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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 November 2017 to 01 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Orthoperiodic acid
EC Number:
233-937-0
EC Name:
Orthoperiodic acid
Cas Number:
10450-60-9
Molecular formula:
H5IO6
IUPAC Name:
tetrahydroxyiodous acid
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: White crystalline powder
- Storage: At room temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended test system in international guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue lot number(s): 27631
- On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM (serum-free; MatTek Corporation).

EXPERIMENTAL DESIGN
- Test for the Interference of the Test Material with the MTT Endpoint: A test material may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test material is present on the tissues when the MTT viability test is performed.
- Test for Colour Interference by the Test Material: The test material was checked for possible colour interference before the study was started. Some non-coloured test materials may change into coloured materials in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, at least 25 mg of the test material or 50 µL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.
- Test for Reduction of MTT by the Test Material: The test material was checked for possible direct MTT reduction before the study was started. To assess the ability of the test material to reduce MTT, at least 25 mg of the test material or 50 µL Milli-Q water as a negative control were added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C. At the end of the exposure time it was checked if a blue / purple colour change or a blue / purple precipitate was observed.
- MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). 

MAIN TEST
- Application/Treatment of the Test Material: The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 2.5 hours at 37.0 ± 1.0 ºC. The medium was replaced with fresh DMEM just before the test material was applied. The test was performed on a total of 4 tissues per test material together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test material and two for a 1-hour exposure. The skin was moistened with 25 µL Milli-Q water to ensure close contact of the test material to the tissue and 29.23 to 35.05 mg of the solid test material was on top of the skin tissues.
- For the negative and positive controls, 2 tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues were treated with 50 µL 8N KOH (positive control) for both the 3-minute and 1-hour time point.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test material. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.

ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS
- The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37 °C in air containing 5 % CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.0 °C
- Temperature of post-treatment incubation (if applicable): 37 °C with MTT

ENVIRONMENTAL CONDITIONS
- All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100 % (actual range 45 - 85 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.2 - 37.3 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity. 

INTERPRETATION
A test material is considered corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50 %.
- In addition, a test material considered non-corrosive (viability ≥50 %) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test material is decreased below 15 %.
A test material is considered non corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50 %.
- In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15 %.

ANALYSIS
- Calculation of Cell Viability:
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw):

ODc = ODraw – ODbl

The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:

%Viability = (ODc/mean ODlt_u+MTT) * 100



Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 29.23 to 35.05 mg. No correction was made for the purity/composition of the test material.

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration (if solution): 8 N
Duration of treatment / exposure:
3 minutes or 60 minutes
Duration of post-treatment incubation (if applicable):
Incubated for 3 hours with MTT
Number of replicates:
2 per exposure time

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
86
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure
Value:
23
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- The test material was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test material to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test material did not interfere with the MTT endpoint.
- Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 86 and 23 % respectively. Because the mean relative tissue viability for the test material was not below 50 % after 3 minutes treatment and not below 15 % after 1 hour treatment the test material is considered to be not corrosive. Since the stability of the test was only confirmed up to a temperature of 30 °C, it could be that some substance degraded at the 1 hour exposure.
- The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 7.6 %.
- In the range of 20 – 100 % viability the Coefficient of Variation between tissue replicates was 28 % for the negative and positive control. The Coefficient of Variation between tissues replicates treated with the test material was 42 % after the 1-hour exposure. However, the individual tissue viabilities after the 1-hour exposure were 29 and 17%. Both viabilities were >15 % and therefore in the same category, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 1: Mean OD570 Values and Viabilities for the Negative Control, Positive Control and Test Material

Tissue

Exposure Period

Mean OD570 of individual tissues

Mean OD570 of duplicate tissues

Standard Deviation

Coefficient of Variation
(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

1.885

1.851

0.049

3.6

100*

1.816

60 Minutes

2.092

1.908

0.260

18

1.724

Positive Control

3 Minutes

0.422

0.363

0.083

28

20

0.304

60 Minutes

0.162

0.145

0.024

21

7.6

0.128

Test Material

3 Minutes

1.559

1.585

0.037

3.2

86

1.611

60 Minutes

0.563

0.445

0.167

42

23

0.327

OD: Optical density

* The mean percentage viability of the negative control tissue is set at 100 %.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified in accordance with EU Criteria
Conclusions:
Under the conditions of this study the test material was not corrosive to the skin.
Executive summary:

The potential of the test material to cause corrosion to skin was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40 bis, under GLP conditions.

The objective of this study was to evaluate the test material for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test material was tested through topical application for 3 minutes and 1 hour.

Skin tissue was moistened with 25 µL of Milli-Q water and at least 25 mg of the test material was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 7.6 % after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit2.8) and the laboratory historical control data range. In the range of 20 – 100 % viability the Coefficient of Variation between tissue replicates was 28 % for the negative and positive control. The Coefficient of Variation between tissues replicates treated with the test item was 42 % after the 1-hour exposure. However, the individual tissue viabilities after the 1-hour exposure were 29 and 17 %. Both viabilities were >15 % and therefore in the same category, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 86 and 23%, respectively. Because the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment the test material is considered to be not corrosive.

Under the conditions of this study the test material was not corrosive to the skin.