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Eye irritation

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eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2016
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 Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: Appendix B3 of "ICCVAM Test Method Evaluation Report: Current Validation Status of In Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals and Products", NIH Publication No. 10-7553, September 2010.
Principles of method if other than guideline:
- Principle of test: The Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) is a test method which implies the use of a complete tissue constituted of blood vessels and proteins that is capable of responding to chemical injury with an inflammatory process similar to the one occuring in the conjunctival tissue of the eye.

- Short description of test conditions: The test substance is applied directly to the chorioallantoic membrane (CAM) of fertilized chicken eggs

- Parameters analysed / observed: acute effects on haemorrhage, lysis of blood vessels and coagulation
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Valeric anhydride
EC Number:
EC Name:
Valeric anhydride
Cas Number:
Molecular formula:
pentanoic anhydride
Constituent 2
Reference substance name:
Pentanoic acid, 1,1¿-anhydride
Pentanoic acid, 1,1¿-anhydride
Constituent 3
Reference substance name:
Test material form:
other: liquid
Details on test material:
- Purity: Not specified

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
- Source: Brinkschulte Josef GmbH & Co.KG, 48308 Senden
- Number of eggs: 4
- Characteristics of donor animals fertile Lohmann Brown hens
- Treatment conditions of eggs prior initiating testing: day 1-7: an incubator with an automatic rotating device (e.g. Ehret GmbH), optimum temperature : 37.5 °C, relative humidity 63%. day 8: with the large end
upward and not rotated for ensuring accessibility to the Chorioallantoic membrane (CAM) region
- Time interval prior to initiating testing: 8 days
- indication of any existing defects or lesions in eggs: After 7 days of incubation, all eggs were candled in order to discard those that were defect and to mark the air bubble.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 300 μl/egg

Duration of treatment / exposure:
300 sec
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
4 eggs
Details on study design:
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. Undilutet test item was applied directly onto the Chorioallantoic membrane (CAM) of each egg in a volume of 300 µL undiluted test item. 4 eggs each were used for the test item, negative and positive controls.

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).

Scoring criteria for the acute effects and calculation of Irritancy Score (IS):

0 = no effect
1 = vasodilatation, slight haemorrhage (H)
2 = vessel lysis, strong haemorrhage (L)
3 = blood-coagulation, albumen-coagulation (C)

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

Data Interpretation:

Irritation Score (IS)

0-0.9 -> Non irritant
1-4.9 -> Slight irritant
5-8.9 -> Moderate irritant
9-21 -> Strong irritant

A test substance is considered to cause severe irritation when the IC value is greater than nine.

Results and discussion

In vitro

Irritation parameter:
other: irritation score (IS)
Run / experiment:
Negative controls validity:
Positive controls validity:
Remarks on result:
other: A test substance is considered to cause severe irritation when the IC value is greater than nine.

Any other information on results incl. tables

Table 1: Summary of results, test item

 Egg  Effect  Effect detected after [sec]  Irritation Score (IS)
 25  1 143  
   2  > 300  
   3  > 300  3
 26  1  > 300  
   2   196  
   3  > 300  2
 27  1  > 300  
   2  > 300  
   3  > 300  0
 28  1 50  
   2 250  
   3  > 300  5

effect: 1 = vasodilatation, slight haemorrhage 2 = vessel lysis, strong haemorrhage 3 = blood-coagulation, albumen-coagulation

Applicant's summary and conclusion

Interpretation of results:
other: negative
Executive summary:

The test item was tested in the in vitro assay applying the Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) test method. This is a method that makes use of the chorioallantoic mambrane (CAM) of fertilized chicken eggs. For determination of acut effects on haemorrhage, lysis of blood vessels and coagulation the undiluted test item was directly applied onto the CAM. for 5 minutes. The Irritation Score (IS) value was calculated to be 2 for the test item which was interpreted as being a slight irritant. The results of the positive ( SDS 1% in physiologic saline) and negative (physiologic saline solution) controls confirmed the validity of the test system.