calcium N2,N6-bis(3-carboxypropanoyl)lysine (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 16th to February 04th, 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: ICCVAM-Recommended Test Method Protocol: Hen’s Egg Test - Chorioallantoic Membrane (HET-CAM) Test Method

Version / remarks:

November, 2006

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Set Retard Plus

IUPAC Name:

Set Retard Plus

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

chicken

Strain:

other: white Leghorn

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- System: hen's egg.
- Number: 4 per sample.
- Source: Couvoir de Cerveloup
- Weight at study initiation: 50 - 60 g

TISSUE PREPARATION
- Incubation: after receipment, eggs were kept for 24 hours at a temperature of 12 ± 2.5 °C, then moved to an incubator at a temperature of 37.8 ± 1°C, and humidity percentage 50-60 %, for a period of 10 days.
- Mambrane extraction: at day 11th of incubation, eggs were moved from that incubator and vertically placed on a specific prop. The shells were cut at empty inner space level and then removed in order to expose the membranes.
- Membrane preparation: membranes were humified with a physiological solution at 37 °C, then gently chipped off. Egges showing imperfections in chorioallantoic membrane were discarded.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

Amount / concentration applied:

0.3 g of test item were gently put on chorioallantoic membrane.

Duration of treatment / exposure:

20 seconds

Number of animals or in vitro replicates:

4 eggs/sample

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: sample was removed, softly washing the membrane with a physiological solution.
- Time after start of exposure: contact of 20 seconds.

OBSERVATIONS
Each possible reaction was observed during a post-exposure period of 5 minutes and the arising time recorded.

The procedure evaluated the development of the response at specific time points after the test substance was applied to the CAM. In these reports, the blood vessels on the CAM were examined and scored at 0.5, 2, and 5 minutes after exposure to the test substance.
In particular, the membranes were checked for the reactions described bolow.
- Hemorrhage: bleeding from the vessels or from areas of mambrane.
- Vascular lysis: capillaries, not visible before the substance treatment, becoming visible, dilated and inflamed. Also vessels with a larger diameter can undergo this reaction.
- Coagulation (opacity and/or thrombosis): opacity can be observed by the membrane that appears as a thin opalescent veil. Thrombosis can determine the interruption of the blood flow in vessels, which appear segmented.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Conc: 100 %; eggs number: 4

Value:

6.8

Positive controls validity:

valid

Other effects / acceptance of results:

POSITIVE CONTROL
Lauryl sulfobetaine (0.8 %) score: 12 (4 eggs).

Applicant's summary and conclusion

Interpretation of results:

other: non corrosives or severe irritants, i.e. the substance does not meet the criteria to be classified as Eye Dam. 1, according to the CLP Regulation

Conclusions:

Non corrosives or severe irritants, i.e. the substance does not meet the criteria to be classified as Eye Dam. 1, according to the CLP Regulation

Executive summary:

Method

A toxicological study on test substance was performed in order to get preliminary foretelling data for in vivo eye irritation test. The used method is Het Cam Test, performed on a chorioallantoic membrane of hen's eggs, between day 10 and 11 of incubation. This type of membrane is vascularized.

The method' principle is based on visual observation, from qualified technicians, of prospective irritations coming out during 5 minutes following the product application on the chorioallantoic membrane.

Observations

Observed irritations were registered not on intensity basis, but on pop up time basis. The reactions observed were vascular conjestion, haemorrhage, coagulation (opacity and/or thrombosis).

Results

Keeping on registered data, the test substance Set Retard Plus, tested with Het Cam Test, can be considered considered as not serious eye damage.