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Toxicological information

Eye irritation

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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP 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:

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(2-hydroxyethoxy)ethan-1-ol; 2-{[2-(2-hydroxyethoxy)ethoxy]carbonyl}benzoic acid
EC Number:
Molecular formula:
2-(2-hydroxyethoxy)ethan-1-ol; 2-{[2-(2-hydroxyethoxy)ethoxy]carbonyl}benzoic acid
Test material form:

Test animals / tissue source

other: hen's egg chorioallantoic membrane (HET-CAM)
Details on test animals or tissues and environmental conditions:
The test system 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: negative control: 0.9 % sodium chloride (300 µl); positive control: 1 % sodium dodecyl sulfate (300 µl)
Amount / concentration applied:
300 µl per egg
Duration of treatment / exposure:
single application
Observation period (in vivo):
300 seconds after substance application
Number of animals or in vitro replicates:
4 eggs per group (negative control, positive control, test substance)
Details on study design:
The fertile Lohmann Brown hen eggs (Brinkschulte Josef GmbH & Co.KG, 48308 Senden) were incubated in an incubator with an automatic rotating device (e.g. Ehret GmbH), optimum temperature : 37.5 °C, relative humidity 63 %. After 7 days of incubation, 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 NaCI 0.9 % and carefully removed with forceps. Only eggs with normally developed embryos and blood vessel systems were used for testing.

This study is used to evaluate the potential ocular irritancy or corrosion of a test item as measured by its ability to induce toxicity in the chorioallantoic membrane of a chicken egg. A 100 % concentration is tested on the chorioallantoic membrane (HET-CAM) of a chicken embryo. Effects are measured by the onset of haemorrhage, vessel lysis or coagulation during the first 300 seconds after application. Times till appearance of each of these endpoints are used to calculate an irritation score. However, there is no clear discrimination between strong irritation and corrosion. 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. These tests are also related to OECD 405, with inclusion of the 'Manual of decisions for implementation of the sixth and seventh amendments to Directive 67/548/EEC on dangerous substances (Directives 79/831/EEC and 92/32/EEC).

Physiological saline solution (0.9 % NaCl, 300 µl) was used as negative control. A SDS (sodium dodecyl sulfate) solution (1 %, 300 µl) was used as positive control (Sigma- Aldrich; Cat.No.: L5750-100G).

300 µl of the test item was applied to the CAM (4 eggs each). NaCI 0.9 % treated eggs were used as negative contrals, SDS 1 % as positive contral (in
quadruplicate each). 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.

0= no effect
1 = vasodilatation, slight haemorrhage (H)
2 = vessel lysis, strang 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.

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

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 >= 9.

Any other information on results incl. tables

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



Irritation Score (IS)


 Negative control

NaCl 0.9%


non irritant

 Positive control

SDS 1%


strong irritant

Test item


moderate irritant

Applicant's summary and conclusion

Interpretation of results:
other: moderate irritant effect in vitro
Executive summary:

The test item was identified as "moderate irritant" to the chorioallantoic membrane (irritation score (IS) = 8 of maximal 21) under the conditions of the HET-CAM test (Hen's Egg Test - Chorioallantoic Membrane) according to a protocol recommended by ICCVAM, November 2006.