Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-FEB-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-5). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed in vitro eye irritation tests is the Hen’s Egg Test - Chorioallantoic Membrane (HET-CAM) test. The HET-CAM assay is especially suited for test compounds which have a low density or are water insoluble. The HET-CAM assay measures three important components which are predictive for conjunctival injury of the eye, heamorrhage, vessel lysis and coagulation. Although the HET-CAM test method is not yet fully validated, the EU national regulatory authorities accept positive outcomes (severe eye irritants) of this test method for classification and labelling severe eye irritants. Where a negative result is obtained, an in vivo test is subsequently required.

Data source

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

Materials and methods

Test guideline
Qualifier:
no guideline available
Guideline:
other:
Principles of method if other than guideline:
The study procedures described in the report are based on the following documents (1-2) and follows international recommendations (3-10):
1. The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Hen’s Egg Test - Chorioallantoic Membrane (HET-CAM) test method, March 2006.
2. INVITTOX protocol 47. The HET-CAM test - Method of Spielmann and Liebsch, January 1992.
3. Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No. 40 "Acute Eye Irritation / Corrosion", Paris Cedex, 2002.
4. UN 2003. Globally Harmonized System of Classification and Labelling of Chemicals (GHS). United Nations Publications: New York &Geneva.
5. EC 2004. Commission directive 2004/73/EC adapting to technical progress for the 29th time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions related to classification, packaging and labelling of dangerous substances. Official Journal of the European Union L152:1-316. April 2004.
6. EC 2004. Manual of Decisions for Implementation of the 6th and 7th amendments to Directive 67/548 on Dangerous Substances (Directives 79/831/EEC and 92/32/EEC). July 2004.
7. EPA 1998. Health Effects Test Guideline, OPPTS 870.2400 Acute Eye Irritation. EPA 712-C-98-195. Washington, DC: U.S. Environmental Protection Agency.
8. Luepke N.P. 1985. Hen's egg chorioallantoic membrane test for irritation potential, Food Chem. Toxicol. 23:287-291.9.
9. Kalweit S., Gerner I. and Spielmann H. 1987. Validation project of alternatives for the Draize eye test. Mol Toxic 1:597-603.
10. Kalweit S., Besoke R., Gerner I., Spielmann H. 1990. A national validation project of alternative methods to the Draize rabbit eye test. Toxic In vitro 4:702-706.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Custom Red #2
- Substance type: Violet powder
- Physical state: solid
- Lot/batch No.: C50APR0805D
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
Fresh, fertilised white leghorn chicken eggs.

Source: Eggs were obtained from a non-GLP chicken-breeding center (Het Anker Ochten, the Netherlands).

Environmental conditions: Temperature of the incubator: 37 ± 1°C; Relative humidity: between 50 and 70%.

Age at study initiation: The eggs were incubated for 9 days.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Custom Red #2: 309 to 328 mg
Negative control (physiological saline): 0.3 ml
Positive control (1% (w/v) Sodium dodecyl sulphate): 0.3 ml
Duration of treatment / exposure:
Custom Red #2, negative and positive control solutions: 20 sec
Observation period (in vivo):
Custom Red #2, negative and positive control solutions: 280 sec
Number of animals or in vitro replicates:
Three eggs per treatment group
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): 5 ml water
- Time after start of exposure: 20 sec

SCORING SYSTEM:
The appearance of haemorrhage, vessel lysis and coagulation on the CAM was monitored and recorded over the following 280 seconds.
Evaluation: Results from the three test method endpoints were evaluated separately for each egg.

The irritancy score (IS) was calculated using the formula.


IS = ( (301-Haemorrhage time)/300) ) x 5 +( (301-Lysis time) /300) x 7 + ( (301-Coagulation time) /300) x 9
IS = irritancy score for total effect
Haemorrhage time = observed start (in seconds) of haemorrhage reactions on CAM
Lysis time = observed start (in seconds) of vessel lysis on CAM
Coagulation time = observed start (in seconds) of coagulation formation on CAM

DATA EVALUATION
The mean score for each treatment was calculated from the final scores obtained for each egg in that treatment and evaluated according to the following irritancy classification scheme of Luepke (1985) and Kalweit et al. (1987).

HET-CAM Irritancy Score (IS) Range Irritation Category
0 – 0.9 Not irritant
1 – 4.9 Slight Irritant
5 – 8.9 Moderate Irritant
9 – 21 Severe Irritant



TOOL USED TO ASSESS SCORE: binoculair

Results and discussion

In vivo

Results
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 300 sec
Max. score:
0
Reversibility:
other: not applicable
Irritant / corrosive response data:
The mean in vitro irritancy score obtained during a 280-second observation period after 20 seconds treatment with Custom Red #2 was 0.

The mean in vitro irritancy score obtained with the negative control was less than 1 indicating that the negative control did not induce irritancy on the chorioallantoic membrane. The mean in vitro irritancy score of the positive control (1% (w/v) Sodium dodecyl sulphate) was within the historical control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

EGG 

Heamorrhage time

(sec)

Lysis time (sec)

Coagulation time

(sec)

Irritancy score (IS)

Mean irritancy score (IS)

Negative control

301

301

301

0.0

0.0

 2

301

301

301

0.0

 3

301

301

301

0.0

1% Sodium dodecyl sulphate

 4

301

25

25

14.7

14.7

 5

301

35

20

14.6

301

27

20

14.8

Custom Red #2

 7

301

301

301

0.0

0.0

301

301

301

0.0

 9

301

301

301

0.0

Note:   301 seconds was noted when no effect was observed after 5 minutes of observation.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
It is concluded that this test is valid and that Custom Red #2 is not an irritant in the Hen’s Egg Test – Chorioallantoic Membrane Test.