Acetamide, N-(2,4-dinitrophenyl)-, reaction products with 1-methyl-2,4-dinitrobenzene and sodium sulfide (Na2(Sx)), 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:

23 August - 10 October, 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:

28 April 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Acetamide, N-(2,4-dinitrophenyl)-, reaction products with 1-methyl-2,4-dinitrobenzene and sodium sulfide (Na2(Sx)), leuco derivatives

EC Number:

288-896-1

EC Name:

Acetamide, N-(2,4-dinitrophenyl)-, reaction products with 1-methyl-2,4-dinitrobenzene and sodium sulfide (Na2(Sx)), leuco derivatives

Cas Number:

85940-08-5

Molecular formula:

not applicable

IUPAC Name:

Reaction product of N-(2,4-dinitrophenyl)acetamide with 1-methyl-2,4-dinitrobenzene and polysulfide, leuco derivatives

Test material form:

solid: particulate/powder

Details on test material:

Test item: Leuco Sulphur Brown 46
Appearance: Brown powder
CAS No: 85940-08-5

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 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:

Human Skin
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.

Supplier: SKINETHIC Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
Batch No.: 17-EKIN-035
Expiry date: 04 September 2017

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.: 17 MAIN3 037; Exp. Date: 06 September 2017)
A flask of sterile “Assay Medium” for use in MTT assays.
(Batch No.: 17 ESSC 034; Exp. Date: 06 September 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

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h)

Number of replicates:

3 replicates per test item, 3 replicates negative controls, 3 replicates positive controls, 2 replicates colour controls, 2 replicates non-specific colour control, 3 killed treated tissues and 3 killed negative control tissues for the MTT evaluation

Test system

Details on study design:

Pre-incubation (day [-1]-0)
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight (18-24h) at 37°C in an incubator with 5 % CO2, ≥95% humidified atmosphere.

Application (day 0)
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.

Test Item
The epidermal surface was first moistened with 5 µL deionised water* in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.

Positive and negative control
A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Additional controls for MTT direct interacting chemicals
In addition to the normal procedure, 3 killed treated tissues and 3 killed negative control tissues were used for the MTT evaluation in one run.

Additional controls for dyes and chemicals able to colour the tissue:
In addition to the normal procedure, two additional test item treated tissues were used for the non-specific OD evaluation.

Additional controls for non-specific colour in killed tissues:
In addition to the normal procedure, two killed treated tissues were used for avoiding a possible double correction for colour interference.

Exposure (day 0)
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.

Rinsing (day 0)
After the incubation time the EpiSkinTMSM 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).
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.

MTT test after 42 hours incubation (day 2)
After the 42 hours incubation period the EpiSkinTMSM 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°C in an incubator with 5 % CO2 protected from light, ≥95% humidified atmosphere.

Formazan extraction (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.

Cell viability measurements (day 2)
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 Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at 570 nm (±10nm; Read out range: 0-3.5 Abs) using acidified isopropanol solution as the blank (6×200 µL).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

88

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

66

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

78

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

1-3

Value:

77

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 1.035. The mean OD value obtained for the positive control was 0.262 and this result corresponds to 25 % 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.

Possible direct MTT reduction with test item:
As the test item has an intrinsic colour (reddish 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 (0%), the correction of viability percentage was not necessary. The calculated NSMTT was -1.541%. However, for the calculation of non-specific MTT reduction, small negative numbers are counted as zero, because the reason of the small negative number is a slight difference between the used killed epidermis (biological variability).

Colouring potential of test item:
As the test item has an intrinsic colour (reddish 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 0.015. The Non Specific Colour % (NSC %) was calculated as 1.5 % (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	0.996	96
	2	0.989	96
	3	1.121	108
	mean	1.035	100
	standard deviation (SD)		7.18
Positive Control: SDS (5 % aq.)	1	0.288	28
	2	0.247	24
	3	0.251	24
	mean	0.262	25
	standard deviation (SD)		2.19

OD values and viability percentages of the test item:

Test Item	Optical Density (OD)		Viability (%)
	1	0.911	88
	2	0.682	66
	3	0.805	78
	mean	0.799	77
	standard deviation (SD)		11.11

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

Additional controls	Optical Density (OD)
Negative control killed tissues: 1x PBS	1	0.110
	2	0.046
	3	0.059
	mean	0.071
Test item treated killed tissues:	1	0.032
	2	0.096
	3	0.038
	mean	0.055

 

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	0.019	1.5
	2	0.011
	mean	0.015

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:

An EpiSkinTMSM test has been performed to predict its irritation potential of the test item 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. 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 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 (reddish 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 (reddish 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. 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, the test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 77 %). 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. 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 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).