Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-01-23 to 2015-04-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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:
2013
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
other: Human skin model EpiDerm
Strain:
other: Human skin model EpiDerm
Details on test animals and environmental conditions:
Epi-200- SIT Kit Lot No.: 21627
MTT-100 Assay Kit Components

Epi-200 SIT kits and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia). 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. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅).

EpiDerm™ tissues were shipped with cool packs on medium-supplemented agarose gels in a 24-well plate and reached Harlan CCR on 17 February 2015. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.

Test system

Type of coverage:
other: In-vitro human skin model Epiderm
Preparation of test site:
other: In-vitro human skin model Epiderm
Vehicle:
unchanged (no vehicle)
Controls:
other: No control animals because Human skin model. Negative and positive control were used in this test.
Amount / concentration applied:
30 µL (47µL/cm^2) of the undiluted test item was dispensed directly atop the EpiDerm tissue and spread to match the surface of the tissue.
Duration of treatment / exposure:
60 min
Observation period:
Not applicable
Number of animals:
Human in vitro skin model, 3 tissues per dose
Details on study design:
Treatment
Time: 60 min
Incubator: Within this period the 6-well plates were put into the incubator for 35 min at 37 +/- °C, 5+/- 0.5 % CO2.
Rinsing: After the end of the treatment interval the inserts were removed immediately with DPBS at least 15 times in order to remove any residual test material.
Incubation time: 42 hours

MTT Assay
A volume of 300 µL of the MTT solution was added to each well and the plates were kept in an incubator (37 +/- 1 °C, 5 +/- % CO2) until further use.
Incubation period: 3 hours
Time: After the 42 hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT plates.
Rinsing: After 3 h incubation, wells were rinsed three times with DPBS and were transferred onto new 24-well plates.
Extraction time: About 68 hours at room temperature the formazan salt was extracted.
Extractant solution: Isopropanol

OD reading: OD was read in a microplate reader (Versamax Molecular Devices, Softmax Pro, version 4.7.1) with 570 +/- nm filter. Mean values were calculated from the 3 wells per tissue.


Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: Tissue viability after exposure compared to negative control in %
Value:
89.1
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 60 min treatment. Max. score: 100.0. Remarks: Not irritant.
Other effects / acceptance of results:
After treatment with the test item the mean relative absorbance value decreased to 89.1% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Any other information on results incl. tables

Dose group

Treatment Inveral

Absorbance
570 nm
Tissue 1 *

Absorbance
570 nm
Tissue 2 *

Absorbance
570 nm
Tissue 3 *

Mean
Absorbance
of 3 Tissues

Rel. Absorbance
[%] Tissue
1, 2 +3 **

Relative
Standard
Deviation [%]

Mean Rel.
Absorbance
[% of
Negative
Control] ***

Negative Control

60 min

1.618

1.628

1.564

1.603

100.9
101.6
97.5

2.2

100

Positive Control

60 min

0.068

0.069

0.062

0.067

4.3
'4.3
'3.9

5.8

4.2

Test item

60 min

1.491

1.461

1.335

1.429

93
91.1
83.3

5.8

89.1

* Mean of three replicate wells after blank correction

** relative absorbance per tissue (rounded values): 100 x (absorbance tissue)/mean absorbance negative control)

*** relative absorbance per treatment group (rounded values): 100 x (mean absorbance test/positive control)/(mean absorbance negative control)

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour and the colour of the test item was not intensive => additional test with viable tissues without MTT addition was not necessary

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour => additional test with freeze-killed tissues without MTT addition was not necessary

The mean relative absorbance value of the test item, corresponding to the cell viability, was not reduced (89.1%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The test item did not prove to be a MTT reducer (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes. Each 30 μL of the test item, of the negative control (DPBS), and of the positive control (5% SLS) were applied to each tissue and spread to match the surface of the tissue.

After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system. After treatment with the test item the mean relative absorbance value decreased to 89.1% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the tes titem is not irritant to skin.