methyl-bis(2-arylpropyl)dihydro-heteropolycycle

Endpoint summary

Description of key information

Skin Sensitisation in vitro

REACH_not classified | DPRA | OECD 442c | #key study#

REACH_sensitizing | KeratinoSens | OECD 442d | #key study#

REACH_not sensitizing | hCLAT | OECD 442e | #key study#

Skin Sensitisation in vivo / Read across

REACH_sensitizing | mouse | OECD 429 | #key study##Analogy#

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-14 to 2019-01-18

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

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:

January 12, 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. It was concluded that the direct peptide reactivity assay (DPRA) showed evidence of being a reliable and relevant method to test for skin sensitisation testing. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use.

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.
In the present study methyl-bis(2-arylpropyl)dihydro-heteropolycycle was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 365.51 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.

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 67.86%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion

Value:

9.14

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value in %; no predicxtion can be made due to phase separation

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

Remarks on result:

other: value in %; no predicxtion can be made due to phase separation

Other effects / acceptance of results:

Acceptance Criteria for Cysteine Peptide
- coefficient of determination R² > 0.99 0.9999 pass
- mean peptide concentration of RC A 0.45 ≤ x ≤ 0.55 mM 0.5097 pass
- mean peptide concentration of RC C (PC) 0.45 ≤ x ≤ 0.55 mM 0.5049 pass
- mean peptide concentration of RC C (TI) 0.45 ≤ x ≤ 0.55 mM 0.5049 pass
- CV of the peak area of RC B < 15% 0.81 pass
- CV of the peak area of RC C (PC) < 15% 0.34 pass
- CV of the peak area of RC C (TI) < 15% 0.34 pass
- mean peptide depletion of the PC 60.8% < x < 100% 69.38 pass
- SD of peptide depletion of the PC replicates < 14.9% 0.58 pass
- SD of peptide depletion of the TI replicates < 14.9% 4.27 pass

Acceptance Criteria for Lysine Peptide
- coefficient of determination R² > 0.99 1.0000 pass
- mean peptide concentration of RC A 0.45 ≤ x ≤ 0.55 mM 0.5014 pass
- mean peptide concentration of RC C (PC) 0.45 ≤ x ≤ 0.55 mM 0.5001 pass
- mean peptide concentration of RC C (TI) 0.45 ≤ x ≤ 0.55 mM 0.5001 pass
- CV of the peak area of RC B < 15% 0.84 pass
- CV of the peak area of RC C (PC) < 15% 0.26 pass
- CV of the peak area of RC C (TI) < 15% 0.26 pass
- mean peptide depletion of the PC 40.2% < x < 69.0% 66.34 pass
- SD of peptide depletion of the PC replicates < 11.6% 0.83 pass
- SD of peptide depletion of the TI replicates < 11.6% 0.00 pass

Precipitation and Phase Separation

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no phase separation or precipitation was observed however turbidity was noted 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. Slight phase separation was observed for all the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item no phase separation or precipitation was observed, however turbidity was noted 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 control including the co-elution control. Slight phase separation was also observed for all 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 was fulfilled, the observed phase separation was regarded as not relevant.

Co-elution with the Peptide Peaks

No co-elution of the test item with any of the peptide peaks was observed. Representative HPLC chromatograms are presented in Appendix 3: Representative HPLC Chromatograms Figure 6 and Figure 12.

To detect a co-elution of the test item with the peptide peak, a ratio of the 220 nm peak area and the 258 nm peak area was calculated too (peak purity indication). If the ratio of the control samples and the test item samples do not differ more than 10% from each other, no sign for a co-elution is given.

For the cysteine measurement the peak purity indication was < 10% (0.3 % test item replicate 1; 0.0 % test item replicate 2; 0.2% test item replicate 3) and for the lysine measurement the peak purity indication was < 10% (1.2 % test item replicate 1; 0.8 % test item replicate 2; 1.1 % test item replicate 3)

Categorization of the Test Item

Based on the results of the peptide depletion, categorization according to the prediction model might be performed.

Since no co-elution was observed, prediction model 1 based on the combination of cysteine and lysine peptide depletion should be considered.

Due to the observed phase separation after the incubation period in the cysteine and the lysine peptide samples containing also test item, no prediction can be made.

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	4.57	Minimal Reactivity	(negative)	9.14	Minimal Reactivity	(negative)
Positive Control	67.86	High Reactivity	positive	69.38	Moderate Reactivity	positive

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	16.4390	0.5340	14.6340	0.5340
STD2	8.2610	0.2670	7.3130	0.2670
STD3	4.0740	0.1335	3.6450	0.1335
STD4	1.9450	0.0667	1.8080	0.0667
STD5	0.9340	0.0334	0.9000	0.0334
STD6	0.4240	0.0167	0.4440	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	4.7360	0.1551	69.56	69.38	0.58	0.84
	4.8660	0.1593	68.73
	4.6910	0.1537	69.85
Test Item	13.3730	0.4342	14.06	9.14	4.27	46.69
	14.5480	0.4721	6.51
	14.4960	0.4705	6.84

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	4.4950	0.1644	67.20	66.34	0.83	1.25
	4.6220	0.1690	66.27
	4.7210	0.1726	65.55
Test Item	13.8210	0.5044	0.00	0.00	0.00	0.00
	13.7520	0.5019	0.00
	13.7710	0.5026	0.00

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

Interpretation of results:

study cannot be used for classification

Remarks:

No prediction can be made

Conclusions:

In this study under the given conditions the test item could not be classified due to the observed phase separation of the test item with both peptide peaks.
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.

Executive summary:

In the present study methyl-bis(2-arylpropyl)dihydro-heteropolycycle was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 365.51 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 phase separation or precipitation was observed however turbidity was noted 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. Slight phase separation was observed for all the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item no phase separation or precipitation was observed, however turbidity was noted 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 control including the co-elution control. Slight phase separation was also observed for all 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 was fulfilled, the observed phase separation was regarded as not relevant.

The stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was <= 6.38% (4.57%). Since a phase separation with both peptides was observed, no firm conclusion on the lack of reactivity should be drawn from a negative result. 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 67.86%.

In this study under the given conditions the test item could not be classified due to the observed phase separation of the test item with both peptide peaks.

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 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-23 to 2019-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”

Version / remarks:

25 June 2018

Qualifier:

according to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158

Version / remarks:

01 July 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conductedunder the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. It was concluded that the human cell line activation test (h-CLAT) showed evidence of being a reliable and relevant method to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling for skin sensitisation testing. However, only combinations of several non-animal testing methodswithin an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use

Details on the study design:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.
In the present study methyl-bis(2-arylpropyl)dihydro-heteropolycycle was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.
A CV75 of 33.23 ± 15.50 μg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps:
39.88, 33.23, 27.70, 23.08, 19.23, 16.03, 13.36, 11.13 μg/mL
In the dose finding assay precipitation and in the main experiments turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.
Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments.
The threshold of 150% for CD86 (280% experiment 1; 394% experiment 2) and 200% for CD54 (321% experiment 1; 281% experiment 2) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

147

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value in %; Concentration: 16.03 µg/mL Viability: 91.3%

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

134

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value in %; Concentration: 39.88 µg/mL Viability: 86.3%

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

134

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value in %; Concentration: 33.23 µg/mL Viability: 91.8%

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

145

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value in %; Concentration: 23.08 µg/mL Viability: 96.0%

Other effects / acceptance of results:

ACCEPTANCE CRITERIA
The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

The test met the acceptance criteria. The controls confirmed the validity of the study for all experiments.

A CV75 of 33.23 ± 15.50 μg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps: 39.88, 33.23, 27.70, 23.08, 19.23, 16.03, 13.36, 11.13 μg/mL

In the dose finding assay precipitation and in the main experiments turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 85.7% (CD86), 86.3% (CD54) and 86.5% (isotype IgG1 control) in the first experiment and to 84.1% (CD86), 84.4% (CD54) and 85.0% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item can be considered as non-sensitiser.

Results of the Dose Finding Assay

Sample		Experiment 1		Experiment 2
		Concentration applied [µg/mL]	Cell Viability [%]	Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	96.00	--	94.60
Solvent Control	DMSO	--	95.80	--	94.30
methyl-bis(2-arylpropyl)dihydro-heteropolycycle	C8	7.81	95.50	7.81	94.10
	C7	15.63	94.30	15.63	90.50
	C6	31.25	94.30	31.25	60.20
	C5	62.50	55.70	62.50	20.80
	C4	125.00	25.20	125.00	12.90
	C3	250.00	24.60	250.00	11.20
	C2	500.00	7.20	500.00	10.30
	C1	1000.00	20.70	1000.00	25.60
Calculated CV75 [µg/mL]		44.19		22.27
Mean CV75 [µg/mL]		33.23
SD CV 75 [µg/mL]		15.50

CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	96.5	96.8	96.8	2033	933	632	1401	301	107	81	322	148
Solvent Control	0.20%	96.2	96.1	95.7	1897	962	589	1308	373	100	100	322	163
DNCB	4.00	82.9	84.5	83.7	4350	1881	683	3667	1198	280	321	637	275
methyl-bis(2-arylpropyl)dihydro-heteropolycycle	39.88	85.7	86.3	86.5	1670	1304	803	867	501	66	134	208	162
	33.23	88.3	86.9	87.4	1695	1059	724	971	335	74	90	234	146
	27.69	91.0	91.0	89.8	1819	1088	698	1121	390	86	105	261	156
	23.08	90.8	91.9	91.0	1665	971	649	1016	322	78	86	257	150
	19.23	90.7	90.7	90.9	2123	958	649	1474	309	113	83	327	148
	16.03	91.3	91.8	91.7	2572	999	645	1927	354	147	95	399	155
	13.36	91.7	91.8	91.6	2473	1038	649	1824	389	139	104	381	160
	11.13	91.3	91.5	91.1	2271	1019	664	1607	355	123	95	342	153

CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	96.8	96.7	96.4	2450	1005	640	1810	365	115	90	383	157
Solvent Control	0.20%	96.9	96.9	96.6	2186	1025	618	1568	407	100	100	354	166
DNCB	4.0	90.9	90.7	91.0	6809	1781	638	6171	1143	394	281	1067	279
methyl-bis(2-arylpropyl)dihydro-heteropolycycle	39.88	84.1	84.4	85.0	3018	1410	983	2035	427	130	105	307	143
	33.23	91.8	92.6	91.9	2987	1337	888	2099	449	134	110	336	151
	27.69	95.9	95.7	95.5	2387	1137	645	1742	492	111	121	370	176
	23.08	95.7	96.0	95.9	2760	1269	680	2080	589	133	145	406	187
	19.23	95.9	96.2	95.7	2348	1151	774	1574	377	100	93	303	149
	16.03	97.0	97.3	96.8	2509	1125	682	1827	443	117	109	368	165
	13.36	96.2	96.5	96.3	2496	1088	692	1804	396	115	97	361	157
	11.13	96.3	96.7	96.6	2619	1054	647	1972	407	126	100	405	163

Interpretation of results:

GHS criteria not met

Remarks:

negative in the hCLAT assay

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore, 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 the test item was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

A CV75 of 33.23 ± 15.50 μg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps:

39.88, 33.23, 27.70, 23.08, 19.23, 16.03, 13.36, 11.13 μg/mL

In the dose finding assay precipitation and in the main experiments turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 85.7% (CD86), 86.3% (CD54) and 86.5% (isotype IgG1 control) in the first experiment and to 84.1% (CD86), 84.4% (CD54) and 85.0% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item can be considered as non-sensitiser.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-23 to 2019-04-12

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:

25 June 2018

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

09 March 2018

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conductedunder the auspices of ECVAM and have been considered scientifically valid for the evaluation of theskin sensitisation hazard of chemicals. It was concluded that the KeratinoSens™ assay showed evidence of being a reliable and relevant method to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling for skinsensitisation testing. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use.

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.
In the present study methyl-bis(2-arylpropyl)dihydro-heteropolycycle was dissolved in DMSO. Based on a molecular weight of 365.51 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. Precipitates were observed in the two highest (exp. 2) and in the three highest concentrations (exp. 3). After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.45 (experiment 1); 3.48 (experiment 2) and 3.84 (experiment 3)).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

1.48

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value = fold change; Concentration: 7.81 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

107.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value in %; Concentration: 7.81 µM

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Value:

8.16

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value in µM

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

2.17

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value = fold change; Concentration: 15.63 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

125

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value in %; Concentration: 15.63 µM

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Value:

9.18

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value in µM

Key result

Run / experiment:

other: 3

Parameter:

other: luciferase activity

Value:

2.62

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value = fold change; Concentration: 15.63 µM

Key result

Run / experiment:

other: 3

Parameter:

other: cell viability [%]

Value:

121.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value in %; Concentration: 15.63 µM

Key result

Run / experiment:

other: 3

Parameter:

other: EC1.5 [µM]

Value:

5.25

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value in µM

Other effects / acceptance of results:

The controls confirmed the validity of the study, all acceptance criteria passed.

In the first experiment, a max luciferase activity (Imax) induction of 2.90 was determined at a test item concentration of 31.25 μM. The corresponding cell viability was 19.5%. The lowest tested concentration with a significant luciferase induction >1.5 (1.88) was found to be 15.63 μM. The corresponding cell viability was <70% (60.6%). The calculated EC1.5 was <1000 μM (8.16 μM). Therefore, in this experiment the test item was observed as non-sensitiser.

In the second experiment, a max luciferase activity (Imax) induction of 4.93 was determined at a test item concentration of 31.25 μM. The corresponding cell viability was 61.6%. The lowest tested concentration with a significant luciferase induction >1.5 (2.17) was found to be 15.63 μM. The corresponding cell viability was >70% (125%). The calculated EC1.5 was <1000 μM (9.18 μM). Therefore, in this experiment the test item was observed as sensitiser.

Since in experiment one the substance was considered as non-sensitiser and experiment two as sensitiser, a decisive experiment had to be performed.

In the third experiment, a max luciferase activity (Imax) induction of 7.16 was determined at a test item concentration of 31.25 μM. The corresponding cell viability was 46.8%. The lowest tested concentration with a significant luciferase induction >1.5 (1.77) was 7.81 μM, followed by 15.63 μM (2.62). The corresponding cell viabilities were >70% (138.7% and 121.4). The calculated EC1.5 was <1000 μM (5.25 μM). Therefore, in this experiment the test item was observed as sensitiser.

Overall, a dose response for luciferase activity induction could be 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 sensitiser.

Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Experiment 3	Mean	SD
Negative Control	-	100	100	100	100	0.0
Positive Control	4.00	95.8	95.7	101.1	97.5	3.1
	8.00	106.7	93.9	105.6	102.0	7.1
	16.00	94.1	95.5	109.6	99.7	8.6
	32.00	78.5	85.0	117.0	93.5	20.6
	64.00	74.8	73.7	88.6	79.0	8.3
Test Item	0.98	96.5	95.8	99.0	97.1	1.7
	1.95	102.6	110.1	121.5	111.4	9.5
	3.91	99.5	115.5	122.0	112.3	11.6
	7.81	107.1	123.2	138.7	123.0	15.8
	15.63	60.6	125.0	121.4	102.3	36.2
	31.25	19.5	61.6	46.8	42.6	21.4
	62.50	1.5	12.5	13.5	9.2	6.7
	125.00	0.0	0.8	2.9	1.2	1.5
	250.00	0.0	0.0	0.8	0.3	0.4
	500.00	0.1	0.0	3.4	1.2	2.0
	1000.00	0.2	0.1	2.8	1.0	1.5
	2000.00	0.1	0.2	1.9	0.7	1.0

Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Negative Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.28	1.17	1.25	1.23	0.06
	8.00	1.35	1.37	1.32	1.35	0.03
	16.00	1.66	1.44	1.63	1.58	0.12	*
	32.00	1.93	2.24	2.07	2.08	0.16	*
	64.00	3.35	3.82	3.18	3.45	0.33	*
Test Item	0.98	1.40	1.33	1.24	1.32	0.08
	1.95	1.19	1.09	1.08	1.12	0.06
	3.91	1.27	1.20	1.19	1.22	0.04
	7.81	1.51	1.34	1.60	1.48	0.13
	15.63	2.13	1.76	1.75	1.88	0.22	*
	31.25	3.26	2.59	2.86	2.90	0.33	*
	62.50	0.87	2.20	0.89	1.32	0.77
	125.00	0.07	0.23	0.02	0.11	0.11
	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

Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Negative Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.13	1.19	1.22	1.18	0.05
	8.00	1.29	1.57	1.40	1.42	0.14
	16.00	1.42	1.56	1.40	1.46	0.09
	32.00	1.32	1.97	2.12	1.81	0.43	*
	64.00	3.01	3.42	4.01	3.48	0.51	*
Test Item	0.98	1.27	1.23	1.20	1.23	0.04
	1.95	1.10	1.19	0.93	1.08	0.13
	3.91	1.25	1.38	1.05	1.22	0.17
	7.81	1.37	1.55	1.15	1.36	0.20
	15.63	2.06	2.63	1.83	2.17	0.41	*
	31.25	4.88	5.86	4.04	4.93	0.91	*
	62.50	4.43	5.46	4.53	4.81	0.57	*
	125.00	1.06	0.42	1.18	0.89	0.41
	250.00	0.03	0.00	0.02	0.02	0.01
	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

Induction of Luciferase Activity Experiment 3

Experiment 3	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Negative Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.23	1.03	1.30	1.19	0.14
	8.00	1.18	1.34	1.44	1.32	0.13
	16.00	1.68	1.50	1.39	1.52	0.15
	32.00	2.13	2.12	1.87	2.04	0.15	*
	64.00	3.99	4.03	3.51	3.84	0.29	*
Test Item	0.98	1.15	1.10	1.29	1.18	0.10
	1.95	0.99	1.08	1.17	1.08	0.09
	3.91	1.28	1.41	1.38	1.36	0.07
	7.81	1.51	1.73	2.09	1.77	0.29	*
	15.63	2.55	2.54	2.78	2.62	0.13	*
	31.25	8.18	6.79	6.50	7.16	0.90	*
	62.50	1.92	2.41	2.51	2.28	0.32	*
	125.00	0.07	0.24	0.38	0.23	0.16
	250.00	0.00	0.00	0.01	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

Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction
		Exp. 1	Exp. 2	Exp. 3	Mean	SD
Negative Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.23	1.18	1.19	1.20	0.03
	8.00	1.35	1.42	1.32	1.36	0.05
	16.00	1.58	1.46	1.52	1.52	0.06
	32.00	2.08	1.81	2.04	1.98	0.15
	64.00	3.45	3.48	3.84	3.59	0.22
Test Item	0.98	1.32	1.23	1.18	1.25	0.07
	1.95	1.12	1.08	1.08	1.09	0.02
	3.91	1.22	1.22	1.36	1.27	0.08
	7.81	1.48	1.36	1.77	1.54	0.21
	15.63	1.88	2.17	2.62	2.23	0.37
	31.25	2.90	4.93	7.16	5.00	2.13
	62.50	1.32	4.81	2.28	2.80	1.80
	125.00	0.11	0.89	0.23	0.41	0.42
	250.00	0.00	0.02	0.00	0.01	0.01
	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

Additional Parameters

Parameter	Exp. 1	Exp. 2	Exp 3	Mean	SD
EC1.5[µM]	8.16	9.18	5.25	7.53	2.04
Imax	2.90	4.93	7.16	5.00	2.13
IC30[µM]	14.04	29.17	26.40	23.20	8.06
IC50[µM]	19.64	38.62	30.59	29.61	9.53

Acceptance Criteria

Criterion	Range	Exp. 1	pass/ fail	Exp.2	pass/ fail	Exp. 3	pass/ fail
CV Negative Control	< 20%	11.9	pass	7.9	pass	14.9	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	3.0	pass	2.0	pass	2.0	pass
EC1.5 PC	± 2 x SD of historical mean	13.25	pass	17.73	pass	15.07	pass
Induction PC at 64 µM	2 .00 < x < 8.00	3.45	pass	3.48	pass	3.84	pass

Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Negative Control	< 20%	11.6	3.5	96
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.4	0.6	96
EC1.5 PC	7 < x < 34 µM	18.5	6.0	96
Induction PC at 64 µM	2.00 < x < 8.00	3.8	1.5	96

Interpretation of results:

other: sensitising

Remarks:

positive in the KeratinSens assay

Conclusions:

In this study under the given conditions the test item did 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 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.

Executive summary:

In the present study methyl-bis(2-arylpropyl)dihydro-heteropolycycle was dissolved in DMSO. Based on a molecular weight of 365.51 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. Precipitates were observed in the two highest (exp. 2) and in the three highest concentrations (exp. 3). After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 2.90 was determined at a test item concentration of 31.25 μM. The corresponding cell viability was 19.5%. The lowest tested concentration with a significant luciferase induction >1.5 (1.88) was found to be 15.63 μM. The corresponding cell viability was <70% (60.6%). The calculated EC1.5 was <1000 μM (8.16 μM). Therefore, in this experiment the test item was observed as non-sensitiser.

In the second experiment, a max luciferase activity (Imax) induction of 4.93 was determined at a test item concentration of 31.25 μM. The corresponding cell viability was 61.6%. The lowest tested concentration with a significant luciferase induction >1.5 (2.17) was found to be 15.63 μM. The corresponding cell viability was >70% (125%). The calculated EC1.5 was <1000 μM (9.18 μM). Therefore, in this experiment the test item was observed as sensitiser.

Since in experiment one the substance was considered as non-sensitiser and experiment two as sensitiser, a decisive experiment had to be performed.

In the third experiment, a max luciferase activity (Imax) induction of 7.16 was determined at a test item concentration of 31.25 μM. The corresponding cell viability was 46.8%. The lowest tested concentration with a significant luciferase induction >1.5 (1.77) was 7.81 μM, followed by 15.63 μM (2.62). The corresponding cell viabilities were >70% (138.7% and 121.4). The calculated EC1.5 was <1000 μM (5.25 μM). Therefore, in this experiment the test item was observed as sensitiser.

Overall, a dose response for luciferase activity induction could be observed for each individual run as well as for an overall luciferase activity induction.

Under the conditions of this study, the test item is therefore considered as sensitiser.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Aug - Sept 2009

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

read-across source

Key result

Parameter:

EC3

Value:

34.4

Parameter:

SI

Remarks:

25%

Value:

2.11

Parameter:

SI

Remarks:

50%

Value:

4.47

Parameter:

SI

Remarks:

100%

Value:

4.8

Interpretation of results:

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

Conclusions:

The analogue test item 2-Undecanal-bi-oxazolidine Sa 190 was found to be a skin sensitiser under the described conditions.
According to the Regulation (EC) No 1272/2008 on classification, labeling and packaging (CLP) of substances and mixtures, the test item should be classified as skin sensitiser (Category 1B).
According to the ECETOC classification scheme for potency (ECETOC Technical Report No. 87, Contact sensitization: Classification according to potency, April 2003, Brussels), the test item would be regarded as weak sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The three in vitro tests DPRA, KeratinoSens and h-CLAT were performed according to OECD principles (OECD 442c,d,e). DPRA was inconclusive, h-CLAT resulted that the substance is not a skin sensitizer and KeratinoSenS considered the substance to be a skin sensitiser. Due to its physchem properties (low solubility in water, high log Pow) the test substance is hard to test in vitro. Thus the in vitro results were inconclusive. That is why a read-across approach was considered using the similar substance Sa 190. For this substance an LLNA was performed (OECD 429) resulting that Sa 190 was a weak sensitizer. Since both substances are similar it is considered that methyl-bis(2-arylpropyl)dihydro-heteropolycycle is a weak skin sensitizer, as well.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin Sensitization:

Based on a read accross to the analogue substance Sa 190 the test item is considered to be a weak skin sensitiser.

According to the Regulation (EC) No 1272/2008 on classification, labeling and packaging (CLP) of substances and mixtures, the test item should be classified as a weak skin sensitiser (Category 1B).