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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015
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:
2015
Report Date:
2015

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.
And to 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.

The Hen´s Egg Test on the Chorioallantoic 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 occurring 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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
additive
Test material form:
liquid

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
The test system used for this study is the fertile Lohmann Brown hen egg which is incubated for 8 days before test item application.
SOURCE OF FERTILIZED CHICKEN EGGS
- Source: Brinkschulte Josef GmbH & Co.KG, 48308 Senden
- Number of eggs: 4
- 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

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
other: The test material is a solution in ethyl acetate. Ethyl acetate was tested independently in parallel to distinguish potential toxic effects of the test item from the effects of the solvent.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 300 µL per egg
Duration of treatment / exposure:
300 seconds (i.e. 5 min) after substance application
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
4 eggs per group (negative control, positive control, test substance, solvent)
Details on study design:
METHODS:
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 are used for testing.
Undiluted test item is applied directly onto the Chorioallantoic membrane (CAM) of each egg in a volume of 300 μL.
4 eggs each were used for the test item, negative and positive controls.

Observations of effects to the blood vessels, albumen or to the 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).

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

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 IS.

IRRITATION SCORE:
Following formula was used to generate an irritation score (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

Assessment of Irritation Scores (IS) according to Luepke, N.P., Fd. Chem. Toxic 23, 287-291, 1985:
Score                Assessment
0 - 0.9               no irritation
1 - 4.9               slight irritation
5 - 8.9               moderate irritation
9 - 21                strong irritation
A test substance is considered to cause severe eye irritation when the Irritation Score is equal to or above 9.

Results and discussion

In vitro

Results
Irritation parameter:
other: Irritation Score (IS)
Run / experiment:
mean of 4 eggs
Value:
11
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: the solvent ethyl acetate was tested in parallel and showed an Irritation Score of 13

In vivo

Irritant / corrosive response data:
The test item was identified as "strong irritant" to the chorioallantoic membrane (irritation score (IS) = 11) under the conditions of this assay. The solvent ethyl acetate was even stronger irritating with an IS of 13.

Any other information on results incl. tables

Table 1: Summary of results from HET-CAM test with the test item

 

 Compound

Irritation Score (IS)

Assessment

 Negative control

NaCl 0.9 %

0

non irritant

 Positive control

SDS 1 %

12

strong irritant

Test item

11

strong irritant

 solvent ethyl acetate  13  strong irritant

Applicant's summary and conclusion

Interpretation of results:
other: positive
Executive summary:

The test item was identified as 'strong irritant' to the chorioallantoic membrane (irritation score (IS) = 11 of maximal 21) to fertilized hen's eggs under the conditions of the HET-CAM test (Hen's Egg Test - Chorioallantoic Membrane) according to a protocol recommended by ICCVAM. The test material is a solution in ethyl acetate. Ethyl acetate was tested independently in parallel to distinguish potential toxic effects of the test item from the effects of the solvent. Ethyl acetate showed stronger effects than the test item with an IS of 13.

The results of the positive (SDS 1 % in physiologic saline) and negative (physiologic saline solution) controls confirmed the validity of the test system.