Acetylcysteine

Endpoint summary

Description of key information

Using Derek Nexus v5.0, the skin sensitising potential of the test item was estimated to be plausible. However, 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 (LuSens, OECD TG 422D, 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:

according to guideline

Guideline:

other: ECHA Guidance on IR/CSA R.6 QSARs and grouping of chemicals

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: O=C(NC(C(=O)O)CS)C

Key result

Parameter:

other: alert number 432

Value:

1

Remarks on result:

other: Skin sensitisation in mammal is PLAUSIBLE. Alert matched: 432 Thiol or thiol exchange agent

Remarks:

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

Interpretation of results:

other: Derek result: plausible

Conclusions:

Using Derek Nexus v5.0, the skin sensitising potential of the test item was estimated to be plausible. 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 plausible 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 2018-03-10

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:

February 04, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

January 12, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

direct peptide reactivity assay (DPRA)

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.

Controls:
Positive control (PC) : Cinnamic aldehyde in acetonitrile (100 mM stock solution)
Sigma Aldrich; Cat. No.: W228613; ≥95%; CAS No.: 104-55-2; Lot. No.: MKBX1392V

Co-elution control: set up in parallel to sample preparation but without respective peptide solution
Reference controls (RCs): set up in parallel to sample preparation in order to verify the validity of the test run. Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run. Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run. Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.
HPLC System: see "Any other information on materials"

Test System
Peptides with > 95 % purity, synthesized by JPT Peptide Technologies GmbH, were used.
Cys-Peptide (Cysteine): Lot. No.: 111016HS_MHeW1017
Lys-Peptide (Lysine): Lot. No.: 120514HSDW_W1117

20.15 mg (experiment 1) and 19.65 mg (experiment 2) cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (39.18 mL: experiment 1; 38.10 mL: experiment 2) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
19.32 mg (experiment 1) and 20.42 mg (experiment 2) lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (36.69 mL: experiment 1; 38.78 mL: experiment 2) to reach a concentration of 0.667 mM

Solubility of test item
The test item was completely soluble in acetonitrile, therefore, acetonitrile was chosen as suitable vehicle for the main experiments.

Incubation of the Test Item with the Cysteine and Lysine Peptide
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 - 400xg) to force precipitates to the bottom of the vial. After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using the following HPLC procedure.

Preparation of the HPLC Standard Calibration Curve
A standard calibration curve was generated for both, the cysteine and the lysine peptide.
Peptide standards were prepared in a solution of 20% acetonitrile: 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM)
using the respective DB was performed, resulting in 7 calibration solutions covering the range 0.534 / 0.267 / 0.134 / 0.067 / 0.033 / 0.017/ 0.00 mM peptide.

HPLC Preparation and Analysis
Peptide depletion was monitored by HPLC coupled with an UV detector at A = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30°C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected.

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% (experiment 1) and 69.46% (experiment 2). No co-elution of the test item with any of the peptide peaks was observed.

Key result

Run / experiment:

other: 1st experiment

Parameter:

other: mean peptide depletion of both peptides [%]

Value:

6.28

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2st experiment

Parameter:

other: peptide depletion of both peptides (%)

Value:

2.16

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS: All acceptance criteria are fulfiled
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Depletion of the Cysteine Peptide (experiment 1)

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	4922.7578	0.5198	0.00	0.00	0.00	-
	4953.7280	0.5231	0.00
	4937.0298	0.5214	0.00

Table 2: Depletion of the Cysteine Peptide (experiment 2)

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	3.7100	0.1138	77.40	77.79	0.34	0.44
	3.6200	0.1110	77.95
	3.6080	0.1106	78.02
Test Item	17.3900	0.5340	0.00	0.00	0.00	-
	16.7550	0.5145	0.00
	16.7130	0.5132	0.00

Table 3: Depletion of the Lysine Peptide (experiment 1)

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	3723.8833	0.4440	11.23	12.57	1.20	9.52
	3652.0933	0.4354	12.94
	3627.1846	0.4324	13.54

Table 4: Depletion of the Lysine Peptide (experiment 2)

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	1548.8514	0.1877	62.22	61.12	1.28	2.09
	1581.2108	0.1916	61.43
	1651.4639	0.2002	59.72
Test Item	3939.2124	0.4789	3.91	4.32	0.76	17.62
	3941.6335	0.4792	3.85
	3886.3860	0.4725	5.20

Table 5: Categorization of the Test Item (experiment 1)

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	6.28	Minimal Reactivity	no sensitiser	0.00	Minimal Reactivity	no sensitiser
Positive Control	67.89	High Reactivity	sensitiser	72.27	Moderate Reactivity	sensitiser

Table 6: Categorization of the Test Item (experiment 2)

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	2.16	Minimal Reactivity	no sensitiser	0.00	Minimal Reactivity	no sensitiser
Positive Control	69.46	High Reactivity	sensitiser	77.79	Moderate Reactivity	sensitiser

Interpretation of results:

other: no peptide binding

Conclusions:

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test item shows minimal reactivity in the DPRA under the test conditions chosen.

Executive summary:

A study was conducted according to OECD TG 442C in order to evaluate the reactivity of the test item N-Acetyl-L-cysteine towards cysteine (Cys-) and lysine (Lys-) containing peptides. The test substance was dissolved in acetonitrile and a stock solution of 100 mM 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. All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide or lysine peptide solution in both independent experiments. 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 in both independent experiments.

Phase separation was observed for the co-elution of the positive control in both independent experiments and for the positive control in the second experiment.

Since the acceptance for the depletion range of the positive control were fulfilled, the observed precipitations, turbidity or phase separation were regarded as insignificant.

Experiment 1:

No co-elution of test item with the peptide peaks was observed. 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% (6.28%). Based on the prediction model 1 the test item can be considered as non-sensitiser. According to the evaluation criteria in the guideline, for test items with a combined cysteine/lysine peptide depletion between 3% and 10% a second run should be considered for both peptides. Therefore, no prediction can be made.

Experiment 2:

No co-elution of test item with the peptide peaks was observed. 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% (2.16%).

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% (experiment 1) and 69.46% (experiment 2).

In this study under the given conditions the test item showed minimal reactivity towards both peptides.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-03-15 to 2018-03-29

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:

February 04, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155

Version / remarks:

July 1st, 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:

Skin sensitisation (In vitro test system) - Details on 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.

Cell line
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 were used. Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2. For test item exposure, cells were cultured in medium.

Test Item Exposure Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with 1% fetal bovine calf serum

Dose Groups
1.  Negative Control:               1% (v/v) dimethyl sulfoxide in test item exposure medium
2.  Positive Control:                 cinnamic aldehyde: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
3.  Test Item:                           12 concentrations of the test item

Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run.

Preparation of the Test Item
All test item solutions were freshly prepared immediately prior to use.
The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. A stable suspension was formed when diluted 1:100 in cell culture medium. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent.These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls. 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

Experimental Procedure
A cell suspension of 8 × 10E4 cells/mL in assay medium was prepared. 125 μL of the cell suspension corresponding to 1 × 10E4 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability. After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 μL test item exposure medium. 50 μL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS (Gibco Life Science). Subsequently 20 μL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light. Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 μL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.

Cell viability
For the cell viability plate the medium was replaced with 200 μL test item exposure medium. 27 μL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 μL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1) and over the weekend (experiment 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

Data Analysis
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consists of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run should be performed.
For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p <0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells. The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC1.5 value.

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: Experiment I

Parameter:

other: fold induction of luciferase activity

Value:

0.97

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment II

Parameter:

other: fold induction of luciferase activity

Value:

1.12

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: 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	98.5	103.7	101.1	3.7
	1.95	108.0	104.6	106.3	2.4
	3.91	103.0	101.3	102.1	1.2
	7.81	97.4	96.9	97.2	0.3
	15.63	103.5	97.9	100.7	3.9
	31.25	99.6	98.6	99.1	0.7
	62.50	107.8	95.7	101.8	8.6
	125.00	100.6	91.4	96.0	6.5
	250.00	92.3	89.2	90.7	2.2
	500.00	84.9	83.6	84.2	1.0
	1000.00	79.0	84.4	81.7	3.8
	2000.00	81.7	82.2	82.0	0.4

Table 2: 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	0.38	1.08	1.00	0.82	0.38
	1.95	0.76	0.87	0.98	0.87	0.11
	3.91	1.05	1.00	0.86	0.97	0.10
	7.81	0.87	0.86	0.88	0.87	0.01
	15.63	1.11	0.87	0.82	0.93	0.15
	31.25	0.94	0.79	0.79	0.84	0.08
	62.50	0.96	0.74	0.72	0.80	0.13
	125.00	0.92	0.64	0.61	0.73	0.17
	250.00	0.89	0.67	0.54	0.70	0.18
	500.00	0.68	0.46	0.45	0.53	0.13
	1000.00	0.60	0.37	0.48	0.48	0.11
	2000.00	0.51	0.29	0.37	0.39	0.12

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

Table 3: 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	1.15	1.15	1.05	1.12	0.05
	1.95	0.97	0.93	0.91	0.94	0.03
	3.91	0.94	1.22	1.04	1.07	0.15
	7.81	0.89	1.05	0.97	0.97	0.08
	15.63	0.86	0.80	0.74	0.80	0.06
	31.25	0.85	0.83	0.73	0.80	0.06
	62.50	0.79	0.82	0.85	0.82	0.03
	125.00	0.82	0.87	0.72	0.80	0.08
	250.00	0.63	0.72	0.64	0.66	0.05
	500.00	0.60	0.62	0.66	0.63	0.03
	1000.00	0.57	0.62	0.71	0.63	0.07
	2000.00	0.61	0.63	0.64	0.63	0.01

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

Table 4: 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	0.82	1.12	0.97	0.21
	1.95	0.87	0.94	0.90	0.05
	3.91	0.97	1.07	1.02	0.07
	7.81	0.87	0.97	0.92	0.07
	15.63	0.93	0.80	0.87	0.09
	31.25	0.84	0.80	0.82	0.03
	62.50	0.80	0.82	0.81	0.01
	125.00	0.73	0.80	0.76	0.05
	250.00	0.70	0.66	0.68	0.03
	500.00	0.53	0.63	0.58	0.07
	1000.00	0.48	0.63	0.56	0.11
	2000.00	0.39	0.63	0.51	0.17

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

Table 5: Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	n.a.	n.a.	n.a.	n.a.
Imax	0.97	1.12	1.04	0.10
IC30[µM]	n.a.	n.a.	n.a.	n.a.
IC50[µM]	n.a.	n.a.	n.a.	n.a.

n.a.: not applicable

Table 6: Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment2	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

Table 7: 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

Interpretation of results:

other: no activation of keratinocytes

Remarks:

The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as IATA.

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.

Executive summary:

In an in vitro skin sensitisation assay according to 442D, the potential of the test item to activate the Nrf2 transcription factor, by using the LuSens cell line was investigated.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 assay was performed in two independent experiments. 12 concentrations of the test item were evaluated. 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.

In the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range and no EC1.5 value could be calculated. In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range and no EC1.5 value could be calculated. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. The controls confirmed the validity of the study.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not have the potential to activate the Nrf2 transcription factor.

 

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.

Using Derek Nexus v5.0, the skin sensitising potential of the test item was estimated to be plausible. The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate (reference 7.4.1 -1).

A study was conducted according to OECD TG 442C in order to evaluate the reactivity of the test item N-Acetyl-L-cysteine towards cysteine (Cys-) and lysine (Lys-) containing peptides. The test substance was dissolved in acetonitrile and a stock solution of 100 mM 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. All test item solutions were freshly prepared immediately prior to use. For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide or lysine peptide solution in both independent experiments. 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 in both independent experiments. Phase separation was observed for the co-elution of the positive control in both independent experiments and for the positive control in the second experiment. Since the acceptance for the depletion range of the positive control were fulfilled, the observed precipitations, turbidity or phase separation were regarded as insignificant.

In Experiment 1 no co-elution of test item with the peptide peaks was observed. 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% (6.28%). Based on the prediction model 1 the test item can be considered as non-sensitiser. According to the evaluation criteria in the guideline, for test items with a combined cysteine/lysine peptide depletion between 3% and 10% a second run should be considered for both peptides. Therefore, no prediction can be made. In Experiment 2 no co-elution of test item with the peptide peaks was observed. 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% (2.16%). 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% (experiment 1) and 69.46% (experiment 2). In this study under the given conditions the test item showed minimal reactivity towards both peptides (reference 7.4.1 -2).

Furthermore, in an in vitro skin sensitisation assay according to 442D, the potential of the test item to activate the Nrf2 transcription factor by using the LuSens cell line was investigated. 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 assay was performed in two independent experiments. 12 concentrations of the test item were evaluated. 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. In the first experiment, no significant luciferase induction (>1.5) was found in the tested concentration range and no EC1.5 value could be calculated. In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range and no EC1.5 value could be calculated. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. The controls confirmed the validity of the study. In conclusion, it can be stated that under the experimental conditions reported, the test item did not have the potential to activate the Nrf2 transcription factor (reference 7.4.2 -3).

Conclusion

Using Derek Nexus v5.0, the skin sensitising potential of the test item was estimated to be plausible (reference 7.4.1 -1). However, 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 (LuSens, OECD TG 422D, reference 7.4.1 -3). Therefore, the substance is not predicted to be a skin sensitizer.

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 skin sensitising according to Regulation (EC) No 1272/2008 (CLP), as amended for the 11th time in Regulation (EU) No 2018/669.