Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine

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

Justification for type of information:

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).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine

EC Number:

290-904-3

EC Name:

Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine

Cas Number:

90268-98-7

Molecular formula:

Molecular formula is not available

IUPAC Name:

Reaction product of aniline, 4-nitrobenzenamine, p-phenylenediamine and p-toluidine with sodium polysulfide

Test material form:

solid: particulate/powder

Details on test material:

Test item: Yellow 22
Appearance: ocher clay, solid
CAS No. 90268-98-7
EC No. 290-904-3
Storage: room temperature

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

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: A flask of sterile “Maintenance Medium” for incubations. (Batch No.: 16 MAIN3 064; Exp. Date: 21 September 2016)
A flask of sterile “Assay Medium” for use in MTT assays.
(Batch No.: 16 ESSC 040; Exp. Date: 21 September 2016)
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:

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.

Duration of treatment / exposure:

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

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.
MTT test after 42 hours incubation
After the 42 hours incubation 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.

Number of replicates:

In this assay 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

Run / experiment:

1-3

Value:

>= 67 - <= 92

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean viability: 76%

Other effects / acceptance of results:

The mean OD value of the three negative control tissues was 0.869. The mean OD value obtained for the positive control was 0.136 and this result corresponds to 16 % 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.

As the test item has an intrinsic colour (ocher clay), 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 -3.672%. 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).

As the test item has an intrinsic colour (ocher clay), 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 0.022. The Non Specific Colour % (NSC %) was calculated as
2.6 %. 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	1.018	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

OD values and viability percentages of the test item:

Test Item	Optical Density (OD)		Viability (%)
	1	0.604	70
	2	0.581	67
	3	0.796	92
	mean	0.660	76
	standard deviation (SD)		13.55

 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	1	0.042
	2	0.037
	3	0.048
	mean	0.042

 

OD values and NSC % of additional control:

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSC %)
test item treated tissueswithout MTT incubation	1	0.025	2.6
	2	0.020
	mean	0.022

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 has been performed to predict the irritation potential of the test item according to the OECD Test Guideline No. 439. Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure 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 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. The test item has an intrinsic colour (ocher clay), therefore two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore further 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. 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: 76 %). 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.