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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

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).

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

850-698-3

Cas Number:

2387913-24-6

Molecular formula:

not applicable

IUPAC Name:

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

Test material form:

other: solified melt

Details on test material:

- Name of test material ((as cited in study report):
Product name: V10069,
Systemic name: Quaternary ammonium compounds, tri-C8-10-alkylmethyl-methylsulfate

Results and discussion

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).

In vitro / in chemico

Results

Results 1

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.

Results 2

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.