Benzoic acid, 5-[(4-aminophenyl)azo]-2-hydroxy-, reaction products with 3-[(4-amino-3-methoxyphenyl)azo]benzenesulfonic acid and carbonic dichloride, sodium salts

Endpoint summary

Description of key information

Validated and accepted in vitro tests for eye corrosion/irritation and in vitro skin irritation have been conducted.

It was In vitro Skin Irritation Test (OECD TG No. 439) and In vitro Eye Irritation Test (OECD TG No.492) and BCOP test (OECD TG No. 437).

- Study No. 388/16/4AI: Direct Yellow 44 - In vitro Skin Irritation Test (EpiDermTM Model); VUOS-CETA Report No. 16-636, 2016.

Result: no category in regard to skin irritation

- Study No. 388/16/5AI: Direct Yellow 44 - In vitro Eye Irritation Test (EpiOcularTM Model); VUOS-CETA Report No. 18-4, 2018.

Result: substance potentially requiring classification and labelling, further testing with other test methods required

Under the above-described experimental design average viability of treated tissues by the test substance Direct Yellow 44 was 2.7 % of negative control average value i.e. viability was ≤ 60 %. The effect of the test substance was positive in EpiOcularTM model (tissues were damaged).

According to the classification criteria, the test substance, Direct Yellow 44, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1). Further testing with other test methods will be required.

- Study No. 388/16/5BCOP: Direct Yellow 44 - Bovine Corneal Opacity and Permeability Test; VUOS-CETA Report No. 17-735, 2017

Result: no category in regard to eye irritation

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:

29.09.2016 - 07.10.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted: 28th July, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 761/2009, 23rd July 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Protocol for: INVITRO EpiDermTM SKIN IRRITATION TEST For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm

Version / remarks:

Model EPI-200-SIT, Rev. 26/3/2012,1-37

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: tissue for research puposes from accredited institutions

Source strain:

other: Keratinocyte strain 00267

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUETissues: the reconstructed human epidermal model EpiDerm (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot No. 23361TEMPERATURE USED FOR TEST SYSTEMculture conditions 37±1°C, 5±1 % CO2, moistened tissueREMOVAL OF TEST MATERIAL AND CONTROLS- Volume and number of washing steps: thoroughly rinsed with PBS, blotted to remove the test substanceMTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 1 mg·mL-1- Incubation time: 180±5 mins- Spectrophotometer: Libra S22 at 570 nm. Isopropyl alcohol serves as a blank. FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATABased on Certificate of Analysis the model passed all parametres for viability, barrier function, sterility.NUMBER OF REPLICATE TISSUES: 3CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE- killed tissues- Procedure used to prepare the killed tissues (if applicable): by freezing- N. of replicates : 2NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:A single testing, composed of three replicate tissues, was run.Direct MTT reduction - functional check in tubes => the test substance is not directly-reducing.1. Colour interference => colorant controls were added to tissues in the MTT test2. MTT testPREDICTION MODEL / DECISION CRITERIAOECD Test Guideline No. 439 (1), par. 36:- In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS (3) Category 2. - The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test substance (25 mg of substance/surface ratio 39.7 mg/cm2) was placed directly on tissue moistened with 25 µL of PBS and spread on the tissue surface. A single testing, composed of three replicate tissues, was run.NC: PBS (phosphate buffered saline), prepared in laboratory 30/08/2016, exp. 02/03/2017PC: 5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 012616TMB, exp. 26/01/2017

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

99.8

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All study acceptance criteria were fulfilled. The mean OD570 of the NC tissue was 2.373 ±0.115 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8.The mean viability of the PC tissues expressed as % of the negative control tissues is 3.3% which meets the acceptance criterion of ≤ 20 %. The SD calculated from individual % tissue viabilities of the 3 identically treated replicates for the positive control, negative control and test substance was < 18 %.

MTT test

OD₅₇₀values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

Treatment	OD570			Mean	SD	%NC
PBS (NC)	2.450	2.458	2.211	2.373	0.115
viability (%NC)	103.2	103.6	93.2	100.0	4.8	100.0
388/16 (C5)	2.314	2.456	2.344	2.371	0.061
viability (%NC)	97.5	103.5	98.8	99.9	2.575	99.9
5% SDS (PC)	0.063	0.087	0.082	0.077	0.010
viability (%NC)	2.7	3.7	3.5	3.3	0.4	3.3

388/16 (C5 CC)	0.004	0.002	-	0.003	0.001
viability (%NC)	0.2	0.1	-	0.1	0.069	0.1

PBS - phosphate buffered saline

SDS - sodium dodecyl sulphate

CC - colorant controls

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design average viability of treated tissues was 99.9 % (99.8 % after correction), i.e. viability was > 50 %. The effect of the test item was negative in EpiDermTM model (tissues were not damaged).According to the classification criteria the test substance, Direct Yellow 44, is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant.

Executive summary:

The test item, Direct Yellow 44, was assayed for the in vitro skin irritation in human epidermal model EpiDermTM. The test was performed according to the OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method test (2015) and Protocol for: In Vitro EpiDerm^TMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (2012).

In preliminary experiment no colour interference with the endpoint was found.

Direct MTT reduction of test substance was not solved by a test in test tube due to its blue colour. Therefore test in frozen tissues was performed directly.

After pre-incubation of tissues, 25 mg of the test substance was placed directly atop to the previously moistened tissue and it was spread on the entire tissue surface. The length of exposure was 60 minutes. Three tissues were used for the test substance and every control.

After removal of the test substance, tissues were post-incubated for approximately 42 hours due to leave of damage reparation. Three-hour incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

At direct reduction assay in a test tube MTT medium was coloured yellow, so direct MTT reduction was excluded. All test conditions were fulfilled. Under the above-described experimental design average viability of treated tissues was 99.9 % (99.8 % after correction), i.e. viability was > 50 %.

The effect of the test substance was negative in EpiDerm^TMmodel (tissues were not damaged).

According to the classification criteria, the test substance, Direct Yellow 44, is considered to have no category in regard to skin irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22.11. – 23. 11. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 14th Feb 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES- Source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic- Characteristics of donor animals (e.g. age, sex, weight): The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 30 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test. - Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).- Time interval prior to initiating testing: The time interval between collection of the eyes and use of corneas in the BCOP was minimized (typically collected and used on the same day). All eyes used in the assay were from the same group of eyes collected on a specific day.- indication of any existing defects or lesions in ocular tissue samples: Only corneas from eyes free of defects including scratched, and neovascularisation were used. The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded.

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL - Amount(s) applied (volume or weight with unit): 2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution. VEHICLE Hank`s Balanced Salts Solution (HBSS) - Lot/batch no. (if required): SLBL5152V, Sigma-Aldrich

Duration of treatment / exposure:

4 hrs

Duration of post- treatment incubation (in vitro):

1.5 hr

Number of animals or in vitro replicates:

The results were based on the selection criteria for the eyes, as well as the positive and negative control responses.Number of corneas per group: Exposed group (test substance) - 3 corneas (No. 7, 8, 9) Positive control group (20% Imidazole) – 3 corneas (No. 4, 5, 6) Negative control group (0.9% NaCl) – 3 corneas (No. 1, 2, 3)

Details on study design:

SELECTION AND PREPARATION OF CORNEASSelection criteria for eyes used in BCOP: Only corneas from eyes free of defects including scratched, and neovascularisation were used. The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded. Preparation: Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium an endothelium. Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ± 1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and a baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded. Each test group (test substance, concurrent negative and positive controls) consisted of the three eyes. The three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups. QUALITY CHECK OF THE ISOLATED CORNEASFrom 19 eyes the 2 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 1, 2, 3, 4, 5, 6, 7, 8 and 9), 8 eyes were superfluous.NUMBER OF REPLICATESNumber of corneas per group:Exposed group (test substance) - 3 corneas (No. 7, 8, 9) Positive control group (20% Imidazole) – 3 corneas (No. 4, 5, 6) Negative control group (0.9% NaCl) – 3 corneas (No. 1, 2, 3) NEGATIVE CONTROL USED0.9% NaClSOLVENT CONTROL USED (if applicable)0.9% NaClPOSITIVE CONTROL USED20% ImidazoleAPPLICATION DOSE AND EXPOSURE TIME2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution. Open-chamber method was used, because the test substance at 20% concentration was viscous. The test substance (the test substance in quantity enough to completely cover the cornea) was applied directly to the epithelial surface of the cornea using the spatula. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system.Exposure time: 4 hrsTREATMENT METHOD: open-chamber method POST-INCUBATION PERIOD: 1.5 hrREMOVAL OF TEST SUBSTANCE, POST-EXPOSURE INCUBATIONAfter the exposure period, the negative control and the positive control substance was removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded. The test substance was removed from the anterior chamber with EMEM – more repeatedly, because the test substance is coloured (yellow colour). The corneas (applied the test substance) were also rinsed with EMEM (containing phenol red). Lastly EMEM (without phenol red) was used for final rinsing. The test substance was complete removal. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.METHODS FOR MEASURED ENDPOINTS: - Corneal opacity: measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale- Corneal permeability: The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYSTM 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.SCORING SYSTEM: In Vitro Irritancy Score (IVIS)IVIS = mean opacity value + (15 x mean permeability OD490 value)DECISION CRITERIA: IVISUN GHS ≤ 3 No Category> 3; ≤ 55 No prediction can be made ≥ 55 Category 1

Irritation parameter:

in vitro irritation score

Value:

2.05

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS: Study acceptance criteria were fulfilled.- Acceptance criteria met for negative control: The value of opacity for negative control (0.9% NaCl) obtained during the study was 0.61 and value of permeability was 0.045. The values obtained during this study not exceeded upper limits, so the study is considered acceptable.- Acceptance criteria met for positive control: The value of IVIS for positive control (20% imidazole) obtained during the study was 79.04. This value is within the acceptance limit (one standard deviations of the current historical mean), so the study is considered acceptable.

The In Vitro Irritancy Score (IVIS) was computed according the following formula:

IVIS = mean opacity value + (15 x mean permeability OD490 value)

Group	IVIS calculation	Result
NC (0.9% NaCl)	0.61 + 15 x 0.045	1.29
PC (20% Imidazole in 0.9% NaCl)	49.91 + 15 x 1.942	79.04
EXP (Direct Yellow 44)	2.05 + 15 x 0.0	2.05

Interpretation of results:

GHS criteria not met

Conclusions:

The In Vitro Irritancy Score (IVIS) for Direct Yellow 44 was 2.05.This value of IVIS is ≤ 3 therefore the classification of test substance effect according to UN GHS criteria for eye irritation or serious eye damage is: No category.

Executive summary:

The test substance, Direct Yellow 44, was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea.

The test was performed according to the Method B.47, 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, Adopted 14th February 2017

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used.

Open-chamber method was used, because the test substance was viscous. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) is to be calculated from the values of opacity and permeability.

The In Vitro Irritancy Score (IVIS) for Direct Yellow 44 was 2.05.

This value of IVIS is ≤ 3 therefore the classification of test substance effect according to UN GHS criteria for eye irritation or serious eye damage is: No category.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07.12.2017 - 14.12.2017

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:

Adopted: 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SOP: EpiOcularTM EIT for the prediction of acute ocular irritation of chemicals

Version / remarks:

Version 9, June 29, 2015, MatTek corp.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: keratinocyte strain 4F1188

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicabilityCell damage (cytotoxicity), playing an important, if not the primary, mechanistic role in determining the overall serious eye damage/eye irritation response of a chemical regardless of the physicochemical processes underlying tissue damage, is followed in this test.This test uses an in vitro procedure allowing the identification of chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS. This test is not able to distiguih between serious eye damage and eye irritation. - Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell liveThe RhCE tissues are reconstructed from primary human cells, which have been cultured for several days to form a stratified, highly differentiated squamous epithelium, morphologically similar to that found in the human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.The reconstructed human cornea-like epithelial model EpiOcular™ comes from MatTek, Bratislava, SK, supplied with Certificate of Analysis. Lot No. of tissues used for this test: 27017 kit B.On the day of receipt, EpiOcularTM tissues were conditioned to release transport stress related compounds and debris by incubation in assay medium delivered by MatTek for test performance for 62 minutes at standard culture conditions and, after media replacement, overnight (following 18 hours 28 minutes) also standard at culture conditions.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test substance (50 mg of substance/surface ratio 39.7 mg/cm2/per tissue) is placed directly atop to the tissue moistened with 20 µL of PBS. The test substance was spread over entire tissue surface.A single testing, composed of two replicate tissues, was run.

Duration of treatment / exposure:

6 ± 0.25 hours

Duration of post- treatment incubation (in vitro):

18±0.25 hours

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure usedDirect MTT reduction - functional check in tubes => change of colour was caused by colour of the test substance; no change towards blue colour was observed.1. Colour interferenceAverage isopropyl alcohol OD570 was 0.0455, average test substance extract OD570 was 0.1005. 0.1005-0.0455=0.0550As the difference OD570 caused by the test substance is <0.08, colour of the test item did not interfere with the endpoint, next steps did not need to be performed2. MTT testA single testing, composed of 2 replicate tissues, is run (plus for the positive control (PC) and negative control (NC)).After treatment the test item swell on tissues and it was irremovable by submersion into PBS and shaking in it. The “plug” arisen was disturbed by a spatula and the test item was partially scraped before rinsing in beaker with PBS. In the end the test item was wiped with a cotton swab, so all the test item was removed.Both tissues were separated from the plastic bottom and No. 1 was ruptured. No part of tissue was lost but it was partially quire fold.Tissues were coloured yellow even after extraction with isopropyl alcohol. - RhCE tissue construct used, including batch numberThe reconstructed human cornea-like epithelial model EpiOcular™ comes from MatTek, Bratislava, SK. Lot No. of tissues used for this test: 27017 kit B- Doses of test chemical and control substances usedThe test substance (50 mg of substance/surface ratio 39.7 mg/cm2) is placed directly atop to the tissue moistened with 20 µL of PBS. PC: Methyl Acetate 99%, MatTek, Lot No. 032817ISANC: water for injection- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)6 hours at standard culture conditions (37±1°C, 5±1 % CO2, humidified incubator)25 ± 2 minute immersion incubation (post-soak) at room temperature18 hours at standard culture conditions (37±1°C, 5±1 % CO2, humidified incubator)- Description of any modifications to the test procedure:Assay acceptance criterion was not fulfilled for absorbancies of extracts from positive control tissues. As all the tissues had viabilities under 50% of negative control viability we consider that this deviation has not impact on study results.- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)OD570 is measured on a plate reader Biotek Epoch. Isopropyl alcohol serves as a blank. No external filter is used.- Description of the method used to quantify MTT formazanOptical density (OD570) of isopropyl alcohol extracts was measured on a plate reader. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues. Then the mean relative tissue viability of two individual tissues exposed to the test substance is calculated – this value is, after correction, used for the comparison with limit value.Tests for colour interference and direct reduction did not demonstrate influence of colour or reductive properties of the test item on study results. Thus, no steps for correction of results were performed.- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction modelThe percentage tissue viability cut-off value distinguishing classified from non-classified test chemicals is 60%. Results should thus be interpreted as follows:- The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.- The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60%. - Positive and negative control means and acceptance ranges based on historical data1) The negative control OD > 0.8 and < 2.5, 2) The mean relative viability of the positive control is below 50% of control viability 3) The difference of viability between the two relating tissues of a single chemical is < 20% in the same run. This applies also to the killed controls and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

other: average viablility

Run / experiment:

1

Value:

2.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: i.e. viability was ≤ 60 %

Other effects / acceptance of results:

All study acceptance criteria were fulfilled.

Table 1: OD₅₇₀values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

The data presented are corrected by subtraction of OD570 isopropyl alcohol itself (blank, 0.082).

Code	Treatment	OD570			mean	SD	Viability %
		Tissue 1	Tissue 2	Tissue 3		%SD
NC	water	1.488	1.516	1.909	1.638	0.175	100.0
	% NC	90.9	92.5	116.6	100.0	11.7
C2	388/16	0.038	0.052	NT	0.045	0.004	2.7
	% NC	2.3	3.2	NT	2.7	16.2
PC	99% MA	0.321	0.247	0.414	0.327	0.069	20.0
	% NC	19.6	15.1	25.3	20.0	21.0

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the above-described experimental design average viability of treated tissues by the test substance Direct Yellow 44 was 2.7 % of negative control average value i.e. viability was ≤ 60 %. The effect of the test substance was positive in EpiOcularTM model (tissues were damaged). According to the classification criteria, the test substance, Direct Yellow 44, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1). Further testing with other test methods will be required.

Executive summary:

The test item, Direct Yellow 44, was assayed for the in vitro eye irritation in human cornea-like model EpiOcular^TM. The test was performed according to the OECD Test Guideline No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Details of the procedure are given in Protocol: EpiOcular^TMEye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals (MatTek 06/29/2015).

After pre-incubation and wetting of tissues, 50 mg of the test item was placed directly atop to the tissue and it was spread on the entire tissue surface. Length of exposition was 6 hours at 37±1°C in humidified CO2 incubator (5±1% CO2). Two tissues were used for the test item and 3 for every control.

After removal of the test item, tissues were post-soaked in medium for approximately 25 minutes and post-incubated for about 18 hours at culture conditions. Three hours incubation with MTT and 2-3 hours extraction period with shaking followed then. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a plate reader. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Tests for colour interference and direct reduction did not demonstrate influence of colour or reductive properties of the test item on study results. Thus, no steps for correction of results were performed.

Under the above-described experimental designaverage viability of treated tissues was 2.7% i.e. viability was ≤60 %.

The effect of the test substancewaspositiveinEpiOcular^TMmodel (tissues were damaged).

According to the classification criteria given in chapter 4.5., the test substance,Direct Yellow 44, isidentified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Further testing with other test methods will be required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on in vitro / ex vivo tests the test substance Direct Yellow 44 was non-irritating for skin and eye.