Registration Dossier

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure according to validation study and GLP

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: HET-CAM Test
Principles of method if other than guideline:
This study was conducted according to the procedures indicated by the following internationally accepted guideline and recomendation:
INVITTOX Protocol No. 47: HET-CAM Test.
GLP compliance:
yes (incl. QA statement)

Test material

Test animals / tissue source

other: HET-CAM
not specified

Test system

unchanged (no vehicle)
Amount / concentration applied:
For each replicate about 100 mg of the test item were applied directly to the chorioallantoic membrane in order to cover at least 50% of the membrane. 300 μL of each of the positive controls (SDS: 1% solution in deion. water and NaOH: 0.1 N) and the negative control (physiological sodium chloride solution (0.9% (w/v))) were applied to three eggs each.
Duration of treatment / exposure:
300 s
Observation period (in vivo):
300 s
Number of animals or in vitro replicates:
Details on study design:
CAM Preparation
On day 9, the day of the performance of the experiment, the eggs were candled once again in order to determine the position of the egg's oxygen bubble. The position was marked on the eggshell. Along the marking the eggshell was sawed with an electric saw. Afterwards, the shell above the oxygen bubble was removed. The egg skin beneath was covered with 0.9% (w/v) NaCl in deion. water (saline, produced in-house) in order to separate the egg skin from the chorioallantoic membrane beneath the skin. After 3 minutes incubation at 37.5 ± 0.5 °C the egg skin was removed from the membrane using forceps.

In this HET-CAM assay the test item was tested pure. Six eggs were treated with the test item. For each replicate about 100 mg of the test item were applied directly to the membrane in order to cover at least 50% of the membrane. 300 μL of each of the positive controls and the negative control were applied to three eggs each. During the observation period of 300 seconds any lesions in close proximity to the covered membrane were monitored and recorded.

The membranes of the eggs were observed for 300 seconds. Lesions of the underlying blood vessels were monitored and noted. In particular three endpoints were observed and the time point at which an effect occurs was recorded. The three endpoints were:
• haemorrhage
• coagulation
• lysis of the blood vessel

Results and discussion

In vitro

Irritation parameter:
other: HET-CAM: Mean Irritancy Index
Vehicle controls validity:
not examined
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Mean irritancy index of 6 eggs: Test item = 0.0

Mean irritancy index of 3 eggs:

Positive control (SDS) = 9.92

Positive Control (NaOH) = 19.12

Negative control = 0.00

Applicant's summary and conclusion

Interpretation of results:
not irritating
Migrated information Criteria used for interpretation of results: other: INVITTOX
Classification: not irritating
Executive summary:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item SAT 080003 does not possess any irritating potential.

This in vitro study was performed to assess the irritating potential of SAT 080003 by detection of damages in blood vessels under the chorioallantoic membrane of chicken eggs incubated for 9 days (Hen's Egg Test - Chorioallantoic Membrane Test, HET - CAM)

The Test item was tested pure.

The observation time was 5 minutes at room temperature.

Physiological sodium chloride solution (0.9 % (w/v)) was used as negative control.

The negative control showed no irritating effect on the blood vessels under the membrane (mean irritancy index 0.00).

1 % solutions of sodium dodecyl sulphate (SDS) and 0.1 N sodium hydroxide (NaOH) were used as positive controls.

The positive controls induced severe irritation on the blood vessels (mean irritancy indices of 9.92 for SDS and 19.12 for NaOH)

The mean irritancy indices of the controls are well comparable with the historical control values and are the acceptance criteria. Therefore, it was concluded that the test valid.

No irritating effects were observed during 5 min incubation with the test substance SAT 080003. The calculated mean irritancy index is 0.00.