Registration Dossier

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
other: HET CAM
Principles of method if other than guideline:
The test was based on the observation, by a trained person, of the irritant effects (hyperhemia, hemorrhage and coagulation) occuring during the five minutes after application of the product to the chorioallantoic membrane (CAM) of embryonic hen's eggs on the tenth day of incubation.
The irritant potential was scored according to a scale from 0 to 21. The product was classified in one of the categories defined according to the mean score obtained.

Different concentrations of the test product are used in the assay in order to determine the
amount inducing a 50% réduction in the NRR (IC50, inhibition concentration 50).
An in vitro/in vivo correlation matrix between the IC50 determined in the PREDISAFE assay
and the Draize irritation data, allows the assessment of the ocular tolerance of the test product (Guyomard et al., 1994)
GLP compliance:
not specified

Test material

Test material form:
Specific details on test material used for the study:
Tested at the use concentration of 100%

Test animals / tissue source

other: Embryonic Hen's ,eggs
other: White Leghorn
Details on test animals or tissues and environmental conditions:
Eggs were graded between 50 and 65 g.
Eggs were identified and put into an incubator, under controled conditions of temperature (37.8°C + 0.5°C) and relative humidity (50 to 60 %).
Eggs were incubated, either in an incubator with oscillating plates (in vertical position), or in an incubator with manual reversal. In tins case, eggs were turned over three times a day and were put in vertical position (air pocket upwards) on the 8th day of incubation.

Test system

Amount / concentration applied:
1 concentration tested : 100%
Duration of treatment / exposure:
20 seconds
Duration of post- treatment incubation (in vitro):
5 minutes of observation
Number of animals or in vitro replicates:
4 eggs
Details on study design:
1)Tested product conditions of use
The product was tested as such.
The product was brought at 37°C before use.

2) preparation of eggs
The different steps of the study were performed quickly under a constant lighting which did not give out too much heat in order to avoid the withering of the chorioallantoic membrane.
On the 10th day of incubation, eggs were taken out of the incubator one by one and were candled with a lamp. The defective eggs (image which did not correspond to the expected stage of development) were eliminated and the selected eggs were put on the holder, « air pocket » upwards.
The shell of the each selected egg was drilled (with a lanceolate needle) and cut up (with scissors with blunt ends or forceps) at air pocket level and until the limits of the shell membrane.
Then the whole surface of the shell membrane was moistened with a 0.9 % sodium chloride solution, warmed up at 37°C (bain-marie). Then, the excess of 0.9 % sodium chloride solution was eliminated by tilting of the egg and the shell membrane was removed delicately with forceps in order to uncover the underlying CAM.
Any egg whose chorioallantoic membrane was damaged (tear, presence of hemorrhage and any other lesion) was immediately rejected.

3) Application of the tested product
The product was tested on 4 eggs.
300 µL were deposited on the CAM, with a micropipette (P1000). lmmediately after application, the chronometer was set off.

After 20 seconds' contact, the CAM was rinsed with 10 to 20 ml of sodium chloride isotonic solution (kept at 37°C in bain-marie), with a syringe avoiding any brutal projection.
The rinse liquid was eliminated by tilting of the egg.
The possible phenomena of irritation were observed during 5 minutes according to the process described in the following paragraph. The accurate rime of each phenomenon appearance was noted.
The 20 seconds' contact were included in the 5 minutes' observation.
At the end of the study, embryos were destroyed by quick cooling (enclosure at -20°C).

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: Hyperhemia
ca. 3
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Irritation parameter:
other: Hemorrhage
ca. 0
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Irritation parameter:
other: Coagulation
ca. 0
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable

Applicant's summary and conclusion

Interpretation of results:
Category 2B (mildly irritating to eyes) based on GHS criteria
Executive summary:

The test substance was judged weakly irritating for the embryonic hen's egg chorioallantoic membrane and therefore is expected to be weakly irritating to the eye.