Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to INVITTOX Protocol No. 47: HET-CAM Test with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: INVITTOX Protocol No. 47: HET-CAM Test
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals / tissue source

Species:
other: chicken eggs
Strain:
other: Lohmann Selected Leghorn chicken
Details on test animals or tissues and environmental conditions:
CHICKEN EGGS:
The HET-CAM test was carried out with Lohmann Selected Leghorn chicken eggs. The Lohmann Selected Leghorn chicken was selected for several reasons: the ability to hatch the eggs of this breed is very consistent and reproducible. There did not appear to be any hereditary defects in this breed.

INCUBATION
The eggs were obtained from the LSL Rhein-Main Geflügelvermehrungsbetriebe (64807 Dieburg, Germany) on day one. Afterwards they were incubated at 37.5 ± 1 °C for eight days.
During incubation the eggs were rotated to prevent an attachment of the embryo to one side of the egg. On day eight the eggs were candled (held in front of a strong lamp in order to determine the position of the chicken fetus) and non-viable eggs (eggs containing no fetus) were discarded.
The remaining eggs were placed upwards in the incubator and incubated for another day without rotation.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
300 µL
Duration of treatment / exposure:
5 min
Duration of post- treatment incubation (in vitro):
5 min (observation time)
Number of animals or in vitro replicates:
CAM of 3 eggs
Details on study design:
Each 300 µL of the undiluted test item, the negative control (physiological saline), or the positive controls (0.1 N NaOH and 1% SDS), respectively were applied to the CAM of each three nine days bred chicken eggs. The CAM of the eggs was exposed to the test item or the controls for 5 minutes. The CAM was observed during the complete treatment period and the time points of the occurence of the three possible reactions haemorrhage, lysis, and coagulation were noted.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
CAM mean irritancy index
Run / experiment:
300 seconds treatment
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.00
Positive controls validity:
valid
Remarks:
18.62 (0.1N NaOH) / 10.54 (1% SDS)
Other effects / acceptance of results:
No irritating effects were observed during 5 min incubation with the test item. The calculated irritancy mean index is 0.00.
No other effects were observed.

Any other information on results incl. tables

INDIVIDUAL RESULTS


Test Group

Time until Haemorrhage [s]

Time until Lysis
[s]

Time until Coagulation [s]

Irritancy Index

Mean Irritancy Index

Evaluation

Negative Control

301

301

301

0.00

0.00

not irritant

301

301

301

0.00

301

301

301

0.00

Positive Control

0.1 N NaOH

14

39

50

18.43

18.62

severe irritant

18

30

47

18.66

16

32

43

18.77

Positive Control

1% SDS

39

47

301

10.29

10.54

severe irritant

26

38

301

10.72

29

41

301

10.60

Test Item

301

301

301

0.00

0.00

not irritant

301

301

301

0.00

301

301

301

0.00

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
INVITTOX Protocol No. 47: HET-CAM Test
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possess any irritating potential to mucous membranes or eyes.
Executive summary:

SUMMARY

 

This in vitro study was performed to assess the irritating potential of test item by detection of damages in blood vessels under the chorioallantoic membrane of 9 day incubated chicken eggs.

 

The test item (warmed to 37°C) was tested pure with three eggs. Each 300 µL of the test item were applied directly to the membrane in order to cover at least 50% of the membrane. Each 300 µL of the positive and negative controls were applied to three eggs each. During the observation period for 300 seconds any lesions in close proximity to the covered membrane were monitored and recorded. The observation time was 5 minutes at room temperature.

Physiological sodium chloride solution (0.9% (w/v)) was used as negative control, a 1% solution of SDS and 0.1 served as positive controls.

 

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

The positive controls induced a severe irritation on the blood vessels. The calculated mean irritancy indices are 10.54 for 1% SDS and 18.62 for 0.1 N NaOH.

No irritating effects were observed during 5 min incubation with test item. The calculated irritancy mean index is 0.00.