Hexamethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation

Remarks:

other: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27 Oct 2014 - 12 Dec 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, in-house validated procedure

Qualifier:

according to guideline

Guideline:

other: Direct peptide binding assay (DPRA) performed as described in Bauch C. et al. (2011), Toxicology in Vitro 25, 1162 – 1168.

Qualifier:

according to guideline

Guideline:

other: OECD: Draft Proposal for a New Guideline on In Vitro Skin Sensitization: Direct Peptide Reactivity Assay (DPRA), assessed on 13 Nov 2013 at http://www.oecd.org

Principles of method if other than guideline:

In the DPRA the reactivity of a test item (=test substance) towards synthetic cysteine (C)- or lysine (K)-containing peptides is evaluated. For this purpose the test substance is incubated with synthetic peptides for 24 hours at room temperature and the remaining non-depleted peptide concentration is determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm. The peptide depletion of test-substance incubated samples is compared to the peptide depletion of the NC samples and expressed as relative peptide depletion.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Species:

other: synthetic peptides (Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH; Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH)

Positive control results:

The mean peptide depletion by the positive control was 31.24%.

The mean C-peptide depletion, caused by the test substance was determined to be 0.44%. The mean K-peptide depletion, caused by the test substance was determined to be -2.72%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.22%. No co-elution of test substance and peptides was noticed. Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that the test article shows a minimal

chemical reactivity in the DPRA under the test conditions chosen.

Interpretation of results:

other: minimal chemical reactivity

Executive summary:

The reactivity of the test article towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, a co-elution control was assessed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity. The following results were obtained in the DPRA: The test substance was soluble in acetonitrile. The samples with the test substance and the peptide stock solutions were suspensions. After 24 hours suspensions were noticed. Thus all samples were centrifuged prior to HPLC analysis. The mean C-peptide depletion, caused by the test substance was determined to be 0.44%. The mean K-peptide depletion, caused by the test substance was determined to be -2.72%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.22%. No co-elution of test substance and peptides was noticed. Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that the test article shows a minimal chemical reactivity in the DPRA under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Due to the complexity of the skin sensitization process a single in vitro assay is not sufficient to adequately assess this toxicological endpoint. Therefore, a combination of several methods addressing key steps of the sensitization process: protein reactivity (DPRA), activation of keratinocytes (LuSens or KeratinoSens) and activation of dendritic cells (MUSST or h-CLAT) were used (test battery).

These in vitro studies cover 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). These study types have initially undergone in-house validation using 54 substances (Bauchet al., 2012, Regul Toxicol Pharmacol. 63: 489-504). Recently, a validation performed with 213 substances has been published (Urbisch et al. 2015, Regul Toxicol Pharmacol. 71: 337-351).

Based on the results of the in house validation (Bauchet 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. This was confirmed in the validation study with more substances (Urbisch 2015): When compared to the sensitization potential of the substances in humans, the classification based on an evaluation scheme using results obtained from theDPRA, LuSens and mMUSST had a sensitivity of 90%, a specificity of 90% and an accuracy of 90%.

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 or KeratinoSens, MUSST or 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/KeratinoSens and 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

The in vitro sensitization test battery was initiated with the DPRA and the LuSens. As an unambiguous result was obtained after these two tests, no further test was performed. Each individual assay was performed under GLP and the cell based assay 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 battery applicability is limited when testing substances 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. The substance under evaluation did not have any of the above mentioned limitations and the outcome of this assay is therefore considered adequate for hazard identification for the endpoint of skin sensitization.

The test substance is not peptide reactive and does not activate keratinocytes. In accordance with the published evaluation scheme (Bauch et al., 2012, Regul Toxicol Pharmacol. 63: 489-504) and Sections 1.2 and 1.4 of Annex XI of EC regulation 1907/2006, the test article is judged to be not a skin sensitizer.

In addition, a Bühler test is available which was performed by a contract research institute that was discredited for manipulating data and falsifying study reports (IBT, 1977). Therefore, the reliability of the available study is questionable. However, the data presented in this report is in line with the reliable reports and provides an additional weight of evidence that the test article is not a skin sensitizer.

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 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.

Justification for selection of skin sensitisation endpoint:
As part of the in vitro skin test battery at least 2 studies are required; one is selected representing the valid and acceptable testing battery.

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 substance does not need to be classified and labelled for skin sensitization under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EC.

 

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 substance does not eed to be classified and labelled for askin sensitization under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation EC No 605/2014.