Disodium 2-(11-oxido-3-oxo-3H-dibenzo[c,h]xanthen-7-yl)benzoate

Endpoint summary

Description of key information

Skin:

In an in vitro skin irritation assay according to OECD Guideline 439 (RhE), the test substance showed skin irritating properties (UN GHS: Category 1 or Category 2).

In an in vitro skin corrosion assay (RhE) according to OECD Guideline 431, the test item is considered as corrosive (UN GHS: Category 1).

Based on the available data the test item is classified as skin corrosive, Cat. 1 according to Regulation (EC) No 1272/2008 (CLP) and no further testing is necessary.

Eye:

Based on the results obtained in an in vitro eye irritation assay according to OECD guideline 492, the test item is categorised as irritant (UN GHS Category 1 or Category 2) as the mean percentage tissue viability was less than 60 % of the negative control. Since the substance is corrosive to the skin based on the available in vitro data, the substance is also to be classified as eye damaging, Category 1.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40Bis (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SCT) was selected as test system to assess the skin corrosion potential of the test item as it represents a recommended in vitro test system according to OECD Guideline No. 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SCT) obtianed from MatTek In Vitro Life Science Laboratories, s.r.o, MlynskéNivy 73, 821 05, Bratislava II, Slovak Republic.
- Tissue batch number(s): 28644

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing with sterile PBS (filling and emptying of insert 20 times in a constant soft stream of PBS)
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Wavelength: 750 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item is considered as MTT reducer as it formed dark black colour solution with MTT. Hence, additional live and freeze killed tissue controls were maintained with and without MTT in duplicates. All assay procedures were performed as for the viable tissue but controls without MTT tissue were incubated with medium instead of MTT solution during the MTT incubation step.
- Method of calculation used:
Calculations for Data Correction Procedure for Colored Test Item:
OD = OD colored tissue (MTT assay) – OD colored tissue (no MTT assay)
In 3 minutes application, as the extract from tissues treated by colored substance (test item detected in step 1) has an OD <5 % of the negative control treated tissue, correction of the OD was not considered.
Correction of results were considered for 1 hour application as the OD of extract from tissues treated by colored substance was >5 % of the negative control treated tissue.

Check for direct MTT reducing substances:
To check whether test item directly reduces MTT or not, 25 mg of the test item was added to 1 mL of the MTT medium in a 6-well plate and incubated in the incubator (37±1 °C, 5±1 % CO2) for 60 minutes. Untreated MTT medium was used as control. Post incubation, MTT solution containing test item turned to dark blue color, hence considered as reducer of MTT. As test item reduced MTT, a functional check using freeze-killed tissue controls (killed controls = KC) was performed in one definitive assay to evaluate whether the test item is not binding to the tissue and leading to a false MTT reduction signal. All assay procedures are performed as for the viable tissue.

Calculations for Data Correction Procedure for MTT reducers:
True viability = Viability of treated tissue – Interference from test chemical
= OD tvt – OD kt
where OD kt = (mean OD tkt – mean OD ukt)
tvt = treated viable tissue
kt = killed tissues
tkt = treated killed tissue
ukt = untreated killed tissue (NC treated tissue)

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

3 min or 1 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure (mean value of tissue 1 and 2)

Value:

108.87

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure (mean value of tissue 1 and 2)

Value:

13.44

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Yes
- Colour interference with MTT: Yes. The test item is considered as MTT reducer as it formed dark black colour solution with MTT.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The technical proficiency of the test method was established by using proficieney chemicals under Bioneeds Study No.: BIO-GT 1000. according to OECD test Guideline No. 431.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Summary of optical density (OD) and viability

3 Minutes Exposure

Treatment		OD	Viability (%)	CV (%)	Classification
Negative Control (Sterile water)	Mean	1.105	100.0	8.68	NC
	±SD	0.096	12.2
	n	2	2	2
Positive Control (Glacial acetic acid)	Mean	0.083	7.5	11.55	C (Category 1A)
	±SD	0.010	1.2
	n	2	2	2
Test Item	Mean	1.203	108.87	-	NC
	±SD	-	-
	n	2	2

 

OD (killed tissue)	=	Mean OD of treated killed tissue	-	Mean OD of untreated killed tissue
0.161	=	0.205	-	0.045
True Viability	=	OD of treated viable tissue	-	OD of killed tissue
1.042	=	1.203	-	0.161

 

1 Hour Exposure

Treatment		OD	Viability (%)	CV (%)	Classification
Negative Control (Sterile water)	Mean	1.838	100.00	0.40	NC
	±SD	0.007	0.6
	n	2	2	2
Positive Control (Glacial acetic acid)	Mean	0.065	3.6	1.08	C (Category 1A)
	±SD	0.001	0.1
	n	2	2	2
Test Item	Mean	0.197	7.40	-	C
	±SD	-	-
	n	2	2

NC = Non Corrosive; C = Corrosive; n = No. of tissues; SD = Standard Deviation; CV = Coefficient of variation

 

 

Data Correction for Colored substance	=	OD of Colored tissue (with MTT)	-	OD of Colored tissue (Without MTT)
1.070	=	1.197	-	0.127
OD (killed tissue)	=	Mean OD of treated killed tissue	-	Mean OD of untreated killed tissue
0.822	=	0.890	-	0.067
True Viability	=	OD of treated viable tissue (MTT Assay)	-	OD of killed tissue
0.147	=	1.203	-	0.161

True viability (%) = 13.44%

Acceptance Criteria:

1. The assay met the acceptance criterion as the mean OD750 of the negative control tissues are 1.105 (after 3 minutes exposure) and 1.838 (after 1 hour exposure), which are in the range of ≥0.8 and ≤2.8.
2. The assay met the acceptance criterion as the mean viability of positive control tissues is 7.5 % (after 3 minutes exposure) and 3.6 % (after 1 hour exposure) which were ≤15 % of the negative control tissues and the SD of the three tissues replicates is 0.0 to 12.2.
3. The assay met the acceptance criterion as the OD of the extraction solvent was ≤0.1.
4. Two tissue replicates per exposure time was in range between 0.40 and 11.55 % viability the coefficient of variation (CV).

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

Based on the results obtained under the conditions of this study, the test item is considered as corrosive to reconstructed Human Epidermis (RhE) in accordance with UN GHS category 1, as the mean percentage tissue viability was greater than 50 % after 3 minutes exposure and less than 15 % after 1 hour exposure of the negative control.

Executive summary:

In an in vitro skin corrosion assay (RhE) according to OECD Guideline 431, the potential of the test item to induce skin corrosion in an in vitro human skin model was investigated. The test item developed dark blue colour when dissolved in distilled water and isopropanol. The test item is considered as MTT reducer as it formed dark black colour solution with MTT. Hence, additional live and freeze killed tissue controls were maintained with and without MTT in duplicates. All assay procedures were performed as for the viable tissue but controls without MTT tissues were incubated with medium instead of MTT solution during the MTT incubation step.

Exposure to the test item was performed for 1 hour and 3 minutes separately. Tissues were treated with 25 mg test item or 50 µL of positive control (glacial acetic acid). At the end of treatment time, tissue inserts were rinsed with sterile PBS to remove any residual test item. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viabilily of tissues was calculated.

For 3 minutes exposure, percentage viability of negative control, positive control and test item was 100±12.2, 7.5±1.2 and 108.87, respectively. Correction procedure for colorant control was not considered as the mean OD of extract from tissues treated by colored test item was less than 5 % of negative control. As the percentage viability of test item was greater than 50 % of the negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 15 % of negative control and clearly represents the irritation potential of positive control.

For 1 hour exposure, percentage viability of negative control, positive control and test item was 100±0.6, 3.6±0.1 and 13.44. The percentage viability of the test item was less than 50 % of negative control. Therefore, the test item is considered as corrosive. The percentage viability of positive control (PC) is less than 15 % of negative control clearly represents the irritation potential of positive control.

Based on the results obtained under the conditions of this study, the test item is considered as corrosive to Reconstructed Human Epidermis (RhE) in accordance with UN GHS category 1, as the mean percentage tissue viability was greater than 50 % after 3 minutes exposure and less than 15 % after 1 hour exposure of the negative control.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 March - 25 April, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

As recommended in OECD Guideline No 439, the Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SIT) has been selected as test system for in vitro skin irritation.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200-SIT), MatTek In Vitro Life Science Laboratories, s.r.o, MlynskéNivy 73, 821 05, Bratislava II, Slovak Republic

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 min at 37 °C, 25 min at room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues was rinsed with sterile DPBS/HBSS by filling and emptying the tissue insert for 15 times to remove any residual test item. The constant stream of DPBS/HBSS was applied from nearest distance from the tissue surface. After the 15th rinse with washing bottle, the insert was completely submerged 3 times in approximately 50 mL of DPBS/HBSS and shaken to remove all traces of test item/NC/PC.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: multiplate reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues and killed tissues
- Procedure used to prepare the killed tissues: freeze-killed
- N. of replicates: 3 each
- Method of calculation used: MTT assay

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

PREDICTION MODEL / DECISION CRITERIA
- The test item may be identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the tissue viability after exposure and post-treatment incubation is ≤ 50 %.
- The test item is considered as non-irritant to skin in accordance with UN GHS No Category, if the tissue viability after exposure and post-treatment incubation is > 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5 %

Duration of treatment / exposure:

1 h

Duration of post-treatment incubation (if applicable):

36 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

2.53

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Yes
- Colour interference with MTT: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Interpretation of results:

other: Category 1 (corrosive) or Category 2 (irritant) based on GHS criteria

Conclusions:

In an in vitro skin irritation assay (RhE) according to OECD Guideline 439, the test item showed skin irritating properties (UN GHS Category 1 or 2).

Executive summary:

In an in vitro irritation assay according to OECD Guideline 439, the skin irritating properties of the test substance was determined using Reconstructed Human Epidermal Model - EpiDerm™ (EP1-200-SIT). The test item developed colour when dissolved in deionized water and isopropanol. Test item was considered as MTT reducer as it formed a dark black coloured solution with MTT. Hence, additional live and freeze killed tissue controls were maintained with and without MTT in triplicates. All assay procedures were performed as for the viable tissue but tissues without MTT were incubated with medium instead of MTT solution during the MTT incubation step. After receipt of the tissues, visual inspection was done to verify no defects. As there were no tissue defects, air bubble or excess moisture observed, all tissue inserts were used for the study. After pre - incubation period, the tissues were topically exposed to 30 µL of DPBS (negative control: NC), 30 µL of 5 % aq. SDS solution (positive control: PC) or 25 mg of test item (Mordant Red 27). All the treatments were maintained in triplicates. After 60 minutes of exposure with negative control, positive control or test item, the tissues were washed using DPBS. The tissue inserts were blotted and transferred to fresh medium and incubated in CO2 incubator for 24 hours. After a media change, tissues were incubated for an additional 21 h in the incubator. After post-incubation period, the bottom of the tissue inserts was blotted and transferred into an MTT solution and incubated for ca. 3 h. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, was extracted for ca. 2 h using extraction solvent (MTT-EXT-100). The optical density of the extracted formazan was measured in a 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated from OD values.

Percentage viability of negative control, positive control and test item was 100±0.53, 5.5±0.08 and 2.53 respectively. As the percentage viability of the test item was not greater than 50 % of the negative control, the test item is considered as "irritant". The percentage of viability in the positive control (PC) was less than 50 %, which shows the irritative potential of the PC and the suitability of the test method.

Based on the results obtained under the laboratory testing conditions, the test item is identified to be irritant to Reconstructed Human Epidermis (RhE) in accordance with UN GHS Category 2 or Category 1, as the mean percentage tissue viability was less than 50 % of the negative control.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

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:

30 January - 03 February, 2018

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:

09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: As recommended in OECD Guideline for Eye Irritation, EpiOcular™ reconstructed human ocular tissue has been selected as test system for in vitro eye irritation. Supplied by MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, Bratislava - Slovakia
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: See "Any other information on materials and methods"

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

30 min post soak period, 18 h post treatment incubation

Number of animals or in vitro replicates:

2

Details on study design:

- RhCE tissue construct used, including batch number: EpiOcular™ cornea epithelial model (OCL-200-EIT). Lot No. 27021
- Doses of test chemical and control substances used:
Test item: 50 mg
Neg. Control: 50 µL
Pos. Control: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Exposure: 6 h at 37 °C
Post-exposure immersion: 30 min at room temperature
Post-exposure incubation: 18 h at 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
Test item developed colour when dissolved in distilled water and isopropanol. Post incubation optical density of samples were measured at 570 nm and the OD readings were >0.08. Test item was considered as possibly interacting with the MTT measurement and an additional test on colorant controls was performed to determine the amount of color bound to and extracted from the tissues. For this purpose the colored test item is applied to two additional tissues, the colorant controls (CC).
The test item was pre checked for direct MTT reduction in quantitative MTT estimation. Post incubation, the test item with MTT formed black color. As there was color change, test item was considered as reducer of MTT.
As test item reduced MTT, a functional check using freeze-killed tissue controls (killed controls = KC) was performed in one definitive assay to evaluate whether the test item is not binding to the tissue and leading to a false MTT reduction signal.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled):
All controls were perfomed in dublicates
- Wavelength of measuring device: 570 nm, 96-well plate reader
- Description of the method used to quantify MTT formazan:
Optical density (OD) measurement of the MTT extracts.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
A test item is labeled as “non-irritant”, if the test item treated percent tissue viability determined by MTT assay is >60 % relative to the negative control treated tissue viability.
A test item is labeled as “irritant”, if the percent tissue viability determined by MTT assay is <60 % relative to the negative control treated tissue viability.

Irritation parameter:

other: cell viability [%]

Value:

-2.75

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

Table 2: Summary of Optical Density (OD) and Viability (%)

Treatment		OD	Viability (%)	Viability difference between tissues	Classification
Negative Control (DBPS)	Mean	1.252	100.00	0.26	NI
	±SD	0.006	0.52
	n	2	2	2
Positive Control (Methyl acetate)	Mean	0.076	6.1	0.14	I
	±SD	0.004	0.28
	n	2	2	2
Test Item	Mean	-	-2.75	-	I
	±SD		-
	n		2
NI = Non Irritant; I = Irritant; n = No. of tissues

Interpretation of results:

other: UN GHS Category 1 or Category 2

Conclusions:

Based on the results obtained in an in vitro eye irritation assay according to OECD guideline 492, the test item is categorised as irritant (UN GHS Category 1 or Category 2) as the mean percentage tissue viability is less than 60 % of the negative control.

Executive summary:

The eye irritating potential of the test item was determined in an in vitro eye irritation assay (RhCE) according to OECD guideline 492. Two replicates of EpiOcular™ tissues were treated with test item (50 mg) , positive control (methyl acetate, 50 µL) or negative control (sterile deionized water, 50 µL), respectively and incubated for 6 hours (± 15 min) at standard culture conditions (37 °C in an incubator with 5 % CO2, 90±10 % humidified atmosphere).

Exposure of test material was terminated by rinsing with Ca++Mg++Free-DPBS solution. After rinsing, the tissues were incubated for a 30 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). The viability of each tissue was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically (570 nm).

The test item has an intrinsic colour (dark black), therefore, two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. Since the test item is a possible MTT-reducer and has an intrinsic colour (dark black) a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.

The test item induced significantly reduced cell viability in comparison to the negative control (mean relative viability: -2.75 %). 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 has an eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, the test item is considered as irritating to the eye (UN GHS Category 2 or 1). For a reliable assessment, further in vitro testing is necessary for a final classification.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/ corrosion:

Weight of evidence approach:

The objective was to assess the skin irritation and corrosion potential of the test substance. By using the currently available methods a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (according to OECD guideline 431) and the Skin Irritation Test (according to OECD 439). The studies were assessed in a weight of evidence approach.

In an in vitro irritation assay according to OECD Guideline 439, the skin irritating properties of the test substance was determined using Reconstructed Human Epidermal Model - EpiDerm™ (EP1-200-SIT). The test item developed colour when dissolved in deionized water and isopropanol. Test item was considered as MTT reducer as it formed a dark black coloured solution with MTT. Hence, additional live and freeze killed tissue controls were maintained with and without MTT in triplicates. All assay procedures were performed as for the viable tissue but tissues without MTT were incubated with medium instead of MTT solution during the MTT incubation step. After receipt of the tissues, visual inspection was done to verify no defects. As there were no tissue defects, air bubble or excess moisture observed, all tissue inserts were used for the study. After pre - incubation period, the tissues were topically exposed to 30 µL of DPBS (negative control: NC), 30 µL of 5 % aq. SDS solution (positive control: PC) or 25 mg of test item (Mordant Red 27). All the treatments were maintained in triplicates. After 60 minutes of exposure with negative control, positive control or test item, the tissues were washed using DPBS. The tissue inserts were blotted and transferred to fresh medium and incubated in CO2 incubator for 24 hours. After a media change, tissues were incubated for an additional 21 h in the incubator. After post-incubation period, the bottom of the tissue inserts was blotted and transferred into an MTT solution and incubated for ca. 3 h. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, was extracted for ca. 2 h using extraction solvent (MTT-EXT-100). The optical density of the extracted formazan was measured in a 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated from OD values.

Percentage viability of negative control, positive control and test item was 100±0.53, 5.5±0.08 and 2.53 respectively. As the percentage viability of the test item was not greater than 50 % of the negative control, the test item is considered as "irritant". The percentage of viability in the positive control (PC) was less than 50 %, which shows the irritative potential of the PC and the suitability of the test method.

Based on the results obtained under the laboratory testing conditions, the test item is identified to be irritant to Reconstructed Human Epidermis (RhE) in accordance with UN GHS Category 2 or Category 1, as the mean percentage tissue viability was less than 50 % of the negative control.

In an in vitro skin corrosion assay (RhE) according to OECD Guideline 431, the potential of the test item to induce skin corrosion in an in vitro human skin model was investigated. The test item developed dark blue colour when dissolved in distilled water and isopropanol. The test item is considered as MTT reducer as it formed dark black colour solution with MTT. Hence, additional live and freeze killed tissue controls were maintained with and without MTT in duplicates. All assay procedures were performed as for the viable tissue but controls without MTT tissues were incubated with medium instead of MTT solution during the MTT incubation step.

Exposure to the test item was performed for 1 hour and 3 minutes separately. Tissues were treated with 25 mg test item or 50 µL of positive control (glacial acetic acid). At the end of treatment time, tissue inserts were rinsed with sterile PBS to remove any residual test item. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viabilily of tissues was calculated.

For 3 minutes exposure, percentage viability of negative control, positive control and test item was 100±12.2, 7.5±1.2 and 108.87, respectively. Correction procedure for colorant control was not considered as the mean OD of extract from tissues treated by colored test item was less than 5 % of negative control. As the percentage viability of test item was greater than 50 % of the negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 15 % of negative control and clearly represents the irritation potential of positive control.

For 1 hour exposure, percentage viability of negative control, positive control and test item was 100±0.6, 3.6±0.1 and 13.44. The percentage viability of the test item was less than 50 % of negative control. Therefore, the test item is considered as corrosive. The percentage viability of positive control (PC) is less than 15 % of negative control clearly represents the irritation potential of positive control.

Based on the results obtained under the conditions of this study, the test item is considered as corrosive to Reconstructed Human Epidermis (RhE) in accordance with UN GHS category 1, as the mean percentage tissue viability was greater than 50 % after 3 minutes exposure and less than 15 % after 1 hour exposure of the negative control.

According to OECD 439 the test item is considered to have skin irritating properties (UN GHS: Category 2 or Category 1). Further testing according to OECD 431 showed that the test item is considered as corrosive to skin. Based on the results of both in vitro tests in the weight of evidence approach, the test item is considered to be corrosive to the skin, cat. 1 (H314).

Eye:

The eye irritating potential of the test item was determined in an in vitro eye irritation assay (RhCE) according to OECD guideline 492. Two replicates of EpiOcular™ tissues were treated with test item (50 mg) , positive control (methyl acetate, 50 µL) or negative control (sterile deionized water, 50 µL), respectively and incubated for 6 hours (± 15 min) at standard culture conditions (37 °C in an incubator with 5 % CO2, 90±10 % humidified atmosphere).

Exposure of test material was terminated by rinsing with Ca++Mg++Free-DPBS solution. After rinsing, the tissues were incubated for a 30 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). The viability of each tissue was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically (570 nm).

The test item has an intrinsic colour (dark black), therefore, two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. Since the test item is a possible MTT-reducer and has an intrinsic colour (dark black) a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.

The test item induced significantly reduced cell viability in comparison to the negative control (mean relative viability: -2.75 %). 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 has an eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, the test item is considered as irritating to the eye (UN GHS Category 2 or 1). For a reliable assessment, further in vitro testing is necessary for a final classification.

Based on the available in vitro data on skin irritation/corrosion, the test substance is corrosive to the skin and is consequently also to be classified with Category 1 (H318, causes serious eye damage) for causing serious damage to the eye.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on irritation/ corrosion, the test item is to be classified as corrosive to the skin, Cat. 1 (H314) and is consequently also to be classified as eye damaging, Cat. 1 (H318) according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.