[x Sodium, y Potassium, z Lithium, x+y+z =2] 4-amino-3-[[[(2,4-diaminophenyl)diazenyl]phenyl]diazenyl]-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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:

(Original Guideline adopted July 28, 2015), and as described in detail in the Protocol for: In Vitro
EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed
Human Epidermal Model EpiDerm (EPI-200).

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UN GHS (published 2003, last (7th) revision 2017)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: In Vitro EpiDermTM Skin Irritation Test (EPI-200-

Version / remarks:

Versión 07 November 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[x Sodium, y Potassium, z Lithium, x+y+z =2] 4-amino-3-[[[(2,4-diaminophenyl)diazenyl]phenyl]diazenyl]-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

EC Number:

824-263-3

Cas Number:

2196165-14-5

Molecular formula:

C28H21N9O7S2.xNa.yK.zLi

IUPAC Name:

[x Sodium, y Potassium, z Lithium, x+y+z =2] 4-amino-3-[[[(2,4-diaminophenyl)diazenyl]phenyl]diazenyl]-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

In vitro test system

Test system:

human skin model

Source species:

other: EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have ben cultured to form a multilayered, highly differenciated model of the human epidermis.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Model used: EpiDerm™
- Tissue batch number(s): Epi-200- SIT Kit ( Lot No.: 28624), Epi-200 SIT kits and MTT-100 assay kit were purchased from MatTek Corporation (82105 Bratislava, Slovakia).
Cell Culture: The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs(R), 10 mm diameter).
- Production date: not indicated
- Shipping date: no date indicated
- Delivery date: EpiDerm™ tissues were shipped with cool packs on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on 12 June 2018
- Date of initiation of testing: On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.
- MTT-Solution: The MTT-solution was prepared freshly on day of use (resulting: 1 mg/mL).


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were gently rinsed with PBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in PBS at least three times. Afterwards the inserts once again rinsed with sterile PBs from the inside and outside. Excess Pbs was by gently shaking the inserts and blotting the bottom with sterile blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
Test for Direct MTT Reduction and Colour Interference
A test item may interfere with the MTT endpoint if: a) it is coloured and/or b) able to directly reduce MTT. The MTT assay is affected only if the test item is present in the tissues when the MTT viability test is performed.
Some non-coloured test items may change into coloured test items in wet or aqueous conditions and thus stain tissues during the 60 min exposure. Therefore, before exposure, a functional check for this possibility has to be performed (step 1).
Step 1: 25 ± 2 mg of the test item were added to 0.3 mL of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated.
Since the solution changes colour significantly, the test item was presumed to have the potential to stain the tissue. A functional check on viable tissues has to be performed (step 2).
Step 2: To check the tissue-binding of a coloured test item, two viable tissues were exposed to 25 ± 2 mg of the test item. In parallel, two tissues were exposed to DPBS (negative control). All procedures were followed as described in chapter 3.6 except the tissue were incubated for 3 hour incubation in culture media without MTT (37 ± 1.5°C, 5 ± 0.5% CO2,) instead of incubating in media containing MTT. After the 3 hour incubation, the tissues were rinsed and the tissues were extracted using 2.0 ml of isopropanol and the optical density (OD) at 570 nm was measured.
Data correction procedure
Since the tissues treated by coloured test item (or test item detected in step 1) had an OD < 5% of the PBS treated control tissue and the tissue viability (determined in MTT assay) was not close to the classification cut-off (50%), correction of the results was not necessary, but was calculated using following formula: OD = ODcolored tissue (MTT assay) – ODcolored tissue (no MTT assay)
Step 3: The test item (including those already evaluated in step 1 and step 2) was further evaluated for its potential to interfere with MTT assay. To test if a test item directly reduces MTT, 25 ± 2 mg of the test item were added to 1 mL of the MTT-solution (1 mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT medium was used as control.
Since the MTT solution turned black, the test item reduced MTT and additional functional check (step 4) had to be performed.
Step 4: The procedure employed freeze-killed tissues that possess no metabolic activity but absorb and bind the test item similar to viable tissues.
The MTT reducing test item was applied to two freeze-killed tissues. In addition, two freeze killed tissues were left untreated (Note: The untreated killed controls show a small amount of MTT reduction due to residual reducing enzymes within the killed tissue). The entire assay protocol is performed on the freeze-killed tissues in parallel to the assay performed with the live EpiDerm tissues.
Data were then corrected as follows:
Data correction procedure
True viability = Viability of treated tissue – Interference from test chemical = ODtvt – ODkt, where ODkt = (mean ODtkt – mean ODukt)
tvt = treated viable tissue kt = killed tissues
tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)
Since the interference by the test item is < 30% of the negative control value, the net OD of the test item treated killed control was subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflected metabolic conversion only.
Addition according to OECD 439:
Although it was not mentioned in the Study Plan and is not required in the MatTek protocol OECD 439 requires a third set of control – the so called NSKC (Non-specific killed control) in order to avoid a false double correction if a test item is both coloured and has MTT reducing capacities. There freeze-killed tissues were treated with the test item and during the MTT Assay incubated only with medium without MTT. It is a mixed control and the data which are obtained from this control are used for Data Correction Procedure as follows:
Corrected mean Viability = 〖Tissue Viability〗_(test item )-(〖Tissue Viability〗_(VT test item) 〖- (Tissue Viability〗_(KC test item)- 〖Tissue Viability〗_(KC negative control))) + mean ViabilityNon specific killed control


EXPERIMENTAL PERFORMANCE
1. Pre-warming of EpiDerm™ Tissues: The plastic bag containing the 24-well plate with epidermal tissues was opened under sterile conditions. Under an airflow using forceps, the gauze was removed and the inserts were taken out. Any remaining agarose that adheres to the outer sides of the inserts was removed by gentle blotting on the sterile filter paper or gauze, and the tissues were placed in the empty, sterile 6-well plate. Prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality: If necessary, it was taken care, that
1) air bubbles between agarose and insert were not > 30% of the total surface,
2) liquid on top of the insert was removed with sterile cotton tips,
3) if again moisture is observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded.
0.9 mL of the assay medium (20 – 25 °C) was pipetted into each well of sterile 6-well plates. The inserts with the EpiDerm™ tissues were placed in the upper wells, and were pre-incubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further about 22 hours (37 ± 1.5 °C, 5 ± 0.5% CO2).

2. Treatment: After pre-incubation of EpiDerm™ tissues was completed, medium was replaced by 0.9 mL of fresh medium per well. The negative control, the positive control, and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues. The treatment time was 60 minutes in total. Within this period the 6-well plates were put into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with PBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in PBS at least three times. Afterwards the inserts were once again rinsed with sterile PBS from the inside and the outside. Excess PBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The inserts were placed in the prepared holding plate. Tissues were incubated for about 24 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2. After incubation medium was changed (0.9 mL of pre-warmed fresh medium). Thereafter tissues were incubated for another about 17 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2. The complete incubation time was about 41 hours.

3. MTT Assay: On the day of testing the MTT concentrate was diluted with the MTT diluent (1 mg/mL). The 24-well plates were prepared before the end of the tissue pre-warming period. A volume of 300 µL of the MTT solution was added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until further use.
After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates. After a 3-hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2), the tissues were rinsed three times with PBS, and carefully dried with blotting paper. The tissues were transferred into new 6-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues are completely covered. The 6-well plate was sealed to inhibit the isopropanol evaporation.
The formazan salt was extracted for about 2.5 hours while shaking at room temperature.
Per each tissue, 3 x 200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax® Molecular Devices, Softmax Pro Enterprise, version 4.7.1) with a 570 nm filter. Mean values were calculated from the 3 wells per tissue.

4. Data Recording: The data generated were recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups with the test item, negative control and positive controls.

NUMBER OF REPLICATE TISSUES:
3

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

PREDICTION MODEL / DECISION CRITERIA
For the current test, an irritation potential of a test item leading to EU classification H315 (according to regulation (EC) 1272/2008), and GHS category 2 according to UN GHS (published 2003, last (7th) revision 2017) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.
in vitro result in vivo prediction EU classification UN GHS classification
mean tissue viability <= 50% irritant (I) H315 category 2
mean tissue viability > 50% non-irritant (NI) non-irritant (NI) non-irritant (NI)
In cases of borderline results, such as non-concordant replicate measurements and/or mean percent viability equal to 50 ± 5%, a second run should be considered, as well as a third one in case of discordant results between the first two runs.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were
applied to the tissues, wetted with 25 µL DPBS prior to application, and spread to match the
surface of the tissue for a complete treatment time of 60 minutes.

NEGATIVE CONTROL:
Concurrent controls were used for several Envigo CRS GmbH studies performed
simultaneously. Each 30 µL were applied to triplicate tissue.
Name: DPBS (MatTek)

POSITIVE CONTROL:
Concurrent controls were used for several Envigo CRS GmbH studies performed
simultaneously. Each 30 µL were applied to triplicate tissue.
Name: 5% SDS solution in deionised water (MatTek)

Duration of treatment / exposure:

The treatment time was 60 minutes in total.

Duration of post-treatment incubation (if applicable):

Tissues were incubated for about 24 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2. After incubation medium was changed (0.9 mL of pre-warmed fresh medium). Thereafter tissues were incubated for another about 17 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2. The complete incubation time was about 41 hours.

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

106.68

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

There were the following deviations from study plan:
Concerning 5.6 Test for Direct MTT Reduction and Colour Interference
(page 6 of the study plan)
Although it was not mentioned in the Study Plan and is not required in the MatTek protocol
OECD 439 requires a third set of control to be performed – the so called NSKC (Non-specific
killed control) in order to avoid a false double correction if a test item is both coloured and
has MTT reducing capacities. There freeze-killed tissues were treated with the test item and
during the MTT Assay incubated only with medium without MTT. It is a mixed control and
the data which are obtained from this control are used for Data Correction Procedure as
follows:
Corrected mean Viability =
Tissue Viabilitytest item − (Tissue ViabilityVT test item− (Tissue ViabilityKC test item −
Tissue ViabilityKC negative control)) + mean ViabilityNon specific killed control



Concerning 6.3 MTT Assay (page 9 of the study plan)
Instead of 24-Well-Plates, 6-Well-Plates were used for extraction with isopropanol since
residual test item was still visible on the inserts after rinsing. With this it is possible to avoid
solubility of residual test item in the isopropanol extract and a falsification for OD
measurement.
These deviations were considered to have not affected the integrity or validity of the study.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue v
iability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is >=0.8 and ≤ 2.8 in accordance with OECD TG 439.

- Acceptance criteria met for positive control:
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is <= 20%.

- Acceptance criteria met for variability between replicate measurements:
The SD of 3 concurrently tested tissue replicates should be < 18.

- Range of historical values if different from the ones specified in the test guideline: OD values should not be below historically established boundaries.
Concurrent negative controls (NC) and positive controls (PC) will be used in each run to demonstrate that viability (with the NC), barrier function and resulting tissue sensitivity (with the PC) of the tissues are within a defined historical acceptance range.
Historical data and the quality certificate of the supplier of the test kit demonstrating its robustness are annexed to the report, including quality control data (determined by MatTek Corporation, 82105 Bratislava, Slovakia) of the respective EpiDermTM lot. According to the OECD TG 439, the acceptance limit of the ET50 should be between 4.77 hours and 8.72 hours after treatment with 1% Triton X-100 (QC batch release criteria).

Any other information on results incl. tables

Results after treatment with the substance and the controls

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	OD 570 nm Well 3	Mean OD of 3/2 Wells	Mean OD of 3/2 Wells blank corrected	Mean OD of 3/2 tissues blank corrected	Rel. Viability [%] Tissue 1,3+3*	Standard Deviation	Mean Rel. Viability [%]**
Blank		0.037	0.038	0.036	0.037			106.629
Negative control	1	1.984	2.004	2.012	2.000	1.963	1.841	104.221	9.5	100.0
	2	1.963	1.960	1.945	1.956	1.919		89.150
	3	1.676	1.674	1.678	1.678	1.642		2.721
Positive Control	1	0.086	0.089	0.086	0.087	0.050	0.049	2.629	0.1	2.65
	2	0.085	0.085	0.085	0.085	0.048		2.601
	3	0.085	0.086	0.084	0.085	0.048		112.894
Test item	1	2.007	2.168	2.172	2.116	2.079	1.970	104.826	5.1	106.68***
	2	1.966	1.962	1.973	1.967	1.930		103.316
	3	1.930	1.936	1.952	1.939	1.902
Blank		0.037	0.038	0.039	0.038
Negative Control Viable Tissues	1	0.037	0.037	0.038	0.037	-0.001	0.000	-0.038	0.1	0.00
	2	0.040	0.038	0.038	0.039	0.001		0.040
Test Item Viable Tissues	1	0.039	0.039	0.038	0.039	0.001	0.005	0.052	0.3	0.030
	2	0.048	0.048	0.048	0.048	0.010		0.539
Negative Control Freeze killed Tissues	1	0.078	0.083	0.083	0.081	0.043	0.044	2.355	0.0	2.39
	2	0.081	0.084	0.083	0.083	0.045		2.426
Test Item Freeze killed Tissues	1	0.113	0.117	0.118	0.116	0.078	0.075	4.242	0.3	4.06
	2	0.108	0.110	0.110	0.110	0.072		3.885
Non-specific killed contorl	1	0.058	0.057	0.060	0.058	0.020	0.030	1.101	0.8	1.64
	2	0.076	0.075	0.083	0.078	0.040		2.172

 

* relative viability [rounded values]:

** Mean relative viability [rounded values]:

*** corrected value

 

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did lead to a change in colour. 
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did show blue/purple colour.
The mean relative viability of the test item, corresponding to the cell viability, decreased to 106.68% (threshold for irritancy: ≤ 50%), consequently the test item was non-irritant to skin.  

 

 

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions
reported, DIRECT BLACK RBK is not irritant to skin according to UN GHS and EU CLP
regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

 The test item did reduce MTT (test for direct MTT reduction). Also, its intrinsic colour was intensive. Conseque ntly, additional tests with freeze-killed, viable tissues and non-specific killed controls were necessary.

Each three tissues of the human skin model EpiDerm™were treated with the test tem, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.

 After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD³0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

 Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

 After treatment with the test item the mean relative viability value was 102.06 %compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%.
Therefore, the test item is not considered to possess an irritant potential.

 In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is non-irritant to skin.