Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Sep - 05 Oct 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

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)
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:
TEST SKIN MODEL EpiDerm™ (Epi-200-SIT Kit)
- Source: MatTek Corporation, Bratislava, Slovakia
- Lot No.: 21697

TEST METHOD
The EpiDerm™ 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. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
0.9 mL assay medium (20 – 25 °C) was pipetted into each well of sterile 6-well plates. The inserts with the EpiDerm™ tissues were placed in the upper wells, and were pre-incubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further 22 - 23 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1.5
- CO2 gas concentration (%): 5 ± 0.5
- Humidity (%): 95 ± 5

TEST SITE
- Area of exposure: 0.6 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: After the end of the treatment interval, tissues were gently rinsed with DPBS at least 15 times. After rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were carefully dried using sterile cotton tipped swap.
- Post-treatment incubation period: 42 h

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, a volume of 300 µL MTT solution was added to each well for 3 h at 37 ± 1 °C and 5 ± 0.5% CO2. After aspiration of the MTT solution, wells were rinsed three times with DPBS. Extraction of the formazan product was carried out in 2 mL isopropanol. The formazan salt was extracted for nearly 70 h without shaking in the refrigerator. The optical density (OD) was measured at 570 ± 1 nm wave length in a microplate reader (Versamax Molecular Devices).

EVALUATION OF RESULTS
The mean OD of the three negative control tissues was calculated after blank correction. This 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 (%) = [(OD-test item/positive control) / (OD-negative control)] x 100

ACCEPTABILITY OF THE ASSAY
1. OD 570 nm of the negative control ≥ 0.8 and ≤ 2.8
2. Mean relative tissue viability of the positive control ≤ 20%
3. Rel. standard deviation of 3 identical replicates < 18%
OD values should not be below historically established boundaries.
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% in deionised water
Duration of treatment / exposure:
60 min (35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2, 95 ± 5% RH; thereafter, plates were removed from the incubator and placed in a sterile bench at room temperature until the end of treatment)
Duration of post-treatment incubation (if applicable):
about 42 h
Number of replicates:
The test was performed in triplicates for each treatment and control group.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
60 min exposure
Value:
60.6
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test substance after 1 h incubation with MTT-reagent did not show blue colour.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test substance’s colour change potential in water did not lead to a change in colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values (1.607, 1.622 and 1.669) 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 5.9 % 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% (2.0, 9.2 and 3.6%), thus ensuring the validity of the study.

Any other information on results incl. tables

Table 2. Results after 60 min incubation time.

Test group

Absorbance at 570 nm

Mean absorbance

Rel. absorbance (%) Tissue 1, 2 and 3**

Rel. SD (%)

Mean rel. absorbance (% of negative control)***

Tissue 1*

Tissue 2*

Tissue 3*

Negative control

1.607

1.622

1.669

1.633

98.4
99.3
102.2

2.0

100.0

Positive control

0.107

0.092

0.091

0.097

6.5
5.7
5.6

9.2

5.9

Test substance

0.994

1.022

0.952

0.989

60.9
62.6
58.3

3.6

60.6

* 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: CLP/EU GHS criteria not met, no classification according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the Reconstructed Human Epidermis test the test substance does not possess any skin irritating potential.
Executive summary:

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a human skin model according to OECD Guideline 439 and in compliance with GLP (2016). Each three human skin tissues (EpiDermTM) with a tissue size of 0.6 cm² were treated with 30 µL of the test substance, the negative control (DPBS buffer) or the positive control (5% sodium lauryl sulphate) for 60 minutes. After a 42-hours post-incubation period, the cytotoxic (irritancy) effect was assessed. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The relative mean tissue viability obtained after 60 minutes treatment with the test substance compared to the negative control tissues was 60.6%. Since, the mean relative tissue viability for the test substance was above the threshold for irritancy of 50% after 60 min treatment the test substance was considered to be non-irritant to the skin. The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the substance in water did not lead to a change in colour. Additionally, optical evaluation of the MTT-reducing capacity of the test substance after 1 hour incubation with MTT-reagent did not show blue colour. The positive control, 5% sodium lauryl sulphate, revealed a mean cell viability of 5.9% (required ≤ 20%) after 60 min exposure and thus ensuring the validity of the test system. All other acceptability criteria were met. Based on the results of this study, the test substance was not irritating to the skin under the conditions of the test.