DL-alanine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August-September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

DL-alanine

EC Number:

206-126-4

EC Name:

DL-alanine

Cas Number:

302-72-7

Molecular formula:

C3H7NO2

IUPAC Name:

DL-alanine

Test material form:

solid: crystalline

Specific details on test material used for the study:

98.5% purity

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult human derived epidermal keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 18 EKIN 035.
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.
Source: SkinEthic Laboratories, Lyon, France.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

14.7-18.1 mg

Duration of treatment / exposure:

15 +/- 0.5 min

Duration of post-treatment incubation (if applicable):

42 h at 37 deg C

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

102

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The positive control had a mean cell viability of 26% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 8%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

DL-alanine is non-irritant in the in vitro skin irritation test

Executive summary:

DL-alanine was tested for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM^TM)). The possible skin irritation potential of the test item was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

DL-alanine is a white crystalline powder with a purity of 98.5%. Skin tissue was moistened with 5 µL of Milli-Q water and at least 10 mg of the test item was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 102%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

The positive control had a mean cell viability of 26% after 15 ± 0.5 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 8%, indicating that the test system functioned properly.

In conclusion, DL-alanine is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.