1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2016 - 01 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

2008

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

EC Number:

309-269-1

EC Name:

1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

Cas Number:

100208-66-0

Molecular formula:

not applicable

IUPAC Name:

1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

Test material form:

solid: particulate/powder

Details on test material:

Test item: Brown 3
Appearance: black powder
CAS No. 100208-66-0
EC No. 309-269-1
Storage: room temperature

Specific details on test material used for the study:

Expiration date: 16 June 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

Source strain:

not specified

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
non-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:

EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model. 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 (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
EpiSkinTMSM kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS).

EpiSkinTMSM KIT Contents
Units:EpiSkinTMSM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EpiSkinTMSM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” for incubations.
(Batch No.: 16 MAIN3 081; Exp. Date: 21 December 2016)
A flask of sterile “Assay Medium” for use in MTT assays.
(Batch No.: 16 ESSC 053; Exp. Date: 21 December 2016)


EpiSkinTMSM KIT Reception Procedure

The colour of the agar medium used for transport was checked for its pH:
- orange colour = good
- yellow or violet colour = not acceptable

The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C:
- the indicator changes from white to grey at 40°C

The kit was found to be in good order at reception.

EpiSkinTMSM KIT Storage Procedure
The EpiSkinTMSM units were kept in their packaging at room temperature until the pre-incubation was started. The maintenance and assay medium were stored at 2-8°C.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

10 mg of the test item was applied evenly to the epidermal surface.

Duration of treatment / exposure:

Exposure time 15 minutes (± 0.5 min) at room temperature

Duration of post-treatment incubation (if applicable):

After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5 % CO2, ≥95% humidified atmosphere.

Number of replicates:

3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls, 2 replicates colour 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

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-3

Value:

>= 80 - <= 97

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

The test item Brown 3 is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category)

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

1-3

Value:

90

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:

Validity of the Test
The mean OD value of the three negative control tissues was 1.048. The mean OD value obtained for the positive control was 0.116 and this result corresponds to 11 % 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.

Indicator for potential false viability
Possible direct MTT reduction with test item:
As the test item has an intrinsic colour (brownish black to dark brown), the check-method for possible direct MTT reduction with test item was impossible. The direct interaction with MTT was not defined. However, to avoid the effect of possible interactions with the MTT, an additional control was necessary.
The non-specific MTT reduction (NSMTT) was determined to be 0.041 %. As the NSMTT were below 50 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.

Colouring potential of test item:
As the test item has an intrinsic colour (brownish black to dark brown), 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 1.2 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

Any other information on results incl. tables

OD values and viability percentages of the controls:

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	1.135917	108
	2	0.909867	87
	3	1.096867	105
	mean	1.047550	100
	standard deviation (SD)		11.53
Positive Control: SDS (5 % aq.)	1	0.053267	5
	2	0.090567	9
	3	0.203817	19
	mean	0.115883	11
	standard deviation (SD)		7.48

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

Test Item	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)
Brown 3	1	0.967267	0.966833	92	92
	2	0.835967	0.835533	80	80
	3	1.019417	1.018983	97	97
	mean	0.940883	0.940450	90	90
	standard deviation (SD)			9.02	9.02

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

Additional controls	Optical Density (OD)
Negative control killed tissues: 1x PBS	1	0.022267
	2	0.026317
	3	0.030267
	mean	0.026283
Test item treated killed tissues: Brown 3	1	0.018867
	2	0.033767
	3	0.027517
	mean	0.026717

 OD values and NSC % of additional control:

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSC %)
Brown 3 (test item treated tissueswithout MTT incubation)	1	0.012117	1.2
	2	0.013467
	mean	0.012792

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential.

Executive summary:

The EpiSkinTMSM test of the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% 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 samples were used as positive and negative controls respectively. For each treated tissue the viability was expressed as a percentage relative to negative control. The test item has an intrinsic colour (brownish black to dark brown), 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. The test item is a possible MTT-reducer and has an intrinsic colour (brownish black to dark brown). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, 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. In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 90 %). Positive and negative controls showed the expected cell viability values within acceptable limits.All obtained test item viability results were 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.