3-methyl-2-butenyl salicylate

Endpoint summary

Description of key information

Skin sensitization (OECD 442D): not sensitizing

Skin sensitization (OECD 442C): not sensitizing

Skin sensitization (OECD 442E): sensitizing

Skin sensitization (Open epicutaneous test, 1985): not sensitizing

Skin sensitization (Open epicutaneous test, 1970): not sensitizing

Based on the available in vitro and in vivo tests (DPRA, KeratinoSens, h-CLAT, 2 Open epicutaneous tests) for the read-across substance, the target substance is not considered to be a skin sensitizer.

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:

January - April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 442E h-CLAT

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

TEST SYSTEM
- Cell line: THP-1 cells; human monocytic leukemia cell line
- Source: American Type Culture Collection Manassas, USA (ATCC, TIB-202)

TEST-SUBSTANCE PREPARATION
- Concentrations: Dose range finder: 7.81, 15.63, 31.25, 62.50, 125, 250, 500, 1000 µg/mL; Main experiment: 97.71, 81.42, 67.85, 56.54, 47.12, 39.27, 32.72, 27.27 µg/mL
- Stock: 2x concentration of the highest concentration stock solution
- Solvent: DMSO

CONTROLS
- Negative control (NC):Medium
- Solvent control (SC): 0.2% DMSO
- Isotype control: In order to help distinguish non-specific staining from specific antibody staining each test-substance concentration and control is additionally incubated with mouse IgG1
- Positive control: 1-chloro-2-4-dinitrobenzene (DNCB); 4.0 µg/mL

MEDIUM
- Culture medium: RPMI 1640: with L-glutamine, 25 mM HEPES (Gibco) + 10% FBS inactivated + 1% Penicillin/Streptomycin + 0.05 mM 2-Mercaptoethanol (Gibco)
- FACS Buffer: Phosphate Buffered Saline (DPBS) without Ca2+ / Mg2+ (Gibco) + 0.1% BSA (Sigma A7030)
- Blocking Solution: 0.01% Globulins Cohn fraction II,III with DPBS
- Reagent for cytotoxicity test: Propidium iodide (Sigma)

EXPERIMENTAL PROCEDURE
- Replicates: 1
- Experiments: 3
- Exposure period: 24 hours
- Preparation of cells: THP-1 cells were thawed and cultured in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 0.05 mM 2 -mercaptoethanol (30 > passage >= 5)

ANALYSIS
- FACS: cell staining and flow cytometric analysis 24 hours after exposure

DATA EVALUATION
- CV75 calculation: Relative survival rate is calculated by linear extrapolation. This value is the substance concentration at which cell viability is 75% compared to the vehicle control.
- Relative cell viability: % relative cell viability = (number of living cells / total number of aquired cells) * 100
- Relative fluorescence intensity: RFI (%) = (MFI of chemical-treated cells - MFI of chemical-treated isotype control cells) / (MFI of solvent control cells - MFI of solvent isotype control cells) * 100
- Calculation of EC 150% and EC 200%: If applicable, the concentration resulting in a positive response (RFI of 150% (CD86) or 200% (CD54) and viability >50%) was calculated for each cell surface marker from each experiment. The calculation is performed by linear regression from the two concentrations directly above and below the EC 150% / EC 200% concentration.

ACCEPTANCE CRITERIA
- A tested concentration is not to be further evaluated when viability is less than 50%
- Cell viability of vehicle control cells must yield at least 90%
- In the positive control (DNCB), RFI (relative fluorescence intensity) values of both CD86 and CD54 should be exceed the positive criteria (RFI CD86 =150% and CD54 =200%) and cell viability should be =50%.
- For all vehicle control, the MFI (mean fluoresence intensity) ratio of both CD86 and CD54 to isotype controls should be =105%.
- A study is considered to be acceptable if the positive, negative and vehicle control data lies within the range of the historic data.

EVALUATION RESULTS
- Positive result: A test is considered to be positive when the dendritic cells are activated meaning that CD86 expression is increased =150% and/or CD54 expression increased =200% in relation to vehicle control in at least 2 independent experiments.
- Negative result: A test is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (=5000 µg/mL for the vehicle culture medium or 2000 µg/mL for 0.2% DMSO in culture medium).

Positive control results:

Positive controls achieved RFI values of both CD86 and CD54 over the positive criteria (CD86= 264% experiment 1; 336% experiment 2; 304% experiment 3) and 200% for CD54 (231% experiment 1; 288% experiment 2; 257% experiment 3).

Key result

Run / experiment:

other: Please see 'Any other information on results incl. tables'.

Parameter:

other: RFI CD54 mean (%) and RFI CD54 mean (%)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	94.7	94.7	94.3	2943	1205	613	2330	592	101	111	480	197
Solvent Control	0.20%	94.8	95.2	94.8	2988	1214	679	2309	535	100	100	440	179
DNCB	4.00	80.9	80.5	80.0	6868	2013	779	6089	1234	264	231	882	258
ISOBUTYL SALICYLATE	97.71	12.4	11.9	12.3	4427	3822	942	3485	2880	151	538	470	406
	81.43	57.3	58.4	57.4	3480	1781	726	2754	1055	119	197	479	245
	67.85	83.9	83.4	84.1	3116	1540	673	2443	867	106	162	463	229
	56.55	88.9	89.3	89.5	3066	1380	658	2408	722	104	135	466	210
	47.12	93.2	93.5	93.6	2778	1235	669	2109	566	91	106	415	185
	39.27	94.5	94.3	93.9	2801	1230	686	2115	544	92	102	408	179
	32.72	94.4	94.8	94.9	2996	1287	660	2336	627	101	117	454	195
	27.27	94.7	94.6	94.7	3063	1276	669	2394	607	104	113	458	191

CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	94.7	94.8	93.9	4619	1554	646	3973	908	102	95	715	241
Solvent Control	0.20%	95.0	95.1	94.9	4571	1620	669	3902	951	100	100	683	242
DNCB	4.0	81.4	80.5	81.2	13735	3370	633	13102	2737	336	288	2170	532
ISOBUTYL SALICYLATE	97.71	13.2	12.8	12.4	7467	5420	935	6532	4485	167	472	799	580
	81.43	45.9	45.5	45.7	6028	3615	1213	4815	2402	123	253	497	298
	67.85	79.3	79.0	79.7	5291	3011	782	4509	2229	116	234	677	385
	56.55	90.0	90.8	89.7	4710	2761	874	3836	1887	98	198	539	316
	47.12	93.5	93.5	92.7	4543	2413	735	3808	1678	98	176	618	328
	39.27	93.4	92.5	92.8	4587	2304	746	3841	1558	98	164	615	309
	32.72	94.6	94.1	94.2	4335	2049	756	3579	1293	92	136	573	271
	27.27	95.1	95.0	94.9	4489	2013	790	3699	1223	95	129	568	255

CD54 and CD86 Expression Experiment 3

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	95.2	94.5	94.3	3732	1289	640	3092	649	88	87	583	201
Solvent Control	0.20%	94.7	94.5	94.5	4176	1395	649	3527	746	100	100	643	215
DNCB	4.0	81.9	81.1	82.4	11303	2502	583	10720	1919	304	257	1939	429
ISOBUTYL SALICYLATE	97.71	40.5	38.6	39.3	4938	2769	816	4122	1953	117	262	605	339
	81.43	64.4	64.9	64.8	4625	2417	806	3819	1611	108	216	574	300
	67.85	86.4	85.4	86.3	4465	2304	739	3726	1565	106	210	604	312
	56.55	93.0	91.8	93.0	4049	1925	742	3307	1183	94	159	546	259
	47.12	94.0	93.8	94.4	3792	1728	740	3052	988	87	132	512	234
	39.27	94.5	94.1	93.9	3707	1636	730	2977	906	84	121	508	224
	32.72	95.0	94.9	95.2	3538	1580	735	2803	845	79	113	481	215
	27.27	95.2	95.6	95.0	4119	1701	811	3308	890	94	119	508	210

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Based on the results, it is concluded that the test substance induces dendritic cell activation (CD54 expression increased =200% in at least one assay).

Executive summary:

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human pro-monocytic cell line THP-1 for ca. 24 hours at 37°C and membrane markers expression measured by flow cytometry. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 10 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

 

In the main test after 24 hour exposure THP-1 cells were stained with FITC labelled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analysed using flow cytometry. A total of 3 valid experiments were performed. The expression of the cell surface marker CD54 clearly exceeded the threshold at one concentration with a cell viability ≥50% in at least two independent runs.

The positive control (DNCB) led to an upregulation of CD54 and CD86 in all three experiments. The threshold of 150% for CD86 (264% experiment 1; 336% experiment 2; 304% experiment 3) and 200% for CD54 (231% experiment 1; 288% experiment 2; 257% experiment 3) were clearly exceeded.

 

In summary, it has to be concluded that the test substance induces dendritic cell activation.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01.06.2017-08.02.2018

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Rathausgasse 4, 91126 Schwabach, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

TEST-SUBSTANCE PREPARATION
- Stock solution: 100 mM
- Vehicle: Acetonitrile main experiment 1 (exp. 1) and methanol main experiment 2 (exp. 2).
- Reason for the vehicle: The test item was not soluble in dist. water and dist. water : acetonitrile 1:1 (v/v).

CONTROLS
- Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
- Co-elution control: buffer and test substance without the peptide
- Positive control: Cinnamic aldehyde in acetonitrile

PEPTIDES
- Synthetic peptides:
-- Cysteine- (C-) containing peptide: 22.61 mg (exp. 1) and 20.15 mg (exp. 2); amino acid sequence: Ac-RFAACAA; dissolved in a defined volume (43.9 mL exp. 1 / 39.18 mL exp. 2) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM
-- Lysine- (K-) containing peptide: 20.94 mg (exp. 1) and 19.32 mg (exp. 2); amino acid sequence: Ac RFAAKAA; dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (36.69 mL exp. 1 / 39.74 mL exp. 2) to reach a concentration of 0.667 mM

EXPERIMENTAL PROCEDURE
- Replicates: 3 for each peptide
- Determination remaining non-depleted peptide concentration: HPLC at 220nm: HPLC analysis started 22 to 26 hours after sample preparation and the analysis time was less than 30 hours.
- Calibration samples: samples of a known peptide concentration are measured in parallel

PREPARATIONS SAMPLES
- Calibration sample was prepared from the peptide stock solution in 20% acetonitrile in the respective buffer using serial concentration: 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 or 0.000 mM peptide
- Test-substance samples: samples were incubated at 25°C +/- 2.5°C in the dark for 24 +/- 2 hours
- Reference controls, co-elution controls as well as the positive control were set up in parallel (see table below)

MEASUREMENT PEPTIDE CONCENTRATIONS
- Method: HPLC Agilent 1200 series
- Wavelength: 220 nm and 258 nm
- Detector: UV detector

DATA EVALUATION
- Concentration of the cysteine and lysine peptide determined from absorbance at λ = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves.
- The percent peptide depletion (PPD) was calculated according to the following formula: PPD = [ 1 – ( Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in the Reference Control C)] * 100

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

EVALUATION RESULTS
Cysteine 1:10/ Lysine 1:50 Prediction Model:
- Chemical reactivity was determined by mean peptide depletion [%] and was rated as
-- high: mean peptide depletion > 42.47
-- moderate: mean peptide depletion > 22.62 ≤ 42.47
-- low: mean peptide depletion > 6.38 ≤ 22.62
-- minimal: mean peptide depletion ≤ 6.38
High, moderate and low reactivity are evaluated as positive.
In case of co-elution of the test item with a peptide peak, the peak cannot be integrated correctly and the calculation of the PPD is not possible.
If severe co-elution occurs with both peptides then the analysis was reported as "inconclusive".

- In cases where the co-elution occurs only with the lysine peptide prediction model 2 can be applied (cysteine 1:10 prediction model) :
-- high: mean peptide depletion > 98.24
-- moderate: mean peptide depletion > 23.09 ≤ 98.24
-- low: mean peptide depletion > 13.89 ≤ 23.09
-- minimal: mean peptide depletion ≤ 13.89
High, moderate and low reactivity are evaluated as positive.

DEVIATIONS FROM THE STUDY PLAN
change of supplier for some reagents: the deviation did not influence the quality or integrity of the present study.

Positive control results:

Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control (Positive control values for cystein: 72.27 %, SD 0.27 %; positive control values for lysine: 63.51 %, SD 0.86 %) were fulfilled, the observed precipitations and phase separation were regarded as insignificant.

Run / experiment:

other: Experiment 1, both peptides

Parameter:

other: mean depletion % of peptide

Value:

0.32

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

no prediction can be made (tested in concentration < 100 mM)

Key result

Run / experiment:

other: Experiment 2, cysteine peptide

Parameter:

other: mean depletion % of peptide

Value:

0.3

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Based on the prediction model 2

Run / experiment:

other: Experiment 2, lysine peptide

Parameter:

other: mean depletion % of peptide

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Phase separation

Other effects / acceptance of results:

Exp. 1: Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant.

Exp. 2: Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.

Results of the Cysteine Peptide Depletion

Depletion of the Cysteine Peptide (Experiment1)

Sample	Peptide concentration (mM)	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV (%)
Positive control	0.1438	71.3	71.38	0.25	0.35
	0.1444	71.18
	0.1420	71.66
Test item	0.5005	0.30	0.64	0.32	50.31
	0.4986	0.68
	0.4976	0.93

Depletion of the Cysteine Peptide (Experiment2)

Sample	Peptide concentration (mM)	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV (%)
Positive control	0.1446	72.07	72.27	0.27	0.38
	0.1441	72.17
	0.1419	72.58
Test item	0.5155	0.00	0.30	0.52	173.21

Results of the Lysine Peptide Depletion

Depletion of the Lysine Peptide (Experiment1)

Sample	Peptide concentration (mM)	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV (%)
Positive control	0.2081	58.68	57.65	1.11	1.93
	0.2125	57.80
	0.2193	56.47
Test item	0.5085	0.00	0.00	0.00	#DIV/0 !
	0.5072	0.00
	0.5074	0.00

Depletion of the Cysteine Peptide (Experiment2)

Sample	Peptide concentration (mM)	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV (%)
Positive control	0.1777	64.50	68.51	0.86	1.35
	0.1848	63.08
	0.1854	62.95
Test item	0.4932	3.47	3.87	0.39	10.08
	0.4911	3.89
	0.4893	4.25

Interpretation of results:

GHS criteria not met

Conclusions:

In the first experiment using acetonitrile as solvent under the given conditions, the test item showed minimal reactivity towards the peptides. Due to the observed precipitation in both experiments and phase separations in the lysine run, the prediction model does not apply and a prediction cannot be made.

In the second experiment using methanol as solvent under the given conditions, the test item showed minimal reactivity towards the cysteine peptide. The test item can be considered as “non-sensitiser”.

Executive summary:

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. The test was performed according to OECD TG 442C and in compliance to GLP.

Experiment 1

In the present study the test item was dissolved in acetonitrile based on the results of the pre-experiments. Based on a molecular weight of 194.23 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 stock 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. A slight turbidity was observed for the standard 1 (0.534 mM). Precipitation at the bottom was noted in the samples of the positive control (excluding the co-elution control) as well as for the samples and co-elution control of the test item. Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item, turbidity 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 samples of the positive controls. However, the turbity noted for the test item sample 24 h before was gone. Instead phase separation was observed for the samples of the test item and their co-elution control. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant.

The stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was < 6.38% (0.32%). However, due to the observed precipitation in both experiments and phase separations in the lysine run prior to the HPLC analysis, a test item concentration of 100 mM as well as the full contact of peptide and test item is not guaranteed. According to the evaluation criteria in the guideline, no firm conclusion on the lack of reactivity should be drawn from a negative result, if a test chemical is tested in concentration < 100 mM.

Therefore, no prediction can be made.

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 64.52%.

Experiment 2:

In the present study the test item was dissolved in methanol based on the results of the pre-experiments. Based on a molecular weight of 194.23 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 stock solution of the test item turbidity 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 stock 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 samples of the test item (including co-elution control) and the co-elution of the positive control. 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.

Phase separation in the lysine experiment was observed. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation.

Sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cmethanol). The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was ≤ 13.89% (0.30%). Based on the prediction model 2 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%.

Conclusions:

In the first experiment using acetonitrile as solvent under the given conditions, the test item showed minimal reactivity towards the peptides. Due to the observed precipitation in both experiments and phase separations in the lysine run, the prediction model does not apply and a prediction cannot be made.

In the second experiment using methanol as solvent under the given conditions, the test item showed minimal reactivity towards the cysteine peptide in the second experiment. The test item can be considered as “non-sensitiser”.

The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01.06.-22.09.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 04, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Rathausgasse 4, 91126 Schwabach, Germany

Type of study:

activation of keratinocytes

Details on the study design:

Vehicle:
dimethyl sulfoxide (DMSO)

Concentrations:
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 pM

Controls:
- negative control: DMSO: 1%
- positive control: Cinnamic aldehyde 4 pM, 8 pM, 16 pM; 32 pM; 64 pM

Cell line:
- transgenic cell line KeratinoSensTM (Givaudan, Switzerland), derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct

Replicates:
- three replicates in every independent run (test item and positive control)
- The negative control was assessed using six replicates per plate in every independent run.

Experimental Procedure:
- After seeding cells were grown for 24 h ± 1 h at 37 °C ± 1 °C and 5% CO2
- Cells were incubated with the test item for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.
- Luciferase activity:
-- Cells were washed once with DPBS, ubsequently 20 pL 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
-- luminescence measurement. in the plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms
- Cell viability:
-- incubated for 4 h at 37 °C ± 1 °C and 5% CO2 with MTT solution
-- after removal of the MTT incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1 and 2)
-- OD was measured at 600 nm.

Prediction Model:
- The test item is considered positive in accordance with UN GHS "Category 1" if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 pM
- an apparent overall dose-response for luciferase induction

If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required.
In addition, a negative result obtained with concentrations <1000 pM is considered as inconclusive.

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 pM 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.

Deviations from the Study Plan:
There were no deviations from the study plan.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 pM was between 2 and 8 (3.39 in experiment 1; 2.27 in experiment 2).

Run / experiment:

other: Exp. 1

Parameter:

other: max luciferase activity (Imax) induction

Value:

1.12

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Exp. 2

Parameter:

other: max luciferase activity (Imax) induction

Value:

1.13

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The controls confirmed the validity of the study. The calculated EC1.5 was between 7 and 34 µM (24.15 µM in experiment 1; 21.59 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (14 % in experiment 1; 6.4% in experiment 2). The luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment the luciferase activity was 3.39 and thus the criteria was fulfilled. In the second experiment the activity was 2.27. Thus the acceptance criteria were met.

Induction of Luciferase Activity - Overall Induction

	Concentration (microM)	Fold induction
		Experiment 1	Experiment 2	Mean	SD
Solvent Control		1.00	1.00	1.00	0.00
Positive Control	4.0	1.09	1.09	1.09	0.00
	8.0	1.12	1.24	1.18	0.09
	16.0	1.28	1.33	1.31	0.04
	32.0	1.71	1.81	1.76*	0.07
	64.0	3.39	2.27	2.83	0.08
Test Item	0.98	1.07	1.13	1.10	0.04
	1.95	1.11	1.10	1.10	0.01
	3.91	1.07	1.04	1.05	0.02
	7.81	1.00	1.09	1.05	0.07
	15.63	0.98	1.03	1.01	0.04
	31.25	1.03	1.08	1.05	0.03
	62.50	1.12	1.00	1.06	0.09
	125.00	0.00	1.05	0.53	0.74
	250.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.01	0.01	0.01
	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

Interpretation of results:

GHS criteria not met

Conclusions:

In this study according to OECD 442D under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSensTM cell line in at least two independant 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.

Executive summary:

The in vitro KeratinoSens^TM assay according to OECD 442D and GLP 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^TM. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and

non-sensitisers.

In the present study the test item was dissolved in DMSO. Based on a molecular weight of 194.23 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. 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 pM.

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 1.12 was determined at a test item concentration of 62.50 pM. The corresponding cell viability was 67.7%. No significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. Cytotoxicity was observed in the concentration range 62.50 pM — 2000 pM.

In the second experiment, a max luciferase activity (Imax) induction of 1.13 was determined at a test item concentration of 0.98 pM. The corresponding cell viability was 95.1%. No significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. Cytotoxicity was observed in the concentration range 125 pM — 2000 pM.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation in vitro (key studies)

In vitro studies covering the three key steps of the adverse outcome pathway for skin sensitization as identified by the OECD (OECD Publication No.168; ENV/JM/MONO(2012)10) were performed. These study types have initially undergone in-house validation in the test facility using 54 substances (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504). Recently, a validation performed with 213 substances has been published (Urbisch et al. 2015 Regul Toxicol Pharmacol. 71: 337-351).

 

Based on the results of the in-house validation (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) the predictive capacity of the evaluation scheme using DPRA (peptide reactivity), LuSens/KeratinoSens™ (keratinocyte activation) and (m)MUSST/h-CLAT (dendritic cell activation) is comparable to that of the local lymph node assay. This was confirmed in the validation study with more substances (Urbisch 2015): When compared to the sensitization potential of the substances in humans, the classification based on an evaluation scheme using results obtained from the DPRA, LuSens and mMUSST had a sensitivity of 90%, a specificity of 90 % and an accuracy of 90 %.

 

In the evaluation scheme used here any two of the three test results determine the overall classification, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauchet al. 2012; Table 1). If two assays (DPRA ,LuSens or KeratinoSens, MUSST or h-CLAT) yield concordant results, the result of the third assay is not necessarily required to determine the overall outcome of the evaluation scheme.

 

Table 1: Decision matrix for combinations of DPRA, LuSens/KeratinoSens and MUSST/ h-CLAT assays.

DPRA	LuSens/ KeratinoSens^TM	MUSST/ h-CLAT	Test battery evaluation
positive	positive	positive	sensitizer
positive	positive	negative	sensitizer
positive	negative	positive	sensitizer
positive	negative	negative	non-sensitizer
negative	positive	positive	sensitizer
negative	positive	negative	non-sensitizer
negative	negative	positive	non-sensitizer
negative	negative	negative	non-sensitizer

Each individual assay was performed under GLP-conditions. All three assays follow the procedures of the respective OECD testing guidelines (OECD 442C, D and E).

 

The test battery applicability is limited when testing substances insoluble in the commonly used vehicles and highly volatile substances. Substances susceptible to base-catalyzed hydrolysis cannot be evaluated reliably for binding to lysine as the incubation is performed at pH 10.2. Also substances that interfere with the experimental measurements (e.g., co-elution with the peptide in the DPRA-assay) are not or only partially suitable for testing using in vitro methods.

In accordance with the published evaluation scheme (Bauchet al., 2012 Regul Toxicol Pharmacol. 63: 489-504) and Sections 1.2 and 1.4 of Annex XI of EC regulation 1907/2006, the test substance is judged not to be a skin sensitizer.

Details on the assessed tests:

OECD 442C:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of the test item. All analytical acceptance criteria for each peptide run were met. Solutions of the test item were successfully analysed by the DPRA analytical method in both Cysteine and Lysine containing synthetic peptides.

In the first experiment using acetonitrile as solventunder the given conditions, the test item showed minimal reactivity towards the peptides. Due to the observed precipitation in both experiments and phase separations in the lysine run,the prediction model does not apply and a prediction cannot be made.In the second experiment using methanol as solvent under the given conditions, the test item showed minimal reactivity (maxium depletion of 0.30%) towards the cysteine peptide in the second experiment. The test item can be considered as “non-sensitiser”.The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

OECD 442D:

The in vitro KeratinoSens™ assay according to OECD 442D 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. In the present study the test substance was dissolved in DMSO. Based on a molecular weight of 194.23 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. 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. Therefore, no EC1.5 value could be calculated. In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, 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. Under the condition of this study the test item is therefore considered as non sensitiser. The controls confirmed the validity of the study. The calculated EC1.5 was between 7 and 34 µM (24.15 µM in experiment 1; 21.59 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (14 % in experiment 1; 6.4% in experiment 2). The luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment the luciferase activity was 3.39, in the second experiment 2.27 and thus the criteria was fulfilled.

OECD 442E

In the third key study the potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human pro-monocytic cell line THP-1 for ca. 24 hours at 37°C and membrane markers expression measured by flow cytometry. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 10 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

In the main test after 24 hour exposure THP-1 cells were stained with FITC labelled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analysed using flow cytometry. A total of 3 valid experiments were performed. The expression of the cell surface marker CD54 clearly exceeded the threshold at oneconcentration with acellviability ≥50%inatleasttwoindependentruns.

The positive control (DNCB) led to an upregulation of CD54 and CD86 in all three experiments. The threshold of 150% for CD86 (264% experiment 1; 336% experiment 2; 304% experiment 3) and 200% for CD54 (231% experiment 1; 288% experiment 2; 257% experiment 3) were clearly exceeded.

Skin sensitisation in vivo (supporting studies)

In the Open Epicutaneous Test (OET, 1985) the skin sensitizing effect of the test item was evaluated.

The study was not according to GLP or OECD, however, it is similar to OECD 406. Various test concentrations were used and per group at least 6 guinea pigs were included.

The maximum applied doses for the induction phase and the challenge are unknown. The test item was considered not to be a sensitiser as no reactions occured.

In the second study (1970) study, the sensitisation propreties of the test item has been determined probing white male guinea pigs. The test item was first dissolved in alcohol and then suspended in distilled water so that a 0.1% suspension was in 5% ethyl alcohol in distilled water.

All injection were intradermal. The first and the Challenge injections were 0.05 mL. The second and the consecutive tenth injections were 0.1 mL. The induction injections was performed on alternative days during 3.5 weeks. The callenge injection was performed 10 days to two weeks after the last induction injection. The reading was conducted 24 hours after the challenge injection. The observation demonstrated that the reaction after the challenge injection did not go over the average reaction of the induction injection.

In conclusion, the results show that the test item does not have sensitising propreties under that test conditions.

Overall conclusion

Considering all studies of the source substance, the target substance is not considered 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 sensitization for the read-across substance, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.