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

Eye irritation

Administrative data

Endpoint:

eye 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 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013-07-26

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

2010-12-08

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

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse (Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG, 49699 Lindern, Germany). To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL . Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used.

The quality of each cornea was also evaluated at later steps in the assay. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.

The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM) , while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.

After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 20 % suspension in 0.9 % sodium chloride solution (w/v)

VEHICLE
- Concentration (if solution): 0.9 % sodium chloride solution (w/v)
- Lot/batch no. (if required): Batch no. 163548002; B. Braun Melsungen AG, 34212 Melsungen, Germany
- Purity: analytical grade

Duration of treatment / exposure:

240 min

Number of animals or in vitro replicates:

3 in vitro replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse . To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL .

QUALITY CHECK OF THE ISOLATED CORNEAS
Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used.
The quality of each cornea was also evaluated at later steps in the assay. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
0.9% sodium chloride solution

POSITIVE CONTROL USED
20% Imidazole (CAS no. 288-32-4) in 0.9% sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME
750 µL of the test or control items as recommended as suitable test volume according to OECD TG 437 were added to completely cover the cornea’s epithelium in the anterior chamber. Exposure time: 240 min.

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 washing steps
The epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

POST-INCUBATION PERIOD:no.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: To determine the corneal permeability 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32±1°C for 90±5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader (Tecan Sunrise Magellan Version 7.2 ). Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer (Tecan Sunrise) using a standard 1 cm path length.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. Yes

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Treatment group

Value:

0.073

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

other: mean opacity compared to the negative control

Run / experiment:

Treatment group

Value:

0.213

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: For hiitorical data 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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions L-(+)-2,5-diaminopentanoic acid (L-ORNITHINE MONOHYDROCHLORIDE) tested in the in vitro BCOP test method, had an IVIS value of 0.073, which is below the cut-off value of 3 (UN GHS no category) and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.

Executive summary:

The aim of the experiment was to determine if L-(+)-2,5-diaminopentanoic acid (L-ORNITHINE MONOHYDROCHLORIDE) has tobe classified as “ocular corrosive and severe irritant” without in vivo testing.

Under the present test conditions L-(+)-2,5-diaminopentanoic acid (L-ORNITHINE MONOHYDROCHLORIDE) tested in the in vitro BCOP test method, had an IVIS value of 0.073, which is below the cut-off value of 3 (UN GHS no category) and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.