Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 October 2015 - 31 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Purity 98.76%
Batch No: 0000120575

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
The model consists of an airlifted, living, multilayered epidermal tissue construct (surface 0.5cm2) reconstructed of normal human epidermal keratinocytes for 17 days, produced in polycarbonate inserts in serum-free and chemically defined medium, featuring normal ultra-structure and functionally equivalent to human epidermis in vivo. The three-dimensional human epidermal model constructs contain basal, spinous and granular layers along with a functional stratum corneum.
Justification for test system used:
Validation studies have shown that tests employing human skin models are able to reliably distinguish between known skin corrosives and non-corrosives (Botham et al, 1995, Barratt et al, 1998 and Fentem et al, 1998). On the basis of peer review (ESAC, 2006) of the results of an inter-laboratory study (Kandarova et al, 2006) with the SkinEthic RHE Model, the ECVAM Scientific Advisory Committee (ESAC) endorsed the conclusion that the SkinEthic RHE Model can be used for distinguishing between skin corrosives and non-corrosive chemicals.
Vehicle:
unchanged (no vehicle)
Details on test system:
On arrival, the SkinEthic RHE tissues (Day 18 cultures), were stored at room temperature prior to transferring into 24-well plates designated ‘arrival plates’ containing 1000 µL of maintenance medium. It was important to ensure that there were no air bubbles present under the tissue inserts. The tissues were incubated overnight at 37 °C, 5% CO2 in air.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
20 mg of test item were applied. 20 μl of sterile distilled water was added for wetting of the solid test item to ensure adequate contact with the tissue culture surface.

Using sterile techniques 0.9 mL of maintenance medium, at room temperature, was dispensed into each well of two pre-labeled 6-well plates designated as treatment plates. One plate contained the tissues to be used in the 3 minute experiment whilst the other plate contained tissues to be used in the 60 minute experiment. The tissues were aseptically transferred into the treatment plates prior to dosing.
One plate was placed into the incubator, whilst the tissues in the other plate were being treated. 40 μL of sterile distilled water (negative control) was applied to the first two tissues, and the timer started. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure, and to ensure that each tissue received an equal exposure time. The test item and the positive control item (8.0N Potassium Hydroxide) were also applied to the corresponding duplicate tissues. 20 mg of the test item was applied. 20 μL of sterile distilled water was added for wetting of the solid test item to ensure adequate contact with the tissue culture surface. It was important to ensure the test item and the control items uniformly covered the tissue surface.
Duration of treatment / exposure:
3 minutes and 60 minutes
Duration of post-treatment incubation (if applicable):
The same treatment procedure was used for both exposure times. After treatment the plate for the 1 hour exposure experiment was placed into the incubator until rinsing. Due to the short exposure time the plate for the 3 minute exposure experiment remained in the safety cabinet during the exposure period. At the end of each exposure period, each tissue was removed from the well of the treatment plate using forceps and rinsed using a wash bottle containing PBS. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item. Excess PBS was removed by blotting the bottom of the tissue culture insert with absorbent paper. The rinsed tissues were placed into a holding plate containing 300 µL of maintenance medium in each well until all tissues had been rinsed, after which each tissue was blotted and transferred to a 24-well plate containing 300 µL of 0.5 mg/mL (v/v) MTT solution in each well for MTT loading. The plate was placed in an incubator at 37 °C, 5% CO2 in air for 3 hours.

At the end of the 3-hour MTT incubation period, the tissue culture inserts were blotted and transferred to pre-labeled 24-well plates for MTT extraction. 1.5 mL of MTT extractant solution (isopropanol) was used to completely immerse each tissue insert, and the plate was covered with a suitable plate sealer to inhibit isopropanol evaporation. The sealed plates (covered to protect from light) stood overnight at room temperature to extract the reduced MTT (formazan dye) from within the MTT loaded tissue.
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute
Value:
ca. 93.1
Vehicle controls valid:
not examined
Negative controls valid:
yes
Remarks:
The mean OD562 for the negative control treated tissues was 1.497 for the 3-Minute exposure period and 1.449 for the 60-Minute exposure period.
Positive controls valid:
yes
Remarks:
The relative mean tissue viability for the positive control treated tissues was 0.6% relative to the negative control treated tissues following the 60-Minute exposure period.
Remarks on result:
other: The test item was considered to be non-corrosive to the skin
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute
Value:
ca. 98.3
Vehicle controls valid:
not examined
Negative controls valid:
yes
Remarks:
The mean OD562 for the negative control treated tissues was 1.497 for the 3-Minute exposure period and 1.449 for the 60-Minute exposure period.
Positive controls valid:
yes
Remarks:
The relative mean tissue viability for the positive control treated tissues was 0.6% relative to the negative control treated tissues following the 60-Minute exposure period.
Remarks on result:
other:
Remarks:
not corrosive to skin
Other effects / acceptance of results:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative Control: The absolute OD562 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD562 of the two negative control tissues should be ≥ 0.8 and ≤ 3.0 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Positive Control: Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criteria if the mean relative tissue viability of the 60 minute positive control is <15%.
Coefficient of Variation: In the range of 20 to 100% viability and for Optical Densities ≥ 0.3, the difference in viability between the two tissue replicates should not exceed 30%.

Applicant's summary and conclusion

Interpretation of results:
other: Not corrosive to skin
Conclusions:
The test item was considered to be non-corrosive to the skin.
Executive summary:

Introduction

The purpose of this test was to evaluate the corrosivity potential of the test item using the SkinEthic in vitro Reconstructed Human Epidermal (RHE, SkinEthic Laboratories, Lyon, France) model after treatment periods of 3 and 60 minutes. Corrosive test items are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase by viable cells in the test item treated tissues relative to the negative control treated tissues.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 1.5 mL Isopropanol for MTT extraction. At the end of the formazan extraction period the tissues were mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viabilities of the test item treated tissues were: 3 minutes exposure : 93.1% 60 minutes exposure : 98.3%

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was considered to be non-corrosive to the skin.