N,N''-naphthalene-1,5-diylbis[N'-[3-[(2-ethylhexyl)oxy]propyl]urea]

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In vitro studies covering three key steps of the adverse outcome pathway for skin sensitization as identified by the OECD (OECD Publication No.168; ENV/JM/MONO(2012)10) were performed. These study types have undergone in-house validation using 54 substances (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) and methods for keratinocyte activation and dendritic cell activation are in the final stages of validation at ECVAM (http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/validation-regulatory-acceptance/topical-toxicity/skin-sensitisation). The ECVAM opinion on the direct peptide binding assay has been published on Dec 13, 2013. Based on the results of the in house validation (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) the predictive capacity of the evaluation scheme using DPRA (peptide reactivity), LuSens/KeratinoSens™ (keratinocyte activation) and (m)MUSST/h-CLAT (dendritic cell activation) is comparable to that of the local lymph node assay. When compared to the sensitization potential of the substances in humans, the classification based on an evaluation scheme using results obtained from the DPRA, LuSens and hClat had a sensitivity of 93%, a specificity of 95% and an accuracy of 94%. This publication was also referenced by the OECD in the AOP guidance document. Furthermore, it is also in line with the recently published strategy by ECVAM (EURL ECVAM, JRC79446, doi:10.2788/84214; 2013).

 

In the evaluation scheme used here any two of the three test results determine the overall classification, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauchet al. 2012; Table 1). If two assays (DPRA, LuSens, h-CLAT) yield concordant results, the result of the third assay is not necessarily required to determine the overall outcome of the evaluation scheme.

 

Table 1: Decision matrix for combinations of DPRA,LuSens/KeratinoSensand MUSST/ h-CLAT assays.

DPRA	LuSens/ KeratinoSens^TM	MUSST/ h-CLAT	Test battery evaluation
positive	positive	positive	sensitizer
positive	positive	negative	sensitizer
positive	negative	positive	sensitizer
positive	negative	negative	non-sensitizer
negative	positive	positive	sensitizer
negative	positive	negative	non-sensitizer
negative	negative	positive	non-sensitizer
negative	negative	negative	non-sensitizer

Each individual assay was performed under GLP/and the cell based assays LuSens consisted of at least two independent experiments. Positive and negative controls were included in each experiment and confirmed the functionality and validity of the individual assays.

The test substance was insoluble in the vehicle and in the incubation medium. Indeed, the hClat assay could not be performed becasuese precipates of the test substance blocked the tubes of the flow cytometer.

Absence of an antioxidant response was reported in the LuSens assay. The DPRA assay showed no depletion of the the Lys-containing peptide and depletion of the Cys-containing peptide, accompanied by a cystin-peak.

 

The test battery applicability is limited when testing substances are insoluble in the commonly used vehicles and highly volatile substances. Substances susceptible to base-catalyzed hydrolysis cannot be evaluated reliably for binding to lysine as the incubation is performed at pH 10.2. Also substances that interfere with the experimental measurements (e.g., co-elution with the peptide in the DPRA-assay) are not or only partially suitable for testing using in vitro methods. Substance catalysing the oxidation of Cystein thiol groups to Cystin are false positve in the DPRA. The substance under evaluation may have been an oxidation catalyst, since the depletion by 20% of the Cys-Peptide was accormpanied by a Cystin Peak.

In the absence of solubility, adduct formation with the model peptides is difficult to imagine. Applying the profilers for protein binding alerts OASIS v1.2 and OECD contained in the OECD QSAR Toolbox v3.2.0.103, no structural alerts were identified. One metabolite was identified by the skin metabolism simulator module of the OECD QSAR Toolbox. This metabolite also did not contain a structural alert for protein binding. QSAR modelling with TIMES-SS, which takes into accounted simulated skin metabolism simulation and autooxidation, resulted in the prediction as a non-sensitizer. The substance contains some fragments unknown in the training set of the model.

No indication of any kind of reactivity was noted in the existing toxicity data (in-vitro clastogenicity, mutagenicity, local toxicity, acute and subacute oral toxicity).

In an overall weight-of-evidence approach based on in-vitro data and (Q)SAR modelling, the substance is judged to be non sensitizing to skin.

Migrated from Short description of key information:
Sensitization involves a number of key steps in order to take place, and can be described in terms of an adverse outcome pathway (AOP). These include reactivity with skin proteins (peptide reactivity), activation of skin cells (keratinocyte activation) and immune cells (dendritic cell activation). The studies carried out for this substance address these three key steps. The results are then used in a predefined evaluation scheme to determine hazard classification as a sensitizer by a weight-of-evidence approach.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substanceis / is not considered to be classified for skin sensitization under Directive 67/548/EEC, as amended for the 31^sttime in Directive 2009/2/EG.

 

Classification,Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substanceis / is notconsidered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the third time in Directive (EC 618/2012).