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

Endpoint summary

Description of key information

Not skin corrosive, not eye corrosive

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / Corrosions

Corrosion characteristics of test item were evaluated according to the OECD Guideline 431, on reconstitued epidermis 3D (EpiDerm™), through MTT test cytotoxicity analysis.

The test item was applied on each epidermis unit in duplicate for 3 minutes and 1 hour at 37°C, 5% CO2. At the end of the exposure, the test item was removed, and cell viability evaluated through MTT test.

The percentage of mean cell viability for treated cells resulted to be:

- after 3 minutes: 105.1 %

- after 60 minutes: 122.5 %

According to guideline OECD 431, the test item has to be considered Non corrosive.

FIRST ADOPTATION OF OECD 439 ON 2010

Eye irritation:

A toxicological study on the target substance Set Retard Plus 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.

Observed irritations were registered not on intensity basis, but on pop up time basis. They were:

- vascular conjestion; haemorrhage; coagulation (opacity and/or thrombosis).

 

 

In general, no in vitro test was found capable of replacing completely the Draize rabbit eye test, but some of the assays showed considerable promise as screens for ocular irritancy. The main reason for this is the difficulty of comparing in vitro test results with historical animal data where the subjective scoring of tissue lesions in the eye in the Draize test provides variable estimates of eye irritancy. Other possible contributing reasons for the outcomes of recently completed validation studies are: a) the in vitro tests only partially modelled the complex in vivo eye irritation response, b) the protocols might have been insufficiently developed, and c) the choice of statistical approaches for analysing the data might not have been appropriate.

One of the alternative in vitro test is the Het Cam Test, performed on a chorioallantoic membrane of hen's eggs. This method is suitable for transparent test materials, for non-transparent insoluble and solid materials the examination of the endpoints shall be conducted after a very careful rinsing to remove the sample.

The HET-CAM method It is not commonly used for hazard evaluations in the sense of labelling and classification, since it has not yet been recognized as a generally accepted alternative to the Draize rabbit eye in vivo test.

However, the HET-CAM assay has been accepted already by the British, French, Dutch and German (in conjunction with the NRU cytotoxicity assay) authorities for the classification of severe irritants.

The majority of the validation studies carried out, showed a useful correlation between the HET-CAM test and the Draize rabbit eye test for the assessment of raw materials and cosmetic products. This in vivo versus in vitro correlation revealed good results in the area of mild and non-irritating test materials. Although the HET-CAM assay in principle is applicable to all types of chemicals regardless of their physico-chemical properties, measurements on solid and insoluble or sticky materials may cause problems in the reproducibility of test results. (EU Guidance “Subgroup 3. Eye Irritation”)

 

 As reported in theNIH Publication No: 06-4515, many protocols developed for the HET-CAM method, that calculated IS values (either by the IS[A] or IS[B] analysis methods) used a similar classification scheme. Ranges of in vitro scores were assigned to one of four irritation categories. In addition to the IS ranges mentioned in the endpoint record, several other irritation classification schemes have been described. In the table below are summarized the IS value which is associated to irritant susbtances.

 

Decision Criteria used by Gettings and Colleagues to Define Test Substances as irritants

 

Literature Source	IS Value for Irritants
Gettings et al. 1991 (HET-CAM I)	> 10
Gettings et al. 1991 (HET-CAM II)	> 3001
Gettings et al. 1994 (HET-CAM)	≥ 5
Gettings et al. 1994 (Chorioallantoic Membrane Assay)	≥ 4.8
Gettings et al. 1996 (HET-CAM I)	≥ 5.1
Gettings et al. 1996 (HET-CAM III)	≥ 4.83

 

Based on the combined test result approach, the HET-CAM test method has the following accuracy in predicting substances classified as corrosives or severe irritants, according to the GHS classification system (UN 2003): 44 % to 85 %. The sensitivity from 25 % to 100 %, the specificity is 40 % to 100 %, the false positive rate of 0 % to 60 % and a false negative rate of 0 % to 75%.

 

Moreover according to the ECHA Guidance r7.a, the HET-CAM test method were not sufficiently accurate for regulatory use or that there was not sufficient data, especially for Category 2 chemicals, to make a final conclusion on their validity.

Justification for classification or non-classification

Skin irritation

According to the interpretation of results and prediction model given in the OECD 431, a substance shall be considered as Non corrosive if the Mean cell viability is ≥ 50 % after 3 min exposure AND ≥ 15 % after 60 min exposure

 

The percentage of mean cell viability for treated cells resulted to be ≥ 50 % both after 3 minutes and after 60 minutes of exposure ( 105.1% and 122.5 % respectively), therefore the substance shall be considered as non corrosive for skin.

 

Eye irritation

According to the classification scale for irritant effect of ICCVAM-Recommended Test Method Protocol, a substance shall be classified as Irritant if the Irritation Grade (N) is greater than 9, if 5 ≤ N < 9, the substance is considered as mildly irritant.

The Irritation Grade of the tested substance is 6.8, therefore the substance is Mildly Irritant.

Based on the test results, the substance does not requires classification for serious eye damage. According to the ECHA Guidance R7.a, the HET-CAM test method were not sufficiently accurate for regulatory use especially for Category 2 chemicals, to make a final conclusion on their validity. Considering the test results, the variable evaluation of irritant categories, the false positive rate of the HET-CAM test and the chemical nature of the substance (Ca-Complex), no conclusion on the eye irritation classification is reachable.