N-octylpyridin-4-amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 04 JULY 18, Experimental end date: 13 JULY18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

N-octylpyridin-4-amine

EC Number:

265-019-0

EC Name:

N-octylpyridin-4-amine

Cas Number:

64690-19-3

Molecular formula:

C13H22N2

IUPAC Name:

N-octylpyridin-4-amine

Results and discussion

In vitro / in chemico

Results

Key result

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

Solubility Assessment

The testconcentrations ofN-Octyl-4-pyridinamine used in the KeratinoSens™methodwere selected onthe basisof solubilitytestcarried out duringthestudy:

Solubility ofthe test item inwas confirmedup to200mMin DMSO.Subsequentdilution incell culturemediumgave a top concentration of 2000μM.

 

Determination of the skin sensitisation potential of N-Octyl-4-pyridinamine

 

Determination criteria for the skin sensitisation potential of the test item

	REP1	REP2	REP3
Does at least one concentration of Test Item induce luciferase activity ≥1.5-fold:	No	No	No
Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%:	N/A	N/A	N/A
Is p value < 0.05 for at least one concentration that yielded ≥1.5-fold induction with viability above 70%	N/A	N/A	N/A
Does EC1.5 value occur at a concentration <1000μM (or <200μglml)	N/A	N/A	N/A
Does the test item induce the luciferase in a dose-dependent manner	N/A	N/A	N/A
Classification	Negative	Negative	Negative

 

Assay Acceptance Criteria (Mean of 3 repetitions)

 

 

Discussion

Thehumanskin sensitisation potential ofN-Octyl-4-pyridinaminewasassessedusing the validatedinvitromethod:the KeratinoSens™test todeterminekeratinocyte activation.Themethodwas adaptedtoanimal product-freeconditionsbyXCellR8 and reference chemicalsdescribedin theguideline and intheperformancestandards wereusedtoconfirmthereliability, accuracy,sensitivityandspecificityvalues.The adaptedmethodshowed fullconcordance with theValidated Reference Method (VRM)-theKeratinoSens™ standard protocol.XCellR8recentlyobtained clarification from theEuropean ChemicalsAgency (ECHA)thatdatausingthe adaptedmethodmaybeused in REACHsubmissions,providedthat thePerformanceStandards data, demonstratingequivalencewiththeVRM,areincludedin thedossier.

In thisstudy,N-Octyl-4-pyridinamine was classified asaNegativeusingtheKeratinoSens predictionmodel.Thesensitisationpotentialof thetestitemwas quantifiedbycalculating2 parameters known astheEC1.5andtheI_MAXvalue:

•TheEC1.svalueistheEffectiveConcentration(EC) oftestitemthat yielded aninductionofluciferase activitygreater than1.5-foldoveruntreated controls.If atleastone concentrationinducesstatistically significant luciferase activity.≥1.5,thenthe productisclassified as positiveprovidedthe cellviability measured by MTT is greater than70%. Thetest item didnotinduce statisticallysignificantluciferaseinduction.≥1.5inany ofthe 3repetitions.The statisticalsignificance,viability,doseresponse and dose acceptancecriteria wereall met andtherefore:

The test itemwas classified asnegativeusing the KeratinoSens predictionmodel.

• The IMAX value is the maximum induction observed within theconcentrationrange tested.Although the KeratinoSens™ test is notvalidatedto predictpotency, theIMAXvalue canprovide a useful tool for preliminary comparison of sensitisationpotentialbetween test items.As shown in Section 13.2, the I_MAXvalues for repetitions 1,2 and3were0.997 (0.977μM and 1.953 μM), 0.734 (1.953 μM) and 0.808(3.906 μM),respectively.Forreference, during testvalidation,sensitising proficiencychemicalsproduced I_MAXvaluesofup to 36-fold over untreated controls.

All of the formal acceptance criteria of thetestswere met.

 

Solubility Assessment

The test concentrations of N-Octyl-4-pyridinamine used in the KeratinoSens™ method were selected on the basis of solubility test carried out during the study:

Solubility of the test item in was confirmed up to 200mM in DMSO. Subsequent dilution in cell culture medium gave a top concentration of 2000μM.

 

Determination of the skin sensitisation potential of N-Octyl-4-pyridinamine

	REP1	REP2	REP3
Does at least one concentration of Test Item induce luciferase activity ≥1.5-fold:	No	No	No
Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%:	N/A	N/A	N/A
Is p value < 0.05 for at least one concentration that yielded ≥1.5-fold induction with viability above 70%	N/A	N/A	N/A
Does EC1.5 value occur at a concentration <1000μM (or <200μglml)	N/A	N/A	N/A
Does the test item induce the luciferase in a dose-dependent manner	N/A	N/A	N/A
Classification	Negative	Negative	Negative

 

Determination criteria for the skin sensitisation potential of the test item

 

Criteria		Result	PASS or FAIL
1	Positive Control (PC) (Cinnamic aldehyde) induction ~1.5-fold in at least one concentration	1.532 at 8.00 μM 1.842 at 16.00 μM 2.844 at 32.00 μM 3.996 at 64.00 μM 12.253 at 128.00 μM	PASS
2	Average induction of PC at 32μM is [1.6-3.0)	2.844	PASS
3	EC1 .5 value is [6-39μM]	7.1 74 μM	PASS
4	CV% of blank values < 20%	9.551	PASS

 

 

Discussion

The human skin sensitisation potential of N-Octyl-4-pyridinamine was assessed using the validated in vitro method: the KeratinoSens™ test to determine keratinocyte activation. The method was adapted to animal product-free conditions by XCellR8 and reference chemicals described in the guideline and in the performance standards were used to confirm the re liability, accuracy, sensitivity and specificity values. The adapted method showed full concordance with the Validated Reference Method (VRM) - the KeratinoSens™ standard protocol. XCellR8 recently obtained clarification from the European Chemicals Agency (ECHA) that data using the adapted method may be used in REACH submissions, provided that the Performance Standards data, demonstrating equivalence with the VRM, are included in the dossier.

In this study, N-Octyl-4-pyridinamine was classified as a Negative using the KeratinoSens prediction model. The sensitisation potential of the test item was quantified by calculating 2 parameters known as the EC_1.5and the IMAX value:

• The EC_1.5value is the Effective Concentration (EC) of test item that yielded an induction of luciferase activity greater than 1.5-fold over untreated controls. If at least one concentration induces statistically significant luciferase activity .≥1.5, then the product is classified as positive provided the cell viability measured by MTT is greater than 70%. The test item did not induce statistically significant luciferase induction .≥1.5 in any of the 3 repetitions. The statistical significance, viability, dose response and dose acceptance criteria were all met and therefore:

The test item was classified as negative using the KeratinoSens prediction model.

• The I_MAXvalue is the maximum induction observed with in the concentration range tested. Although the KeratinoSens™ test is not validated to predict potency, the I_MAXvalue can provide a useful tool for preliminary comparison of sensitisation potential between test items. As shown in Section 13.2, the I_MAXvalues for repetitions 1, 2 and 3 were 0.997 (0.977μM and 1.953 μM), 0.734 (1.953 μM) and 0.808 (3.906 μM), respectively. For reference, during test validation, sensitising proficiency chemicals produced I_MAXvalues of up to 36-fold over untreated controls.

All of the formal acceptance criteria of the tests were met.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

After 48h exposure of cells with 12 concentrations of N-Octyl-4-pyridinamine, Luciferase measurements and MTT viability testing were performed.
N-Octyl-4-pyridinamine was classified as Negative according to the KeratinoSens prediction model.