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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:
January 2016 - March 2016
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:
July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
July 2012
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: off-white powder
- Storage condition of test material: At room temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. Broomdex has been tested previously in the Skin corrosion test using EpiDerm as a skin model and was found to be not corrosive. To evaluate Broomdex for its ability to induce skin irritation EPISKIN is used as a skin model, as recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
EPISKIN Standard Model™ (EPISKIN-SM™, 0.38 cm², Lot no.: 16-EKIN-007). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which resulted in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

APPLICATION TEST SUBSTANCE:
The skin was moistened with 5 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

ACCEPTABILITY OF THE ASSAY:
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.

A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
22.5 to 30.4 mg
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
three replicates

Test system

Vehicle:
unchanged (no vehicle)

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
96
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that the test substance did not interact with MTT.
Negative and positive controls were within historical data range.

The mean absorption at 570 nm measured after treatment with Broomdex was 0.856 (negative control = 0.892, positive control = 0.086).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
An in vitro skin irritation test was conducted with Broomdex according to OECD 439 guideline and in compliance with GLP principles.
It is concluded that this test is valid and that the test substance is not irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model), the influence of the test substance on the viability of human skin was tested according to OECD guideline and in compliance with GLP principles. The test substance was applied directly to 0.38 cm2 cultured skin (22.5 to 30.4 mg, in presence of 5 μl Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 10% whereas the test substance showed cell viability of 96%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is not irritating in the in vitro skin irritation test.