Leuco polysulfided 4-[(2,4-dinitrophenyl)amino]phenol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August - 10 October, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July, 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Version / remarks:

2009 February

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Leuco polysulfided 4-[(2,4-dinitrophenyl)amino]phenol

EC Number:

310-132-3

EC Name:

Leuco polysulfided 4-[(2,4-dinitrophenyl)amino]phenol

Cas Number:

61902-31-6

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 4-[(2,4-dinitrophenyl)amino]phenol with sodium polysulfide, leuco derivatives

Test material form:

solid: particulate/powder

Details on test material:

Test item: Leuco Sulfur Blue 11
Appearance: dark black powder
CAS No. 61902-31-6
EC No. 310-132-3
Storage: room temperature

Specific details on test material used for the study:

Expiration date: 29 April 2020
Storage: Room temperature (15-25°C)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

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 TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) 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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model.
- Tissue batch number: 16-EKIN-037
- Date of initiation of testing: 2016-09-14

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the EpiSkinTMSM units were 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
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h ± 15 min
- Spectrophotometer: 96-well plate spectrophotometer
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls, 2 replicates color controls and 2 replicates non-specific colour control were used.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: incubation in distilled water followed by freezing.
- N. of replicates: 3 killed treated tissues and 3 killed negative control tissues are used for the MTT evaluation.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating/corrosive to skin if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.
- The test substance is considered to be non-irritant to skin if 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

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 %

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 hours ± 1 hour

Number of replicates:

3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls, 2 replicates color controls and 2 replicates non-specific colour control were used. Furthermore, 3 killed treated tissues and 3 killed negative control tissues are used for the MTT evaluation.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

1-3

Value:

104

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:
- Visible damage on test system: No
- Direct-MTT reduction: The non-specific MTT reduction (NSMTT) was determined to be 0.017 %. 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: As the test item has an intrinsic colour (dark black), 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 0.013. The Non Specific Colour % (NSC %) was calculated as 1.5 %. Therefore additional data calculation was not necessary. A false estimation of viability can be precluded

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

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

Any other information on results incl. tables

Table 1: Summary of neg. and pos. control results

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	1.018	117
	2	0.823	95
	3	0.765	88
	mean	0.869	100
	standard deviation (SD)		15.22
Positive Control: SDS (5 % aq.)	1	0.167	19
	2	0.168	19
	3	0.072	8
	mean	0.136	16
	standard deviation (SD)		6.34

Table 2: OD values and viability percentages of the test item (including corrected values)

Test Item	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)
Leuco Sulfur Blue 11	1	1.059	1.059	122	122
	2	0.811	0.811	93	93
	3	0.845	0.844	97	97
	mean	0.905	0.905	104	104
	standard deviation (SD)			15.49	15.49

TOD_TT :true MTT metabolic conversion

Table 3: OD values of additional controls for MTT-interacting test item

Additional controls	Optical Density (OD)
Negative control killed tissues: 1x PBS	1	0.065
	2	0.078
	3	0.079
	mean	0.074
Test item treated killed tissues: Leuco Sulfur Blue 11	1	0.066
	2	0.059
	3	0.099
	mean	0.075

 

 

Table 4: OD values and NSC % of additional control

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSC %)
Leuco Sulfur Blue 11 (test item treated tissueswithout MTT incubation)	1	0.008	1.5
	2	0.019
	mean	0.013

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vivo skin irritation assay (recontructed human epidermis) according to OECD 439, a mean cell viability of 104 % was determined. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Executive summary:

An in vitro skin irritation assay (recontructed human epidermis, RhE) has been performed according to the OECD Test Guideline No. 439, to determine the skin irritation potential by measurement of the cytotoxic effect of the test item, as reflected in the MTT assay. Disks of EPISKIN (three units) were treated with 10 mg of the test item and incubated for 15 minutes at room temperature. Exposure to the test item was terminated by rinsing with PBS 1x solution. The tissues were then incubated at 37 °C for 42 hours at 37 °C. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. 10 µL SDS (5 % aq.) or 10 µL 1x PBS treated tissues (three replicatrees each) 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 (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 (NSC_killed) was performed to avoid a possible double correction [TODTT (MTT and NSC)] for colour interference. Two killed treated tissues were used to avoid a possible double correction for colour interference. Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid

In this in vitro skin irritation test using the EPISKIN model, cell viability was not significantly reduced (corrected value (mean relative viability: 104 %) after test item treatment compared to the negative control. Therefore the test item was considered to be not irritanting to the skin.

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