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: study was preformed according to GLP

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
other: ICCVAm Test Method Evaluation Report: Appendix G ICCVAM recommended HET-CAM Method Protocol (Nov. 2006)
Principles of method if other than guideline:
This "in vitro study" is preformed to assess the irritating potential of Blei-cyanamid by detection of damages in blood vessels under the chorioallantoic membrane of nine day incubated chicken eggs.
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Lead cyanamidate
EC Number:
EC Name:
Lead cyanamidate
Cas Number:
Molecular formula:
lambda2-lead(2+) (azanidylenemethylidene)azanide

Test animals / tissue source

other: Lohman Leghorn chicken eggs
not specified
Details on test animals or tissues and environmental conditions:
Freshly laid Lohmann Leghorn chicken eggs will be obtained from the LSL Rhein Main Geflügelvermehrungsbetrieb (Dieburg, Germany) nine days before the start of the test (the day of delivery will be stated in the final report).
The eggs are incubated at 38.3 ± 0.2 °C and 58 ± 2% humidity for eight days in a still air incubator. During incubation, the eggs are rotated to prevent an attachment of the embryo to one side of the egg.

Test system

Details on study design:
Preparation of Test System
24 hours before the start of the experiment (eight days after obtaining the eggs), the chicken eggs are candled and non-viable eggs are discarded. The rest of the eggs is placed upward in the incubator until the next day.
On the day of the test, the eggs are removed from the incubator for use in the assay.
At first, the eggs are weighed and only eggs with a weight between 50g and 60g are used. All other eggs are discarded. Then, the eggs are candled and the air bubble is marked. The egg is opened on the air bubble with a rotating dentist’s saw blade or tweezers. The inner membrane is moistened with 0.9 % NaCl; then, the egg is left to stand into the incubator for 30 minutes. After this period, the solution is decanted and the inner membrane is carefully removed with a forceps.
Description of the Method
The test item resp. the negative control resp. the positive control is given directly onto the CAM surface.
For each replicate of the liquid negative and positive controls, 300 µL are measured using an adjustable pipette with single-use tips.
For each test item replicate, 300 µL are measured using a centrifuge vial and weighed. If the weight surpasses 300 mg, six replicates with 300 ± 30 mg each are weighed for the test. If the weight is less than 300 mg, six replicates with 300 µL each are weighed for the test.
Number of eggs which are used for the test item: 6
Number of eggs which are used for the negative control: 3
Number of eggs which are used for each positive control: 3
The reactions on the CAM are observed over a period of 301 seconds.
The following endpoints are described in the guideline:
• haemorrhage (bleeding from the vessels)
• vascular lysis (blood vessel disintegration)
• coagulation (intra- and extra-vascular protein denaturation)
The time for the appearance of each of the noted endpoints is monitored and recorded in seconds.

Results and discussion

Any other information on results incl. tables

The substance lead cyanamid can be regarded as severely irritant.

Applicant's summary and conclusion

Interpretation of results:
other: severly irritant
The presented data are reliable and adequate. The EU-Method is an international accepted method. This method had been chosen to avoid unnecessary animal experiments.
Executive summary:

The substance lead cyanamid can be regarded as severely irritant.