N,N,N-tri-C8-C10-alkyl-N-methyl-1-aminium sulfate

Endpoint summary

Description of key information

The potential for the substance to cause skin sensitisation has been investigated using a battery of in vitro methods, in accordance with Section 8.3.1 of Annex VII of the REACH Regulation which respectively address the key events of the adverse outcome pathway (AOP) associated with skin sensitisation: molecular interaction with skin proteins (key event 1); inflammatory response in keratinocytes (key event 2) and activation of dendritic cells (key effect 3). Three respective assays: the Direct Peptide Reactivity Assay (DPRA) conducted according to OECD TG 442C, the ARE-Nrf2 Luciferase (KeratinoSens) assay conducted according to OECD TG 442D and the U937 cell line activation Test (U-Sens) assay conducted according to OECD 442E were planned as part of this assessment. While the U-Sens gave positive results, negative results were obtained in ARE-Nrf2 Luciferase and the DPRA assays could not be performed due to the UVCB properties of the substance. Due to the contradictive results of the two performable tests, the KeratinoSens and the U-Sens), the substance is the substance is regarded to be a skin sensitiser as a worst case conclusion.

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:

Experimental Start Date: 03 April 2020, Experimental Completion Date: 02 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Annex VIII Data Requirement

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

In Chemico / in vitro tests are required to be performed prior to the performance of in vivo studies (Section 8.3.1 of Annex VII of the REACH Regulation).

Positive control results:

The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.

The EC1.5 of the positive control (37 µM) was within two standard deviations of the historical mean in the second experiment. The EC1.5 of the positive control (125 µM) was not within two standard deviations of the historical mean (-4.3 – 123 µM) in the first experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system is sensitive enough. A clear dose-response with increasing luciferase activity induction at increasing concentrations was observed. Furthermore, the value was only slightly higher, all other acceptability criteria were met and an independent repeat experiment was performed to determine the effect of the assay in the KeratinoSens assay.
A dose response was observed and the induction at 250 µM was higher than 2-fold
(2.16-fold and 3.74-fold in experiment 1 and 2, respectively).

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The Imax was 0.81 and therefore no EC1.5 could be calculated.

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The Imax was 1.16 and therefore no EC1.5 could be calculated.

Two independent experiments were performed.The cells were in these experiments incubated withthe test itemin a concentration range0.098– 200 µg/mL (2-fold dilution series, first experiment) andin test concentrations of 0.005– 10 µg/mL (2-fold dilution series, second experiment) for 48 hours± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

Experiment 1

·          Precipitation was observed at the start of the incubation period at test concentrations of 100 µg/mL and upwards and at the end of the incubation period at test concentrations
200 µg/mL in the 96-well plates.

·          The test item showed toxicity. The calculated IC₃₀was 0.38 µg/mL and the calculated IC₅₀was 0.52 µg/mL.

·          No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The I_maxwas 0.81 and therefore no EC_1.5could be calculated.

·          The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The I_maxwas 2.16 and the EC_1.5125 µM.

Experiment 2

·          No precipitation was observed at the start and end of the incubation period in the 96-well plates.

·          The test item showed toxicity. The calculated IC₃₀was 0.37 µg/mL and the calculated IC₅₀was 0.48 µg/mL.

·          No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The I_maxwas 1.16 and therefore no EC_1.5could be calculated.

·          The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The I_maxwas 3.74 and the EC_1.537 µM.

Both tests passed the acceptance criteria:

·          The luciferase activity induction obtained with the positive control,Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.

·          The EC_1.5of the positive control (37 µM) was within two standard deviations of the historical mean in the second experiment. The EC_1.5of the positive control (125 µM) was not within two standard deviations of the historical mean (-4.3 – 123 µM) in the first experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system is sensitive enough. A clear dose-response with increasing luciferase activity induction at increasing concentrations was observed. Furthermore, the value was only slightly higher, all other acceptability criteria were met and an independent repeat experiment was performed to determine the effect of the assay in the KeratinoSensÔassay.

A dose response was observed and the induction at 250 µM was higher than 2-fold
(2.16-fold and 3.74-fold in experiment 1 and 2, respectively).

·           Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (10% in experiment 1 and 2).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

 

      

    Overview Luminescence Induction and Cell Viability of test item in Experiment 1 and 2

Concentration (µg/mL)	0.098	0.20	0.39	0.78	1.6	3.1	6.3	12.5	25	50	100	200
Exp 1 luminescence	0.77	0.73	0.81	0.61	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Exp 1 viability (%)	114.8	115.9	68.2	12.0	-0.1	-0.1	0.0	0.0	0.0	0.1	0.2	0.6
Concentration (µg/mL)	0.005	0.01	0.02	0.04	0.08	0.16	0.31	0.63	1.3	2.5	5	10
Exp 2 luminescence	0.87	1.11	1.16	1.11	1.08	1.14	0.95	1.01	0.00	0.00	0.00	0.00
Exp 2 viability (%)	89.8	98.1	100.0	101.8	110.2	103.6	79.3	24.8	-0.8	-0.7	-0.8	-0.7

 

      
Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)	7.8	16	31	63	125	250
Exp 1 luminescence	0.84	0.95	1.00	1.20	1.50^***	2.16^***
Exp 1 viability (%)	116.2	107.9	108.3	102.4	92.0	86.9
Exp 2 luminescence	0.91	1.20	1.46	1.69^***	2.47^***	3.74^***
Exp 2 viability (%)	92.1	92.0	81.6	74.4	77.5	67.5

^***p<0.001 Student’s t test

 

          
Overview EC_1.5, I_max, IC₃₀and IC₅₀Values

	EC1.5(µg/mL)	Imax	IC30(µg/mL)	IC50(µg/mL)
Test item Experiment 1	NA	0.81	0.38	0.52
Test item Experiment 2	NA	1.16	0.37	0.48
	EC1.5(µM)	Imax	IC30(µM)	IC50(µM)
Pos Control Experiment 1	125	2.16	NA	NA
Pos Control Experiment 2	37	3.74	219	NA

NA = Not applicable

 

Interpretation of results:

GHS criteria not met

Conclusions:

It was concluded that the test item gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is not a skin sensitizer.

Executive summary:

The test item showed toxicity (IC₃₀values of 0.38µg/mL and 0.37µg/mL and IC₅₀values of 0.52µg/mL and 0.48 µg/mL in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC_1.5value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 0.81-fold and 1.16-fold in experiment 1 and 2 respectively. The test itemis classified as negative in the KeratinoSens^TMassaysince negative results (<1.5-fold induction) were observed at test concentrations up to 200µg/mL.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start Date: 09 Mar 2020, Experimental Completion Date: 03 Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Annex VIII Data Requirement

Qualifier:

according to guideline

Guideline:

other: OECD 442E: In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays addressing the Key Event on Activation of Dendritic Cells on the Adverse Outcome Pathway for Skin Sensitisation

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

In Chemico / in vitro tests are required to be performed prior to the performance of in vivo studies (Section 8.3.1 of Annex VII of the REACH Regulation).

Positive control results:

In both experiments the positive control were considered valid and fell within the historical control data.

Experiment 2:
The positive control (TNBS) showed a S.I. ≥ 717% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 91% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Experiment 1:
The positive control (TNBS) showed a S.I. ≥ 213% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 112% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Key result

Run / experiment:

other: 1

Parameter:

other: EC150 in µg/mL, expression of CD86

Value:

0.11

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: EC150 in µg/mL, expression of CD86

Value:

0.08

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Overview EC₁₅₀and CV₇₀Values

	EC150(µg/mL)	CV70(µg/mL)
Test item Experiment 1	0.11	0.87
Test item Experiment 2	0.08	6.6

                             NA = Not applicable

Overview Stimulation index of CD86 and Cell Viability in
Experiment 1 and 2

Test items	Dose (µg/mL)	% Viability (Mean)*		CD86-IgG1 S.I.*		Colour Interference S.I.*
		Experiment		Experiment		Experiment
		1	2	1	2	1	2
Test item
	0.1	-	100	-	189	-	189
	0.5	-	97	-	487	-	487
	1	66	90	1308	224	91	224
	3	-	91	-	176	-	176
	5	-	80	-	106	-	106
	10	28	49	1031	506	129	506
	20	24	-	-55	-	110	-
	50	4	-	789	-	120	-
	100	2	-	705	-	163	-
	200	9	-	186	-	167	-

*     Red values are either below 70% viability or above 150 S.I..

-       Not Applicable

Overview Stimulation index of CD86 and Cell Viability in Experiment 1 and 2 of the Positive, Negative and Vehicle Control

Controls	% Viability (Mean)*			CD86-IgG1 S.I.*
	Experiment			Experiment
	1	2		1	2
LA1	100	100		80	85
LA2	100	100		112	91
LA3	100	99		47	83
TNBS1	100	100		394	717
TNBS2	100	99		477	833
TNBS3	99	100		213	828
DMSO (mean)	100	100		124	164
	IgG1 value (%)			CD86 basal expression (%)		CD86-IgG1 expression (%)
	Experiment			Experiment		Experiment
	1	2		1	2	1	2
RPMI1	1.4	1.2		3.6	5.4	2.2	4.2
RPMI2	0.9	1.3		4.2	6.7	3.3	5.4
RPMI3	1.3	1.3		4.1	5.5	2.8	4.2
DMSO1						3.4	6.8
DMSO2						3.3	10.0^1
DMSO3						3.6	5.8
RPMI Mean Viability				99	100
RPMI Drift				2%	-13%
LA Drift				-51%	-6%

*            Red values are either below 70% viability, above 150 S.I..

¹                 Excluded from analysis, value > 25% from mean

 

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item showed toxicity (CV70 values of 0.87 µg/mL and 6.6 µg/mL in experiment 1 and 2, respectively). A biologically relevant, induction of the CD86 activity (EC150 values of 0.11 µg/mL and 0.08 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The test item is classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.

Executive summary:

The objective of this study is to evaluate the ability of the test item to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay.

The study procedures described in this report were based on the most recent OECD guideline.

The test item was a light yellow solidified melt. The test item was dissolved in dimethyl sulfoxide at 50 mg/mL (first experiment) and 2.5 mg/mL (second experiment).In the first experiment the stock was diluted to six test concentrations (1, 10, 20, 50, 100 and 200μg/mL).In the second experiment, a more narrow dose-response analysis was performed to investigate the increase in expression of experiment 1 up to 1 µg/mL in more detail.The test item precipitated at the dose levels of 100 and 200μg/mL. Two independent experiments were performed.

Both experiments passed the acceptance criteria:

·      At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (99% in experiment 1 and 100% in experiment 2).

·      The mean viability of the triplicate DMSO vehicle control cells was above the threshold of 90% (100% in experiment 1 and 2).

·      The DMSO vehicle control mean value of its triplicate CD86 S.I. was smaller than 250% of the mean of the triplicate CD86 S.I. of untreated U937 cells in both experiments.

·      The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in both experiments.

·      At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5% in both experiments.

·      No drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls.

In both experiments the positive (TNBS) and negative (LA) control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The test item showed toxicity (CV₇₀values of 0.87µg/mLand 6.6µg/mLin experiment 1 and 2, respectively). A biologically relevant, induction of the CD86 activity (EC₁₅₀values of 0.11µg/mL and 0.08µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The test itemis classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.

In conclusion, the test item is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

An investigation of the potential for the substance to cause skin sensitisation was planned using a battery of in vitro methods, in accordance with Section 8.3.1 of Annex VII of the REACH Regulation which respectively address the key events of the adverse outcome pathway (AOP) associated with skin sensitisation: molecular interaction with skin proteins (key event 1); inflammatory response in keratinocytes (key event 2) and activation of dendritic cells (key effect 3).

The reactivity and sensitising properties, and the potential for the substance to initiate key event 1 of the skin sensitisation AOP (the molecular initiating event involving the covalent binding of electrophilic substances to nucleophilic centres in skin proteins), could not be investigated using the Direct Peptide Reactivity Assay (DPRA), due to the UVCB properties of the substance.

The second key event of the skin sensitisation AOP relates to events taking place in keratinocytes including inflammatory responses as well as gene expression associated with specific cell signalling pathways such as antioxidant/electrophilic response (ARE)-dependent pathways. The potential for the substance to initiate key event 2, by inducing genes regulated by the antioxidant response element (ARE) was investigated using the ARE-Nrf2 Luciferase (KeratinoSens) method in a study conducted according to OECD TG 442D. In two initial tests performed, negative and positive results were obtained respectively. The test item showed toxicity (IC30values of 0.38µg/mL and 0.37µg/mL and IC50values of 0.52µg/mL and 0.48 µg/mL in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 0.81-fold and 1.16-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 200µg/mL.

The potential for the substance to induce the third key event of the skin sensitisation AOP (activation of dendritic cells) was investigated using the U937 cell line activation Test (U-Sens) assay in a study conducted according to OECD 442E. The objective of this study is to evaluate the ability of the test item to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay. The test item was dissolved in dimethyl sulfoxide at 50 mg/mL (first experiment) and 2.5 mg/mL (second experiment).In the first experiment the stock was diluted to six test concentrations (1, 10, 20, 50, 100 and 200μg/mL).In the second experiment, a more narrow dose-response analysis was performed to investigate the increase in expression of experiment 1 up to 1 µg/mL in more detail. The test item precipitated at the dose levels of 100 and 200μg/mL. Two independent experiments were performed. The test item showed toxicity (CV70values of 0.87µg/mLand 6.6µg/mLin experiment 1 and 2, respectively). A biologically relevant, induction of the CD86 activity (EC150values of 0.11µg/mL and 0.08µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The test itemis classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control. Based on these findings, the U-Sens prediction was considered to be positive for the substance; indicating a potential to initiate the third key event of the skin sensitisation Adverse Outcome Pathway (AOP); activation of dendritic cells.

The evaluation of the skin sensitisation potential of the substance is based on two in vitro tests (KeratinoSens and U-Sens), each respectively assessing the potential of the substance to initiate a key event in the adverse outcome pathway for skin sensitisation. While negative results were obtained in the ARE-dependent keratinocyte activation assay, positive results were obtained in the dendritic cell activation assay. Since the findings overall were conflicting, a worst case assumption has been used to characterise the potential skin sensitisation properties of the substance.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

No data are available on the potential of the substance to cause respiratory sensitisation. There are no standard tests available to investigate the respiratory sensitisation hazard of the substance.

Justification for classification or non-classification

The skin sensitisation potential of the substance has been characterised using two in vitro studies that respectively assess the potential induction of two key events in the Adverse Outcomes Pathway (AOP) for skin sensitisation. Based on the described findings and a worst case assumption, the substance is considered to have potential to cause skin sensitisation and classification according to Regulation (EC) 1272/2008 in respect of this endpoint is required.