Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-10-20 to 2017-01-17
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
Qualifier:
according to
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)
Version / remarks:
28th July, 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability:
This in vitro method is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS “No Category”) without further testing within a tiered testing strategy from those requiring classification and labelling (UN GHS categories 1 and 2). It therefore can be used for regulatory purposes as an initial step in the Bottom-Up approach or as one of the last steps in a Top-Down approach. It is not intended to differentiate between UN GHS “Category 1” (serious eye damage) and UN GHS “Category 2” (eye irritation) which would require additional testing. Ocular irritation potential is predicted by the relative viability of the tissue after a single exposure to the test substance. Relative viability is determined by measuring the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye conversion by the EpiOcular™ tissue construct after topical exposure to the test substance.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg
Duration of treatment / exposure:
6 ± 0.25 h
Duration of post- treatment incubation (in vitro):
18 ± 0.25 h
Number of animals or in vitro replicates:
2

Results and discussion

In vitro

Results
Irritation parameter:
other: Mean Tissue Viability [%]
Run / experiment:
Mean value
Value:
79.2
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Mean Absolute OD570nm NK: Value: 1.389, Cut off: 0.8- Acceptance criteria met for positive control: Mean Relative Viability [%]: Value: 26.1, Cut off: <50%
- Range of historical values if different from the ones specified in the test guideline: Relative Viability PC[%]: Mean: 26.3, SD: 12.75, n = 9

Any other information on results incl. tables

Table 1: Result of the Test Item L-Asparagine

Name

NK

PC

TM

Tissue

1

2

1

2

1

2

OD 570 Values (Blank-Corrected)

1.325

1.326

0.390

0.298

0.906

1.182

1.310

1.420

0.420

0.298

0.973

1.199

Mean of the Duplicates

1.318

1.373

0.405

0.298

0.939

1.190

Mean OD 570 

1.345*

0.351

1.065

Mean SD OD 570 

0.05

0.06

0.15

Tissue Viability [%]

97.9

102.1

30.1

22.2

69.8

88.5

Relative Tissue Viability

Difference [%]***

4.1

7.9

18.7

Mean Tissue Viability

[%]

100.0

26.1**

79.2

*Corrected mean OD 570 of the negative control corresponds to 100% absolute tissue viability

**mean relative tissue viability of the positive control is < 50%

***relative tissue viability difference of replicate tissues is < 20%.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The present study was conducted in order to determine the irritating potential of L-Asparagine on the eye. The test was performed according to OECD Test Guideline 492. The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The measured mean relative viability was 79.2 %. Thus, L-Asparagine is not considered to be irritating to the eye and therefore not classified according to GHS criteria.