Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vitro test system

Test system:
human skin model
Remarks:
EpiDerm
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: normal human keratinocytes, not further specified
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
This in vitro study was performed to assess the irritation potential of test item by means of the Human Skin Model Test.

CELL CULTURE:
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. lt consists of organized basal, spinous and granular layers, and a multilayered 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 diameter).
EpiDerm™ tissues were shipped at 4°C on medium-supplemented agarose gels in a 24-well plate and reached Harlan CCR on 16 July 2013. On day of receipt EpiDerm™ tissues were kept in the refrigerator at 4 °C until use.

STUDY DESIGN:
Each three tissues of the human skin model EpiDerm TM were treated with the test item, the negative or the positive control for 60 minutes.
The test item was warmed in a water bath to 37°C. 30 μL of the test item were applied to each tissue and spread to match the surface of the tissue.
30 μL of either the negative control (DPBS) or the positive control (5% Sodium lauryl sulfate) were applied to each tissue.
The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following 69.5 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
30 µL
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42 h post-treatment incubation
3 h MTT incubation
Number of replicates:
Test substance, negative control and positive control were tested in triplicate.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: % mean relative absorbance
Run / experiment:
test item, 60 min treatment
Value:
96.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100.0
Positive controls validity:
valid
Remarks:
3.0
Other effects / acceptance of results:
Compared to the relative absorbance value of the negative control the mean relative absorbance value decreased to 96.4% after exposure of the test item to the skin tissues. This value is well 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

After treatment with the negative control (DPBS) the absorbance values were well within the required acceptability criterion of mean OD between 1.0 and 2.5 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control (5% SLES) induced a decrease in the relative absorbance as compared to the negative control to 3.0% thus ensuring the validity of the test system.

The standard deviations between the % variabilities of the test item, the positive and negative controls were below 10% (threshold of OECD 439: 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative control the mean relative absorbance value decreased to 96.4% after exposure of the test item to the skin tissues. This value is well above the threshold for irritancy of <=50% . Therefore, the test item is not considered to possess an irritant potential.

Dose Group

Treatment Interval

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

1.800

1.863

1.875

1.846

97.5
100.9
101.6

2.2

100.0

Positive Control

60 min

0.050

0.060

0.055

0.055

2.7
3.2
3.0

9.2

3.0

Test Item

60 min

1.605

1.841

1.892

1.779

86.9
99.7
102.5

8.6

96.4

 

*      Mean of three replicate wells after blank correction
**
       relative absorbance per tissue [rounded values]
***
     relative absorbance per treatment group [rounded values]

 

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 96.4% (threshold for irritancy: ≤50%), consequently the test item was not irritant to skin. 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, test item is not irritant to skin.
Executive summary:

SUMMARY

 

This in vitro study was performed to assess the irritation potential of test item by means of the Human Skin Model Test.

Each three tissues of the human skin model EpiDermwere treated with the test item, the negative or the positive control for 60 minutes.

The test item was warmed in a water bath to 37°C. 30 µL of the test item were applied to each tissue and spread to match the surface of the tissue.

 

30 µL of either the negative control (DPBS) or the positive control (5% Sodium lauryl sulfate) were applied to each tissue.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following 69.5 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

 

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD greater or equal than 1.0 and less or equal than 2.5 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.0% thus ensuring the validity of the test system.

 

The standard deviations between the % variabilities of the test item, the positive and negative controls were below 10% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

 

Compared to the relative absorbance value of the negative control the mean relative absorbance value decreased to 96.4% after exposure of the test item to the skin tissues. This value is well above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.