Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
HET-CAM Test. No guideline available. However, the method is based on scientifically accepted publications; moreover, the study was conducted according to internal SOPs and was documented well

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
no guideline available
Version / remarks:
There are no official national or international guidelines for the HET-CAM Test; however, the study is performed according to the methods described in publications cited in the study protocol.
Principles of method if other than guideline:
- Principle of test: With the HET-CAM Test the potential of severe eye/mucous membrane damage using the chorionallantoic membrane (CAM) of fertilized, incubated hen eggs is tested. In the test, incubated hen’s eggs are opened carefully on day 9 and the CAM is exposed. Solid test substances are applied, if possible, on approximately the half of the membrane area, starting from the center. The visible parts of the membrane are observed. The time until appearance of unambiguous reactions is recorded. The reaction outcomes are coagulation and other reactions like e.g. haemorrhage or vessel lysis. lf reactions cannot be identified due to the nature of the test substance, the test substance will be removed by washing with 0.9% aqueous NaCl-solution after 3.5 minutes or. A final assessment of the CAM is performed immediately after washing.
- Parameters analysed / observed:Coagulation, Haemorrhagia
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 33470-153 FK
- Expiration date of the lot/batch: The sponsor guarantees the storage stability at least until 30 Apr 2009.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
- Stability under test conditions: Stability is guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: Since the test substance was determined to be not soluble in doubly distilled water or in olive oil at the concentration of 10%, a study protocol based on the undiluted solid test substance was followed. Because the test substance was applied undiluted, no analysis of the test substance in a vehicle was required.

Test animals / tissue source

Species:
chicken
Strain:
other: White Leghorn, SPAFAS lnc., USA, SPF Premium
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: The HET-CAM Test is an alternative in vitro method for testing of severe eye irritation to the eye of the rabbit. With the HET-CAM Test the potential of severe eye/mucous membrane damage using the chorionallantoic membrane (CAM) of fertilized, incubated hen eggs is tested. The test method does not yet allow for the evaluation of eye irritation. The result does not exclude an eye irritation potential of the test substance.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: Fresh, fertilized hen eggs produced under controlled SPF conditions were used. Hen eggs are recommended as preferred type of eggs in the references describing the method. Analogous to test animals, controlled SPF housing guarantees a defined microbiological status of the eggs. Contaminations also cause adverse effects or lack of comparability on the results of the study.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
Duration of treatment / exposure:
210 seconds
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done):Yes, 0.9% NaCl solution
- Time after start of exposure: 3.5 min

SCORING SYSTEM: 0 no visible chnage; 1 slight reaction; 2 moderate reaction; 3 severe reaction

TOOL USED TO ASSESS SCORE: stereomicroscope

Results and discussion

In vitro

Results
Irritation parameter:
morphological effects
Run / experiment:
run 1; 3 eggs evaluated
Vehicle controls valid:
not applicable
Negative controls valid:
not applicable
Positive controls valid:
not specified
Remarks on result:
other: no risk of serious eye damage
Remarks:
no reaction
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: CAMs with signs of pre-existing damage were excluded from the assay.

Any other information on results incl. tables

Table 1: Summarized Results

Concentration:

Egg-No.: Observation period
(seconds):
Grading of effects observed after
test substance removal by washing:
Additional findings
Haemorrhagia: Coagulation: Haemorrhagia: Coagulation:

Undiluted
test substance

1 210 210 0 0 none
2 210 210 0 0 none
3 210 210 0 0 none

Mean:

n = 3 210 210 0 0  

Grading: 0 = no reaction; 1 = slight; 2 = moderate; 3 = severe

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
No risk of serious damage to the eyes. However, the test method does not yet allow for the evaluation of eye irritation. The result does not exclude an eye irritation potential of the test substance.
Executive summary:

The potential of severe eye/mucous membrane damage was tested using the HET-CAM Test.

Therefore, the chorionallantoic membrane (CAM) of fertilized, incubated hen eggs was exposed with the undiluted test substance.

No haemorrhagia or coagulation was identified during an observation period of 210 seconds.

Therefore, the test substance is considered to cause no risk of serious damage to the eyes.

However, the test method does not yet allow for the evaluation of eye irritation. The result does not exclude an eye irritation potential of the test substance.