L 131

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 20, 2014 - October 27, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

(2012)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

(2013)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): L 131
- Appearance: Faint light-green powder

Test animals

Species:

human

Details on test animals or test system and environmental conditions:

ENVIRONMENTAL CONDITIONS
- All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 77 - 88%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.1 – 36.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
- Temporary deviations may have occurred and were caused by opening and closing of the incubator door.

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amounts applied: 13.6 to 16.0 mg
- The test material was spread to match the size of the tissue

NEGATIVE CONTOL:
- Amount applied: 25 µL Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 µL
- Concentration: 5% (aq) Sodium dodecyl sulphate in Phosphate buffered saline

Duration of treatment / exposure:

15 minutes

Details on study design:

TEST SITE
- EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 14-EKIN-040). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

TEST FOR REDUCTION OF MTT
The substance was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 14.8 mg of the test substance was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

APPLICATION/TREATMENT
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 15 μL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and the solid test substance (13.6 to 16.0 mg; with a small glass weight boat) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.

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

POST INCUBATION PERIOD
- 42 hours

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.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: tissue viability

Value:

97

Remarks on result:

other:

Remarks:

Basis: other: percentage of control. Time point: 15 minutes. Remarks: Negative control = 100%; Positive control = 27%. (migrated information)

In vivo

Irritant / corrosive response data:

Mean tissue viability for the test substance was > 50% (97%), therefore the test substance is considered not to be irritant to the skin.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and 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 Small Model), conducted according to OECD 439 and GLP principles, the influence of L 131 on the viability of human skin was tested. The test substance was applied directly to 0.38 cm² cultured skin (13.6 to 16.0 mg, in presence of 15 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 27% whereas the test substance showed cell viability of 97%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it is concluded that the test substance is not irritating in the in vitro skin irritation test.