dialkyl-methyldihydro-heteropolycycle

Endpoint summary

Description of key information

Skin Sensitization

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

REACH_not sensitizing | KeratinoSens | OECD 442d | #key study#

REACH_sensitizing | h-CLAT | OECD 442e | #key study#

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:

04 February 2015

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

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.

Experimental Procedure
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 - 400x g) 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 HPLC procedure.

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:

1.55

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value in %

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion

Value:

0.76

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value in %

Other effects / acceptance of results:

Acceptance Criteria
Cysteine Peptide Run
- 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: 0.4945 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%: 1.97 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%: 1.40 pass

Lysine Peptide Run
- 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: 0.5067 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%: 0.43 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%: 1.20 pass

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

In the present study the test item was dissolved in isopropanol, based on the results of the pre-experiments. Based on a molecular weight of 395.66 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.

All test item solutions were freshly prepared immediately prior to use. 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 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. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution 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 not relevant.

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

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% (1.15%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86%.

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

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	14.8270	0.4812	2.72	1.55	1.40	90.32
	15.4360	0.5008	0.00
	14.9480	0.4851	1.92

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	13.9810	0.5102	0.00	0.76	1.20	158.59
	13.8660	0.5060	0.13
	13.5870	0.4959	2.14

Prediction Model 1

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

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

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

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

Prediction Model 2

Cysteine 1:10 Prediction Model

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

Categorization of the Test Item

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	1.15	Minimal Reactivity	negative	1.55	Minimal Reactivity	negative
Positive Control	67.86	High Reactivity	positive	69.38	Moderate Reactivity	positive

Interpretation of results:

GHS criteria not met

Remarks:

DPRA

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is 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 isopropanol, based on the results of the pre-experiments. Based on a molecular weight of 395.66 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.

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

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 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. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution 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 not relevant.

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

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% (1.15%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86%.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-23 to 2019-03-01

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 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 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 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 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 (P 02 in experiment 1; P 04 in experiment 2) were used.
Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2 in a humidified incubator. For test item exposure, cells were cultured in medium for test item exposure.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.59 (experiment 1); 4.74 (experiment 2)).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

1.23

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value = fold induction; Concentration: 0.98 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability

Value:

124.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value in %; Concentration: 0.98 µM

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

1.18

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value = fold induction; Concentration: 1.95 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability

Value:

92.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value in %; Concentration: 1.95 µM

Other effects / acceptance of results:

The controls confirmed the validity of the study.

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.

Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent / Neg. Control	-	100	100	100	0.0
Positive Control	4.00	94.8	116.6	105.7	15.4
	8.00	103.7	122.0	112.9	12.9
	16.00	101.1	112.0	106.6	7.7
	32.00	88.2	129.8	109.0	29.4
	64.00	107.5	126.0	116.7	13.1
Test Item	0.98	124.5	75.5	100.0	34.6
	1.95	100.3	92.9	96.6	5.2
	3.91	106.0	105.1	105.5	0.6
	7.81	96.7	105.4	101.1	6.1
	15.63	111.1	144.4	127.8	23.6
	31.25	105.1	130.5	117.8	18.0
	62.50	107.8	125.2	116.5	12.3
	125.00	102.7	151.2	126.9	34.3
	250.00	97.5	125.7	111.6	19.9
	500.00	98.0	106.2	102.1	5.8
	1000.00	99.4	97.5	98.5	1.3
	2000.00	111.1	101.3	106.2	6.9

Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent / Neg. Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.14	1.21	1.16	1.17	0.04
	8.00	1.21	1.22	1.31	1.25	0.05
	16.00	1.41	1.58	1.71	1.57	0.15	*
	32.00	2.29	2.21	2.17	2.23	0.06	*
	64.00	4.24	4.60	4.91	4.59	0.34	*
Test Item	0.98	1.18	1.30	1.23	1.23	0.06
	1.95	1.10	1.16	1.06	1.11	0.05
	3.91	1.03	1.11	1.13	1.09	0.05
	7.81	1.08	1.10	1.11	1.10	0.02
	15.63	1.13	1.28	1.21	1.21	0.07
	31.25	1.21	1.24	1.21	1.22	0.02
	62.50	1.08	1.17	1.21	1.15	0.07
	125.00	1.08	1.20	1.08	1.12	0.07
	250.00	1.11	1.09	1.14	1.11	0.03
	500.00	1.11	1.13	1.01	1.08	0.06
	1000.00	1.07	1.14	0.92	1.04	0.11
	2000.00	1.11	1.19	1.06	1.12	0.07

Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent / Neg. Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.10	1.27	1.22	1.19	0.09
	8.00	1.20	1.49	1.07	1.25	0.22
	16.00	1.60	1.88	1.39	1.62	0.25	*
	32.00	2.02	2.76	1.64	2.14	0.57	*
	64.00	3.72	6.81	3.68	4.74	1.80	*
Test Item	0.98	1.31	1.15	1.06	1.17	0.13
	1.95	1.09	1.41	1.04	1.18	0.20
	3.91	1.12	1.09	1.01	1.08	0.05
	7.81	1.09	1.05	1.02	1.05	0.04
	15.63	1.18	1.05	1.14	1.12	0.07
	31.25	1.05	1.02	0.97	1.02	0.04
	62.50	1.07	0.95	0.93	0.99	0.07
	125.00	1.07	0.99	1.05	1.04	0.04
	250.00	1.06	0.96	1.02	1.02	0.05
	500.00	1.02	1.02	1.02	1.02	0.00
	1000.00	1.09	1.10	0.92	1.03	0.10
	2000.00	1.07	1.21	1.04	1.10	0.09

Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent / Neg. Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.17	1.19	1.18	0.02
	8.00	1.25	1.25	1.25	0.00
	16.00	1.57	1.62	1.60	0.04	*
	32.00	2.23	2.14	2.18	0.06	*
	64.00	4.59	4.74	4.66	0.11	*
Test Item	0.98	1.23	1.17	1.20	0.04
	1.95	1.11	1.18	1.14	0.05
	3.91	1.09	1.08	1.08	0.01
	7.81	1.10	1.05	1.08	0.03
	15.63	1.21	1.12	1.16	0.06
	31.25	1.22	1.02	1.12	0.15
	62.50	1.15	0.99	1.07	0.12
	125.00	1.12	1.04	1.08	0.06
	250.00	1.11	1.02	1.07	0.07
	500.00	1.08	1.02	1.05	0.04
	1000.00	1.04	1.03	1.04	0.01
	2000.00	1.12	1.10	1.11	0.01

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	n.a.	n.a.	n.a.	n.a.
Imax	1.23	1.18	1.21	0.04
IC30[µM]	n.a.	n.a.	n.a.	n.a.
IC50[µM]	n.a.	n.a.	n.a.	n.a.

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Negative Control	< 20%	8.2	pass	15.5	pass
CV Solvent Control	<20%	10.2	pass	8.3	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	3.0	pass	3.0	pass
EC1.5 PC	7 < x < 34 µM	14.31	pass	13.32	pass
Induction PC at 64 µM	2.00 < x < 8.00	4.59	pass	4.74	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:

GHS criteria not met

Remarks:

KeratinoSens

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In the present study the test item was dissolved in THF. Based on a molecular weight of 395.66 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, nosignificant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5value could be calculated.

In the second experiment,nosignificant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5value 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.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-23 to 2019-03-28

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 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 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 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 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.

CELL LINE
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 10E6 cells/mL. Cells were cultured in 175 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 μg/mL streptomycin at 37 ± 1°C and 5% CO2.

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 (351% experiment 1; 364% experiment 2) and 200% for CD54 (301% experiment 1; 401% experiment 2) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

160

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value in %; Concentration: 1000 µg/mL Cytotoxicity: 95.9%

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

129

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value in %; Concentration: 279.08 µg/mL Cytotoxicity: 95.3%

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

377

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value in %; Concentration: 401.88 µg/mL Cytotoxicity: 96.10%

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

625

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value in %; Concentration: 1000 µg/mL Cytotoxicity: 95.40%

Other effects / acceptance of results:

The controls confirmed the validity of the study for all experiments.

In the main experiments precipitation 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 95.9% (CD86), 96.1% (CD54) and 96.5% (isotype IgG1 control) in the first experiment and 97.3% (CD86), 95.4% (CD54) and 94.6% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was upregulated to 160% (1000 μM) in the first experiment and up to 377% (401.88 μM) in the second experiment. The expression of the cell surface marker CD54 was upregulated up to 625% (1000 μM) only in the second experiment.

Since the expression of both cell surface markers exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.

Results of the Dose Finding Assay

Sample		Experiment 1
		Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	96.20
Solvent Control (THF)	THF	--	95.70
dialkyl-methyldihydro-heteropolycycle	C8	7.81	96.30
	C7	15.63	96.00
	C6	31.25	95.80
	C5	62.50	95.90
	C4	125.00	95.80
	C3	250.00	95.70
	C2	500.00	96.20
	C1	1000.00	95.50
Calculated CV75 [µg/mL]		No CV75
Mean CV75 [µg/mL]		No CV75
SD CV 75 [µg/mL]		No SD

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	-	96.2	95.9	95.6	1456	924	590	866	334	81	80	247	157
Solvent Control 2(THF)	0.20%	96.0	95.7	96.1	1371.0	918.0	637.0	734	281	100	100	215	144
Solvent Control 1 (DMSO)	0.20%	94.1	95.3	94.8	1639	983	565	1074	418	100	100	290	174
DNCB	4.00	89.9	90.1	89.1	4449	1938	680	3769	1258	351	301	654	285
dialkyl-methyldihydro-heteropolycycle	1000	95.9	96.1	96.5	1817	902	639	1178	263	160	94	284	141
	833.33	95.9	96.2	96.3	1298	812	603	695	209	95	74	215	135
	694.44	95.9	96.0	96.0	1443	854	624	819	230	112	82	231	137
	578.70	97.1	96.7	96.9	1368	859	613	755	246	103	88	223	140
	482.25	96.3	96.0	95.7	1427	832	606	821	226	112	80	235	137
	401.88	96.5	96.6	96.5	1283	839	604	679	235	93	84	212	139
	334.90	96.3	96.0	95.9	1305	819	602	703	217	96	77	217	136
	279.08	95.8	95.3	95.9	1625	1018	655	970	363	132	129	248	155

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	-	96.5	97.0	97.4	1614	838	619	995	219	109	127	261	135
Solvent Control 2 (THF)	0.20%	96.1	97.0	97.1	1495	816	630	865	186	100	100	237	130
Solvent Control 1 (DMSO)	0.20%	97.6	97.1	97.6	1568	826	654	914	172	100	100	240	126
DNCB	4.0	88.7	88.9	88.1	4012	1377	688	3324	689	364	401	583	200
dialkyl-methyldihydro-heteropolycycle	1000.00	97.30	95.40	94.60	2352	1913	751	1601	1162	185	625	313	255
	833.33	97.00	97.20	97.10	1725	878	637	1088	241	126	130	271	138
	694.44	97.60	97.60	96.90	1570	855	626	944	229	109	123	251	137
	578.70	97.10	96.80	97.10	1571	856	613	958	243	111	131	256	140
	482.25	97.30	97.40	96.50	1715	848	678	1037	170	120	91	253	125
	401.88	96.10	96.90	96.70	3903	1174	645	3258	529	377	284	605	182
	334.90	97.10	97.00	96.70	3672	889	633	3039	256	351	138	580	140
	279.08	96.70	97.30	96.60	1776	852	619	1157	233	134	125	287	138

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

hCLAT

Conclusions:

In this study under the given conditions the test item did upregulate the cell surface markers in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser.
The data generated with this method may be not sufficient to conclude on 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 THF. 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.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:

1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL

In the main experiments precipitation 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 95.9% (CD86), 96.1% (CD54) and 96.5% (isotype IgG1 control) in the first experiment and 97.3% (CD86), 95.4% (CD54) and 94.6% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was upregulated to 160% (1000 µM) in the first experiment and up to 377% (401.88 µM) in the second experiment. The expression of the cell surface marker CD54 was upregulated up to 625% (1000 µM) only in the second experiment.

Since the expression of both cell surface markers exceeded the threshold in two independent experiments the test item is considered to be a skin 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). Two tests (DPRA, KeratinoSens) resulted that the substance is not a skin sensitiser, the third test (h-CLAT) considered the substance to be a skin sensitiser. With the 2 out of 3 approach and considering the results of the OECD QSAR toolbox (V 4.4), which did not find any alerts for skin sensitization, it could be believed that, the substance might not be a skin sensitizer. But due to its physchem properties (low solubility in water, high log P_ow) the test substance is hard to test in vitro. Furthermore, the substance can hydrolize, forming an aldehyde, which is a skin sensitizer. Thus, the results of the three in vitro tests are not considered reliable. That is why a read-across approach was considered using the very 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 very similar it is considered that dialkyl-methyldihydro-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).