DL-alanine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

DL-alanine

EC Number:

206-126-4

EC Name:

DL-alanine

Cas Number:

302-72-7

Molecular formula:

C3H7NO2

IUPAC Name:

DL-alanine

Test material form:

solid: crystalline

Specific details on test material used for the study:

98.5% purity

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27438 Kit B).
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.
Source
MatTek Corporation, Ashland MA, U.S.A.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

52.2-52.6 mg

Duration of treatment / exposure:

6 h +/- 15 min

Duration of post- treatment incubation (in vitro):

18 h +/- 15 min

Number of animals or in vitro replicates:

2

Details on study design:

No correction was made for the purity/composition of the test item.
The solid test item (52.2 to 52.6 mg) was applied directly on top of the skin tissue.
Amino acid FC-C 13387 was spread to match the size of the tissue. Any residual volumes were discarded.
The test was performed on a total of 2 tissues per test item together with a negative control and positive control.
Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. Two tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues with
50 µL Methyl Acetate (positive control) respectively.
At least 50 mg solid was added into the 6-well plates on top of the tissues. After the exposure period with the test item (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C.
After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 mL isopropanol for 2 - 3 hours at room temperature with gentle shaking.
The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

Results and discussion

In vitro

Results

Irritation parameter:

other: cell viability (%)

Value:

82

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

DL-alanine is non-irritant in the EpiOcular(TM) test.

Executive summary:

The eye hazard potential of DL-alanine was tested. For this purpose DL-alanine was topically applied on the Reconstructed Human EpiOcular™ Model. The possible eye hazard potential was tested through topical application for 6 hours. The study procedures described in this report were based on the most recent OECD guideline.

DL-alanine is a white crystalline powder. The test item (52.2 to 52.6 mg) was applied directly on top of the tissue for 6 hours±15 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

The positive control had a mean cell viability of 21% after 6 hours±15 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 11%, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours±15 minutes treatment with the test item compared to the negative control tissues was 82%. Since the mean relative tissue viability for DL-alanine was above 60% after 6 hours±15 minutes treatment the test item is considered to be non-irritant.

In conclusion, DL-alanine is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.