Registration Dossier

Administrative data

Description of key information

Acid Orange 94 Refined: Determination of Skin Irritation Potential using the EPISKIN™ Reconstructed Human Epidermis Model

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours (Fentem et al., 2001, Zuang et al., 2002, Cotovio et al., 2005, Portes et al., 2002 and Hartung, 2007). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay.

The study was performed in compliance with the: OECD Guideline for the Testing of Chemicals No. 439 (updated 28 July 2015) and Method B.46. in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), in accordance with GLP.

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

Acid Orange 94 Refined: The Bovine Corneal Opacity and Permeability (BCOP) Assay

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The study was performed in compliance with: OECD Guideline for the Testing of Chemicals No. 437 (updated 09 October 2017) “Bovine Corneal Opacity and Permeability Assay” and Method B.47 of Commission Regulation (EC) No. 440/2008, in accordance with GLP.

The corneas treated with the test item appeared clear with no staining, however some test item remained adhered to the sides of all chambers causing the clear MEM to turn pale orange post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

No prediction of eye irritation can be made.

Acid Orange 94 Refined: In vitro Eye Irritation Test: Human Cornea Model Test – OECD 492

This in vitro study was performed to assess the eye irritation potential of Acid Orange 94 Refined by means of the Human Cornea Model Test. the test was performed according to OECD Study Guidline 492, in accordance with GLP.

Irritating effects were not observed following incubation with Acid Orange 94 Refined. Compared with the value of the negative control, the mean absorption value corresponding to the tissue viability did not decrease below 60% (determined value for the test item 75.69%).

Under the test conditions reported for this studyAcid Orange 94 Refined does notpossess any eye irritating potential.

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:
25 October 2018 to 30 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
The test item was found to have the potential to cause color interference with the MTT endpoint, therefore additional color correction tissues were incorporated into the testing procedure.
Qualifier:
according to
Guideline:
other: Method B.46. in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008
Deviations:
yes
Remarks:
The test item was found to have the potential to cause color interference with the MTT endpoint, therefore additional color correction tissues were incorporated into the testing procedure.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test item: Acid Orange 94 Refined
Alternative name: Disodium 5, 5’-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]
CAS number: 70161-18-1
EC number: 274-354-1
Intended use: Industrial chemical
Appearance: Reddish brown crystals
Storage conditions: Room temperature (10 – 30C), in the dark
Lot number: 8009
Expiry/Retest date: 31 December 2019
Purity: 97%
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: The EPISKINTM model is a three-dimensional reconstructed human epidermis model
Source strain:
other: adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
Details on animal used as source of test system:
Not applicable, In-Vitro assay
Justification for test system used:
Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.
Vehicle:
unchanged (no vehicle)
Details on test system:
The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
10 mg (26.3 mg/cm2)
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
EpiSkin, Skin irritation
Value:
5.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Interpretation of results:
GHS criteria not met
Conclusions:
The test item was classified as non-irritant
Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours (Fentem et al., 2001, Zuang et al., 2002, Cotovio et al., 2005, Portes et al., 2002 and Hartung, 2007). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay.

The study was performed in compliance with the: OECD Guideline for the Testing of Chemicals No. 439 (updated 28 July 2015) and Method B.46. in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), in accordance with GLP.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 November 2018 to 01 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test item: Acid Orange 94 Refined
Alternative name: Disodium 5, 5’-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]
CAS number: 70161-18-1
EC number: 274-354-1
Intended use: Industrial chemical
Appearance: Reddish brown crystals
Storage conditions: Room temperature (10 – 30C), in the dark
Lot number: 8009
Expiry/Retest date: 31 December 2019
Purity: 97%
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 ml
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
3 corneas per test material, positive control and negative control respectively.
Details on study design:
Study Design

3.4.1 Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 70 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

3.4.2 Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer (Annex 1).
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

3.4.3 Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

3.4.4 Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mLof sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

3.4.5 Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

3.4.6 Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

3.5 Data Evaluation
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

3.5.1 Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

3.5.2 Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

3.5.3 In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

3.5.4 Visual Observation
The condition of the cornea was visually assessed post treatment.
Irritation parameter:
in vitro irritation score
Run / experiment:
BCOP
Value:
6.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
No prediction of eye irritation can be made.
Other effects / acceptance of results:
The positive control In Vitro Irritancy Score was within the range of 71.2 to 132.9. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤2.3 and permeability ≤0.44. The negative control acceptance criteria were therefore satisfied.
Interpretation of results:
other:
Remarks:
No prediction of eye irritation can be made.
Conclusions:
No prediction of eye irritation can be made.
Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The study was performed in compliance with: OECD Guideline for the Testing of Chemicals No. 437 (updated 09 October 2017) “Bovine Corneal Opacity and Permeability Assay” and Method B.47 of Commission Regulation (EC) No. 440/2008, in accordance with GLP.

The corneas treated with the test item appeared clear with no staining, however some test item remained adhered to the sides of all chambers causing the clear MEM to turn pale orange post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

No prediction of eye irritation can be made.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
16 January 2019 to 24 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 29 June 2015.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test item: Acid Orange 94 Refined
Alternative name: Disodium 5, 5’-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]
CAS number: 70161-18-1
EC number: 274-354-1
Intended use: Industrial chemical
Appearance: Reddish brown crystals
Storage conditions: Room temperature (10 – 30C), in the dark
Lot number: 8009
Expiry/Retest date: 31 December 2019
Purity: 97%
Species:
human
Details on test animals or tissues and environmental conditions:
3.4.4 Cell Culture
EpiOcular™ kits and MTT-100 kits were 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 ∅).
EpiOcular™ tissues were received 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. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 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 fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) overnight (approximately 18.5 hours).
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
50 mg test item
50 µl controls
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
EXPERIMENTAL DESIGN AND STUDY CONDUCT

4.1 Experimental Performance
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes. After the 30 minute Ca2+Mg2+free-DPBS pre-treatment, the test and control item were 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 °C, 5 ± 0.5% CO2) for 6 hours.
At the end of the 6 hours treatment time, the test item was removed by extensively rinsing the tissues with Ca2+Mg2+-free DPBS (brought to room temperature).
Three clean beakers containing a minimum of 100 mL each of Ca2+Mg2+-free DPBS were used per test item. The test item utilized a different set of three beakers. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts are mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beaker of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
Since it was not possible to remove the visible test item completely, this was noted in the study file. No further rinsing was done.
After rinsing, the 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 minutes 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-labelled 6-well plate containing 1 mL of warm Assay medium. The tissues were incubated for 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

4.2 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.
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-labelled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. The plates were sealed with parafilm (between the plate cover and upper edge of the wells) or a standard plate sealer, and were immediately extracted (shaken for approximately 2 hours at room temperature). 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.
The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

4.3 Killed Controls (KC) for Assessment of Residual Test Item Reduction of MTT
Since the test item proved to reduce MTT, only test item that remained bound to the tissue after rinsing, resulting in a false MTT reduction signal, presented a problem. To demonstrate that residual test item was not acting to directly reduce the MTT, a functional check on killed controls (KC) was performed in the definitive assay to show that the test material is not binding to the tissue and leading to a false MTT reduction signal.
Freeze killed tissues are prepared by placing untreated EpiOcular™ constructs (in a 24-well plate) in the -20°C freezer overnight, thawing to room temperature, and then refreezing (two freeze-thaw cycles). Once frozen, the tissue may have been stored indefinitely in the freezer.
Two freeze-killed tissues were treated with the test item in parallel to the main experiment since a small amount of MTT reduction is expected from the residual NADH and associated enzymes within the killed tissue.
Although it was not necessary the data correction procedure was performed according to the MatTek protocol.

4.4 Colorant Controls for Assessment of Coloured 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 4.1 and 4.2. 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 4.2, 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 according to chapter 5.
Although it was not necessary the data correction procedure was performed according to the MatTek protocol.

4.5 Non-Specific Killed Control (NSK) Controls for Assessment of Colored and Direct MTT Test Items
Since the test item was identified as producing both colour interference and direct MTT reduction, a third set of controls was needed to be performed. This is usually the case with darkly coloured test items absorbing light in the range of 570±30 nm (e.g., blue, purple, black) because their intrinsic colour impeded the assessment of their capacity to directly reduce MTT. This forced by default the use of both controls, KC and CC, which led to double correction for colour interference. In NSKC, the test chemical was applied on two killed tissue replicates, which underwent the entire testing procedure but were incubated with medium instead of MTT solution during the MTT incubation step. NSKC were performed concurrently to the KC control and with the same tissue batch. To calculate true tissue viability the percent non-specific colour obtained with NSKC was added to relative tissue viability.

4.6 Data Recording
The data generated were recorded in the raw data file. The results are presented in tabular form, including experimental groups with the test item and the controls.
Irritation parameter:
cornea opacity score
Run / experiment:
EpiOccular
Vehicle controls validity:
not applicable
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 led to a change in colour. Therefore, an additional test with viable tissues without MTT addition was necessary.
After mixing water with the Test Item a black precipitate was built. Therefore, an additional test with freeze-killed tissues was necessary.

The mean absorbance value of the test item, corresponding to the relative cell viability, decreased to 75.69% (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 (2.082 and 2.166).
• The tissue viability of the positive control is below 50% of the negative control viability (18.94%).
The difference of viability between the two relating tissues of a single item is < 20% (values between 0.04 p.p and 10.90 p.p) in the same run (for positive and negative control tissues and tissues of single test items). This applied also to the killed controls (Test Item and negative control) and the additional viable tissues (without MTT addition) which were calculated as percent values related to the viability of the relating negative control.
Interpretation of results:
GHS criteria not met
Remarks:
Does not need to be classified according UN GHS.
Conclusions:
This in vitro study was performed to assess the eye irritation potential of Acid Orange 94 Refined by means of the Human Cornea Model Test. Irritating effects were not observed following incubation with Acid Orange 94 Refined. Compared with the value of the negative control, the mean absorption value corresponding to the tissue viability did not decrease below 60% (determined value for the test item 75.69%).
Under the test conditions reported for this study Acid Orange 94 Refined does not possess any eye irritating potential.
Executive summary:

This in vitrostudy was performed to assess the eye irritation potential of Acid Orange 94 Refined by means of the Human Cornea Model Test. the test was performed according to OECD Study Guidline 492, in accordance with GLP.

Irritating effects were not observed following incubation with Acid Orange 94 Refined. Compared with the value of the negative control, the mean absorption value corresponding to the tissue viability did not decrease below 60% (determined value for the test item 75.69%).

Under the test conditions reported for this study Acid Orange 94 Refined does not possess any eye irritating potential.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification

EpiSkin

The results of the assay are considered acceptable if the following assay acceptance criteria are achieved: Positive Control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.

Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean OD570for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.

Test Item: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

The relative mean tissue viability for the positive control treated tissues was 6.6% relative to the negative control treated tissues and the standard deviation value of the viability was 2.3%. The positive control acceptance criteria were therefore satisfied.

The mean OD570for the negative control treated tissues was 0.957 and the standard deviation value of the viability was 1.4%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 5.7%. The test item acceptance criterion was therefore satisfied.

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 cannot be determined).

BCOP

The corneas treated with the test item appeared clear with no staining, however some test item remained adhered to the sides of all chambers causing the clear MEM to turn pale orange post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

Therefore, no prediction of eye irritation can be made.

EpiOccular

Thisin vitrostudy was performed to assess the eye irritation potential of Acid Orange 94 Refined by means of the Human Cornea Model Test.

The test item proved to be an MTT reducer in the MTT pre-test. Also, its intrinsic colour was intensive and it proved to dye water and isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed tissues, viable tissues and NSKC had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the normal tests.

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

Treatment with the positive control induced a decrease in the mean tissue viability compared with the negative control to 18.94%, thus the validity of the test system is ensured.

The acceptance criteria were met.

Since the mean viability of the test item exposed tissues was 75.69% and hence did not decrease below 60%, the test item is not considered to possess an eye irritating potential.

If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is identified as not requiring classification and labelling according to UN GHS (No Category).

If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, no prediction can be made from this result in isolation and requires additional information for classification purpose.

According to OECD guideline 492 a single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Acid Orange 94 Refined does notneed to be classified according UN GHS.