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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:
04 July to 29 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 439 without any deviation.

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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
23 July 2009
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz (inspected on July 13-16, 2015 / signed on September 14, 2015)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state/Appearance: Colourless to pale yellow liquid, slightly viscous.
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Stable under normal temperatures and pressure

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: human-derived epidermal keratinocytes
Justification for test system used:
Following the REACH top-down strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France).
- Tissue batch number(s): 17-EKIN-030
- Expiry date: 31 July 2017
- Date of initiation of testing: 04 July 2017
The EpiSkin™ tissue consists of NHEK, 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 25 July 2017. EpiSkin™ tissues were transferred to 12-well plates with maintenance medium and the pre-incubation phase of the EpiSkin™ tissues started.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the end of the treatment interval the inserts were removed immediately from the 12-well plate. The tissues were be gently rinsed with PBS to remove any residual test material. Excess PBS were 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. Tissues were incubated for 42 ± 1 hour at 37 ± 1.5 °C, 5 ± 0.5% CO2.Number of washing steps not reported.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none

IL-1 ALPHA IMMUNOASSAY
If the results obtained by means of the MTT assay are unclear or borderline, additionally the IL-1 α concentration in the medium after 42 hours incubation can be determined by order of the Sponsor. For this purpose, samples of all treatment groups are taken from the wells. The plates are shaken for approximately 15 minutes to homogenise the released mediators in the medium before sampling. At least 1.6 mL medium from each well is taken and is stored in the freezer at ≤ -18 °C until analysis.
Following the instruction from the QuantikineTM kit the amount of released IL-1 α will be determined. Therefore 200 μL of each sample and the standard solutions is mixed with 50 μL Assay Diluent RD1C in the wells of the IL-1 α Microplate. After 2 hours incubation and washing steps 200 μL IL-1 α Conjugate is added to each well. Meanwhile the substrate solution is prepared by mixing Colour Reagent A and B in equal volumes. After 1 hour incubation and washing steps 200 μL of the substrate solution is added to each well. The plates are covered with the adhesive strips and incubated for further 20 min under light protection. After 20 min 50 μL of the stop solution is added to each well. The plates are read at 450 nm in a photometer (Versamax® Molecular Devices, software version 4.7.1) within 30 minutes. Each sample is tested in duplicate.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, software version 4.7.1
- Wavelength: 570 nm
- Filter: with 570 nm filter
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: 2.1 mg/L (1.5 mg/L < IC50 < 3;0 mg/L)
- Morphology: well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: on blood of the same donors: absence of HIV1 and 2 antibodies, of hepatitis C antibodies, and hepatitis B antigen HBs. On epidermal cells of the same donors: absence of bacteria, fungus and mycoplasma.
- Reproducibility: the results for the positive and negative controls are within the historical ranges obtained by Envigo CRS GmbH in the previous eleven months (means. rel. standard deviation. and ranges) of Envigo CRS GmbH

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
Not needed

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if relative mean tissue viability is ≤ 50% after 15 minutes of exposure.
- The test substance is considered to be non-irritating to skin if relative mean tissue viability is > 50% after 15 minutes of exposure.
- For the current test, an irritation potential of a test item is predicted if the release of IL-1 α is above the threshold of 60 pg/mL.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- The test item was used as supplied (undiluted).
- Amount(s) applied (volume or weight with unit): approximately 10 μL (26.3 μL/cm² according to guideline)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL (in DPBS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Triplicate tissues for test substance, negative and positive controls

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
52.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
6.9%
Remarks on result:
no indication of irritation
Remarks:
> 50 %
Irritation / corrosion parameter:
other: IL-alpha concentration (pg/L)
Run / experiment:
1
Value:
26.15
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
3.14
Positive controls validity:
valid
Remarks:
129.21
Remarks on result:
no indication of irritation
Remarks:
< 50 pg/mL
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.672 and the standard deviation value of the viability was 2.8%.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 6.9% relative to the negative control treated tissues and the standard deviation value of the viability was 11.9%.
- 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 5.6%. - Reference to historical values: All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system.

Any other information on results incl. tables

Table 7.3.1/1: Results of the MTT assay

Test Group

Absor-bance 570 nm
Tissue Well 1

Absor-bance 570 nm
Tissue Well 2

Mean Absor-bance 570 nm

Mean Absor-bance 570 nm
blank corrected

Mean Absor-bance of 3 Tissues

Relative Viability [%] Tissue
1, 2, 3*

Relative Standard Deviation [%]

Mean Rel. viability

[%]**

Blank

0.038

0.039

0.038

 

 

Negative Control

0.718

0.684

0.701

0.663

0.672

 

98.7

2.8

 

100.0

0.759

0.705

0.732

0.694

103.2

0.717

0.677

0.697

0.659

98.1

Positive Control

0.089

0.087

0.088

0.050

0.047

 

7.4

11.9

 

6.9

0.086

0.090

0.088

0.050

7.4

0.075

0.082

0.078

0.040

6.0

Test Item

0.406

0.406

0.406

0.368

0.351

54.8

5.6

52.2

0.395

0.392

0.394

0.355

52.9

0.376

0.360

0.368

0.329

49.0

* Relative absorbance [rounded values]

**  Mean relative absorbance [rounded values]

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 52.2% (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

This value is very close to the threshold for irritancy (≤ 50%), consequently, the results of the MTT test were considered borderline. It was considered necessary to perform IL-1α analysis.

Table 7.3.1/2: Results of the IL1 ELISA

Abs. 1
(OD)

Abs. 2
(OD)

Mean
Abs. 1-2 (OD)

IL1αConc.
[pg/ml]*

Medium

0.020

0.017

0.019

0.903

42h Negative Ctrl, Tissue 1

0.060

0.078

0.069

4.507

42h Negative Ctrl, Tissue 2

0.059

0.055

0.057

3.641

42h Negative Ctrl, Tissue 3

0.105

0.115

0.110

7.510

42h Positive Ctrl, Tissue 1

1.447

1.554

1.501

150.458

42h Positive Ctrl, Tissue 2

1.051

1.360

1.206

113.458

42h Positive Ctrl, Tissue 3

1.189

1.482

1.336

129.320

42h Test Item, Tissue 1

0.264

0.236

0.250

18.287

42h Test Item, Tissue 2

0.503

0.451

0.477

37.482

42h Test Item, Tissue 3

0.406

0.270

0.338

25.476

*the parallel entrainment of a definite IL1α standard led to a curve with the following formula:

y = 20,654x2+ 69,547x - 0,3903


The IL1α concentration of the samples (y) was calculated by setting the value
Mean Abs. 1-2 (OD) as x.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. 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 570 nm.

 

The relative mean viability of the test item treated tissues was 52.2% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period. The amount of mean IL1 -alpha release is 26.15 pg/mL.

The relative mean tissue viability for the positive control treated tissues was 6.9% relative to the negative control treated tissues and the standard deviation value of the viability was 11.9%. The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.672 and the standard deviation value of the viability was 2.8%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 5.6%. The test item acceptance criterion was therefore satisfied.

All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system.

Under the experimental conditions of this study, the viability being above 50% and the IL1 -alpha release below 50 pg/mL, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.