[Name confidential or not available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 20, 2008 to February 16, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: in vitro test methods for identifying ocular corrosives and severe irritants: The Hen’s egg Test – Chorioallantoic Membrane (HET-CAM) test method, March 2006.

Version / remarks:

The Ocular Toxicity Working Group (OTWG) of the interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Intergency Centre from the Evaluation of Alternative Toxicological Methods (NICEATM):

Principles of method if other than guideline:

HET-CAM Test

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: White powder
- Analytical purity: 99%
- Lot/batch No.: 08-018
- Expiration date of the batch: 30 April 2009
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature protected from light

Test animals / tissue source

Species:

other: Hen Egg

Details on test animals or tissues and environmental conditions:

CAM preparation:
Before use the eggs were stored in the dark at 36.5C for approximately 45 minutes. The number of eggs used in the study was 3 per treatment group. On day 9 of incubation the air cells of the eggs were pierced and the egg shells surrounding the air cells was removed by useing a pair of tweezers. The uncovered inner membrane was moistened with physiological saline and subsequently the egg was kept at room temperature for approximately 2 minutes. Subsequently the physiological saline was removed and the inner egg membrane carefully eliminated using forceps to expose the CAM.

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 300 mg per egg
NEGATIVE CONTROL
- Name: physiological saline
- Amount(s) applied (volume or weight with unit): 0.3 mL per egg
- Concentration (if solution): 0.9%(w/v) NaCl
- Supplier: B.Braun, Melsungen AG, Germany

POSITIVE CONTROL
- Name: Sodium dodecyl sulphate (CASNo. 151-21-3)
- Amount(s) applied (volume or weight with unit): 0.3 mL per egg
- Concentration (if solution): 1% (w/v)
- Lot/batch no.: 114K0214 (Sigma-Aldrich Chemie GmbH, Germany)
- Purity: 99.5%

Duration of treatment / exposure:

20 seconds
(After the treatment period the eggs were carefully rinsed with approximately 5 mL of physiological saline.)

Observation period (in vivo):

CAM was monitored and recorded over the following 280 seconds.

Number of animals or in vitro replicates:

three eggs each for the test substance, negative control substance and positive control substance

Results and discussion

In vitro

Results

Irritation parameter:

other: Mean Irritancy score

Run / experiment:

Mean of 3 runs

Value:

0

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Table 1 The results from the three test method endpoints and irritancy scores of the individual eggs.

 

Egg	Haemorrahage time (sec)	Lysys time (sec)	Coagulation time (sec)	Irritancy score (IS)	Mean irritancy score (IS)
Negative control
1	301	301	301	0	0
2	301	301	301	0
3	301	301	301	0
1% Sodium dodecyl sulphate
4	301	20	20	15.0	14.9
5	301	20	20	15.0
6	301	32	20	14.7
Test substance
7	301	301	301	0	0
8	301	301	301	0
9	301	301	301	0

Note: 301 seconds was noted when no effect was observed after 5 minutes of observation.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

It is concluded that this test is valid and that the test substance is not irritant in the Hen's Egg Test - Chorioallantoic Membrane Test under the experimental conditions described in the report.