Dodecyltrimethylammonium bromide

Endpoint summary

Description of key information

Using Derek Nexus v5.0, the skin sensitising potential of the test item was estimated to be equivocal (reference 7.4.1-1). However, basaed on the results of an in chemico/in vitro test strategy the test item is not peptide reactive (DPRA,OECD TG 442C, reference 7.4.1-2) and does not activate keratinocytes (Keratinosens, OECD TG 442D, reference 7.4.1-3). Therefore, the substance is not predicted to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Remarks:

in silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

Please refer to the QMRF and QPRF files provided under the section attached justification.

Qualifier:

no guideline available

Principles of method if other than guideline:

Estimates the skin sensitising properties of chemicals using structural alert relationships.

GLP compliance:

no

Specific details on test material used for the study:

SMILES: [Br-].CCCCCCCCCCCC[N+](C)(C)C

Key result

Parameter:

other: alerts

Value:

1

Remarks on result:

other: Skin sensitisation in mammal is equivocal. Alert matched: 436 Quaternary ammonium salt

Remarks:

QSAR predicted value. The substance is within the applicability domain of the model.

Interpretation of results:

other: Derek result: equivocal

Conclusions:

Using Derek Nexus v5.0, the skin sensitising potential of the test item was estimated to be equivocal. The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate.

Executive summary:

The skin sensitising properties were estimated using Derek Nexus v5.0. The skin sensitising potential of the test item was estimated to be equivocal based on the described QSAR method (Derek, 2017).

The adequacy of a prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for the regulatory purpose.

For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.

 

Description of the prediction Model

The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 

 

Assessment of estimation domain

The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-01-24 to 2018-02-08

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))

Version / remarks:

04 February 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

Version / remarks:

12 January 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

Positive control results:

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 67.89%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

0.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptance Criteria

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the
positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control
replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine
percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine
percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	5045.1484	0.5340	4484.7075	0.5340
STD2	2548.3921	0.2670	2226.4971	0.2670
STD3	1279.3019	0.1335	1125.6809	0.1335
STD4	637.0319	0.0667	549.5435	0.0667
STD5	311.3560	0.0334	277.2835	0.0334
STD6	153.8533	0.0167	145.1036	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Depletion of the Cysteine Peptide

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	1371.9458	0.1446	72.07	72.27	0.27	0.38
	1367.3362	0.1441	72.17
	1346.7943	0.1419	72.58
Test Item	4949.3926	0.5227	0.00	0.10	0.11	114.56
	4901.3276	0.5176	0.22
	4908.6392	0.5184	0.07

 

Depletion of the Lysine Peptide

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	1489.3650	0.1777	64.50	63.51	0.86	1.35
	1548.7262	0.1848	63.08
	1554.0549	0.1854	62.95
Test Item	4220.2124	0.5031	0.00	0.00	0.00	n/a
	4239.8545	0.5054	0.00
	4222.2485	0.5033	0.00

n/a:        not applicable

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model ¹

Mean Cysteine andLysine PPD	Reactivity Class	DPRA Prediction²
0.00% ≤ PPD ≤ 6.38%	No or Minimal Reactivity	Negative
6.38% < PPD ≤ 22.62%	Low Reactivity	Positive
22.62% < PPD ≤ 42.47%	Moderate Reactivity
42.47% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 13.89%	No or Minimal Reactivity	Negative
13.89% < PPD ≤ 23.09%	Low Reactivity	Positive
23.09% < PPD ≤ 98.24%	Moderate Reactivity
98.24% < PPD ≤ 100%	High Reactivity

Categorization of the Test Item

Prediction 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	0.05	Minimal Reactivity	no sensitiser	0.10	Minimal Reactivity	no sensitiser
Positive Control	67.89	High Reactivity	sensitiser	72.27	Moderate Reactivity	sensitiser

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item might be considered as “non-sensitiser”.
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.

Executive summary:

In the present study dodecyltrimethylammonium bromide was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 308.34 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the co-elution control of the positive control. No precipitation, turbidity or phase separation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.

No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C _acetonitrile).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (0.05%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

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 67.89%.

The controls confirmed the validity of the study for both the cysteine and lysine run.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-03-15 to 2018-03-26

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)

Version / remarks:

04 February 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

Version / remarks:

01 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Details on the study design:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (5.80 (experiment 1); 3.67 (experiment 2)).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

1.34

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 15.63µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

12.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

2.71

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 7.81 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

46.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Value:

2.36

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptance Criteria

The test meets acceptance criteria if:
- 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.

The controls fullfilled the validity criteria of the test.

Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100	100	100	0.0
Positive Control	4.00	102.3	102.7	102.5	0.2
	8.00	101.2	104.2	102.7	2.1
	16.00	96.5	102.3	99.4	4.1
	32.00	94.2	102.3	98.3	5.7
	64.00	81.3	105.9	93.6	17.4
Test Item	0.98	81.1	98.7	89.9	12.4
	1.95	63.5	83.8	73.7	14.4
	3.91	55.6	72.4	64.0	11.9
	7.81	35.9	46.9	41.4	7.8
	15.63	12.9	20.6	16.7	5.5
	31.25	0.2	0.5	0.4	0.2
	62.50	0.1	0.0	0.0	0.1
	125.00	0.1	-0.1	0.0	0.1
	250.00	0.1	0.1	0.1	0.0
	500.00	0.3	0.3	0.3	0.0
	1000.00	0.3	0.3	0.3	0.0
	2000.00	0.4	0.9	0.6	0.3

Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.14	1.01	1.00	1.05	0.08
	8.00	1.52	1.19	1.20	1.30	0.19
	16.00	1.82	1.69	1.33	1.61	0.26	*
	32.00	2.94	2.80	2.21	2.65	0.39	*
	64.00	5.54	7.93	3.94	5.80	2.01	*
Test Item	0.98	0.37	0.85	0.88	0.70	0.28
	1.95	0.59	0.95	0.92	0.82	0.20
	3.91	0.96	1.42	1.23	1.20	0.23
	7.81	1.06	1.25	1.11	1.14	0.10
	15.63	1.33	1.35	1.32	1.34	0.02
	31.25	0.02	0.02	0.02	0.02	0.00
	62.50	0.00	0.00	0.00	0.00	0.00
	125.00	0.00	0.00	0.00	0.00	0.00
	250.00	0.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

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

Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.01	0.93	1.10	1.01	0.09
	8.00	1.07	1.10	1.06	1.08	0.02
	16.00	1.31	1.33	1.51	1.38	0.11
	32.00	2.02	2.22	2.02	2.08	0.12	*
	64.00	3.52	3.78	3.71	3.67	0.13	*
Test Item	0.98	1.08	1.07	1.07	1.07	0.00
	1.95	1.24	1.38	1.35	1.33	0.07
	3.91	2.17	2.09	2.20	2.15	0.05	*
	7.81	2.67	2.67	2.79	2.71	0.07	*
	15.63	2.69	2.47	2.74	2.63	0.14	*
	31.25	0.07	0.09	0.08	0.08	0.01
	62.50	0.00	0.00	0.00	0.00	0.00
	125.00	0.00	0.00	0.00	0.00	0.00
	250.00	0.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

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

Induction of Luciferase Activity – 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	1.05	1.01	1.03	0.02
	8.00	1.30	1.08	1.19	0.16
	16.00	1.61	1.38	1.50	0.16
	32.00	2.65	2.08	2.36	0.40	*
	64.00	5.80	3.67	4.74	1.51
Test Item	0.98	0.70	1.07	0.89	0.26
	1.95	0.82	1.33	1.07	0.36
	3.91	1.20	2.15	1.68	0.67
	7.81	1.14	2.71	1.93	1.11
	15.63	1.34	2.63	1.98	0.92
	31.25	0.02	0.08	0.05	0.04
	62.50	0.00	0.00	0.00	0.00
	125.00	0.00	0.00	0.00	0.00
	250.00	0.00	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

  Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	n.a.	2.36	n.a.	n.a.
Imax	1.34	2.71	2.02	0.97
IC30[µM]	1.59	4.28	2.93	1.90
IC50[µM]	5.01	7.34	6.17	1.65

n.a.:not applicable

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	14.0	pass	9.0	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	3.0	pass	2.0	pass
EC1.5 PC	7 < x < 34 µM	13.11	pass	18.67	pass
Induction PC at 64 µM	2.00 < x < 8.00	5.80	pass	3.67	pass

Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.3	3.3	41
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.3	0.6	41
EC1.5 PC	7 < x < 34 µM	20.4	6.7	41
Induction PC at 64 µM	2.00 < x < 8.00	3.3	1.1	41

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
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.

Executive summary:

In the present study dodecyltrimethylammonium bromide was dissolved in DMSO. Based on a molecular weight of 308.34 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium and a stable suspension was formed. 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°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

Microscopically, cytotoxic effects were observed in the whole tested concentration range.

In the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range andno EC_1.5 value could be calculated.

In the second experiment, a max luciferase activity (I_max) induction of 2.71 was determined at a test item concentration of 7.81 µM, but the corresponding cell viability was 46.9%. The lowest tested concentration with a significant luciferase induction >1.5 (2.15) was found to be 3.91 µM. The corresponding cell viability was > 70% (72.4%).The calculated EC_1.5 was < 1000 µM (2.36 µM).
However, due to the observed cytotoxicity in the whole tested concentration range, it was assumed that the induction above the threshold was caused by cellular stress.

A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. However, this effect was below the threshold value of 1.5.

Under the condition of this study the test item is considered as non sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

For the evaluation of the skin sensitisation potential of the test substance a Weight of Evidence approach was used. An in silico assessment using Derek Nexus v5.0 was carried out. The protein reactivity of the test item was assessed in a Direct Peptide Reactivity Assay (DPRA) and a Keratinocyte Activation Assay (KeratinoSens) was performed to evaluate the keratinocyte activation potential.

In silico assessment (reference 7.4.1-1)

For in silico assessment, Derek Nexus v5.0 was used. Skin sensitising properties were regarded as equivocal based on the described QSAR method (Derek, 2018).

DPRA (reference 7.4.1-2)

In the present study dodecyltrimethylammonium bromide was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 308.34 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the co-elution control of the positive control. No precipitation, turbidity or phase separation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.

No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C _acetonitrile).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (0.05%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

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 67.89%.

The controls confirmed the validity of the study for both the cysteine and lysine run.

KeratinoSens (reference 7.4.1-3)

In the present study dodecyltrimethylammonium bromide was dissolved in DMSO. Based on a molecular weight of 308.34 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium and a stable suspension was formed. 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°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

Microscopically, cytotoxic effects were observed in the whole tested concentration range.

In the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range and no EC_1.5 value could be calculated.

In the second experiment, a max luciferase activity (I_max) induction of 2.71 was determined at a test item concentration of 7.81 µM, but the corresponding cell viability was 46.9%. The lowest tested concentration with a significant luciferase induction >1.5 (2.15) was found to be 3.91 µM. The corresponding cell viability was > 70% (72.4%).The calculated EC_1.5 was < 1000 µM (2.36 µM).
However, due to the observed cytotoxicity in the whole tested concentration range, it was assumed that the induction above the threshold was caused by cellular stress.

A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. However, this effect was below the threshold value of 1.5.

Under the condition of this study the test item is considered as non sensitiser.

Conclusion
Skin sensitising properties were regarded as equivocal based on the described QSAR method (Derek, 2018). Based on the results of an in chemico/in vitro test strategy the test item is not peptide reactive (DPRA,OECD TG 442C,reference 7.4.1-2) and does not activate keratinocytes (Keratinosens, OECD TG 442D, reference 7.4.1-1). Therefore, the substance is not predicted to be a skin sensitizer. The overall result is based on two concordant findings. Considering an AOP “2 out of 3” the test item is predicted as non-skin sensitizer and further tests are not necessary.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item is not classified as a skin sensitizer according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.