Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
At the time of test conduct (and to date) there are no relevant OECD/EC guidelines for this test type.

However, the study was conducted according to the following publications and is considered reliable:
- Lüpke N.P. (1985): Hen’s Egg Chorio allantoic Membrane Test for Irritation Potential. Fd. Chem. Toxic. 23, pp. 287 – 291.
- Spielmann, H. (1995): HET-CAM Test. In: Methods in Molecular Biology, 43 (eds.: O’Hare, S. and Atterwill, C. K.) pp. 199 – 204.
- Spielmann, H. et al. (1996): Results of a Validation Study in Germany on Two In Vitro Alternatives to the Draize Eye Irritation Test the HET-CAM Test and the 3T3 NRU Cytotoxicity Test. ATLA 24, pp. 741 – 858.

The occurance of vascular injury or coagulation in response to a compound is the basis for employing this technique as an indication of the potential of a chemical to damage mucous membranes (in particular for the eye) in vivo. The data are internally validated using reference substances tested in parallel.
GLP compliance:
yes (incl. certificate)

Test material

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

Test animals / tissue source

Species:
chicken
Strain:
other: White leghorn
Details on test animals or tissues and environmental conditions:
Egg source: freshly fertilised white eggs were obtained from a chicken breeding centre. The eggs were candled before use and any defective eggs were discarded.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The test item was applied to an area of 25% of the chorioallantoic membrane (CAM) using a 100% concentration.
Duration of treatment / exposure:
The reactions of the CAM were observed over a period of 5 minutes from the start of exposure
Number of animals or in vitro replicates:
6 eggs were used for the test item, 2 for the controls (with physiological saline) and 4 eggs were used as positive controls (2 eggs treated with NaOH and 2 with SDS)
Details on study design:
Assay preparation:
Initially the egg shell was removed from the air chamber. This was then moistened with physiological saline and replaced into an incubator for the start of the assay (no longer than 30 minutes from when shell removed).

Assay procedure:
The egg was removed from the incubator and the membrane carefully removed using tapered forceps. Any eggs with injured blood vessels were discarded. A volume of 0.3 mL of standard solution was applied to the CAM alongside the test material. The reactions of the CAM were monitored over 5 minutes and scored based on the incidence of haemorrhage, vascular lysis and coagulation.

Results and discussion

In vitro

Results
Irritation parameter:
other: Mean Irritation Score
Run / experiment:
1
Value:
0
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Other effects / acceptance of results:
The time for each reaction to occur (if any) and an irritation score was calculated. The mean score was calculated from irritation scores for each egg for each test group. A score of > 2 would indicate that the substance potentially possess irritant properties to eyes (with severe irritants possessing a score > 11).

The results of the reference substances were considered acceptable based upon historical laboratory data.

Any other information on results incl. tables

The irritation score was calculated as follows:

IS = [(301 -sec H)/300] x 5 x S + [(301 -sec L)/300] x 7 x S + [(301 -sec C)/300] x 9 x S

sec = second of first occurence of reaction

H = Haemorrhage

l = Vascular lysis

C = Coagulation

S = 0.1 if H, C, L is grade 1 (weak), 0.5 if H, C, L is grade 2 (moderate) or 1 if H, C, L is grade 3 (strong).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Although non-standard, the HET-CAM study can provide an indication of the possible irritation effects of a substance in eyes. According to the evaluation criteria, the test item would be considered non-irritant to mucous membranes. Based on this, no irritation would be expected if the substance were to come into contact with eyes.