4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

Endpoint summary

Description of key information

The substance was tested in the DPRA test according to OECD 442C (first key event in AOP). Samples prepared for cysteine and lysine depletion assessment co-eluted with the peptides and therefore the results were considered inconclusive (Eurofins 2018)

In an in vitro KeratinoSens™ assay the sensitising potential of the substance by addressing the second molecular key event of the AOP for skin sensitization, namely activation of keratinocytes. Maximum luciferase activity (I_max) induction of 16.82 and 11.29 was determined at a substance concentration of 125 µM at 62.5 mM. The lowest tested concentration with a significant luciferase induction >1.5 (2.11 and 2.52) was found to be 15.63 µM in both experiments. The calculated EC_1.5 was < 1000 µM (11.53 µM) and < 1000 µM (9.56 µM) (Eurofins 2018).

A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

The result is an indication for skin sensitizing properties of the substance. However, as this involves only the second key event in the AOP for skin sensitization, no definitive conclusion can be drawn.

In the hCLAT assay (3rd key event) the substance showed a strong shift of the fluorescence signal compared to the solvent control. Hereby, the plot is shifted rightwards out of the graphical diagram. Furthermore, a strong cytotoxic effect in the whole concentration range was observed (PI negative as percentage 0, actual cell number 5-37). Thus the cytotoxic effect of the test item cannot be evaluated (see Annex). Therefore, the study was stopped after the first dose finding experiment and no conclusion can be drawn (Eurofins 2018).

In an LLNA test (Eurofins 2018) 3 mice were treated by topical application of 25 µL of the substance at concentrations of 6.25, 12.5 or 25% w/w in DMSO on the entire dorsal surface of each ear once daily over three consecutive days.

Five days after the first topical application all mice were dosed with 20 µCi³H-methyl thymidine by intravenous injection

Approximately 5 hours after the injection all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, weighed individually, pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared and the ³H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM).

All of the tested concentrations exceeded the stimulation index of 3 (3.9,3.9 and 3.8 at 6.25, 12.5 and 25%). The EC3 value could not be calculated as the stimulation indices of all concentrations were above 3.

The results of the radioactivity determination are supported by the second endpoint, the means of the lymph node weights per group, which showed increased values compared to the negative control values.

The substance is considered to be sensitizing to the skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 2017 to 13 October 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

Details on the study design:

Skin sensitisation (In vitro test system) - transgenic cell line KeratinoSens™ (Givaudan, Switzerland)
Based on a stock solution (40 mg/L arbitrary set at 200 mM) a set of twelve master solutions in 100% DMSO was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium (Dulbecco’s Modified Eagle Medium). The following concentration range was tested in the assay:
2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM
Cells were incubated with the test item for 48 h at 37 ± 1°C and 5% CO2. . After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

Positive control results:

Cinnamic aldehyde (4 µM, 8 µM, 16 µM; 32 µM; 64 µM): Induction PC at 64 µM: 3.62 and 5.74 (within 2 times SD of historical control values)

Run / experiment:

other: 1

Parameter:

other: IC 1.5

Value:

11.53

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

IC 1.5 15.89 uM

Remarks on result:

positive indication of skin sensitisation

Remarks:

cell viability at lowest concentration inducing > 1.5 fold increase 109.4%

Run / experiment:

other: 2

Parameter:

other: IC 1.5

Value:

9.56

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

IC 1.5 10.36 uM

Remarks on result:

positive indication of skin sensitisation

Remarks:

cell viability at lowest concentration inducing > 1.5 fold increase 105.9%

Induction of Luciferase Activity – Overall Induction

Overall Induction	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.19	1.18	1.18	0.01
	8.00	1.37	1.38	1.37	0.00
	16.00	1.50	1.79	1.65	0.21	*
	32.00	2.11	2.54	2.33	0.31	*
	64.00	3.62	5.74	4.68	1.50	*
Test Item	0.98	1.06	1.14	1.10	0.06
	1.95	0.98	1.01	1.00	0.02
	3.91	0.87	0.89	0.88	0.02
	7.81	0.95	1.21	1.08	0.18
	15.63	2.11	2.52	2.31	0.29	*
	31.25	4.82	5.83	5.33	0.72	*
	62.50	9.65	11.09	10.37	1.02	*
	125.00	16.82	10.18	13.50	4.70
	250.00	0.01	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00

* = significant induction according to Student’s t-test, p<0.05

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	6.4	pass	8.2	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	3.0	pass	3.0	pass
EC1.5 PC	7 < x < 34 µM	15.89	pass	10.36	pass
Induction PC at 64 µM	2.00 < x < 8.00	3.62	pass	5.74	pass

 

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the in vitro KeratinoSens™ assay the substance is considered sensitizing to the skin.

Executive summary:

In an in vitro KeratinoSens™ assay the sensitising potential of the substance by addressing the second molecular key event of the adverse outcome pathway (AOP) for skin sensitization, namely activation of keratinocytes, cells were incubated with the test item 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 (I_max) induction of 16.82 was determined at a substance concentration of 125 µM. The corresponding cell viability was 6.6%. The lowest tested concentration with a significant luciferase induction >1.5 (2.11) was found to be 15.63 µM. The corresponding cell viability was >70% (109.4%).The calculated EC_1.5 was < 1000 µM (11.53 µM).

In the second experiment, a max luciferase activity (I_max) induction of 11.09 was determined at a substance concentration of 62.5 µM. The corresponding cell viability was 45.8%. The lowest tested concentration with a significant luciferase induction >1.5 (2.52) was found to be 15.63 µM. The corresponding cell viability was >70% (105.9%).The calculated EC_1.5 was < 1000 µM (9.56 µM).

A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

The result is an indication for skin sensitizing properties of the substance. However, as this involves only the second key event in the AOP for skin sensitization, no definitive conclusion can be drawn.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 August 2017 to 30 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 substance was applied as such.

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 %

Remarks on result:

not determinable

Remarks:

co-elution with cysteine

Key result

Parameter:

other: lysine depletion %

Remarks on result:

not determinable

Remarks:

coelution with lysine

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. No precipitation, turbidity or phase separation was observed for the samples of the test item. 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).

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	1268.3999	0.1469	72.35	73.05	0.61	0.83
	1222.3235	0.1418	73.35
	1217.9059	0.1413	73.45
Test Item	n/a	n/a	n/a	n/a	n/a	n/a
	n/a	n/a	n/a
	n/a	n/a	n/a

n/a:        not applicable

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	2198.9507	0.2087	57.98	58.30	1.46	2.50
	2249.3828	0.2136	57.02
	2099.5659	0.1992	59.88
Test Item	n/a	n/a	n/a	n/a	n/a	n/a
	n/a	n/a	n/a
	n/a	n/a	n/a

n/a:        not applicable

Interpretation of results:

other: inconclusive

Conclusions:

Based on the outcome of the test the preliminary conclusion is that due to co-elution of test item with both peptide peaks, sensitizing potential of the test item could not be predicted and the test result must be considered as inconclusive.
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.57%.

Executive summary:

The substance was tested in the DPRA test according to OECD 442C. Samples prepared for cysteine and lysine depletion assessment co-eluted with the peptides and therefore the results were considered inconclusive. The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.57% for the positive control.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Based on the outcome of the in vitro tests it was decided to perform an in vivo LLNA test. Only one of the three in vitro tests was positive, but both others were inconclusive. As it could not be excluded that the substance is a absorbed via the skin (see also under toxicokinetics), an LNNA test was performed that confirmed the outcome of the Keratinosens test and showed stimulation indices > 3. An EC3 value could not be calculated due to the lack of a dose response.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information the substance needs ot be classified as sensitizing to the skin (H317) according to EC Regulation No 1272/2008 (CLP).