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Eye irritation

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eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June 25, 2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Principles of method if other than guideline:
The protocol for this study is executed according to the ICCVAM Test Method Evaluation Report: Appendix G: ICCVAM Recommended Protocol for Future Studies Using the Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) Test method, November 2006.
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
solid: particulate/powder

Test animals / tissue source

other: Assessment of the irritant/corrosive potential of the test substance by observing its effect on the extra-embryonal blood vessel system (e.g. haemorrhage, vessel lysis, coagulation) of the fertile 8 days incubated chicken egg.
Details on test animals or tissues and environmental conditions:
The analysis model used for this study is the fertile Lohmann Brown hen egg (Brinkschulte Josef GmbH & Co.KG, 48308 Senden, Germany), which is incubated for 8 days before test item application.

Test system

unchanged (no vehicle)
other: as negative control: physiological saline solution (0.9 % NaCl, 300 µL)
Details on study design:
After 7 days of incubation (37.5 °C, relative humidity 63 %) in an incubator with an automatic rotating device, all eggs were candled in order to discard those that were defect and to mark the air bubble. The eggs were replaced into the incubator with the large end upward but not rotated, thus ensuring accessibility to the CAM region. At day 8 of incubation the sections marked for the air bubble were sawed out of the shell. The inner membrane was moistened with NaCl 0.9 % and carefully removed with forceps. Only eggs with normally developed embryos and blood vessel systems were used for testing. A 100 % concentration (300 mg) is tested on the chorioallantoic membrane (HET-CAM) of a chicken embryo. 4 eggs each were used for negative and positive controls and for the test substance.
As positive control 1 % sodium dodecyl sulfate was applied.
Observations of effects to the blood vessels, albumen or embryo over a period of 300 seconds after substance application are determined for each single egg. The time to the appearance of each of the observations mentioned above has been monitored and recorded. If no effect appeared during the observation period of 300 seconds (observation = 0) the result was assigned as negative for the related endpoint, and the factor set to 0 for this endpoint when calculating the Irritation Score (IS).
Formula for calculation of IS:
IS = 5 x (301-sec H)/300 + 7 x (301- sec L)/300 + 9 x (301- sec C)/ 300
H= observed start in seconds of haemorrhage reactions; L= observed start in seconds of vessel lysis, strong haemorrhage; C= observed start in seconds of blood - coagulation, albumen - coagulation

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation

Any other information on results incl. tables

result with test substance, 300 mg

 egg  effect

 effects detected after




1 >300   0

2 >300  0

3 >300  0


1 >300


   2  >300  0
   3  >300  0
 3  1  >300  0
 2  >300  0
   3  >300  0
 4  1  >300  0
   2  >300  0
   3  >300  0

effects: 1 = vasodilatation, slight haemorrhage (H), 2 = vessel lysis, strong haemorrhage (L), blood-coagulation, albumen-coagulation (C)     

The test substance induced no irritant effects to the chorioallantoic membrane of hen eggs under the conditions of this assay.

The positive and negative controls were functional.

Applicant's summary and conclusion

Interpretation of results:
other: no corrosive property