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

Endpoint summary

Description of key information

Two tests of the AOP for skin sensitization were performed on the substance:

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.

In the in vitro KeratinoSens™ assay cells were incubated with the substance for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In two experiments maximum luciferase activity (Imax) induction of 3.69 and 1.75 were determined at a substance concentration of 500 µM with calculated EC1.5 values of < 200 µg/mL (56.09 µg/mL) and < 200 µg/mL (81.12 µg/mL) respectively. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non-sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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

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

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

Key result

Parameter:

other: cysteine depletion %

Value:

0.23

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.

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

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

 

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 2017 to 17 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

in vitro study

Details on the study design:

- cells line: transgenic cell line KeratinoSens™ , a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct.
- source: Givaudan, Switzerland
- culturing: in 75 cm2 culture flasks (Greiner) in maintenance medium (Dulbecco’s Modified Eagle Medium with 1.0 g/L D-glucose and Na-Pyruvate, supplemented with 10% fetal bovine calf serum and 1% geneticin)
- storage conditions: at 37 ± 1°C and 5% CO2
- Passage number: exp 1: 10 P; exp 2: 12P
- test medium: DMEM with 1.0 g/L D-glucose and Na-Pyruvate, supplemented with 10% fetal bovine calf serum
- vehicle for test substance: DMSO: 1% (v/v) in DMEM with 1.0 g/L D-glucose and Na-Pyruvate, supplemented with 1% fetal bovine calf serum
- Concentrations: 2000, 1000, 500, 250, 125, 61.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM (1% DMSO)
- Negative control: vehicle
- Positive control: Cinnamic aldehyde: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM (1% DMSO)
- luciferase substrate used: kit from Promega containing 10 x Luciferase Assay Substrate (lyophilized), 10 x 10 mL Luciferase Assay Buffer and 30 mL Luciferase Cell Culture Lysis 5x Reagent
- Luminometer used (e.g. model), including instrument settings: no data
- Historical controls: data included


Acceptability criteria:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.


Positive result when:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is < 200 µg/mL
- an apparent overall dose-response for luciferase induction

Positive control results:

within ranges required

Key result

Run / experiment:

other: exp 1

Parameter:

other: Imax > 1.5

Remarks:

at 500 µM

Value:

3.08

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: exp 2

Parameter:

other: Imax >1.5

Remarks:

at 500 µM

Value:

1.69

Vehicle controls validity:

valid

Positive controls validity:

valid

Parameter:

other: viability at 0.98-250 µM

Value:

118.5

Vehicle controls validity:

not applicable

Remarks:

100% default

Positive controls validity:

valid

Remarks:

viability 110-139%

Parameter:

other: viability at 500 µM

Value:

87.7

Parameter:

other: viability at 1000 and 2000 µM

Value:

0

Other effects / acceptance of results:

see tables below

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	280.44	405.62	343.03	88.51
Imax	3.69	1.75	2.72	1.37
IC30 [µM]	510.11	661.12	585.62	106.78
IC50 [µM]	650.58	758.17	704.37	76.08

Interpretation of results:

study cannot be used for classification

Conclusions:

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Under the condition of this study the substance is considered as non sensitiser.

Executive summary:

In the in vitro KeratinoSens™ assay cells were incubated with 2000, 1000, 500, 250, 125, 61.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM of the substance for 48 h at 37°C. After exposure cells were lysed and

luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 3.69 was determined at a substance concentration of 500 µM. The corresponding cell viability was 71.4%. %. Only at a substance concentration of 500 µM a significant luciferase induction >1.5 was found. The calculated EC1.5 was < 200 µg/mL (56.09 µg/mL).

In the second experiment, a max luciferase activity (Imax) induction of 1.75 was determined at a substance concentration of 500 µM. The corresponding cell viability was 103.2%. Only at a substance oncentration of 500 µM a significant luciferase induction >1.5 was found. The calculated EC1.5 was < 200 µg/mL (81.12 µg/mL).

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

As the substance is corrosive, no further testing for the sensitization endpoint was performed.

According to column II of Annex VII sensitization testing can be waived if the substance is corrosive to the skin.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the information available the substance does not need to be classified for sensitization according to Regulation (EC) No 1907/2006.