L-(+)-2,5-diaminopentanoic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-09 to 2017-08-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Version / remarks:

2015-07-28

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012-07-06

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

L-(+)-2,5-diaminopentanoic acid

EC Number:

221-678-6

EC Name:

L-(+)-2,5-diaminopentanoic acid

Cas Number:

3184-13-2

Molecular formula:

C5H12N2O2.ClH

IUPAC Name:

L-(+)-2,5-diaminopentanoic acid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Kyowa Hakko Bio Co., Ltd. / lot 160060
- Purity test date: 2016-12-01

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a tightly closed container, in a dry and well-ventilated place in a cool and dry place at +10°C to +25°C.
- Stability under test conditions: yes
- Solubility and stability of the test substance in the solvent/vehicle: highly soluble

In vitro test system

Test system:

human skin model

Source species:

human

Details on animal used as source of test system:

The test method is based on reconstructed human epidermis models, which in their overall design (the use of human derived epidermal keratinocytes as cell source, representative tissue and cyto-architecture) closely mimic the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Justification for test system used:

The procedure described under this test method allows the hazard identification of irritant substances in accordance with UN GHS Category 1 or Category 2.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm TM (EPI-200)
- Tissue batch number(s): Lot no. 25815
- Date of initiation of testing: May 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 1 washing step Dulbecco's phosphate buffered saline (D-PBS)
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
Negative control (DPBS = Dulbecco's Phosphate-Buffered Saline). 100 % mean
Positive control (5 % SDS): 5.1 % mean
- Reproducibility: acceptable

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
According to the EU and GHS classification (H314 or H315 / Category 1/2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or equal to 50% of the mean viability of the negative controls.
mean tissue viability ≤ 50% Irritant (I), (H314 or H315 or GHS Category 1 or 2 )
mean tissue viability > 50% non-irritant (NI).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

VEHICLE
No vehicle. :
For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl Dulbecco’s phosphate buffered saline.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5 % SDS

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test material group

Value:

77.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

other: mean optical density

Run / experiment:

Treatment group

Value:

1.794

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: Histrorical values see "Any other information on materials and methods incl. tables"

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: yes

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions, L-(+)-2,5-diaminopentanoic acid tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, predicted to be non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin. Hence, the test item did not show irritant properties and is therefore not classified as irritant (UN GHS no category).

Executive summary:

The purpose of this study was to determine cytotoxic properties of L-(+)-2,5-diaminopentanoic acidL (-ORNITHINE MONOHYDROCHLORIDE) to skin cells, which might lead to irritation ofhuman skin, by using an artificial three-dimensional model of human skin. The EpiDerm^TM model was employed.

Under the present test conditions, L-(+)-2,5-diaminopentanoic acid tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, predicted to be non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin. Hence, the test item did not show irritant properties and is therefore not classified as irritant (UN GHS no category).