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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:
08 - 29 Aug 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(2015)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(Commission Regulation(EC) No 440/2008 of 30 May, 1st ATP 2009)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm™ (EPI-200)
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 23351
- Delivery date: 23 Aug 2016
- Date of initiation of testing: 23 Aug 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min in the incubator; thereafter at room temperature for 25 min in a sterile bench

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least 3 times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, Softmax Pro v.4.7.1)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 2.031 ± 0.069 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.77 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance showed Color Interference 1h after incubation in deionised water, an additional test with one viable tissue without MTT addition was necessary.
- N. of replicates: One for each untreated and test susbtance treated group
- Method of calculation used: Data were corrected as follows:
OD = ODcoloured tissue (MTT assay) – ODcoloured tissue (no MTT assay)

Since the test substance showed reducing capacity 1 h after MTT incubation, an additional test with freeze-killed tissues had to be performed to determine a correction factor for calculating the true viability in the main test.
- N. of replicates: 2 for each untreated and test susbtance treated group
- Method of calculation used: Data were corrected as follows:
True viability = Viability of treated tissue – Interference from test chemical = ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt)
tvt = treated viable tissue kt = killed tissues tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)


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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 hour exposure is less or equal than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% aqueous solution
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
approx. 43 h
Number of replicates:
triplicates for each treatment and control group

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 3 tissues
Run / experiment:
60 min exposure
Value:
6.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: viability corrected for color interference and direct MTT reduction of the test substance
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent showed blue colour. An additional test with freeze-killed tissues was performed. The result was used for data correction of the results in the main experiment.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water led to a colour change of water. An additional test with one viable tissue was performed to gain the correction factor for calculation of the true viability.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values (1.745, 1.842, 1.717) were in the range of the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 min treatment.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.2% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations of the 3 identical replicates of the test substance, the positive and negative control, were < 18% (1.3, 1.8, 3.7%), thus ensuring the validity of the study.

Any other information on results incl. tables

Table 2. Results after treatment with the test substance and controls

 

 

Absorbance at 570 nm *

 

 

Mean absorbance of 3 tissues

Rel. absorbance (%) **

Rel. SD (%)

Rel. absorbance (% of negative control)***

Mean absorbance blank corrected viable tissue without MTT

Mean absorbance blank corrected freeze-killed tissue minus mean absorbance blank corrected freeze-killed negative control tissue

Mean rel. absorbance (% of negative control) after complete correction procedure

Tissue 1

Tissue 2

Tissue 3

Tissue 1

Tissue 2

Tissue 3

Negative control

1.745

1.842

1.717

1.768

98.7

104.2

97.1

3.7

100.0

 

Positive control

0.055

0.057

0.056

0.056

3.1

3.2

3.2

1.8

3.2

 

Test substance

0.116

0.117

0.114

0.116

6.6

6.6

6.4

1.3

6.5

0.003

0.074

6.4

Additional test with one viable tissue without MTT addition

Negative Control

0.001

 

100.0

 

Test substance

0.003

 

218.9

 

Additional test with two freeze-killed tissues

Negative Control

0.064

0.049

 

0.056

 

100.0

 

Test substance

0.125

0.135

 

0.130

 

230.7

 

* Mean of 3 replicate wells after blank correction

** Relative absorbance per tissue (rounded values): 100 × (absorbance tissue) / (mean absorbance negative control)

*** Relative absorbance per treatment group (rounded values): 100 × (mean absorbance test item/positive control) / (mean absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:
other: Skin Irrit. 2 or Skin Corr. 1 according to Regulation (EC) No 1272/2008
Conclusions:
Under conditions of the reconstructed human epidermis test, a tissue viability of 6.4% was determined (threshold for classification ≤ 50%). Therefore, the test substance is considred to possess an irritant potential to the skin.
Executive summary:

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a reconstructed human skin model according to OECD Guideline 439 and in compliance with GLP (2017). After treatment with the test substance for 60 min the tissue viability decreased to 6.4% compared to the negative control (threshold for irritancy ≤ 50%). Therefore, the test substance is considered to possess an irritant potential.