(+)-L-arginine hydrochloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

Three-dimensional reconstructed human epidermis model.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-01-11 to 2012-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(+)-L-arginine hydrochloride

EC Number:

214-275-1

EC Name:

(+)-L-arginine hydrochloride

Cas Number:

1119-34-2

Molecular formula:

C6H14N4O2.ClH

IUPAC Name:

(2S)-2-amino-5-carbamimidamidopentanoic acid hydrochloride

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: reconstructed human epidermis

Test animals

Species:

other: reconstructed human epidermis model (EST1000)

Strain:

other: Not applicable

Test system

Type of coverage:

other: The test substance was applied to the skin model to uniformly cover the skin surface.

Preparation of test site:

other: Not applicable as three-dimensional reconstructed human epidermis model EST1000.

Vehicle:

unchanged (no vehicle)

Remarks:

But the epidermis surface was moistened with Dulbecco's phosphate buffered saline (D-PBS) before application to ensure good contact with the skin.

Controls:

other: Concurrent negative (Dulbecco's phospahe buffered sline) and positive control (SDS) each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are acceptance range.

Amount / concentration applied:

30 mg on the surface area of 0.6 cm².

Duration of treatment / exposure:

See "Details on study design"

Observation period:

See "Details on study design"

Number of animals:

3 samples were applied to the skin model

Details on study design:

1. General model conditions
Normal human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) were present under a functional stratum corneum. Stratum corneum was multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of the cytotoxic marker substance sodium dodecyl sulphate (SDS). The barrier function is assessed either by determination of the concentration at which a marker substance reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50% (ET50) upon application of the marker substance at a specified, fixed concentration. The containment properties of the model prevented the passage of material around the stratum corneum to the viable tissue, which would lead to poor modelling of skin exposure. The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.

2. Functional model conditions
Viability: The preferred assay for determining the magnitude of viability was the MTT. The optical density (OD) of the extracted (solubilised) dye from the tissue treated with the negative control (NC) should be at least 20-fold greater than the OD of the extraction solvent alone. The tissue treated with NC should exhibit stability in culture (provide similar viability measurements) for the duration of the test exposure period.

3. Barrier function and quality controls (QC) of the model
Each batch of the epidermal model used meets defined production release criteria, set by the supplier, among which those for viability and for barrier function are the most relevant (MTT, 2 hours Triton X-100: target > 50%). The barrier properties of the tissues were verified by the supplier.

4. Administration of the test, negative and positive reference items
Three samples of 30 mg L-Arginine hydrochloride were applied to the skin model with a surface area of 0.6 cm2 to uniformly cover the skin surface. The epidermis surface was moistened with Dulbecco's phosphate buffered saline (D-PBS) before application to ensure good contact with the skin. A minimum of 25 mg substance applied per cm2 is required by the guidelines. At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS).
The models were cultivated at 21°C for 20 minutes according to the instructions of the EST1000 supplier CellSystems®. An incubation time with the test item for 20 minutes was recommended by the European Centre for the Validation of Alternative Methods (ECVAM).

5. Cell viability measurements
The MTT conversion assay is a quantitative validated method which is used to measure cell viability. It is compatible with use in a three-dimensional tissue construct. The most important element of the test procedure was that viability measurements were not performed immediately after the exposure to the test item, but after a post-treatment incubation period of the rinsed tissues in fresh medium of 42 hours. This period allows both for recovery from weakly irritant effects and for appearance of clear cytotoxic effects. Each skin sample was placed in a MTT solution of 1 mg/mL (37°C incubation temperature, 5% CO2, 95% humidity) for 3 hours. The precipitated blue formazan product was extracted using the solvent propanol-2, and the concentration of the formazan was measured by determining the optical density (OD) at a wavelength of 540 nm in a spectrophotometer. The measurements were made for each of the three tissues in duplicate.

6. Assay acceptability criteria
For each assay using valid batches, tissues treated with the NC exhibit OD reflecting the quality of the tissue that followed all shipment and receipt steps and all the irritation protocol processes. The OD values of controls should not be below historical established lower boundaries. Similarly, tissues treated with the PC, i.e. 5% aqueous SDS, should reflect the sensitivity retained by tissues and their ability to respond to an irritant substance in the conditions of each individual assay (e.g. viability ≤ 50% for the validated method, see Appendix 2). Associated and appropriate measures of variability between tissue replicates are defined (e.g. if standard deviations should be ≤ 18%).

7. Interpretation of results
The OD values obtained for each test sample were used to calculate a mean percentage viability relative to the negative control, which is arbitrarily set at 100%. The cut-off mean percentage cell viability value that distinguishes irritant from non-classified test substances is given below:
The test item is to be considered to be irritant to skin in accordance with UN GHS category 2 if the tissue viability after exposure and post-treatment incubation is ≤ 50%.
The test item is to be considered to have no category if the tissue viability after exposure and post-treatment incubation was > 50%.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

98

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean 1

Value:

75.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean 2

Value:

85.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean 3

Value:

134.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. An exposure time of 20 minutes was employed.

The mean viability of the cells exposed to the test item was 98.0% of the mean negative control value. The OD540 values were well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of >50% for a 20-minute exposure.

The test item was considered to be non-cytotoxic and predicted to be not irritant to skin.

The viability of cells treated with the positive reference item, 5% SDS, was 6.9% of the negative controls and was below the cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.

All quality criteria required were fulfilled.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In a GLP gudeline study according to OECD 439 (three-dimensional reconstructed human epidermis model) L-arginine-HCl did not show to be irritatting to the skin.