50 µL of dionised water in 1 mL of 1.0 mg/mL MTT solution) was run concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
Since the MTT solution color did not turn blue/purple, the test item was not presumed to be a MTT reducer, and an additional test with freeze-killed tissues was not necessary.
2. Assessment of Coloured or staining materials
Coloured test items or test items which become coloured after application to the tissues may interfere with the quantitative photometric MTT measurement, if the colorant binds to the tissue and is extracted together with MTT. Therefore, each test item has to be checked for its colorant properties.
For this purpose each approximately 50 mg of the test item was aded to 1.0 ml of water and to 2 ml isopropanol in 6-well plates. the water mixture was incubated in the dark at 37 +-1.5ºC in a humidified atmosphere of 5+-0.5% CO2 in air for at least 1 hour, the isopropanol mixture or for 2 to 3 hours at room temperature.
Since the test item stained water and isopropanol, it had to be considered as possibly interacting with the MTT measurement and an additional test on colorant controls had to be performed.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
EpiOcular kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia) received under sterile conditions.
The certificate of analysis is included in the report.

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test material was crushed and ground in a mortar with pestle for improving its consistency. Approximately 50 mg of the test item was tested topically on duplicate EpiOcularTM tisses.

Duration of treatment / exposure:

6 hours (37+-1.5ºC, 5+-0.5% CO2, 95% RH)

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

Test system: Lot No.: 23719
1. EpiOcular Kit Components Needed for the Assay
Sealed 24-well plate: Contains 12/24 inserts with EpiOcular™ tissues on agarose
Serum-free test medium: DMEM-Medium
Positive control: Methyl Acetate (CAS#79-20-9)
12-well plate Holding plate
24-well plates For MTI viability assay
6-well plates For storing inserts, or for topically applying test agents
Serum-free assay medium DMEM-based medium

2. MTT-I00 Assay Kit Components
1 vial, 2 mL: MTT concentrate
1 vial, 8 mL: MTI diluent (supplemented DMEM), For diluting MTT concentrate prior to use in the MTT assay
60mL: Extractant solution (Isopropanol), For extraction of formazan crystals

3. MTT -Solution
For the pre-test for "Assessment of Direct Test Item Reduction by MTT" a 1.0 mg/mL MTT solution in DMEM was prepared directly prior to the pre-test.
MTT concentrate and MTT diluent together with the extractant solution was ordered as MTT test kit (MTT-100, MatTek) and was used for the main experiment. A MTT solution was prepared. MTT concentrate was diluted in MTT diluent to produce a 1.0 mg/mL solution.

3.1. Cell culture
EpiOcular™ kits and MTT -100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm2) are cultured on specially prepared cell culture inserts (MILLICELL ®, 10 mm 0).
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24- well plate on Tuesday. On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. An appropriate volume of EpiOcular™ Assay Medium was warmed to approximately 3 7 CC. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-Iabeled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper.
The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6- well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37°C and the EpiOcular™ tissues were incubated at standard culture conditions overnight (about 16.5 hours).

4 Experimental Performance
After the overnight incubation, the tissues were pre-wetted with 20 µL ofCa++Mg++free DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
Test item exposure: After the 30 minute Ca ++Mg++free-DPBS pre-treatment, the test and control item was tested by applying approximately 50 mg (test item) or 50 µL (controls) topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions (37 ± 1.5 QC, 5 ± 0.5% C02, 95% RH) for 6 hours.
At the end of the 6 hours treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously-warmed assay medium (room temperature) in a pre-Iabeled 12-well plate for a 25 minute immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test item or control absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material and transferred to the appropriate well of the pre-Iabeled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 18 hours at standard culture conditions (posttreatment incubation).

5 MTT Assay
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 ml of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions and rinsed 3 times with DPBS afterwards.
Inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-Iabelled 6-well plate containing 2 ml isopropanol in each well so that no isopropanol was flowing into the insert. The corresponding negative and positive controls had to be treated identically without piercing. For this procedure it was necessary to seal the plates particularly thorough since a higher evaporation rate had to be expected due to the larger surface of wells in 6-well plates.
Extraction was performed for 20 hours in the refrigerator.
The extract solution was mixed and two 200 ilL aliquots were transferred to the appropriate wells of a pre-Iabeled 96-well plate(s).
The absorbance at 570 nm (OD570) of each well was measured with a plate reader reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

6 Colorant Controls for Assessment of Colored or Staining Test Items
Since the test item showed to have or to develop relevant colour, which could interact with the MTT measurement, an additional test had to be performed to determine the amount of colour bound to and extracted from the tissues. For this purpose the coloured test item was applied to two additional tissues (= colorant controls (CC», and were treated in the same way as described in 4 and 5. In contrast to the normal viability test, no MTT assay was performed. The bound colour was extracted and the absorbance of the isopropanol extracts was measured identically as in the MTT assay for coloured test items (according to 4, as described for the MTT assay with 2 ml extraction solution in 6-well plates without piercing the tissue, and starting with a 180 min incubation in medium instead of MTT solution addition). The amount of extracted colour was subtracted from the results of the viability assay.

7 Data evaluation
1) The mean OD value of the blank control wells (ODBlk) for each experiment was calculated.
2) The mean value of the two aliquots for each tissue (= corrected test item OD) was calculated.
3) The mean ODBlk from the mean OD value of the same experiment (blank corrected values) was subtracted.
4) The mean value of the two relating tissues for each control and test item (= corrected mean OD) was calculated. For further calculations only the corrected mean negative control OD value was needed.
5) The corrected OD value of the negative control corresponds to 100% viability.
Corrected negative control OD = Negative Control OD - ODBlk = 100% Viability

7.1 Calculations for Viability Tests only
1) The percent viability of each ofthe two relating tissues for each control and test item relative to the negative control (100% control) was calculated:

Viability [%] = 100 * ( corrected test item OD / corrected mean negative control OD)

2) The difference of the viability between duplicate tissues was calculated. If the difference was >20% the test is considered as non-qualified.
3) The mean test item viability (TI viability) was calculated and the test item is classified according to the prediction model.

7.2. Calculation for Viability plus Colorant Control Tests
1) The OD values of the colorant control test (blank corrected) were calculated as described at the beginning of chapter 7.
2) The colorant control OD for the two relating tissues was transferred to a percentage value relative to the viability scale (colorant control "viability") by comparison to the corrected viability negative control of the same experiment.

Colorant control viability [%] = 100 * (OD colorant control / corrected mean negative control OD)

3) The difference of the viability of the two tissues was calculated. If the difference is >20%, the colorant control test is considered as non-qualified.
4) The mean viability for the colorant control was calculated and this mean colorant control "viability" value (CC "viability") was subtracted from the relating mean viability of the same test item (TI viability) to determine the colorant control corrected viability (CC corrected viability).

CC corrected viability = TI viability - CC viability

5) The test item was classified regarding the colorant control corrected viability according to the prediction model.

7.3 Prediction Model
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ~ 60% relative to negative control treated tissue viability, the test item is labeled irritant.

7.4 Acceptability of the Assay
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is> 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

in vitro irritation score

Remarks:

The indicated value is the mean relative absorbance value, corresponding to the cell viability (%)

Run / experiment:

test item

Value:

89.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item's colour change potential in water or isopropanol did lead to a change in colour. Therefore, an additional test with viable tissues without MTT addition was necessary.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour. Therefore, an additional test with freeze-killed tissues was not necessary.
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 89.9% (threshold for irritancy ≤ 60%), consequently the test item was not
irritant to eye.

Concerning acceptance criteria:
• The negative control OD is > 0.8 and < 2.5 (1.248).
• The mean relative viability of the positive control is below 50% of the negative control viability (42.3%).
• The difference of viability between the two relating tissues of a single item is < 20% (values between 1.3% to 8.2%) in the same run (for positive and negative control tissues and tissues of single test items). This applied also to the colorant controls which were calculated as percent values related to the viability of the relating negative control.

Table: Results after treatment for 6 hours with Direct Red 254 sodium salt and the controls

Dose group		absorbance well 1 (tissue 1/2)	absorbance well 2 (tissue 1/2)	mean absorbance (tissue 1/2)	mean absorbance *	mean absorbance of 2 tissues *	rel. absorbance [%] tissue 1 and 2**	absolute value of the difference of the Rel. absorbances [%] tissue 1 and 2	mean rel. absorbance [% of negative control]**	corrected viability
negative control		1.267 1.297	1.282 1.284	1.275 1.290	1.240 1.256	1.248	99.4 100.6	1.3	100.0
positive control		0.531 0.594	0.532 0.595	0.531 0.594	0.497 0.560	0.528	39.8 44.9	5.0	42.3
test item		1.128 1.161	1.170 1.179	1.149 1.170	1.114 1.135	1.125	89.3 91.0	1.7	90.1	89.9
negative control viable tissue		0.039 0.039	0.038 0.038	0.038 0.038	0.004 0.004	0.004	95.9 104.1	8.2	100.0
test item viable tissue		0.037 0.038	0.038 0.037	0.037 0.037	0.003 0.003	0.003	71.2 75.3	4.1	73.3

* Mean of two replicate wells after blank correction

** Relative absorbance [rounded values]: 100 x (absorbance test item/positive control) / (absorbance negative control)

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test substance does not possess any eye irritating potential.

Executive summary:

This in vitro study was performed to assess the eye irritation potential of the substance Direct Red 254 sodium salt by means of the Human Cornea Model Test.

Additional tests with viable tissues were performed, since the test item dyed water or isopropanol. Additional tests with freeze killed tissues were not necessary, since the test item did not prove to be a MTT reducer.

About 50 mg of the test item and each 50 µL of he controls, respectively, were applied to each of duplicate EpiOcular TM tissue for 6 hours.

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 42.3%, thus the validity of the test system is ensured.

The acceptance criteria were met.

Since the viability value of the test item exposed tissues did not decrease below 60%, the test item is not considered to possess an eye irritating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the available data and studies on skin irritation, the substance has not to be classified for this hazard class following the classification criteria of the EU Regulation 12/2008 (CLP Regulation).

Based on the results of the available data and studies on eye irritation, the substance has not to be classified for this hazard class following the classification criteria of the EU Regulation 12/2008 (CLP Regulation).

Regarding respiratory irritation, there are no data available. Based on the non-irritant behaviour of the substance, both in skin and eyes, it is not expected that the substance could be a respiratory irritant.