2-Propanol, 1,3-bis[(3-methyl-2-buten-1-yl)oxy]-

Endpoint summary

Description of key information

DPNG is classified as sin sensitiser category 1B according to methods outlined in the Guideline on Defined Approaches (DA) for Skin Sensitization (Guideline 497) supplemented by Read-Across prediction models within OECD Toolbox (v4.5).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2021

Reliability:

1 (reliable without restriction)

Justification for type of information:

Software: QSAR Toolbox 4.5
Version: Data base version 4.5

SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: CC(C)=CCOCC(O)COCC=C(C)C

for further information see report

Interpretation of results:

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

Executive summary:

Predicted endpoint: EC3, S M W N, Skin sensitisation; No effect specified; No species specified; No
duration specified; No guideline specified
Predicted value: Positive
Unit/scale: Skin sensitisation II (ECETOC)
Data gap filling method: Read-across analysis
Summary: 
The predicted Log Kow value is outside the domain of applicability. However, the target molecule does
contain structural elements with alerts for at least one key element for dermal sensitization - formation
of radial reactions and protein binding and is within the domain of applicability from at least one
metabolic profiler. Therefore, within the limitations of the available information, the target molecule is
predicted to be positive for dermal sensitization based on read-across.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-05-03 to 2021-06-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

23 July 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

442E

PREPARATION OF TEST SOLUTIONS
The test item was freshly prepared immediately prior to use. The test item was soluble in dimethyl sulfoxide (DMSO) at a concentration of 500 mg/mL.

Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2 in the dose-finding assay and factor 1:1.2 in the main experiments.

The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium.

Only in the dose finding assay 2 phase separation was observed in the two highest concentrations when diluted 1:250 in cell culture medium. Sonication was used to aid solubilisation.

The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.


DOSE Groups:
1. Medium Control: cell culture medium
2. Solvent Control: 0.2% DMSO (v/v) in cell culture medium
3. Positive Control: 4 µg/mL DNCB
4. Test Item: 8 concentrations of the test item
(dose finding assay/ main experiment)
dose finding assay 1 and 2: 1000, 500, 250, 125, 62.50, 31.25, 15.63, 7.81 µg/mL - A CV75 of 358.04 ± 55.37 µg/mL was derived in the dose finding assay
Based on the CV75, the main experiment was performed covering the following concentration steps:
main experiment 1 and 2: 429.65, 358.04, 298.37, 248.64, 207.20, 172.67, 143.89, 119.91 µg/m


APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
A medium control, a solvent control, and a positive control were set up in parallel in order to confirm the validity of the test.

Medium Control
A medium control was included in the test.

Solvent Controls
Solvent controls were included in the test. The solvent controls were set up depending on the appropriate solvent previously determined.
Since the test item was solubilized in DMSO, a DMSO control served as solvent control for the test item.
Since the positive control was solubilized in DMSO, a DMSO control was included and served as solvent control for the positive control.
The solvent controls were diluted, resulting in a final concentration of 0.2% (v/v) for DMSO.

Positive Control
2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 µg/mL (alternatively at the concentration of the CV75) was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted, resulting in a final DMSO concentration of 0.2% (v/v).


MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT
Dose Finding assay:
The PI (propidium iodide) uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of lambda = 488 nm and an emission wavelength of lambda > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration.
Main experiment:
The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of lambda = 488 nm and an emission wavelength of lambda = 530 nm ± 15 nm for FITC and lambda > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated.


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%.



DATA EVALUATION
- Cytotoxicity assessment
Cell viability were analysed by flow cytometry
- Prediction model used
For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day or on the same day provided that for each run: independent fresh stock solutions and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used. Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability = 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability = 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability = 50% in at least two independent runs. In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 µg/mL for 0.9% NaCl solution; 1000 µg/mL for DMSO or a different organic solvent) even if the cell viability is >90%.
A negative result for test items with a Log KOW > 3.5 should be considered as inconclusive.

Vehicle / solvent control:

DMSO

Negative control:

other: Medium control

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

Refer to the result tables in 'Any other information on results incl. tables'

Group:

test chemical

Run / experiment:

other: run/experiment 1 and 2

Parameter:

other: RFI CD54

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments

Remarks:

Refer to the result tables in 'Any other information on results incl. tables'

Group:

test chemical

Run / experiment:

other: run/experiment 1 and 2

Parameter:

other: RFI CD86

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments.

Remarks:

Refer to the result tables in 'Any other information on results incl. tables'

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: see below table

Discussion

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 sensitiser and non[1]sensitisers.

Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.

In the present study 1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane 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 358.04 ± 55.37 µg/mL was derived in the dose finding assay.

Based on the CV75, the main experiment was performed covering the following concentration steps: 429.65, 358.04, 298.37, 248.64, 207.20, 172.66, 143.89, 119.91 µg/mL

Phase separation of the test item was observed for the two highest concentration steps in the dose-finding study 2 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.

Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 35.4% (CD86), 38.0% (CD54) and 43.8% (isotype IgG1 control) in the first experiment and to 34.9% (CD86), 36.2% (CD54) and 47.3% (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 was negative in this assay.

The controls confirmed the validity of the study for all experiments as shown in Table 6.

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

 

Results

Reactivity Check of the Cell Stock

Doubling time of the cells was monitored and found to be 32.2 h which is within the doubling time range specified by the manufacturer (30 - 55 h).

Table 2: Results of the Cell Batch Activation Test

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	87	276	>150	86	413	>200	yes	pass
NiSO4	100 µg/mL	88	351	>150	87	596	>200	yes	pass
LA	1000 µg/mL	97	103	=150	98	109	=200	no	pass

 

The positive controls DNCB and NiSO₄ led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86.

The cell batch was accepted for further testing.

 

Solvent Finding

All test item solutions were freshly prepared immediately prior to use. The test item was soluble in DMSO at a concentration of 500 mg/mL.

 

 

Dose Finding Assay

The dose finding assay was performed using stock solutions with a concentration of 500 mg/mL (applied concentration 1000 µg/mL).

Table 3: 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	--	--	94.54	--	95.59
Solvent Control	DMSO	--	92.47	--	94.92
1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane	C8	7.81	92.73	7.81	93.61
	C7	15.63	93.79	15.63	93.08
	C6	31.25	93.24	31.25	94.57
	C5	62.50	92.72	62.50	95.86
	C4	125.00	92.35	125.00	95.63
	C3	250.00	91.21	250.00	93.70
	C2	500.00	66.94	500.00	40.44
	C1	1000.00	3.14	1000.00	1.95
Calculated CV75 [µg/mL]		397.19		318.88
Mean CV75 [µg/mL]		358.04
SD CV 75 [µg/mL]		55.37

 

 

Results CD54 and CD86 Expression

For determination of the cell surface markers CD54 and CD86 two independent experiments were performed using separate cultivated cells at passage 19 (first experiment) and 25 (second experiment). For each experiment separately weighted samples and preparations were used.

Table 4: 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	-	96.0	93.9	92.0	1144	693	490	654	203	81	53	233	141
Solvent Control	0.20%	96.7	91.9	94.6	1211	780	400	811	380	100	100	303	195
DNCB	4.00	85.4	73.8	87.9	2017	1180	395	1622	785	200	207	511	299
1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane	429.65	35.4	38.0	43.8	852	783	494	358	289	44	76	172	159
	358.04	74.7	64.8	80.5	952	806	411	541	395	67	104	232	196
	298.37	85.9	86.9	90.5	1066	724	399	667	325	82	86	267	181
	248.64	89.6	90.9	89.4	979	645	395	584	250	72	66	248	163
	207.20	92.2	90.5	94.3	1005	703	397	608	306	75	81	253	177
	172.67	92.9	92.9	93.9	905	658	393	512	265	63	70	230	167
	143.89	93.7	91.6	94.1	967	639	396	571	243	70	64	244	161
	119.91	94.8	93.9	90.3	972	605	393	579	212	71	56	247	154

 

 

 

Table 5: 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	-	91.2	93.0	93.0	1234	742	473	761	269	79	82	261	157
Solvent Control	0.20%	92.5	90.4	92.8	1387	753	425	962	328	100	100	326	177
DNCB	4.0	80.3	77.1	76.9	3093	2070	725	2368	1345	246	410	427	286
1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane	429.65	34.9	36.2	47.3	1503	1023	517	986	506	102	154	291	198
	358.04	56.0	61.5	66.4	1587	994	426	1161	568	121	173	373	233
	298.37	84.5	82.9	86.9	1540	1033	423	1117	610	116	186	364	244
	248.64	88.2	89.3	91.3	1604	916	425	1179	491	123	150	377	216
	207.20	91.6	90.9	93.1	1591	810	401	1190	409	124	125	397	202
	172.67	92.2	92.2	94.0	1554	848	493	1061	355	110	108	315	172
	143.89	88.9	92.7	92.7	1428	835	554	874	281	91	86	258	151
	119.91	93.1	93.6	91.0	1427	979	401	1026	578	107	176	356	244

 

 

 

Acceptance Criteria

Table 6: Acceptance Criteria

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	91.9	-	96.7	pass	90.4	-	93.0	pass
number of test dosed with viability >50% CD86	>=4	7			pass	7			pass
number of test dosed with viability >50% CD54	>=4	7			pass	7			pass
number of test dosed with viability >50% IgG1	>=4	7			pass	7			pass
RFI of positive control of CD86	>=150	200			pass	246			pass
RFI of positive control of CD54	=200	207			pass	410			pass
RFI of solvent control of CD86	<150	124			pass	126			pass
RFI of solvent control of CD54	<200	187			pass	122			pass
MFI ratio CD86/IgG1 for medium control [%]	>105	233			pass	261			pass
MFI ratio CD86/IgG1 for DMSO control [%]	>105	303			pass	326			pass
MFI ratio CD54/IgG1for medium control [%]	>105	141			pass	157			pass
MFI ratio CD54/IgG1for DMSO control [%]	>105	195			pass	177			pass

 

 Historical Data

Table 7: Historical Data

Criterion	mean	SD	N
cell viability solvent controls [%]	96.3	1.5	1554
number of test doses with viability >50%	-	-	4026
RFI of positive control of CD86	355.0	122.9	259
RFI of positive control of CD54	435.4	255.5	259
RFI of solvent control of CD86	111.1	31.3	259
RFI of solvent control of CD54	114.4	43.2	259
MFI ratio IgG1/CD86 for medium control [%]	309.0	154.2	259
MFI ratio IgG1/CD86 for DMSO control [%]	353.1	364.2	259
MFI ratio IgG1/CD54 for medium control [%]	179.0	150.3	259
MFI ratio IgG1/CD54 for DMSO control [%]	176.5	54.3	259

 

 

 

Interpretation of results:

GHS criteria not met

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 substance was negative in this study.
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:

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.

Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.

In the present study 1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane 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 358.04 ± 55.37 µg/mL was derived in the dose finding assay.

Based on the CV75, the main experiment was performed covering the following concentration steps: 429.65, 358.04, 298.37, 248.64, 207.20, 172.66, 143.89, 119.91 µg/mL

Phase separation of the test item was observed for the two highest concentration steps in the dose-finding study 2 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. Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 35.4% (CD86), 38.0% (CD54) and 43.8% (isotype IgG1 control) in the first experiment and to 34.9% (CD86), 36.2% (CD54) and 47.3% (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 “negative” in this assay.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-06-23 to 2021-07-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

442D

PREPARATION OF TEST SOLUTIONS
All test item solutions were freshly prepared immediately prior to use.
The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity =99%; AppliChem; Lot No.: 0001886811). A stock solution of 200 mM was prepared by pre-weighing the test material into a suitable tube.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent.
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.



DOSE Groups:
1. Negative Control: 1% (v/v) DMSO in test item exposure medium
2. Positive Control: CA: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
3. Test Item: 12 concentrations of the test item
Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per 96-well plate in every independent run.


APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
A cell suspension of 8 × 10000 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 10000 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.


- CONTROL -
A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.
Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.
Negative Control
DMSO (AppliChem; Lot No.: 0001886811) at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.
Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 14371-10-9; >98%; Alfa Aesar; Lot No.: MKCJ4653) was used as positive control. CA was dissolved in DMSO (AppliChem; Lot No.: 0001886811) at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells. (CA: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM)


LUCIFERASE ACTIVITY MEASUREMENTS
Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.

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


DATA EVALUATION
- Prediction model used
A KeratinoSens™ prediction is considered positive if the following conditions will be 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 µM
- 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 µM is considered as inconclusive.

A negative result for test items with a log KOW > 7 has to be interpreted with care due to the applicability of the test method.

Vehicle / solvent control:

DMSO

Negative control:

other: Blank Control

Positive control:

cinnamic aldehyde [442D]

Positive control results:

Refer to the experiment 1-3 results in the pdf attachment.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

2.91

At concentration:

2 000 other: µM

Cell viability:

93.9%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

5.55

At concentration:

2 000 other: µM

Cell viability:

64.7%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

256.25 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The lowest tested concentration with a significant luciferase induction >1.5 (1.80) was found to be 500 µM. The corresponding cell viability was >70% (121.0%).

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

253.54 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The lowest tested concentration with a significant luciferase induction >1.5 (1.86) was found to be 500 µM. The corresponding cell viability was >70% (97.2%).

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: see table below

Discussion

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 1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane was dissolved in DMSO.

Based on a molecular weight of 228.33 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, a max luciferase activity (I_max) induction of 2.91 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 93.9%. The lowest tested concentration with a significant luciferase induction >1.5 (1.80) was found to be 500 µM. The corresponding cell viability was >70% (121.0%). The calculated EC_1.5 was <1000 µM (256.25 µM).

In the second experiment, a max luciferase activity (I_max) induction of 5.55 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 64.7%. The lowest tested concentration with a significant luciferase induction >1.5 (1.86) was found to be 500 µM. The corresponding cell viability was >70% (97.2%). The calculated EC_1.5 was <1000 µM (253.54 µM).

A 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 sensitiser.

The controls confirmed the validity of the study (see Table 6).

 

Luciferase Activity - Overall Induction

Table 4: Induction of Luciferase Activity – Overall Induction

Overall Induction	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.23	1.25	1.24	0.01
	8.00	1.28	1.43	1.36	0.10
	16.00	1.41	1.49	1.45	0.05
	32.00	1.72	1.85	1.79	0.09	*
	64.00	2.05	6.65	4.35	3.25
Test Item	0.98	1.06	0.82	0.94	0.17
	1.95	1.27	0.95	1.11	0.23
	3.91	1.18	1.02	1.10	0.11
	7.81	1.28	1.00	1.14	0.20
	15.63	1.31	1.13	1.22	0.13
	31.25	1.41	1.25	1.33	0.11
	62.50	1.32	1.11	1.22	0.15
	125.00	1.34	1.28	1.31	0.04
	250.00	1.49	1.49	1.49	0.00
	500.00	1.80	1.86	1.83	0.05	*
	1000.00	2.19	2.72	2.45	0.37	*
	2000.00	2.91	5.55	4.23	1.87

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

 

Additional Parameters

Table 5: Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5	256.25	253.54	254.89	1.92
Imax	2.91	5.55	4.23	1.87
IC30	n.a	1833.97	1833.97	n.a.
IC50	n.a	n.a	n.a	n.a

n.a=not applicable

Acceptance Criteria

Table 6: Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control [%]	< 20%	15.4	pass	9.6	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	= 1	2.0	pass	2.0	pass
EC1.5 PC [µM]	± 2 x SD of historical mean	20.57	pass	16.63	pass
Induction PC at 64 µM	2 .00 < x < 8.00	2.05	pass	6.65	pass

 

Historical Data

Table 7: Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.6	3.4	186
No. of positive control concentration steps with significant luciferase activity induction >1.5	= 1	2.3	0.6	186
EC1.5 PC	7 < x < 34 µM	19.1	5.9	186
Induction PC at 64 µM	2.00 < x < 8.00	3.8	1.4	186

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item induced the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item might 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:

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 first experiment, a max luciferase activity (I_max) induction of 1.66 was determined at a test item concentration of 500 µM. The corresponding cell viability determined by MTT staining was 149.8%. Microscopically, a clear cytotoxic effect was observed at the three highest test item concentrations (500, 1000 and 2000 µM) and a slight cytotoxic effect at a test item concentration of 250 µM. The increase in % viability may be caused by a slight MTT reducing potential of the test item, as determined in a MTT interference test. Therefore, the slight induction above the threshold of 1.5 may be caused by the cytotoxic effect.

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

To verify the results and as the test item induced the gene activity very close to the cytotoxic level in experiment 1, a third experiment with an adapted concentration range and a narrower dose-response analysis with dilution of 1.15-fold was performed to decide if induction is at cytotoxic levels or not.

In the third experiment, a max luciferase activity (I_max) induction of 2.61 was determined at a test item concentration of 756.14 µM. The corresponding cell viability was 87.6 %. The lowest tested concentration with a significant luciferase induction >1.5 (1.55) was found to be 326.90 µM. The corresponding cell viability was >70% (97.9%). The calculated EC_1.5 was <1000 µM (313.33 µM).

A dose response for luciferase activity induction was observed for experiment 1 and experiment 3.

Under the condition of this study the test item is therefore considered positive.

Reference 4

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-03-22 to 2021-06-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013-01-12

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

2019-06-18

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Justification for the Selection of the Test System: The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for assessing the sensitising potential of a chemical.

Justification for the Selection of the Test Method: This test method is able to detect chemicals that cause skin sensitisation and may be used on its own to classify a chemical into UN GHS “Category 1”. 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 an integrated approach such as IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the AOP.

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions:
20.2 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (40.319 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
20.55 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (39.672 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

- Preparation of the test chemical solutions:
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.

- Preparation of the positive controls, reference controls and co-elution controls:
Positive Control:
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
Co-elution Control:
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
Reference Control:
Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run.
Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each
HPLC run.
Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally, reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.

INCUBATION
- Incubation conditions: 25 ± 2.5 °C for 24 ± 2 h
- Precipitation noted: No precipitation was observed

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys:
A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile: 80% buffer ( v / v ) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions covering the range 0.000 - 0.534 Cys and Lys Peptide [mM].

- Verification of the suitability of the HPLC for test chemical and control substances: A single HPLC analysis for both the cysteine and the lysine peptide should be sufficient for a test chemical when the result is unequivocal. However, in cases of results close to the threshold used to discriminate between positive and negative results (i.e. borderline results), additional testing may be necessary. In situations where the mean percent depletion falls in the range of 3% to 10% for the cysteine, 1:10/lysine, 1:50 prediction model or the cysteine percent depletion falls in the range of 9% to 17% for the cysteine 1:10 prediction model, a second run should be considered, as well as a third one in case of discordant results between the first two runs.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

Turbidity and phase separation was observed for the samples of the co-elution control of the positive control. Samples were not centrifuged prior to the HPLC analysis. Since the depletion range of the positive control were fulfilled, the observed turbidity and phase separation were regarded as not relevant.
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 67.16%.

Key result

Group:

test chemical

Parameter:

other: mean deletion of cysteine and lysine

Value:

28.07 %

At concentration:

100 mM

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for reference controls A to C: yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: see tables below

The in chemico direct peptide reactivity assay (DPRA) enables detection of the hapten forming potential of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity to form haptens, by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers.

In the present study 1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 228.33 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 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. Slight turbidity was observed for the samples of the test item (excluding the co-elution control), but was judged to have negligible effects on the interpretation of the assay. Samples were not centrifuged prior to the HPLC analysis.

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. No precipitation, turbidity or phase separation was observed for the samples of the test item.

Turbidity and phase separation was observed for the samples of the co-elution control of the positive control. Samples were not centrifuged prior to the HPLC analysis. Since the depletion range of the positive control were fulfilled, the observed turbidity and phase separation were regarded as not relevant.

A slight turbidity in the cysteine experiment was observed. Since it cannot be determined if the precipitate resulted from the test item or the peptide, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

The 100 mM stock solution of the test item showed moderate reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (28.07%). Even though turbidity was observed a positive result can still be used. Based on the prediction model 1 the test item can be considered as positive in this assay.

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 67.16%.

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	17.0730	0.5340	14.2900	0.5340
STD2	8.6330	0.2670	7.2390	0.2670
STD3	4.3570	0.1335	3.5950	0.1335
STD4	2.1380	0.0667	1.7760	0.0667
STD5	1.0580	0.0334	0.8830	0.0334
STD6	0.4820	0.0167	0.4370	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	4.8680	0.1517	69.47
	4.8600	0.1514	69.52	69.73	0.41	0.59
	4.7500	0.1480	70.21
Test Item	7.1270	0.2222	55.30
	6.9980	0.2181	56.11	55.71	0.40	0.73
	7.0600	0.2201	55.72

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	4.6890	0.1747	64.84
	4.6500	0.1732	65.13	64.58	0.72	1.12
	4.8330	0.1801	63.76
Test Item	13.2970	0.4956	0.29
	13.2930	0.4955	0.32	0.43	0.21	49.47
	13.2460	0.4937	0.67

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	28.07	Moderate Reactivity	positive	55.71	Moderate Reactivity	positive
Positive Control	67.16	High Reactivity	positive	69.73	Moderate Reactivity	positive

Acceptance Criteria for Lysine Peptide

 

Lysine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² > 0.99	1.0000	pass
mean peptide concentration of RC A	0.45 ≤ x ≤ 0.55 mM	0.4998	pass
mean peptide concentration of RC C (PC)	0.45 ≤ x ≤ 0.55 mM	0.4971	pass
mean peptide concentration of RC C (TI)	0.45 ≤ x ≤ 0.55 mM	0.4971	pass
CV of the peak area of RC B	< 15%	0.31	pass
CV of the peak area of RC C (PC)	< 15%	0.12	pass
CV of the peak area of RC C (TI)	< 15%	0.12	pass
mean peptide depletion of the PC	40.2% < x < 69.0%	64.58	pass
SD of peptide depletion of the PC replicates	< 11.6%	0.72	pass
SD of peptide depletion of the TI replicates	< 11.6%	0.21	pass

Historical Data Cysteine Peptide

 

Cysteine Peptide
	mean	SD	N
linearity of the calibration curve	0.9996	0.0009	75
mean peptide concentration of reference A [mM]	0.5096	0.0156	75
mean peptide concentration of reference C [mM]	0.5042	0.0151	75
CV of the peak area of control B [%]	1.71	1.45	75
CV of the peak area of control C [%]	1.17	1.05	75
mean peptide depletion of the PC [%]	71.07	4.21	75
SD of peptide depletion of the PC replicates [%]	1.10	4.82	75
SD of peptide depletion of the test items [%]	1.17	2.26	251

Historical Data Lysine Peptide

 

Lysine Peptide
	mean	SD	N
linearity of the calibration curve	0.9999	0.0001	64
mean peptide concentration of reference A [mM]	0.4989	0.0110	64
mean peptide concentration of reference C [mM]	0.4978	0.0115	64
CV of the peak area of control B [%]	0.77	1.08	64
CV of the peak area of control C [%]	0.59	0.74	64
mean peptide depletion of the PC [%]	60.17	4.23	65
SD of peptide depletion of the PC replicates [%]	1.29	1.44	65
SD of peptide depletion of the test items [%]	1.17	6.97	215

Results of the Reference Controls for the Cysteine Peptide

 

Cysteine Peptide Run
Sample	Peptide Peak Area				Peptide Concentration [mM]
	PA	Mean	SD	CV [%]	Peptide Concentration	Mean	SD	CV [%]
Reference A 1	16.4220	16.2250	0.1708	1.05	0.5122	0.5060	0.0053	1.05
Reference A 2	16.1350				0.5032
Reference A 3	16.1180				0.5027
Reference B 1	15.5440	15.6530	0.2608	1.67	0.4848	0.4882	0.0081	1.67
Reference B 2	16.1020				0.5022
Reference B 3	[9.758]				-
Reference B 4	15.6450				0.4879
Reference B 5	15.4460				0.4817
Reference B 6	15.5280				0.4843
Reference C 1 (PC solvent)	16.1340	15.9457	0.1905	1.19	0.5032	0.4973	0.0059	1.20
Reference C 2 (PC solvent)	15.9500				0.4974
Reference C 3 (PC solvent)	15.7530				0.4913
Reference C 1 (TI solvent)	16.1340	15.9457	0.1905	1.19	0.5032	0.4973	0.0059	1.20
Reference C 2 (TI solvent)	15.9500				0.4974
Reference C 3 (TI solvent)	15.7530				0.4913

Results of the Reference Controls for the Lysine Peptide

Lysine Peptide Run
Sample	Peptide Peak Area				Peptide Concentration [mM]
	PA	Mean	SD	CV [%]	Peptide Concentration	Mean	SD	CV [%]
Reference A 1	13.3930	13.4103	0.0551	0.41	0.4992	0.4998	0.0021	0.41
Reference A 2	13.4720				0.5021
Reference A 3	13.3660				0.4982
Reference B 1	13.4160	13.3627	0.0413	0.31	0.5001	0.4981	0.0015	0.31
Reference B 2	13.3670				0.4982
Reference B 3	13.4080				0.4998
Reference B 4	13.3210				0.4965
Reference B 5	13.3310				0.4969
Reference B 6	13.3330				0.4970
Reference C 1 (PC solvent)	13.3400	13.3360	0.0154	0.12	0.4972	0.4971	0.0006	0.12
Reference C 2 (PC solvent)	13.3190				0.4964
Reference C 3 (PC solvent)	13.3490				0.4976
Reference C 1 (TI solvent)	13.3400	13.3360	0.0154	0.12	0.4972	0.4971	0.0006	0.12
Reference C 2 (TI solvent)	13.3190				0.4964
Reference C 3 (TI solvent)	13.3490				0.4976

Interpretation of results:

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

Conclusions:

In this study under the given conditions the test item showed moderate reactivity towards both peptides. The test item is considered as positive in this assay.
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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

DPNG is classified as sensitizers according to methods outlined in the Guideline on Defined Approaches (DA) for Skin Sensitization (Guideline 497). 

This conclusion was based on a positive response in the Direct Peptide Reactivity Assay (DPRA) (OECD 422C)  and the Kerotinosens® (OECD 422E) assay and applying the 2o3 DA logic. 

The DPRA assay assesses the potential for a test substance to react with proteins and form haptens.  This is Key Element (KE) 1 of the Adverse Outcome Pathway for dermal sensitization.  In the DPRA assay, depletion of target peptides signals chemical reactivity which may lead to immunoreactivity.  Higher depletion levels signifies greater potency.  DPNG depletion of the cysteine peptide was 28%.  This values exceeds the minimum cutoff of 6.38% and has moderate reactivity according to the test guidelines for the DPRA. 

The Kerotinose® assay assesses the potential to activate keratinocytes, KE2 in the AOP for dermal sensitization.  In the Kerotinsens® assay activation of keratinocytes, is  measured by luminescence from luciferase in a transgenic cell line.  The luminescence activity is defined as the concentration of the test substance causing a 50% increase above background.  This concentration is referred to and the EC1.5.  Test substances with lower EC1.5 values are more potent in this assay than those with higher EC1.5 values.  The EC1.5 concentrations for DPNG was 253 micromolar.  THis is less than the 1000 micromolar cutoff and therefore positive in this assay. 

As a result of these two assays, DPNG is considered sensitizer. The studies used are all Klimisch Category 1 (reliable without restrictions), internally consistent with the positive and negative controls used and all had clearly positive results (no borderline findings). DPNG was negative in the h-CLAT assay, however, two positive assays lead to the conclusion that DPNG is a sensitiser.

We supplemented support for these findings by using Read-Across prediction models within OECD Toolbox (v4.5).  By Read-Across, DPNG is predicted to be sensitizer.  It should be noted that this prediction is limited because the Kow value for DPNG is outside the range of Kow values for the read-across categories generated for each molecule. However, by using the metabolic profilers in the OECD Toolbox, reactive subgroups were identified that identified relevant structural alerts that were in common with the analogs in the respective categories and a prediction could be made – see attached prediction reports.

To determine potency according to the DA, the combined normalized scores from the DPRA and hCLAT scores are used to discriminate between Cat 1A and 1B.  For h-CLAT, the minimum induction threshold (MIT) is converted to a score from 0 to 3 based on the cutoffs of 10 and 150 μg/ml. For DPRA, the mean percent depletion for the cysteine and lysine peptides, or the cysteine peptide alone, is converted to a score from 0 to 3, based on the threshold values associated with reactivity.  The scores to be assigned are identified in Table 3.1 (page 25 of 54) in the DA.  A positive prediction from the OECD Toolbox application generates a score of 1 and a negative prediction would generate a score of 0.

Based on the updated DIP, a total battery score is assigned into three ranks:

• score of 6-7 is defined as a strong (UN GHS Cat. 1A) sensitiser;


• score of 2-5 as moderate/weak (UN GHS Cat. 1B) sensitiser;


• score of 1 or 0, as not classified (i.e. a non-sensitiser).

The combined normalized scores for DPNG;

 

Endpoint	DPNG
h-CLAT (mg/mL)	NC
Normalized Score	0
DPRA (Mean Cysteine & Lyseine % Depletion)	28.07
Normalized Score	2
DPRA (Cysteine % Depletion)	55.71
Normalized Score	NC
In Silico Prediction (OECD Toolbox v 4.5)	1
Total Normalized Score	3

 

Based on the normalized scores from the DA, DPNG is classified as 1B sensitizers.