L-Arginine, N2-(2,3-dihydroxypropyl)-, monohydrochloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 February 2012 - 03 March 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted according to international guideline and line with ECVAM recommendation under GLP albeit with slight deviation i.e. the optical density measurement was 540 nm instead of 570 nm.

Justification for type of information:

Refer to the read-across justification attached in Section 13.2.

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:

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

Version / remarks:

24 August 2009

Deviations:

yes

Remarks:

The optical density measurement was 540 nm instead of 570 nm

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

yes

Remarks:

The optical density measurement was 540 nm instead of 570 nm

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

IUPAC Name:

(+)- L-arginine hydrochloride

Test material form:

solid: crystalline

Details on test material:

CAS no. 1119-34-2
EC no. 214-275-1
Batch no. 10110839
Receipt no. 50080
Date of receipt: February 09, 2012
Characteristics: White crystalline powder
Storage conditions: +10°C - +25°C
Stability (retest date): December 15, 2013
Assay (dry basis): 100.0%

In vitro test system

Test system:

human skin model

Remarks:

EST1000

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human derived epidermis keratinocytes

Source strain:

not specified

Details on animal used as source of test system:

N/A

Justification for test system used:

The principle of the reconstructed human epidermis model test is based on the premise that irritant substances are able to penetrate the stratum corneum by diffusion and are cytotoxic to the cells in the underlying layers. Cell viability was measured by dehydrogenase conversion of the vital dye MTT (3-[4,5- Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue1 ; EINECS number 206-069-5, CAS number: 298-93-1), into a blue formazan salt that was quantitatively measured after extraction from tissues. Irritant substances were identified by their ability to decrease cell viability below defined threshold levels (i.e.≤ 50% for UN GHS category 2 irritants). Substances that produce cell viabilities above the defined threshold level will not be classified (i.e.> 50%, no category).
The reconstructed human epidermis model systems may be used to test solids, liquids, semi-solids and waxes. Solids may be soluble or insoluble in water. Whenever possible, solids should be tested as a fine powder. The test item is a solid white powder and therefore the test system was deemed suitable.

Vehicle:

unchanged (no vehicle)

Remarks:

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

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Skin model EST1000 (CellSytems® Biotechnology GmbH, 53562 Katharinen, Germany), insert size 0.6 cm^2
- Tissue batch number(s): Batch no. EST-120213-001 (Catalogue no. CS-1001)
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: Not specified
- Date of initiation of testing: 10 February 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The models were cultivated at 21°C for 20 minutes according to the instructions of the EST1000 supplier CellSystems®.
- Temperature of post-treatment incubation (if applicable): 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 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).
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: N/A

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Yes
- Wavelength: 540 nm
- Filter: Not specified
- Filter bandwidth: Not specified
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT conversion assay. 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. Viability was performed after a post-treatment incubation period of the rinsed tissues in fresh medium of 42 h. 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 h. 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.

- Barrier function: 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. 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.

- Morphology: The test item was applied topically to a three-dimensional reconstructed human epidermis model, comprised of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised 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 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.

- Contamination: The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.

- Reproducibility: Concurrent negative and positive controls were used, each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are within a defined historical acceptance range. The NC item was Dulbecco's phosphate buffered saline (D-PBS) and the PC item was 5% aqueous sodium dodecyl sulphate (SDS)

NUMBER OF REPLICATE TISSUES: 3 tissues in duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable): N/A
- No. of replicates: 3 tissues in duplicate
- Method of calculation used:
The MTT conversion assay was used to measure cell viability. 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%.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA
The test item is to be considered to be an irritant to skin in accordance with 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%.

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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Three samples of 30 mg test item were applied to the skin model with a surface area of 0.6 cm^2 to uniformly cover the skin surface.
- Concentration (if solution): N/A

VEHICLE: N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Dulbecco’s phosphate buffered saline (D-PBS) in triplicate (concentration not specified)
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): sodium dodecyl sulphate
- Concentration (if solution): 5 % aqueous

Concurrent negative and positive controls were used, each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are within a defined historical acceptance range.

Duration of treatment / exposure:

20 m

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3 tissues in duplicate

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 3 (mean)

Value:

134

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2 (mean)

Value:

76

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1 (mean)

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: None reported
- Colour interference with MTT: None reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: N/A, model variability criteria not provided in OECD Guideline
- Range of historical values if different from the ones specified in the test guideline: N/A

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.

Any other information on results incl. tables

Table 1. Summary results of in vitro skin irritation optical density (OD)

Formazan extraction solution	Extinction# (n= 3 tissues)	SD	% extinction compared to control
Negative control D-PBS	0.548	0.027	-
Test item	0.537	0.152	98.0
Positive control 5 % SDS	0.038	0.002	6.9

# : at λ = 540 nm

D-PBS: Dulbecco's phosphate buffered saline

SD: standard deviation

SDS: 5 % aqueous deviation sodium dodecyl sulphate

 

Table 2. Individual values optical density (OD)

                                   Extinction at λ = 540nm

Replicates (duplicate measurements per tissue)	Negative control (D-PBS)	Test item	Postive control 5 % SDS
Tissue 1	0.592	0.412	0.035
	0.566	0.464	0.036
Tissue 2	0.537	0.447	0.042
	0.534	0.472	0.034
Tissue 3	0.533	0.727	0.040
	0.526	0.697	0.038
Mean	0.548	0.537	0.038
SD	0.027	0.152	0.002

D-PBS: Dulbecco's phosphate buffered saline

SD: standard deviation

SDS: 5 % aqueous deviation sodium dodecyl sulphate

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the conditions of the study, the test item was concluded not to be irritating to the skin.

Executive summary:

An in vitro study was performed in accordance with OECD 439 (2010), in order to assess the skin irritation potential of the test item.

The EST1000 three-dimensional reconstructed human epidermis skin model was utilised. Cell viability was determined by measuring the optical density (OD) at a wavelength of 540 nm.

The test item was applied to the model's skin surface for an exposure time of 20 minutes. D-PBS was used as a negative control and 5 % aqueous SDS was used as a positive reference control.

The mean viability of cells exposed to the test item was 98 % of the negative controls. The OD₅₄₀ value was above the percentage cut-off value for cell viability of 50 %, following 20 minutes exposure. The negative control and positive controls produced valid results (positive control: 6.9 % OD₅₄₀; i.e. below the 50 % cut off for cell viability).

It was concluded based on the conditions of the test, that the test item was not cytotoxic and not irritating to the skin and would not be classified under CLP Regulation (EC) 1272/2008.