Formamide, N,N'-1,4-phenylenebis-, reaction products with 4-methyl-1,3-benzenediamine and sulfur, leuco derivatives

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2017 - 05 July 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:

06 July, 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Formamide, N,N'-1,4-phenylenebis-, reaction products with 4-methyl-1,3-benzenediamine and sulfur, leuco derivatives

EC Number:

276-059-3

EC Name:

Formamide, N,N'-1,4-phenylenebis-, reaction products with 4-methyl-1,3-benzenediamine and sulfur, leuco derivatives

Cas Number:

71838-68-1

Molecular formula:

not applicable

IUPAC Name:

Reaction product of formamide, N,N'-1,4-phenylenebis- with 4-methyl-1,3-benzenediamine and sulfur, leuco derivatives

Test material form:

solid: particulate/powder

Details on test material:

Test Item: Leuco Sulfur Brown 26
Appearance: Dark, near black powder

In vitro test system

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 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:

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: sterile “Maintenance Medium” for incubations (Batch No.: 17 MAIN3 020; Exp. Date: 24 May 2017)
sterile “Assay Medium” for use in MTT assays (Batch No.: 17 ESSC 019; Exp. Date: 24 May 2017)
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:

The plates with the treated epidermis units were incubated for the exposure time of 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:

In this assay 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

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

1-3

Value:

81

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean OD value of the three negative control tissues was 0.614. Furthermore, the individual OD values of the three negative control tissues were in the acceptable range (0.603, 0.637 and 0.602). The mean OD value obtained for the positive control was 0.125 and this result corresponds to 20 % 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.

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	0.603	98
	2	0.637	104
	3	0.602	98
	mean	0.614	100
	standard deviation (SD)		3.23
Positive Control: SDS (5 % aq.)	1	0.139	23
	2	0.096	16
	3	0.138	23
	mean	0.125	20
	standard deviation (SD)		3.99

OD values and viability percentages of the test item:

Test Item	Optical Density (OD)		Viability (%)
	1	0.478	78
	2	0.526	86
	3	0.481	78
	mean	0.495	81
	standard deviation (SD)		4.39

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

Additional controls	Optical Density (OD)
Negative control killed tissues: 1x PBS	1	0.083
	2	0.039
	3	0.073
	mean	0.065
Test item treated killed tissues:	1	0.058
	2	0.058
	3	0.053
	mean	0.056

OD values and NSC % of additional control:

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSC %)
Test item (test item treated tissues without MTT incubation)	1	0.021	3.9
	2	0.027
	mean	0.024

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 under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Executive summary:

EpiSkinTMSM test has been performed to predict the irritation potential of the test item by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test guideline 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. 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% 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 (dark ochre), 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 (dark ochre). 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. 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.

In this in vitro skin irritation test using the EPISKIN model, positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 81 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).