Disodium 3-[[2,2'-dimethyl-4'-[[4-[[(p-tolyl)sulphonyl]oxy]phenyl]azo][1,1'-biphenyl]-4-yl]azo]-4-hydroxynaphthalene-2,7-disulphonate

Endpoint summary

Description of key information

Not irritant to skin.

Not irritant to eye.

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:

From June 19th to June 21th, 2019

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:

2015

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

GLP compliance:

yes (incl. QA statement)

Test system:

isolated skin discs

Source species:

human

Cell type:

other: Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purpose of distinguishing between skin irritating and no-skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

-Pre-incubation: the "maintenance medium" was pre-warmed to 37°C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 ml per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37±1°C in an incubator with 5±1 % CO2, >/=95 % humidified atmosphere.

-Application: three replicates were used for the test item and positive and negative controls respectively. Test item: the epidermal surface was first moistened with 5 µl deionised water in order to improve further contact between powder and epidermis. Subsequently, approximately 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necesssary. Positive and negative control: a volume of 10 µl positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette trip in order to cover evenly all the epidermal surface if necessary. Additional controls for dyes and chemicals able to colour the tissue: in addition to the normal procedure, two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).

-Exposure: the plates with the treated epidermis units were incubated for the exposure time of 15 minutes (±0.5 min) at room temperature.

-Rinsing: after the incubation time, the EpiSkin SM units were removed and rinsed thoroughly with approximately 25 ml PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

-Post-incubation: after rinsing the units were placed into the plate wells with fresh pre-warmed "maintenance medium" (2ml/well) below them and then incubated for 42 hours (±1 h) at 37±1°C in an incubator with 5±1 % CO2 , >/= 95 % humidified atmosphere.

-MTT test after 42 hours incubation: after the 42 hours (±1 h) incubation, the EpiSkin Sm units were transferred into the MTT solution filled wells ( 2 ml of 0.3 mg/ml MTT per well) and then incubated for 3 hours (± 5 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, >/=95 % himidified atmosphere.

-Formazan extraction: at the end of incubation with MTT a formazan extraction was undertaken: a disk of epidermis was cut from the units (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µl acidified isopropanol (one tube corresponding to one well of the tissue culture plate). The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for approximately four hours at room temperature protected from light with gentle agitation (ca. 150 rpm) for formazan extraction. At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

-Cell viability measurements: following the formazan extraction, 2x200 µl sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance/Optical Density) of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at 570 nm (±10 nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using acidified isopropanol solution as the blank (6×200 µl).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg

Duration of treatment / exposure:

15 minutes ±0.5 min

Duration of post-treatment incubation (if applicable):

Epidermis units were incubated for 42 hours (±1 h)

Number of replicates:

three

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate 1

Value:

92

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate 2

Value:

97

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate 3

Value:

93

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The mean OD value of the three negative controls tissues was 0.951. The mean OD value obtained for the positive control was 0.204 and this result corresponds to 21 % viability when compared to the reults obtained from the negative control. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.
Possible direct MTT reduction with test item: no colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore addittional controls and data calculations were not necessary. A false estimation of viability can be precluded.
Colouring potential of test item: the test item has an intrinsic colour red. If the test item is not white, off white, almost white and/or colorless the addtional controls are automatically used and based on the OD results of additional controls the Non Specific Colour % (NSCliving %) is determined. This step prevents the false viability, which can possibly be caused by the stained surface of the tissues by the test item. Two addtional test item-treated tissues were used for the non.specific OD evaluation. The mean OD (measured at 570 nm) of these tissues was determined as 0.060. The NSCliving % was calculated as 6.3 %. Therefore, additional data calculation was necessary.

OD values and viability percentages of the controls

Substance	Optical Density (OD)		Viability (%)
Negative Control:	1	1.002	105
1x PBS	2	0.899	94
	3	0.952	100
	mean	0.951	100
	standard deviation (SD)		5.46
Positive Control:	1	0.238	25
SDS (5 % aq.)	2	0.197	21
	3	0.178	19
	mean	0.204	21
	standard deviation (SD)		3.2

OD values and viability percentages of the test item (including corrected values)

Test Item	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)
Acid Red 111	1	0.871	0.811	92	85
	2	0.921	0.861	97	90
	3	0.885	0.824	93	87
	mean	0.892	0.832	94	87
	standard deviation (SD)			2.68	2.68

TOD_TT: OD true MTT metabolic conversion for treated tissues

OD values and NSC_living% of additional control

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSCliving %)
Acid Red 111 (test item treated tissues without MTT incubation)	1	0.052	6.3
	2	0.068
	mean	0.06

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (</=) to 50 % of the negative control.

Interpretation of results:

other: According to CLP Regulation (EC) 1272/2008 criteria the test item do not need to be classified.

Conclusions:

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean corrected value:87 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non.irritant to skin. Positive and negative controls showed the expected ODs, cell viability values were within acceptable limits and standard deviations of all calculated viability values (positive and negative control, test item) were below 18. The experiment was considered to be valid.
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilisied testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified.
According to CLP Regulation (EC) 1272/2008 criteria the test item do not need to be classified.

Executive summary:

EpiSkin SM test of the test item has been performed to predict its irritation potential by measurements of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes (±0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1 °C for 42 hours (±1 h) in an incubator with 5±1 % CO2, >/= 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (±5 min) with MTT solution at 37±1°C in 5±1 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

The test item has an intrisic colour (red), therefore two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).

SDS (5 % aq.) and 1x PBS treated (three units/positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test chemical is identified as requiring classification and labelling according to UN GHS (category 2 or category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (</=) to 50 % of the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean corrected value: 87 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected ODs, cell viability values were within acceptable limits and standars deviations of all calculated viability values (positive and negative control, test item) were below 18. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified.

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:

weight of evidence

Study period:

From October 16th to 17th, 2019

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:

2019

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

EpiOcular™ (OCL-200-EIT)
The EpiOcular™ human cell construct (MatTek Corporation) is used in this assay. This three-dimensional human cornea model allows the identification of test items with the potential to induce eye irritation or serious eye damage by assessing cell viability after treatment. The model is composed of stratified human keratinocytes in a three-dimensional structure, consisting of at least three viable layers of cells. Test materials can be applied topically to the model so that also water insoluble materials may be tested. Prior to use, each plate (6, 12, and 24-well) and its cover will be uniquely identified with a permanent marker by a plate number and/or test item number.
The cytotoxicity of the test item (and thus the ocular irritation potential) is evaluated by the relative viability of the treated tissues in comparison to the negative control-treated tissues. Viability is determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, by the succinate dehydrogenase reduction of MTT) in control and test item treated cultures (Berridge, et al., 1996). Data are presented in the form of relative survival (relative MTT conversion).

Justification for selection of the test system
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.

Demonstration of proficiency
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392-492-1722) using the fifteen Proficiency Chemicals according to OECD Test Guideline No. 492.

Quality control
The EpiOcular™ (OCL-200-EIT) kits are manufactured according to defined quality assurance procedures. All biological components of the EpiOcular™ tissue and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with Triton X-100 (100 μl of 0.3% (v/v) Triton X-100). A certificate of quality as provided by the supplier is annexed to this report.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

approximately 50 mg on top of 0.6 cm2 tissue; approx. 83.3 mg/cm2

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

2 replicates per test item and 2 replicates of the negative control, 2 replicates of the positive control and 2 replicates of colour controls (NSCliving) were used.

Details on study design:

Indicator for potential false viability
Optical properties of the test item or its chemical action on MTT, isopropanol and water may interfere with the assay leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the EpiOcular™ tissue. If the test material acts directly on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement. In order to check if the test item is a MTT-reducer or becomes coloured during tissue treatment the following verifications (see 5.5.1 and 5.5.2) are carried out prior to the main experiment.

Check-method for Possible Direct MTT Reduction by the Test Item
Approximately 50 mg test item was added to 1 mL MTT 1 mg/mL solution in a 6-well plate or tube and the mixture is incubated in the dark at 37 ±2°C, 5±1% CO2, ≥95% humidified atmosphere. The mixture is incubated for approximately 3 hours (±15 min) and then any colour change observed:
- Test item which do not interact with MTT: yellow
- Test item interacting with MTT: blue or purple
If the MTT solution’s colour becomes blue or purple, the test item interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non-specific reduction of the MTT (i.e. by using killed EpiOcular tissues).
Results of this check are detailed in section 10.2.

Check-method to detect the colouring potential of test item
The test item has an intrinsic colour (red). If the test item is not white, off white, almost white and/or colorless the additional controls are automatically used and based on the OD results of additional controls the Non Specific Colour % (NSCliving %) is determined. This step prevents the false viability which can possibly be caused by the stained surface of the tissues by the test item.

Additional controls for dyes and chemicals able to colour the tissue:
In addition to the normal procedure, two additional test item-treated tissues were used for the non-specific OD evaluation. These tissues followed the same treatment steps as the other tissues except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. OD reading was made following the same conditions as for the other tissues.

Preparation of EpiOcular™ tissues for treatment
After the test kit arrival, the tissues were equilibrated to room temperature for about 15 minutes. The Assay Medium was pre-warmed to 37±1°C. The appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed medium (1 ml per well). The insert was transferred aseptically into the 6-well plates and pre-incubated at 37±2 °C in an incubator with 5±1 % CO2, ≥95 % humidified atmosphere 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 was incubated at standard culture conditions overnight (16 - 24 hours).

APPLICATION
Pre-treatment
After the overnight incubation, the tissues were pre-wetted with approximately 20 μl of Ca++Mg++Free-DPBS. If the Ca++Mg++Free-DPBS did not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.

Test item
Approximately 50 mg test item was applied topically onto the EpiOcular™ tissues. To avoid that test item is spilled into the medium under the tissue inserts, the tissue insert was removed from the medium, placed onto a sterile surface (e.g. the lid of a microtiter plate) and dosed by pouring the solid test article onto the tissue surface so that the surface of the tissue was completely covered by the test item. The insert was gently shaken from side to side to ensure that tissue was completely covered by the test item. After this procedure the tissue was then returned to its medium.

Positive and negative control
A volume of 50 µl positive control (methyl acetate) or negative control (sterile deionized water) was applied on the tissues surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the EpiOcular™ tissues surface if necessary.
Additional controls for dyes and chemicals able to colour the tissue:
In addition to the normal procedure, two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).

EXPOSURE
The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions (37±2 °C in an incubator with 5±1 % CO2, ≥95 % humidified atmosphere).

RINSING
After the incubation time the plastic films were removed and the EpiOcular™ units were removed and rinsed thoroughly with Ca++Mg++ Free-DPBS as follows: 3 clean beakers (glass or plastic with minimal 150 ml capacity), containing a minimum of 100 ml each of Ca++Mg++ Free-DPBS were used per test item. Each test item utilizes a different set of three beakers. The inserts containing the tissue was lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. Use of curved forceps facilitates handling and decanting. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps (taking care of not to damage the tissues by the forceps). The test or control items were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the tissues was dipped into the first beaker of DPBS and was swirled in a circular motion in the liquid for approximately 2 seconds and after was lifted out so that the inserts were 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 tissue was rinsed in the second and third beakers 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 and touching the upper lip to the absorbent material (to break the surface tension).

POST-SOAK AND POST-INCUBATION
After rinsing, the tissues were transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation (Post-Soak) at room temperature. This incubation in Assay Medium was intended to remove any test item 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 afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm (37°C) Assay Medium. The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation).

MTT TEST AFTER POST-INCUBATION
After the post-incubation the EpiOcular™ units were transferred into the MTT ready to use solution filled 24-well plate (300 µl of 1 mg/ml MTT per well) and then incubated for 3 hours (± 15 min) at 37±2 °C in an incubator with 5±1 % CO2 protected from light, ≥95 % humidified atmosphere.

FORMAZAN EXTRACTION
Inserts were removed from the 24-well plate after 3 hours ± 15 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 2 ml isopropanol in each well so that no isopropanol was flowing into the insert. The plate was sealed with parafilm (between the plate cover and upper edge of the wells).
To extract the MTT, the plates were placed on an orbital plate shaker and shaken (120 rpm) for approximately 2 hours at room temperature. At the end of the extraction period, the tissue was not pierced. The corresponding negative, positive, and colorant controls were treated identically.

CELL VIABILITY MEASUREMENTS
Following the formazan extraction, 200 µl sample(s) from each tube (preferably 2×200 µl if possible) was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at the wavelength of 570 nm ((±10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2908 – 2.6589) using isopropanol solutions as the blank (8×200 µl).

Irritation parameter:

other: OD

Run / experiment:

mean

Value:

1.818

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: % corrected final tissue viability

Remarks:

test item

Run / experiment:

mean

Value:

71

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Validity of the test
- The mean OD value of the two negative control tissues was: 2.538
- The positive control result showed 5 % viability at 6 hours exposure.
- The difference of viability between the two tissue replicates 0.5 % to 5.6 %
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Indicator for potential false viability
- Possible direct MTT reduction with test item
No colour change was observed after three hours of incubation during the check for possible direct MTT reduction by the test item. The test item did not interact with MTT, therefore additional controls and data corrections were not necessary. A false estimation of viability can be precluded.

- Colouring potential of test item
The test item has an intrinsic colour (red). If the test item is not white, off white, almost white and/or colorless the additional controls are automatically used and based on the OD results of additional controls the Non Specific Colour % (NSCliving %) is determined. Two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.018. The Non Specific Colourliving % (NSCliving %) was calculated as 0.69 %.

Cell viability
The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:
OD values and viability percentages of the controls:
Controls Optical Density (OD) Viability (%) Δ%
NC: sterile deionized water 1 2.467 97 5.6
2 2.609 103
mean 2.538 100
PC: methyl acetate 1 0.088 3 2.1
2 0.142 6
mean 0.115 5

OD values and viability percentages of the test item:
Test Item Optical Density (OD) Viability (%) CFV NSCliving Δ%
Acid Red 111 1 1.758 69 68 4.7
2 1.878 74 74
mean 1.818 72 71

OD values and NSCliving % of additional control:
Additional colour control Optical Density (OD) Non Specific Colour % (NSCliving %) Δ%
Acid Red 111 1 0.024 0.69 0.5
2 0.011
mean 0.018

Remark: Δ%: The difference of viability between the two relating tissues
CFV NSCliving: Corrected Final Viability
Mean blank value was 0.0384

Interpretation of results:

other: not classified according to the CLP Regulation (EC 1272/2008)

Conclusions:

Not irritant to eye.

Executive summary:

Before treatment the tissues were pre-wetted with approximately 20 μl of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with test item and incubated for 6 hours (± 15 min) at standard culture conditions (37 ± 2 °C in an incubator with 5 ± 1 % CO2, ≥95 % humidified atmosphere).

Exposure of test material was terminated by rinsing with Ca++Mg++ Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours (± 15 min) with MTT solution at 37 ± 2 °C in an incubator with 5 ± 1 % CO2 protected from light, ≥95 % humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

The test item has an intrinsic colour (red), thus two additional test item treated tissues were used for the non-specific OD (Optical Density) evaluation (NSCliving).

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively.

The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes.

The test item Acid Red 111 did not show significantly reduced cell viability in comparison to the negative control (corrected final tissue viability: 71 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to eye.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The skin irritation potential of test item was assessed using the EPISKIN model, according to OECD guideline 439.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes (±0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1 °C for 42 hours (±1 h) in an incubator with 5±1 % CO2, >/= 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (±5 min) with MTT solution at 37±1 °C in 5±1 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

The test item has an intrisic colour (red), therefore two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).

SDS (5 % aq.) and 1x PBS treated (three units/positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

Test item did not show significantly reduced cell viability in comparison to the negative control (mean corrected value:87 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non irritant to skin. Positive and negative controls were valid.

Based on these results, test item has no skin irritation potential under test conditions.

The first eye irritation test was the Isolated Chicken Eye Test (ICET), according to OECD guideline 438.

Test item was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

The test item and positive control (imidazole) applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used; one negative control eye was treated with 30 µl saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 ml saline solution at ambient temperature and this procedure was repeated for each eye.

In this ICET, Acid Red 111 did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1×II, 2×III. Positive and negative controls were valid. 

According to the OECD guideline 438, based on this result, no prediction can be made on classiification.

A second in vitro eye irritation test was run according to OECD guideline 492, using the three-dimensional RhCE tissue in the EpiOcular™ model in vitro.

Before treatment the tissues were pre-wetted and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with test item and incubated for 6 hours (± 15 min) at standard culture conditions (37 ± 2 °C in an incubator with 5 ± 1% CO₂, ≥95% humidified atmosphere). Exposure of test material was terminated by rinsing with Ca⁺⁺Mg⁺⁺Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours (± 15 min) with MTT solution at 37 ± 2 °C in an incubator with 5 ± 1% CO₂ protected from light, ≥95% humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

The test item has an intrinsic colour (red), thus two additional test item treated tissues were used for the non-specific OD (Optical Density) evaluation (NSC_living).

Positive (methyl acetate) and negative (sterile deionized water) controls were valid.

Test item did not show significantly reduced cell viability in comparison to the negative control (corrected final tissue viability: 71 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore, test item was considered to be non-irritant to eye.

Test results revealed no eye irritation potential under the applied testing conditions.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), skin corrosion means the production of irreversible damage to the skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test substance for up to 4 hours. Corrosive reactions are typified by ulcers, bleeding, bloody scabs, and, by the end of observation at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia, and scars. Histopathology shall be considered to evaluate questionable lesions.

Skin irritation means the production of reversible damage to the skin following the application of a test substance for up to 4 hours.

Serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application.

Eye irritation means the production of changes in the eye following the application of test substance to the anterior surface of the eye, which are fully reversible within 21 days of application.

According to the relevant testing guidelines, i.e. OECD guideline 439 and 492, Acid Red 111 has no classification as skin irritant/skin corrosive or eye irritant/eye damaging according to the CLP Regulation (EC 1272/2008).