2,5-cyclohexadien-1-one, 4-[[4-(phenylamino)phenyl]imino]-, reaction products with 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:

December 12, 2016- March 01, 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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,5-cyclohexadien-1-one, 4-[[4-(phenylamino)phenyl]imino]-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

EC Number:

235-518-8

EC Name:

2,5-cyclohexadien-1-one, 4-[[4-(phenylamino)phenyl]imino]-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

Cas Number:

12262-26-9

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 2,5-cyclohexadien-1-one, 4-[[4-(phenylamino)phenyl]imino]- with polysulfide, leuco derivatives

Test material form:

solid

Details on test material:

Test item: Leuco Sulfur Blue 13
Appearance: Bluish black, solid
CAS No: 12262-26-9

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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin SM
- Tissue batch number: 16-EKIN-050

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure: roomt temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation time the EpiSkin SM 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
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC)
- Wavelength: 570 nm (± 10 nm; Read out range: 0-3.5 Abs)

NUMBER OF REPLICATE TISSUES:
3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues and killed tissues
- Procedure used to prepare the killed tissues: freeting
- No of replicates: 2 replicates color controls and 2 replicates non-specific colour control were used. Furthermore, 3 killed treated tissues and 3 killed negative control tissues were used for the MTT evaluation.
- Method of calculation used:
Data calculation for normal test items
Blank
– The mean of the 6 blank OD values is calculated:
Negative control
– Individual negative control OD values are corrected with the mean blank OD: :
OD Negative Control (ODNC) = ODNCraw – ODblank mean
– The corrected mean OD of the 3 negative control values are calculated: this corresponds to 100% viability
Positive control
– Individual positive control OD values are corrected with the mean blank OD: :
OD Positive Control (ODPC) = ODPCraw – ODblank mean
– The corrected mean OD of the 3 positive control values are calculated
– The % viability for each positive control replicate is calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
% Positive Control 2 = (ODPC2 / mean ODNC) ×100
% Positive Control 3 = (ODPC3 / mean ODNC) ×100
– The mean value of the 3 individual viability % for positive control is calculated:
Mean PC % = (%PC1 + %PC2 + %PC3) / 3
Test item
– Individual test item OD values are corrected with the mean blank OD: :
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The corrected mean OD of the 3 test item values are calculated
– The % viability for each test item replicate is calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
– The mean value of the 3 individual viability % for test item is calculated
Mean TT % = (%TT1 + %TT2 + %TT3) / 3

Data calculation for MTT-interacting items
Test items that interfere with MTT can produce non-specific reduction of the MTT. It is necessary to evaluate the OD due to non-specific reduction and to subtract it before calculations of viability %.
– Non-specific MTT reduction calculation (NSMTT):
NSMTT = [(ODKT - ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD (mean)
ODKT: test item treated killed tissues OD (mean)
ODNC: negative control OD (mean)
If NSMTT is > 50% relative to the negative control: additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.
– True MTT metabolic conversion (TODTT) is undertaken if NSMTT is < 50%:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test item treated viable tissues
– The % relative viability (% RV) for each test item replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / ODNC] × 100
% RV 2= [TODTT2 / ODNC] × 100
% RV 3 = [TODTT3 / ODNC] × 100
– The mean value of the 3 individual relative viability % for test item is calculated
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3

Data calculation for dyes and chemicals able to colour the tissue
For test items detected as able to stain the tissues the non-specific OD is evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability %.
– Non Specific Colour % (NSC %):
NSC % = (mean ODCT / mean ODNC) × 100
ODCT: test item treated tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
– True MTT metabolic conversion (TODTT) is undertaken if NSC % is > 5 % and ≤ 50 %.
TODTT = [ODTV – mean ODCT]
ODTV: test item-treated tissues (incubated with MTT)
ODCT: test item-treated tissues (not incubated with MTT)
– The % relative viability (% RV) for each test substance replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / ODNC] × 100
% RV 2= [TODTT2 / ODNC] × 100
% RV 3 = [TODTT3 / ODNC] × 100
– The mean value of the 3 individual relative viability % for test substance is calculated:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3
– If NSC % is ≤ 5 % then the normal calculation mode is used.
– If NSC % is > 50 % relative to the negative control, additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability exposure is or equal to or less than 50 %.
- The test substance is considered to be non-corrosive to skin if the viability after exposure is greater than 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

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.

TEST MATERIAL
- Amount applied: 10 mg

NEGATIVE CONTROL
- Amount applied: 10 µL, PBS
- Concentration: 1x PBS

POSITIVE CONTROL
- Amount applied: 10 µL, SDS
- Concentration: 5 % aq.

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h)

Number of replicates:

Three replicates were used for the test item and controls, respectively.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

>= 95 - <= 103

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: The non-specific MTT reduction (NSMTT) was determined to be 1.476 %. As the NSMTT were below 50 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.
- Colour interference with MTT: As the test item has an intrinsic colour (bluish 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 0.022. The Non Specific Colour % (NSC %) was calculated as 2.1 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:

 

Table 1: 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

 

Table 2: OD values and viability percentages of the test item (including corrected values):

Test Item	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)
Leuco Sulfur Blue 13	1	1.015117	0.999650	97	95
	2	1.077467	1.062000	103	101
	3	1.094567	1.079100	104	103
	mean	1.062383	1.046917	101	100
	standard deviation (SD)			3.99	3.99

 

Table 3: 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: Leuco Sulfur Blue 13	1	0.049317
	2	0.041667
	3	0.034267
	mean	0.041750

 

Table 4: OD values and NSC % of additional control:

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSC %)
Leuco Sulfur Blue 13 (test item treated tissueswithout MTT incubation)	1	0.024067	2.1
	2	0.020117
	mean	0.022092

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin irritation assay according to OECD 439, the test item did nit show skin-irritating properties and is therefore not classified (UN GHS No Category).

Executive summary:

An in vitro skin irritation assay (EpiSkin SM) has been performed according to OECD 439 to predict the skin irritation potential of the test item.

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. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The test item has an intrinsic colour (bluish 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 (bluish 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, the test item did not significantly reduce cell viability in comparison to the negative control (mean viability: 100 %). 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.

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 is considered to be not irritating to the skin and is therefore not classified (UN GHS No Category).