Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Endpoint summary

Description of key information

Skin irritation/corrosion: There is one test in vitro OECD 439 conducted with the substance. In the main experiment additional viable, freeze killed and NSKC tissues were necessary.  Two additional tests with freeze killed and viable tissues were done too. The test item obtained a corrected viability of 102.6%. This is above the threshold for Skin Irritancy of 50% viability and therefore the test item has to be considered as non-irritant for the skin.

Eye irritation: There is one test in vitro OECD 437 conducted with the substance where two duplicates have been done. The IVIS of both repliacates support the classification of the substance as Serious eye damaging, Eye Dam 1.

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:

May-June 2018

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:

(Original Guideline adopted July 28, 2015), and as described in detail in the Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200).

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Qualifier:

according to guideline

Guideline:

other: UN GHS (published 2003, last (7th) revision 2017)

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

Version 07 November 2014.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other: The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Model used: EpiDerm™
- Tissue batch number(s): Epi-200- SIT Kit ( Lot No.: 28624), Epi-200 SIT kits and MTT-100 assay kit were purchased from MatTek Corporation (82105 Bratislava, Slovakia).
Cell Culture: The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs(R), 10 mm diameter).
- Production date: not indicated
- Shipping date: no date indicated
- Delivery date: EpiDerm™ tissues were shipped with cool packs on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on 12 June 2018
- Date of initiation of testing: On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.
- MTT-Solution: The MTT-solution was prepared freshly on day of use (resulting: 1 mg/mL).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
Test for Direct MTT Reduction and Colour Interference
A test item may interfere with the MTT endpoint if: a) it is coloured and/or b) able to directly reduce MTT. The MTT assay is affected only if the test item is present in the tissues when the MTT viability test is performed.
Some non-coloured test items may change into coloured test items in wet or aqueous conditions and thus stain tissues during the 60 min exposure. Therefore, before exposure, a functional check for this possibility has to be performed (step 1).
Step 1: 25 ± 2 mg of the test item were added to 0.3 mL of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated.
Since the solution changes colour significantly, the test item was presumed to have the potential to stain the tissue. A functional check on viable tissues has to be performed (step 2).
Step 2: To check the tissue-binding of a coloured test item, two viable tissues were exposed to 25 ± 2 mg of the test item. In parallel, two tissues were exposed to DPBS (negative control). All procedures were followed as described in chapter 3.6 except the tissue were incubated for 3 hour incubation in culture media without MTT (37 ± 1.5°C, 5 ± 0.5% CO2,) instead of incubating in media containing MTT. After the 3 hour incubation, the tissues were rinsed and the tissues were extracted using 2.0 ml of isopropanol and the optical density (OD) at 570 nm was measured.
Data correction procedure
Since the tissues treated by coloured test item (or test item detected in step 1) had an OD < 5% of the PBS treated control tissue and the tissue viability (determined in MTT assay) was not close to the classification cut-off (50%), correction of the results was not necessary, but was calculated using following formula: OD = ODcolored tissue (MTT assay) – ODcolored tissue (no MTT assay)
Step 3: The test item (including those already evaluated in step 1 and step 2) was further evaluated for its potential to interfere with MTT assay. To test if a test item directly reduces MTT, 25 ± 2 mg of the test item were added to 1 mL of the MTT-solution (1 mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT medium was used as control.
Since the MTT solution turned black, the test item reduced MTT and additional functional check (step 4) had to be performed.
Step 4: The procedure employed freeze-killed tissues that possess no metabolic activity but absorb and bind the test item similar to viable tissues.
The MTT reducing test item was applied to two freeze-killed tissues. In addition, two freeze killed tissues were left untreated (Note: The untreated killed controls show a small amount of MTT reduction due to residual reducing enzymes within the killed tissue). The entire assay protocol is performed on the freeze-killed tissues in parallel to the assay performed with the live EpiDerm tissues.
Data were then corrected as follows:
Data correction procedure
True viability = Viability of treated tissue – Interference from test chemical = ODtvt – ODkt, where ODkt = (mean ODtkt – mean ODukt)
tvt = treated viable tissue kt = killed tissues
tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)
Since the interference by the test item is < 30% of the negative control value, the net OD of the test item treated killed control was subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflected metabolic conversion only.
Addition according to OECD 439:
Although it was not mentioned in the Study Plan and is not required in the MatTek protocol OECD 439 requires a third set of control – the so called NSKC (Non-specific killed control) in order to avoid a false double correction if a test item is both coloured and has MTT reducing capacities. There freeze-killed tissues were treated with the test item and during the MTT Assay incubated only with medium without MTT. It is a mixed control and the data which are obtained from this control are used for Data Correction Procedure as follows:
Corrected mean Viability = 〖Tissue Viability〗_(test item )-(〖Tissue Viability〗_(VT test item) 〖- (Tissue Viability〗_(KC test item)- 〖Tissue Viability〗_(KC negative control))) + mean ViabilityNon specific killed control

EXPERIMENTAL PERFORMANCE
1. Pre-warming of EpiDerm™ Tissues: The plastic bag containing the 24-well plate with epidermal tissues was opened under sterile conditions. Under an airflow using forceps, the gauze was removed and the inserts were taken out. Any remaining agarose that adheres to the outer sides of the inserts was removed by gentle blotting on the sterile filter paper or gauze, and the tissues were placed in the empty, sterile 6-well plate. Prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality: If necessary, it was taken care, that
1) air bubbles between agarose and insert were not > 30% of the total surface,
2) liquid on top of the insert was removed with sterile cotton tips,
3) if again moisture is observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded.
0.9 mL of the assay medium (20 – 25 °C) was pipetted into each well of sterile 6-well plates. The inserts with the EpiDerm™ tissues were placed in the upper wells, and were pre-incubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further about 22 hours (37 ± 1.5 °C, 5 ± 0.5% CO2).

2. Treatment: After pre-incubation of EpiDerm™ tissues was completed, medium was replaced by 0.9 mL of fresh medium per well. The negative control, the positive control, and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues. The treatment time was 60 minutes in total. Within this period the 6-well plates were put into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with PBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in PBS at least three times. Afterwards the inserts were once again rinsed with sterile PBS from the inside and the outside. Excess PBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The inserts were placed in the prepared holding plate. Tissues were incubated for about 24 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2. After incubation medium was changed (0.9 mL of pre-warmed fresh medium). Thereafter tissues were incubated for another about 17 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2. The complete incubation time was about 41 hours.

3. MTT Assay: On the day of testing the MTT concentrate was diluted with the MTT diluent (1 mg/mL). The 24-well plates were prepared before the end of the tissue pre-warming period. A volume of 300 µL of the MTT solution was added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until further use.
After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates. After a 3-hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2), the tissues were rinsed three times with PBS, and carefully dried with blotting paper. The tissues were transferred into new 6-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues are completely covered. The 6-well plate was sealed to inhibit the isopropanol evaporation.
The formazan salt was extracted for about 2.5 hours while shaking at room temperature.
Per each tissue, 3 x 200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax® Molecular Devices, Softmax Pro Enterprise, version 4.7.1) with a 570 nm filter. Mean values were calculated from the 3 wells per tissue.

4. Data Recording: The data generated were recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups with the test item, negative control and positive controls.

NUMBER OF REPLICATE TISSUES:
3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1

PREDICTION MODEL / DECISION CRITERIA
For the current test, an irritation potential of a test item leading to EU classification H315 (according to regulation (EC) 1272/2008), and GHS category 2 according to UN GHS (published 2003, last (7th) revision 2017) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.
in vitro result in vivo prediction EU classification UN GHS classification
mean tissue viability <= 50% irritant (I) H315 category 2
mean tissue viability > 50% non-irritant (NI) non-irritant (NI) non-irritant (NI)
In cases of borderline results, such as non-concordant replicate measurements and/or mean percent viability equal to 50 ± 5%, a second run should be considered, as well as a third one in case of discordant results between the first two runs.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL : Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL DPBS prior to application, and spread to match the surface of the tissue for a complete treatment time of 60 minutes
NEGATIVE CONTROL: Concurrent controls were used for several Envigo CRS GmbH studies performed simultaneously. Each 30 µL were applied to triplicate tissue. Name: DPBS (MatTek)
POSITIVE CONTROL: Concurrent controls were used for several Envigo CRS GmbH studies performed simultaneously. Each 30 µL were applied to triplicate tissue. Name: 5% SDS solution in deionised water (MatTek)

Duration of treatment / exposure:

The treatment time was 60 minutes in total.

Duration of post-treatment incubation (if applicable):

Tissues were incubated for about 24 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2. After incubation medium was changed (0.9 mL of pre-warmed fresh medium). Thereafter tissues were incubated for another about 17 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2. The complete incubation time was about 41 hours.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

102.06

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
There was the following deviation from study plan:
Concerning 6.3 MTT Assay (page 9 of the study plan). Instead of 24-Well-Plates, 6-Well-Plates were used for extraction with isopropanol since residual test item was still visible on the inserts after rinsing. With this it is possible to avoid solubility of residual test item in the isopropanol extract and a falsification for OD measurement. This deviation was considered to have not affected the integrity or validity of the study.

Concerning 5.6 Cell culture: Although it was not mentioned in the Study Plan and is not required in the MatTek protocol OECD 439 requires a third set of control was performed – the so called NSKC (Non-specific killed control) in order to avoid a false double correction if a test item is both coloured and has MTT reducing capacities. There freeze-killed tissues were treated with the test item and during the MTT Assay incubated only with medium without MTT. It is a mixed control and the data which are obtained from this control are used for Data Correction Procedure as follows:
Corrected mean Viability = Tissue Viabilitytest item −(Tissue ViabilityVT test item− (Tissue ViabilityKC test item − Tissue ViabilityKC negative control)) + mean ViabilityNon specific killed control

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is >=0.8 and ≤ 2.8 in accordance with OECD TG 439.
- Acceptance criteria met for positive control: An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is <= 20%.
- Acceptance criteria met for variability between replicate measurements: The SD of 3 concurrently tested tissue replicates should be < 18.
- Range of historical values if different from the ones specified in the test guideline: OD values should not be below historically established boundaries.
Concurrent negative controls (NC) and positive controls (PC) will be used in each run to demonstrate that viability (with the NC), barrier function and resulting tissue sensitivity (with the PC) of the tissues are within a defined historical acceptance range.
Historical data and the quality certificate of the supplier of the test kit demonstrating its robustness are annexed to the report, including quality control data (determined by MatTek Corporation, 82105 Bratislava, Slovakia) of the respective EpiDermTM lot. According to the OECD TG 439, the acceptance limit of the ET50 should be between 4.77 hours and 8.72 hours after treatment with 1% Triton X-100 (QC batch release criteria).

Results after treatment with the substance and the controls

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	OD 570 nm Well 3	Mean OD of 3/2 Wells	Mean OD of 3/2 Wells blank corrected	Mean OD of 3/2 tissues blank corrected	Rel. Viability [%] Tissue 1, 2 + 3*	Standard Deviation	Mean Rel. Viability [%]**
Blank		0.037	0.038	0.036	0.037
Negative Control	1	1.984	2.004	2.012	2.000	1.963	1.841	106.629	9.5	100.0
	2	1.963	1.960	1.945	1.956	1.919		104.221
	3	1.676	1.674	1.686	1.678	1.642		89.150
Positive Control	1	0.086	0.089	0.086	0.087	0.050	0.049	2.721	0.1	2.65
	2	0.085	0.085	0.085	0.085	0.048		2.629
	3	0.085	0.086	0.084	0.085	0.048		2.601
Test Item	1	1.761	1.902	1.905	1.856	1.819	1.897	98.806	4.1	102.06***
	2	1.991	2.003	2.032	2.009	1.972		107.081
	3	1.939	1.925	1.944	1.936	1.899		103.128
Blank		0.037	0.038	0.039	0.038
Negative Control Viable Tissues	1	0.037	0.037	0.038	0.037	-0.001	0.000	-0.038	0.1	0.00
	2	0.040	0.038	0.038	0.039	0.001		0.040
Test Item Viable Tissues	1	0.041	0.040	0.039	0.040	0.002	0.004	0.098	0.2	0.23
	2	0.045	0.045	0.044	0.045	0.007		0.358
Negative Control Freeze killed Tissues	1	0.078	0.083	0.083	0.081	0.043	0.044	2.355	0.0	2.39
	2	0.081	0.084	0.083	0.083	0.045		2.426
Test Item Freeze killed Tissues	1	0.092	0.089	0.086	0.089	0.051	0.079	2.761	2.2	4.31
	2	0.145	0.144	0.149	0.146	0.108		5.853
Non-specific killed control	1	0.052	0.053	0.052	0.053	0.015	0.022	0.793	0.6	1.20
	2	0.068	0.067	0.068	0.068	0.030		1.606

 

*  Relative viability [rounded values]:

 

**  Mean relative viability [rounded values]:

***  corrected value

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did lead to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did show black colour.

The mean relative viability of the test item, corresponding to the cell viability, increased to 102.06% (threshold for irritancy:≤50%), consequently the test item was non-irritant to skin. 

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 item is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

The test item did reduce MTT (test for direct MTT reduction). Also, its intrinsic colour was intensive.Consequently, additional tests with freeze-killed, viable tissues and non-specific killed controls were necessary.

Each three tissues of the human skin model EpiDerm^™were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD³0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item the mean relative viability value was 102.06 %compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is non-irritant to skin.

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:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

9th October 2017

Deviations:

yes

Remarks:

The positive control is 10% (w/v) Benzalkonium chloride (purity not indicated by the producer) in saline since the laboratory historical control data is established with this chemical.

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS (Hank’s Buffered Salt Solution) containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes. The corneae were directly used in the BCOP test on the same day.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

NEGATIVE CONTROL USED, Saline (0.9% NaCl in deionised water)
POSITIVE CONTROL USED, 10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl (saline) using sonication for 10 minutes.
TEST ITEM: The test item was tested as a 20% suspension (w/v) in saline using sonication for 10 minutes.

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 for each Negative control, Positive control and Test item

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments. For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed before basal opacity measurement (t0).
At the end of the incubation period, the basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Only corneae with a value of the basal opacity < 7 were used. Sets of three corneae were used for treatment with the test item and for the negative and positive controls, respectively.

NUMBER OF REPLICATES : 3 for each Negative control, Positive control and Test item

NEGATIVE CONTROL USED : Name: Saline (0.9% NaCl in deionised water)

POSITIVE CONTROL USED : Name: 10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl (saline) using sonication for 10 minutes.

APPLICATION DOSE AND EXPOSURE TIME
The anterior compartment received the test item suspension or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneae, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted 240 minutes.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
After exposure, the test item or the control items, respectively, were each rinsed off from the according application sides with EMEM containing phenol red at least three times or more if phenol red was still discoloured (yellow or purple), or the test item was still visible. Once the medium was free of the test item the corneae were given a final rinse with cMEM without phenol red. Fresh cMEM was added into the anterior compartment and opacity was measured (t240). The opacity measurement is described in chapter 3.5.
The corneae treated with the test item became dyed black after incubation.
In the second step of the assay, permeability of the cornea was determined. The permeability measurement is described in chapter 3.6.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Measures taken at the end of the incubation period, the medium was changed before basal opacity measurement (t0). After exposure of the corneae to the different test groups and after rinsing the opacity value was determined again (t240).
- Corneal permeability: Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value of permeability determination)
The following formula is used to determine the IVIS of the positive control and the test item: IVIS = (opacity value – mean opacity of the negative control) + (15 x (permeability value – mean permeability of the negative control))
The mean IVIS value of each treated group is calculated from the respective individual IVIS values.
Depending on the IVIS score obtained, the test item is classified into the following Category according to OECD guideline 437:
IVIS ≤ 3 UN GHS / EU CLP No Category
IVIS > 3; ≤ 55 UN GHS / EU CLP No prediction can be made
IVIS > 55 UN GHS / EU CLP Category 1

DATA EVALUATION
Opacity: The change of the opacity value of each treated cornea or of the positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
Permeability: The corrected OD490 value of each cornea treated with positive control or test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea

DECISION CRITERIA:
The test will be acceptable if :
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

A single testing run composed of at least three corneae should be sufficient for a test chemical when the resulting classification is unequivocal. In cases of borderline results in the first testing run, a second testing run will be considered, as well as a third one in case of discordant mean IVIS results between the first two testing runs. A result in the first testing run is considered borderline if the predictions from the 3 corneae are non-concordant, such that:
• 2 of the 3 corneae give discordant predictions from the mean of all corneae, or,
• 1 of the 3 corneae gives a discordant prediction from the mean of all 3 corneae, and the discordant result is >10 IVIS units from the cut-off threshold of 55.
• If the repeat testing run corroborates the prediction of the initial testing run (based upon the mean IVIS value), then a final decision can be taken without further testing.

If the repeat testing run results in a non-concordant prediction from the initial testing run (based upon the mean IVIS value), then a third and final testing run should be conducted to resolve equivocal predictions, and to classify the test chemical. It may be permissible to waive further testing for classification and labelling in the event any testing run results in a UN GHS Category 1 prediction.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

72.08

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

300.41

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results of the first experiment after 240 Minutes Treatment Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed in vitro Irritancy Score
		Mean		Mean
Negative Control	0	0.33	0.088	0.080	1.32	1.54	No Category
	1		0.080		2.20
	0		0.073		1.10
Positive Control	77.67*		0.581*		86.38	87.39	Category 1
	78.67*		0.633*		88.16
	82.67*		0.331*		87.63
test item	63.67*		-0.013*		63.47	72.08	Category 1
	71.67*		-0.021*		71.35
	80.67*		0.050*		81.41

*corrected values

Results of the second experiment after 240 Minutes Treatment Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed in vitro Irritancy Score
		Mean		Mean
Negative Control	0.00	0.33	0.079	0.076	1.19	1.48	No Category
	1.00		0.060		1.90
	0.00		0.090		1.35
Positive Control	96.67*		0.438*		103.23	109.51	Category 1
	107.67*		0.559*		116.05
	101.67*		0.505*		109.24
test item	299.67*^#		0.081*		300.88	300.41	Category 1
	299.67*^#		0.100*		301.16
	297.67*		0.101*		299.18

*corrected values

^#Values were set on 300 to enable the analysis. The measured values were 334 and 423.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item is serious eye damaging (EU CLP/UN GHS Category 1).

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of the substance by means of the BCOP assay using fresh bovine corneae.

The experiment was performed twice on sponsor’s request due to an unexpected result. The result of the first experiment could be confirmed. Both experiments will be stated in this report.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item as well as the positive and the negative controls were each applied to different corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240).  

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.  

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed in both experiments.

In the first experiment the positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (EU CLP/UN GHS Category 1). The IVIS of the positive control (IVIS of 87.39) was below two standard deviations (SD = 11.82) of the current historical mean (116.38) but within the range of all obtained historical control data for the positive control (80.38—146.55).  

In the second experiment the positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (EU CLP/UN GHS Category 1) with an IVIS of 109.51.

Relative to the negative control, the test item caused an increase of the corneal opacity. In the first experiment the calculated mean in vitro irritancy score was 72.08 and in the second experiment 300.41. According to OECD 437 (see table in chapter 3.8.3) the test item is classified as serious eye damaging (EU CLP/UN GHS Category 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information and applying the criteria of Annex I of EU Regulation 1272/2008, the substance is not classifed as skin irritant.

Based on the available information and applying the criteria of Annex I of EU Regulation 1272/2008, the substance is classified as Serious eye damaging, Eye Dam 1.