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Administrative data

Description of key information

L-argine-HCl is neither irritating or corrosive to skin nor irritating to eyes as well as to the respiratory system.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
other: 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
Qualifier:
according to
Guideline:
other: OECD Guideline 439 In vitro Skin Irritation: Reconstructed Human Epidermis Test Method, adopted July 22, 2010
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Method B.46. In vitro Skin Irritation: Reconstructed Human Epidermis Model Test, adopted August 24, 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
other: reconstructed human epidermis
Species:
other: reconstructed human epidermis model (EST1000)
Strain:
other: Not applicable
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%.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
98
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
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
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
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

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.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
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:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
Himalayan
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Branch Löhndorf, D-24601 Löhndorf/Post Wankendorf
- Age at study initiation: Approx. 6.5 to 8.5 months at dosing
- Weight at study initiation: 2.4 kg, 2.5 kg. 2.7 kg
- Housing: During the acclimatisation period and after the 8-hour period in restrainers after application, the animals were kept singly in cages with dimensions of 380 mm x 425 mm x 600 mm (manufacturer: Dipl.Ing. W. EHRET GmbH, 16352 Schönwalde, Germany).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
According to guideline

IN-LIFE DATES: From: 2012-01-24 To: 2012-02-17
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg
Duration of treatment / exposure:
1 hour.
Observation period (in vivo):
1 h, 24 h, 48 h, 72 h.
Number of animals or in vitro replicates:
Total of 3. In accordance with the guideline the test was performed initially using one animal. As no corrosive or severe irritant effects were observed in this animal, 2 further animals were employed 24 hours after start of the initial test.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): The eyes were rinsed with portions of 20 mL 0.9% aqueous NaCl solution, each.
- Time after start of exposure: 1 h.

SCORING SYSTEM:
- Cornea score: Opacity - degree of density (area most dense taken for reading)
- Iris score
- Conjunctivae score, redness: Refers to palpebral and bulbar conjunctivae, excluding cornea and iris
- Chemosis score: Swelling - refers to lids and/or nictitating membranes

TOOL USED TO ASSESS SCORE: Hand-slit lamp. 24 h after administration, fluorescein was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions.
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 1 h, 24 h, 48 h, 72 h
Score:
0
Max. score:
4
Remarks on result:
other: Score for each animal was 0 at any observation time.
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 1 h, 24 h, 48 h, 72 h
Score:
0
Max. score:
2
Remarks on result:
other: Score for each animal was 0 at any observation time.
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
mean
Time point:
other: 1 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 24 h
Remarks on result:
other: Score for each animal was 1 after 1 h.
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
mean
Time point:
other: 24 h, 48 h, 72 h
Score:
0
Max. score:
3
Remarks on result:
other: Score for each animal was 0 after 24 h, 48 h and 72 h.
Irritation parameter:
chemosis score
Basis:
mean
Time point:
other: 1 h, 24 h, 48 h, 72 h
Score:
0
Max. score:
4
Remarks on result:
other: Score for each animal was 0 at any observation time.
Other effects:
No pathological findings with the fluorescin test after 24 h.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
L-arginine-HCl did not show eye irritating properties in an in in vivo GLP guideline study according to OECD 405.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Skin

The dermal irritant potential of L-arginine-HCl was assessed in a GLP gudeline study according to OECD 439 (three-dimensional reconstructed human epidermis model) by application of the product to the test system. L-arginine-HCl did not show to be irritatting to the skin and is not classified as irritating or corrosive to human skin.

  

 Eye

 The eye irritant potential of L-arginine-HCl was assessed by application of the dry product to the eyes of three rabbits in accordance with OECD guideline 405. L-argine-HCl did not show any eye irritancy potential in an in vitro GLP study using fertile chicken eggs (HET-CAM test). The substance did not show any eye irritation. The product is classified as not-irritating to human eye.

  Respiratory System

There are no data available which indicate that L-arginine-HCl is irritant to the respiratory system.

 

Justification for selection of skin irritation / corrosion endpoint:

Key study

Justification for selection of eye irritation endpoint:

Key study

Justification for classification or non-classification

L-arginine-HCl does not show any irritating or corrosive properties towards both the skin and the eyes. Thus classification is not required.