Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 December, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
(2013)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
(2010)
Deviations:
no
Principles of method if other than guideline:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): NS-1000
- Appearance: Clear colourless liquid

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL per cornea

NEGATIVE CONTROL
- Amount applied: 750 µL of physiological saline per cornea

POSITIVE CONTROL
Amount applied: 750 µL per cornea
Concentration (if solution): 10% (w/v) Benzalkonium Chloride


Duration of treatment / exposure:
10 minutes
Details on study design:
TEST SITE
- Isolated bovine cornea
- Three corneas were used for each treatment group
- The corneas were incubated at 32 ± 1°C

REMOVAL OF TEST SUBSTANCE
- Washing: yes (with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM)
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
- After 10 minutes of exposure, the cornea is thoroughly rinsed to remove the test substance. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed.
- Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) was evaluated (90 minutes incubation)
- After the incubation period, permeability evaluation of the cornea was performed
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate the mean in vitro score:
Mean in vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).
- Opacity and permeability values were also evaluated individually

TOOL USED TO ASSESS SCORE:
- Opacitymeter and microplate reader

DATA EVALUATION:
- A test substance that induces a mean IVIS >55 is classified with Eye Irr. Cat. 1 in accordance with the CLP Regulation
- A test substance that induces a mean IVIS ≤ 3 is not classified for Eye irritation in accordance with the CLP Regulation
- For a test substance that induces a mean IVIS of >3 - ≤55, it cannot be concluded whether the substance needs to be classified or not and for these substances, more (in vivo) information is needed

Results and discussion

In vivo

Results
Irritation parameter:
other: In vitro irritancy score (IVIS)
Basis:
mean
Time point:
other: 10 minutes
Score:
0.7
Remarks on result:
other: Negative control: 0; Positive control: 125.3

Any other information on results incl. tables

Summary of opacity, permeability and in vitro scores:

Treatment

Mean

Opacity

Mean

Permeability

MeanIn vitroIrritation Score

Negative control

0

0.000

0.0

Positive control

(Benzalkonium Chloride)

79

3.109

125.3

NS-1000

1

0.003

0.7

Individual In Vitro irritancy scores:

Eye

Negative control correctedFinal Opacity

Negative control correctedFinal OD490

In vitroIrritancy Score1

Negative control

1

-0.3

-0.001

-0.3

2

0.7

-0.001

0.7

3

-0.3

0.002

-0.3

Positive control(Benzalkonium Chloride)

4

78.7

2.375

114.3

5

81.7

1.959

111.1

6

75.7

4.991

150.5

NS-1000

7

3.7

0.009

3.8

8

-0.3

0.000

-0.3

9

-1.3

0.002

-1.3

1 In vitro irritancy score (IVIS) = opacity value + (15 x OD490value).

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on a mean in vitro irritancy score of 0.7 in a Bovine Corneal Opacity and Permeability test with NS-1000, no classification is required for eye irritation or serious eye damage.

Executive summary:

Using the Bovine Corneal Opacity and Permeability test (BCOP test) NS-1000 was screened for its eye irritancy potential in accordance with OECD 437 and according to GLP principles. The substance was applied as such directly on top of the corneasin such a way that the cornea was completely covered (750 µL per cornea). Adequate negative and positive controls were included. NS-1000 did not induce ocular irritation through both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score of 0.7 after 10 minutes of treatment. Since NS-1000 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Based on the results of this study, the substance does not need to be classified for eye irritation according to Regulation (EC) No 1272/2008 on classification, labelling and packaging.