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Eye irritation

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

Test material

Details on test material:
SAT 080004
Batch-No.: Huc-Sa-9370-190
Purity: > 98% (NMR)

Test animals / tissue source

other: HET-CAM
not specified

Test system

unchanged (no vehicle)
Amount / concentration applied:
For each replicate about 300 µl 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 ( 1% solution in deion. water) and the negative control (physiological sodium chloride solution, 0.9% w/w) 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 300 μL 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 vesse

Results and discussion

In vivo

Irritation parameter:
other: HET-CAM irritaation score
Time point:
other: 301 s
Max. score:
not specified
Remarks on result:
other: The Irritancy index of all eggs is 0
Other effects:
no other effects

Any other information on results incl. tables

Mean irritancy index of 6 eggs: Test Item = 0.00

Mean irritancy index of 3 eggs:

Positive control (Sodium dodecyl sulphate) = 9.92

Positive control (Sodium hydoxide solution) = 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 080004 does not possess any irritating potential.

This in vitro study was performed to assess the irritating potential of SAT 080004 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 % solution of sodium dodecyl sulphate (SDS) and 0.1 N sodium hydroxid (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 within the acceptance criteria. Therefore, it was concluded that the test was valid.

No irritating effects were observed during 5 min incubation with the test substance SAT 080004. The calculated mean irritancy index is 0.00.