Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 October 2016 - 25 November 2016
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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Original Guideline adopted July 28, 2015
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: normal, human-derived epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories (69007 Lyon, France)
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM: human skin model EpiSkin™
EpiSkin™ Kit
Supplier: SkinEthic Laboratories (69007 Lyon, France)
Date received: 22 November 2016
EpiSkin™ Kit Lot No.: 16-EKIN-047

The EpiSkin™ 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 EpiSkin™ tissues (surface 0.38 cm2) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 22 November 2016. On the day of receipt EpiSkin™ tissues were transferred to 12-well plates with maintenance medium and the pre-incubation phase of the EpiSkin™ tissues started.

Test for Direct MTT Reduction and Colour Interference:
Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose the colourless test item (10 µL) was mixed with 90 µL of deionised water in a pre-experiment. The test item/water mixture was gently shaken for 15 minutes at room temperature.
The colour of the test item/water mixture did not change during the incubation period compared with the colour of the pure test item. Therefore, the measurement of the OD of the test item in water at 570 nm was not required and consequently not performed. An additional test with viable tissues (normal test procedure but without MTT addition) did not have to be performed.
For correct interpretation of results it is necessary to assess the ability of the test item to directly reduce MTT. To test for this ability 10 µL of the test item was added to 2 mL of MTT solution (0.3 mg/mL) and the mixture will be incubated in the dark at 37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT solution with 10 µL of DMEM was used as the control
Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer, and an additional test with freeze-killed tissues did not have to be performed.

Pre-warming of EpiSkin™ Tissues:
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for about 23 hours.

Treatment:
The negative control, positive control and the test item were added to the inserts atop the concerning EpiSkin™ triplicate tissues. Exposure period of the tissues in 12-well plates was 15 minutes at room temperature.
After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42.25 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

IL-1 α Immunoassay:
Samples of all treatment groups were taken from the wells. The plates were shaken for approximately 15 minutes to homogenise the released mediators in the medium before sampling. At least 1.6 mL medium from each well was taken and was stored in the freezer at –20 °C. Since the results derived from the MTT assay were not unclear or borderline, the IL-1 α concentration in the medium was not determined and the taken samples will be discarded after report finalization.

MTT Assay:
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the treatment procedure was completed for all tissues the cell 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) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for nearly 3 hours while shaking at room temperature.
Per tissue sample 2 x 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. Isopropanol was used for blank measurement. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1) with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.

DATA EVALUATION:
The mean OD of the three tissues per group (negative control, positive control and test item) was calculated after blank correction. The mean OD of the negative control value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = (mean OD test item or positive control / mean OD negative control) *100
For the test item and the positive control, the mean relative viability +/- rel. standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model:
For the current test, a test item is considered irritant if the mean relative tissue viability of three individual tissues is reduced to =< 50% of the negative control.
For the current test, a test item is considered non-irritant if the mean relative tissue viability of three individual tissues is > 50% of the negative control.
in vitro result : in vivo prediction
mean tissue viability =< 50%: irritant
mean tissue viability > 50%: non-irritant

Acceptability of the Assay:
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is >= 0.6 till <= 1.5.
The rel. standard deviations in between tissues of the same treatment group should be <= 18%.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is <= 40%.
The data of the quality control (determined by SkinEthic Laboratories, 69007 Lyon, France) of the respective EpiSkin™ lot is mentioned in the report (the acceptance limit of the IC50 should be between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS).



Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 µL
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
approximately 42.25 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: mean relative tissue viability (%)
Value:
59.6
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Quality control data (determined by SkinEthic Laboratories, 69007 Lyon, France) of the respective EpiSkinTM lot: the acceptance limit of the IC50 should be between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (actual value: 1.9 mg/mL)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD >= 0.6 till <= 1.5 (0.612-0.617) for the 15 minutes treatment interval thus showing the quality of the tissues.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.3% (<= 40%) thus ensuring the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The rel. standard deviations between the % variability values of the test item, the positive and negative controls were below 13% (<= 18%) thus ensuring the validity of the study.
- Range of historical values: Results of the positive control and negative control are within historical data obtained at Envigo CRS GmbH.

Any other information on results incl. tables

IL-1 α Immunoassay:

Since the results derived from the MTT assay were not unclear or borderline, the IL-1 α concentration in the medium was not determined.

Results after treatment with the test item and controls:

Dose Group

Absor-bance 570 nm
Tissue 1*

Absor-bance 570 nm
Tissue 2*

Absor-bance 570 nm
Tissue 3*

Mean Absor-bance of 3 Tissues

Relative Absor-bance [%] Tissue 1, 2 + 3**

Relative Standard Deviation [%]***

Rel. Absor-bance
[% of Negative Control]****

Blank

 

 

 

 

 

 

 

Negative Control

0.612

0.612

0.617

0.614

99.8
99.7
100.5

0.5

100.0

Positive Control

0.023

0.026

0.029

0.026

3.7
4.3
4.8

12.5

4.3

Test Item

0.363

0.411

0.324

0.366

59.1
66.9
52.8

11.9

59.6

*      Mean of two replicate wells after blank correction

**     relative absorbance per tissue [rounded values]: (100 x (absorbance tissue) ) / mean absorbance negative control

***   relative Standard Deviation: (Standard Deviation / Mean Absorbance of 3 tissues ) x 100

****  relative absorbance per treatment group [rounded values]: (100 x (mean absorbance test item)) / mean absorbance negative control

Applicant's summary and conclusion

Interpretation of results:
other: Not a skin irritant
Remarks:
according to EU CLP 1272/2008 and its amendments
Conclusions:
The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissues was 59.6%. This value is above the threshold for irritancy of <= 50%. Therefore, the test item is not considered to be a skin irritant under the conditions of this study.
Executive summary:

The possible skin irritation potential of the substance was tested in compliance with OECD TG 439 and GLP principles. Each three tissues of the human skin model EpiSkin™ were treated by topical application of 10 µl of the undiluted test item, the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate) for 15 minutes. After about 42.25 hour post-exposure incubation period, determination of the cell viability was performed. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissues was 59.6%. This value is above the threshold for irritancy of <= 50%. Therefore, the test item is not considered to be a skin irritant under the conditions of this study. After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD >= 0.6 till <= 1.5 (actual values 0.612-0.617), the mean relative tissue viability of the positive control was  <= 40% (4.3%) and the rel. standard deviations in between tissues of the same treatment group were <= 18% (below 13%) indicating that the test system functioned properly.