[2-[[2-cyano-3-[4-[ethylbenzylamino]phenyl]-1-oxoallyl]oxy]ethyl]trimethylammonium chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 February 2017 to 4 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

the average molecular weight used for the calculation of the 100 mM stock concentration was not correct

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Principles of method if other than guideline:

The average molecular weight (278.19 g/mol) was used to calculate the doses tested.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

in chemico method as required according to ECHA guidelines

Test material

Test material information

Constituent 1

Reference substance name:

[2-[[2-cyano-3-[4-[ethylbenzylamino]phenyl]-1-oxoallyl]oxy]ethyl]trimethylammonium chloride

EC Number:

275-604-2

EC Name:

[2-[[2-cyano-3-[4-[ethylbenzylamino]phenyl]-1-oxoallyl]oxy]ethyl]trimethylammonium chloride

Cas Number:

71550-24-8

Molecular formula:

C24H30N3O2.Cl

IUPAC Name:

[2-[[2-cyano-3-[4-[ethylbenzylamino]phenyl]-1-oxoallyl]oxy]ethyl]trimethylammonium chloride

Test material form:

liquid

Details on test material:

Storage : -15 to +40 °C
Form: yellow liquid

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system): according to OECD 442C with the appropriate controls
RC A = accuracy control
RC B = stability control
RC C = solvent control

Results and discussion

Positive control results:

cysteine:
SD of peptide depletion of the PC replicates < 14.9% actual 0.58
SD of peptide depletion of the TI replicates < 14.9% actual 0.40

lysine:
SD of peptide depletion of the PC replicates < 11.6% actual 0.72
SD of peptide depletion of the TI replicates < 11.6% actual 0.47

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: cysteine depletion %

Value:

0.23

Results 2

Key result

Parameter:

other: lysine depletion %

Value:

4.11

Remarks on result:

other: not valid because of phase separation

Other effects / acceptance of results:

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Precipitation was observed for the samples of the positive control. Samples were not centrifuged prior to the HPLC analysis (no interference with the system).
After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the test item, samples were not centrifuged prior to the HPLC analysis. Turbidity and phase separation was observed for the samples of the positive control. Samples were not centrifuged prior to the HPLC analysis (no interference with the system).

The average molecular weight used for the calculation of the 100 mM stock concentration was not correct. It is expected that the concentration tested was too high.

Any other information on results incl. tables

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	906.9033	0.1111	78.63	78.90	0.58	0.74
	867.1926	0.1064	79.57
	912.4229	0.1118	78.50
Test Item	4312.5039	0.5147	0.00*	0.23	0.40	173.21
	4211.7358	0.5027	0.00*
	4154.0147	0.4959	0.69

* Values were set to Zero due do negative depletion

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	2051.3940	0.2418	52.31	51.62	0.72	1.39
	2078.4993	0.2450	51.68
	2113.1423	0.2491	50.87
Test Item	4059.3049	0.4790	3.65	4.11	0.47	11.32
	4020.0920	0.4744	4.58
	4040.4526	0.4768	4.10

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the outcome of the test the preliminary conclusion is that the substance is non-sensitizing. The mean depletion of the cysteine peptide was ≤ 13.89% (0.23%). Based on the prediction model 2 the test item can be considered as non-sensitiser (since turbidity was observed immediately after mixing of the test item solution with the lysine peptide and this may lead to an underestimation of the lysine reactivity)

Executive summary:

The substance was tested in the DPRA test according to OECD 442C. Samples prepared for lysine depletion assessment showed phase separation and therefore these results were considered less reliable. Based on prediction model 2 the substance is considered a non-sensitizer.

Predicition Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	n.a	n.a	n.a	0.23	Minimal Reactivity	no sensitizer
Positive Control	65.26	High Reactivity	sensitizer	78.90	Moderate Reactivity	sensitizer