[2-[4-(dihexylamino)-2-methylbenzylidene]benzo[b]thien-3(2H)-ylidene]malononitrile S,S-dioxide

Endpoint summary

Description of key information

Not irritant to skin

Not irritant to eyes

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 September 25th to 27th, 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:

2019

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 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 irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD 404) for the purposes 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:

SKIN DISK PREPARATION
- Procedure used: adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum
- Quality control for skin discs: quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature during pre-icubation (day -1 to 0): epidermis units were placed with maintenance medium, 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.
- Temperature used during treatment / exposure (day 0): 23.1 - 23.8 °C during incubation (15 ± 0.5 min).
- Temperature of post-treatment incubation (day 0-2): 37 °C for 42 ± 1 h in an incubator with 5±1 % CO2, ≥95 % humidified atmosphere.
- Temperature in MTT test after 42 h incubation (day 2): EpiSkin 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 % humidified atmosphere.

REMOVAL OF TEST MATERIAL AND CONTROLS (day 0)
- Number of washing steps: EpiSkin 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).

FORMAZAN EXTRACTION AND CELL VIABILITY MEASUREMENTS (day 2)
At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from the unit (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 (~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.

Following the formazan extraction, 2×200 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).

ASSAY ACCEPTANCE CRITERIA
- The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be ≤ 18.
- The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be ≤ 18.
- For test chemicals, the standard deviation value (SD) of the % viability should be ≤ 18.

PREDICTION MODEL / DECISION CRITERIA
The irritancy potential of test substances is predicted by mean tissue viability of tissues exposed to the test substance. 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. However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion (OECD 431) will be required to decide on its final classification. In case the test chemical is found to be non-corrosive, 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 Category 2.
Depending on the regulatory framework in member countries, 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 %.
The prediction model (PM) is described below:

Criteria for In Vitro interpretation Classification
Mean tissue viability % is ≤ 50 % Category 2 or Category 1
Mean tissue viability % is > 50 % No Category

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: controls for MTT direct interacting chemicals

Amount/concentration applied:

TEST MATERIAL : 10 mg
NEGATIVE CONTROL : 10 µl
POSITIVE CONTROL : 10 µl

Duration of treatment / exposure:

15 ± 0.5 min.

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3 replicates of test item, 3 replicates of negative control, 3 replicates of positive control, 2 replicates of colour controls (NSCliving), 2 replicates of non-specific colour control (NSCkilled), 3 killed test item treated tissues and 3 killed negative control treated tissues used for the MTT evaluation.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

true tissue viability (TTV)

Value:

89.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: as the test item has an intrinsic colour (blue), the check-method for possible direct MTT reduction with test item was not completed for the following reason: The color of the MTT solution was close to black after it was mixed with test item. So the interaction of the test item with MTT solution could not be determined. Based on this information the additional controls were automatically used. This step prevents the false viability which is the possible interaction with MTT, and it occurs if the test item cannot be removed totally from the surface of the tissues. In this situation the direct interaction with MTT was defined based on the OD results of additional controls.
The non-specific MTT reduction (NSMTT) was determined to be 36.4 %. As the NSMTT were below 50 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.

- Colour interference with MTT: test item has an intrinsic colour (blue). 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. 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 0.235. The Non Specific Colourliving % (NSCliving %) was calculated as 24.0 %.
In order to avoid a possible double correction [TODTT (MTT and NSCliving)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was determined. Two additional test item-treated killed tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.475. The Non Specific Colourkilled % (NSCkilled %) was calculated as 48.5 %.

ACCEPTANCE OF RESULTS:
The mean OD value of the three negative control tissues was 0.978. The mean OD value obtained for the positive control was 0.160 and this result corresponds to 16.4 % viability when compared to the results 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.

OD values and viability percentages of controls and test item

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	0.978	100.1
	2	0.993	101.5
	3	0.962	98.4
	mean	0.978	100.0
	standard deviation		1.59
Positive Control: SDS (5 % aq.)	1	0.182	18.6
	2	0.111	11.4
	3	0.187	19.1
	mean	0.160	16.4
	standard deviation		4.33
Test Item:	1	1.033	105.6
	2	1.002	102.5
	3	0.951	97.3
	mean	0.995	101.8
	standard deviation		4.20

OD values of additionale controls for MTT-interacting test item

Additional controls	Optical Density (OD)
Negative control killed tissues: 1x PBS	1	0.124
	2	0.111
	3	0.078
	mean	0.104
Test item treated killed tissues:	1	0.476
	2	0.453
	3	0.451
	mean	0.460

OD values and NCS_living % of additional control

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSCliving %)
test item treated tissues without MTT incubation	1	0.194	24.0
	2	0.276
	mean	0.235

OD values and NCS_killed % of additional control

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSCkilled %)
test item treated killed tissues without MTT incubation	1	0.468	48.5
	2	0.481
	mean	0.475

True tissue viability % (TTV %)

Parameters	Percent value (%)
Mean[%Viability test item]	101.8
Mean[%NSMTT]	36.4
Mean[%NSCliving]	24.0
Mean[%NSCkilled]	48.5
MeanTTV %	89.9

Remark:

NSMTT: Non-Specific MTT reduction

NSC_living: Non-Specific Colour in living tissues

NSC_killed: Non-Specific Colour in killed tissues

TTV%: True tissue viability

Interpretation of results:

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

Conclusions:

Not irritant to skin.

Executive summary:

Disks of EPISKIN (three units) were treated with 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.

SDS (5 % aq.) and 1×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 item has an intrinsic colour (blue), therefore two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).

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.

The test item is a possible MTT-reducer and has an intrinsic colour (blue). To avoid a possible double correction [TODTT (MTT and NSCliving)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed.

A test chemical is identified as requiring classification and labelling according to CLP Regulation (EC 1272/2008) in cat. 2 or cat. 1, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is ≤ to 50 % of the negative control.

In this in vitro skin irritation test using the EPISKIN model, test item did not show significantly reduced cell viability in comparison to the negative control (mean true tissue viability: 89.9%). 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 OD and cell viability values within acceptable limits and standard deviation of all calculated viability values (positive and negative control, test item) was 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 test conditions. Test item is considered to be non-irritant to skin and is not classified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation/corrosion

Disks of EPISKIN (three units) were treated with 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 % CO₂protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5 % aq.) and 1×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 item has an intrinsic colour (blue), therefore two additional test item treated tissues were used for the non-specific OD evaluation (NSC_living).

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.

The test item is a possible MTT-reducer and has an intrinsic colour (blue). To avoid a possible double correction [TODTT (MTT and NSC_living)] for colour interference, a third control for non-specific colour in killed tissues (NSC_killed) was performed.

In this skin irritation test using the EPISKIN model, test item did not show significantly reduced cell viability in comparison to the negative control (mean true tissue viability: 89.9%). 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.

Based on acceptance criteria, the experiment was considered to be valid.

Eye irritation/corrosion

The purpose of this study was to determine the acute ocular irritation potential of the test item Disperse Blue 354 on three-dimensional RhCE tissue in the EpiOcular™ Modelin vitro,according to the procedure described on the OECD guideline 492.

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 (blue), therefore two additional test item treated tissues were used for the non-specific OD (Optical Density) evaluation (NSC_living).

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 item interference with the viability measurement.

The test item is a possible MTT-reducer and has an intrinsic colour (blue). To avoid a possible double correction [TODTT*(MTT and NSC_living)] for colour interference, a third control for non-specific colour in killed tissues (NSC_killed) was performed. Two killed test item treated tissues were used to avoid a possible double correction for colour interference.

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 Disperse Blue 354 did not show significantly reduced cell viability in comparison to the negative control [true tissue viability (TTV%): 97 %]. 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 control viability results (mean tissue viability: 24 %) were below 50% when compared to the viability values obtained from the negative control. Furthermore, the mean OD value of the two negative control tissues (mean OD value: 1.342) were between 0.8 and 2.8. So the positive and negative controls showed the expected 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.

* TODTT: OD true MTT metabolic conversion for treated tissues

Justification for classification or non-classification

According to OECD guideline 439, a test chemical is identified as requiring classification according to the CLP Regulation (EC 1272/2008) in cat. 2 or cat. 1, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is ≤ to 50 % of the negative control.

Under test conditions, 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 and is thus not classified (no category).

According to the OECD guideline 492, a test chemical is identified as reqquiring classification according to the CLP Regulation (EC 1272/2008) in cat. 2 or cat. 1, if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60% of the negative control.

Under test conditions, the test item was considered to be non-irritant to the eyes and is thus not classified (no category).