Phenol, 4-amino-, reaction products with 4-(2-naphthalenylamino)phenol 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:

23 August 2017 - 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:

Phenol, 4-amino-, reaction products with 4-(2-naphthalenylamino)phenol and sodium sulfide (Na2(Sx))

EC Number:

291-833-0

EC Name:

Phenol, 4-amino-, reaction products with 4-(2-naphthalenylamino)phenol and sodium sulfide (Na2(Sx))

Cas Number:

90480-94-7

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 4-aminophenol with 4-​(2-​naphthalenylamino)​phenol and sodium polysulfide

Test material form:

solid

Details on test material:

Test item: Leuco Sulphur Black 11
Appearance: black solid
CAS No: 90480-94-7
EC No: 291-833-0

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:

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

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

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.

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.

Indicator for potential false viability

Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material acts directly on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test substance interference with the viability measurement.

Check-method for possible direct MTT reduction with test item

As the test item has an intrinsic colour (black), the check-method for possible direct MTT reduction with test item is impossible. The direct interaction with MTT was not defined. However, to avoid the effect of possible interactions with MTT, an additional control was used.

Additional controls for MTT direct interacting chemicals:
In addition to the normal procedure, three killed treated tissues and three killed negative control tissues were used for the MTT evaluation in one run (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batch of the living tissues (batch no. of killed epidermis: 17-EKIN-032). The same treatment steps were followed with these tissues and the living tissues.

Water-killed epidermis for MTT-interaction items:
– Place the living epidermis in a 12 well plate with 2 mL of distilled water (replacing the culture medium).
– Incubate at 37 °C, 5% CO2, ≥ 95% humidified atmosphere for 48h +/- 1 hours. At the end of the incubation, discard the water.
– Keep dead epidermis frozen (dry) in freezer at -18°C to -20°C (killed epidermis can be stored and used up to 6 months).
– Before use, the killed tissues are de-frozen at room temperature (app. 1 hour in 2 mL of maintenance medium).
Further use of killed tissues is similar to living tissues.

Check-method to detect the colouring potential of test item

As the test item has an intrinsic colour (black), further evaluation to detect colouring potential was not necessary. Non Specific Colour % (NSC %) was determined in order to evaluate the ability of test item to stain the epidermis by using additional control tissues.

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. These tissues followed the same treatment steps as the other tissues except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. OD reading was made following the same conditions as for the other tissues.

Additional controls for non-specific colour in killed tissues:
The test item is a possible MTT-reducer and has an intrinsic colour (black). 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. However, the NSCliving % was 1.7 % (below 5 %), so the (NSCkilled) was not determined and used during the calculation of true MTT metabolic conversion.

In addition to the normal procedure, two killed treated tissues were used to avoid a possible double correction for colour interference. In this additional control, the test item was applied on two killed tissue replicates, which undergo the entire testing procedure but were incubated with medium instead of MTT solution during the MTT incubation step.

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:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h)

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

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

80

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:

94

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:

80

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:

85

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.

Indicator for potential false viability

Possible direct MTT reduction with test item:
As the test item has an intrinsic colour (black), 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 -1.182%. 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 (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 0.017. The Non Specific Colour % (NSC %) was calculated as 1.7 % (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	0.832	80
	2	0.973	94
	3	0.831	80
	mean	0.878	85
	standard deviation (SD)		7.88

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	0.102
	2	0.037
	3	0.039
	mean	0.059

 

OD values and NSC % of additional control:

Additional colour control	Optical Density (OD)		Non Specific Colour % (NSC %)
Test item (test item treated tissueswithout MTT incubation)	1	0.020	1.7
	2	0.014
	mean	0.017

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 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 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 (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. The test item is a possible MTT-reducer and has an intrinsic colour (black). 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: 85 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. The test item was considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).