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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September to November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
yes
Remarks:
3 deviations from the Study Plan occurred but were considered to have not affected the integrity or validity of the study. See detailed explanations in the section "Overall remarks, attachments"
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
3 deviations from the Study Plan occurred but were considered to have not affected the integrity or validity of the study. See detailed explanations in the section "Overall remarks, attachments"
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 7-amino-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate
EC Number:
228-589-1
EC Name:
Disodium 7-amino-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate
Cas Number:
6300-50-1
Molecular formula:
C22H17N5O7S2.2Na
IUPAC Name:
disodium 7-amino-4-hydroxy-3-({4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-2-sulfonate
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
The test item was used as supplied.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM reconstructed human epidermis model Kit: SkinEthic Laboratories, Lyon, France (27-09-2016),
- Tissue batch number(s): EpiSkin Tissues (0.38 cm2) lot number 16-EKIN-039; Maintenance Medium lot number: 16-MIAN3-066; Assay Medium lot number: 16-ESSC-042
- Production date: nd
- Shipping date: nd
- Delivery date: 27 september 2016
- Date of initiation of testing:

Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C / room temperature
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 μL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface.

- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2 in air for 42 hours
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium
- Incubation time: The tissues were incubated for 3 hours at 37 °C, 5%
- Spectrophotometer:Anthos 2001 microplate reader
- Wavelength:562 nm
- Filter: without reference filter
- Filter bandwidth: nd
- Linear OD range of spectrophotometer: nd

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Water-killed tissues
- Procedure used to prepare the killed tissues (if applicable): Water-killed tissues were prepared prior to the study by placing untreated EPISKINTM tissues in a 12-well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5% CO2 in air for 48 ± 1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (−14 to −30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.
- N. of replicates : the MTT reducing test item was applied to three water-killed tissues. In addition, three water-killed tissues remained untreated.
- Method of calculation used:
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
Approximately 10 mg of test item was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.
The test item was also found to have the potential to produce color interference with the MTT endpoint if residual test item remained on the tissues after rinsing.
Therefore a parallel test was performed using living tissues that was identical to the main test with the exception of the MTT loading step being replaced with a 3 hour incubation in assay medium. Three living tissues were treated with the test item and three tissues remained untreated.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: pre-test followed by main test

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean tissue viability following the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period is equal or less than 50%.
The test substance is considered to be non-irritant to skin if the mean tissue viability following the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period is higher than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (26.3 mg/cm2)
- Concentration (if solution): n.a.

VEHICLE
- Amount(s) applied (volume or weight with unit): 5 microL
- Concentration (if solution): n.a.
- Lot/batch no. (if required): n.a.
- Purity: n.a.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 microL
- Concentration (if solution): 100%

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 microL
- Concentration (if solution): 5% w/w

The negative control item, DPBS, was used as supplied.
The positive control item, SDS, was prepared as a 5% w/v aqueous solution.
A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.
Duration of treatment / exposure:
15 min (room temperature)
Duration of post-treatment incubation (if applicable):
Incubation at 37ºC, 5% CO2 in air for 42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of 3 experiments
Value:
110.6
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The relative mean viability of the test item treated tissues was 110.6% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.
Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

- OTHER EFFECTS:
- Visible damage on test system:
- Direct-MTT reduction: The test item was found to be a possible direct MTT reducer. The MTT solution did not turn blue or purple but became a dark red color; therefore, as a precaution the test item was treated as if it was a direct MTT reducer and an additional procedure using water-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.
- Colour interference with MTT: The solution containing the test item was a dark red color. Therefore, an additional procedure using living tissues with no MTT loading step was performed. However, the results obtained showed that negligible color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The relative mean viability of the test item treated tissues was 110.6% after a 15-Minute exposure period and 42-Hour post-exposure incubation period.
It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 0.874 and the standard deviation value of the viability was 5.7%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 5.5% relative to the negative control treated tissues and the standard deviation value of the viability was 1.4%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 5.5%. The test item acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline:

Any other information on results incl. tables

Deviations from the study plan:

Deviation 1 An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

Deviation 2 An assessment found the test item had the potential to cause color interference with the MTT endpoint. Therefore, an additional procedure using living tissues with no MTT loading step was performed. However, the results obtained showed that negligible color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

Deviation 3 To clarify Deviation 1. The test item was found to be a possible direct MTT reducer.

Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item:

OD = Optical Density

SD = Standard deviation

∗ = The mean viability of the negative control tissues is set at 100%

 Item  OD562 of tissues  Mean OD562 of triplicate tissues  +-SD of OD562  relative individual tissue viability (%)  relative mean viability (%)   +-SD of relative mean viability (%)
 negative control item

 0.927

0.828

0.867

 0.874  0.050

 106.1

94.7

99.2

 100* 5.7 
 positive control item

 0.058

0.034

0.051

 0.048  0.012

 6.6

3.9

5.8

 5.5  1.4
 test item

 0.991

0.912

0.998

 0.967  0.048

 113.4

104.3

114.2

 110.6  5.5

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was classified as non-irritant. The following classification criteria apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).
Executive summary:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was found to be a possible direct MTT reducer and therefore additional non-viable tissues were incorporated into the testing for correction purposes. The test item was also found to have the potential to produce color interference with the MTT endpoint and therefore additional living tissues were incorporated into the testing for correction purposes.

At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 562 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).