4-hydroxybenzaldehyde

Endpoint summary

Description of key information

Skin sensitisation

Based on the results of an in chemico/in vitro test strategy the test item is not peptide reactive (DPRA test, OECD TG 442C) and does not activate keratinocytes ( ARE-Nrf2 luciferase test, OECD TG 422D). Therefore, the substance is predicted to be no skin sensitizer (reference 7.4.1 -1 and -2).

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:

2017-10-19 to 2018-01-18

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))

Qualifier:

according to guideline

Guideline:

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

Details on the study design:

Skin sensitisation (In chemico test system)
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. In the present study the test material was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 122.12 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.

Preparation of the cysteine or lysine-containing peptides:
Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides of purity higher than 95% were freshly prepared just before their incubation with the test item. The final concentration of the cysteine peptide was 0.666 mM in pH 7.5 phosphate buffer, whereas the final concentration of the lysine peptide was 0.668 mM in pH 10.2 ammonium acetate buffer

Positive control
Cinnamic aldehyde (CAS no. 14371-10-9) was used as positive control (PC) at a concentration of 100 mM in acetonitrile.

HPLC measurement
see "Other information on material and methods"

Acceptance Criteria
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.


 

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 63.81% (experiment 1) and 68.63% (experiment 2).

Key result

Run / experiment:

other: cysteine run (experiment 1)

Parameter:

other: mean peptide depletion [%]

Value:

0.12

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysine run (experiment 1)

Parameter:

other: mean peptide depletion [%]

Value:

13.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: cysteine run (experiment 2)

Parameter:

other: mean peptide depletion [%]

Value:

0.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysine run (experiment 2)

Parameter:

other: mean peptide depletion [%]

Value:

4.43

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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

Cysteine and Lysine Values of the Calibration Curve Experiment 1

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	4523.9644	0.5340	4169.8604	0.5340
STD2	2255.1311	0.2670	2102.4409	0.2670
STD3	1081.2599	0.1335	1026.2726	0.1335
STD4	538.3588	0.0667	515.4506	0.0667
STD5	264.6839	0.0334	262.0216	0.0334
STD6	131.3584	0.0167	131.2270	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Cysteine and Lysine Values of the Calibration Curve Experiment 2

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	17.870	0.5340	4663.5073	0.5340
STD2	9.012	0.2670	2361.9275	0.2670
STD3	4.518	0.1335	1192.6749	0.1335
STD4	2.252	0.0667	602.5394	0.0667
STD5	1.101	0.0334	302.3090	0.0334
STD6	0.530	0.0167	151.3044	0.0167
STD7	0.000	0.0000	0.0000	0.0000

Depletion of the Cysteine Peptide Experiment 1

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1320.4471	0.1577	70.95	71.10	0.18	0.25
	1315.4908	0.1571	71.06
	1304.7782	0.1559	71.29
Test Item	4600.2661	0.5437	0.00	0.12	0.20	173.21
	4583.2500	0.5417	0.00
	4529.0562	0.5353	0.35

Depletion of the Cysteine Peptide Experiment 2

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.447	0.1325	74.33	74.15	0.18	0.25
	4.477	0.1334	74.15
	4.510	0.1344	73.96
Test Item	17.353	0.5175	0.00	0.30	0.52	173.21
	17.167	0.5120	0.89
	17.396	0.5188	0.00

Depletion of the Lysine Peptide Experiment 1

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1690.0214	0.2164	56.37	56.51	0.47	0.83
	1699.1774	0.2175	56.14
	1664.2920	0.2131	57.04
Test Item	3573.1260	0.4572	7.76	13.20	5.57	42.20
	3372.0542	0.4315	12.95
	3141.8584	0.4020	18.89

Depletion of the Lysine Peptide Experiment 2

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1539.7554	0.1748	64.31	63.12	1.08	1.71
	1602.9802	0.1820	62.84
	1630.8385	0.1852	62.20
Test Item	4162.2075	0.4752	3.52	4.43	0.86	19.40
	4118.6553	0.4702	4.53
	4088.5010	0.4667	5.23

Prediction Model 1

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

Mean Cystein and Lysine PPD	Reactivity Class	DPRA Prediction (2)
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	Reactivity Class	DPRA Prediction (2)
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 Experiment 1

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	6.66	Low Reactivity	sensitiser	0.12	Minimal Reactivity	no sensitiser
Positive Control	63.81	High Reactivity	sensitiser	71.10	Moderate Reactivity	sensitiser

Categorization of the Test Item Experiment 2

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	2.36	Minimal Reactivity	no sensitiser	0.30	Minimal Reactivity	no sensitiser
Positive Control	68.63	High Reactivity	sensitiser	74.15	Moderate Reactivity	sensitiser

Interpretation of results:

other: peptide depletion negative

Remarks:

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.

Conclusions:

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

Executive summary:

A study was conducted according to OECD TG 442C in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C. The test item was dissolved at a concentration of 100 mM in acetonitrile. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (6.66%). Based on the prediction model 1 the test item might be considered as sensitiser. According to the evaluation criteria in the guideline, for test items with combined cysteine/lysine peptide depletion between 3% and 10% a second run was performed

In the second experiment 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (2.36%). 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 68.63%. The controls confirmed the validity of the study for both, the cysteine and lysine run. As this experiment shows that the test item exhibits minimal reactivity (2.36%), the slightly positive result (6.66%) in the former experiment is not regarded as having a higher impact but a lower one. Therefore, the test item can be considered as non-sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-10-25 to 2017-11-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 04, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Details on the study design:

Details on study design:
The test described in the OECD Guideline 442D is proposed to address the second key event in the Adverse Outcome Pathway (AOP) underlying skin sensitisation. The KeratinoSens™ assay addresses the effect on the antioxidant response element (ARE)-dependent pathway in the KeratinoSens™ cell line by measuring the induction of an ARE dependent gene product, the luciferase gene.

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 8 in experiment 1; P 11 in experiment 2) were used.
Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2. For test item exposure, cells were cultured in medium for test item exposure.

Preparation of the test and control items:
The test item was dissolved in dimethyl sulfoxide (DMSO). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. 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.
A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test. DMSO 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. Cinnamic aldehyde was used as positive control. CA was dissolved in DMSO 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.

Dose Groups
Negative Control:              
DMSO: 1% (v/v) in test item exposure medium
Positive Control: cinnamic aldehyde: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
Test Item: 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

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

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

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

Data evaluation:
The following parameters were calculated:
-the maximal average fold induction of the luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control;
-the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained; and
-the IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p <0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells. The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30% reduction on cellular viability at the EC1.5 determining concentration.

Acceptance criteria:
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 μM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

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

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was 3.95 in experiment 1 and 3.17 in experiment 2.

Key result

Run / experiment:

other: Experiment 1 (62.50 µM)

Parameter:

other: luciferase activity

Value:

1.31

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 2 (62.50 µM)

Parameter:

other: luciferase activity

Value:

1.42

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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

Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100	100	100	0.0
Positive Control	4.00	100.2	99.2	99.7	0.7
	8.00	112.2	104.1	108.2	5.7
	16.00	124.3	106.0	115.1	12.9
	32.00	129.6	111.4	120.5	12.9
	64.00	135.1	112.8	124.0	15.7
Test Item	0.98	93.4	99.5	96.4	4.3
	1.95	88.1	92.9	90.5	3.4
	3.91	101.8	103.2	102.5	0.9
	7.81	99.6	99.8	99.7	0.1
	15.63	108.6	101.1	104.8	5.3
	31.25	112.0	105.4	108.7	4.7
	62.50	114.0	104.3	109.1	6.9
	125.00	113.9	109.0	111.4	3.5
	250.00	113.8	105.1	109.4	6.2
	500.00	106.3	104.0	105.1	1.6
	1000.00	107.6	98.3	102.9	6.6
	2000.00	113.2	96.9	105.1	11.5

Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.12	1.18	1.22	1.17	0.05
	8.00	1.31	1.29	1.18	1.26	0.07
	16.00	1.47	1.55	1.47	1.50	0.04
	32.00	2.12	1.98	2.17	2.09	0.10	*
	64.00	3.92	3.82	4.10	3.95	0.14	*
Test Item	0.98	0.83	0.88	0.88	0.86	0.03
	1.95	0.99	0.87	0.84	0.90	0.08
	3.91	1.05	0.96	0.99	1.00	0.05
	7.81	1.12	1.14	1.04	1.10	0.05
	15.63	1.10	1.04	1.12	1.08	0.04
	31.25	1.08	1.02	1.23	1.11	0.11
	62.50	1.17	1.23	1.52	1.31	0.19
	125.00	1.22	1.11	1.22	1.18	0.07
	250.00	1.46	1.32	1.13	1.30	0.17
	500.00	1.20	1.07	1.23	1.17	0.09
	1000.00	0.95	1.16	1.05	1.05	0.11
	2000.00	0.92	0.92	0.99	0.94	0.04

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

 

Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.38	1.32	1.31	1.34	0.04
	8.00	1.33	1.35	0.78	1.16	0.33
	16.00	1.68	1.69	1.62	1.66	0.04	*
	32.00	2.15	2.19	2.13	2.16	0.03	*
	64.00	4.07	4.17	1.27	3.17	1.65
Test Item	0.98	1.16	1.12	0.50	0.93	0.37
	1.95	1.25	1.14	0.31	0.90	0.51
	3.91	1.16	1.09	0.17	0.81	0.55
	7.81	1.16	1.10	0.85	1.04	0.17
	15.63	1.17	1.34	1.16	1.22	0.10
	31.25	1.47	1.09	1.33	1.30	0.19
	62.50	1.50	1.50	1.25	1.42	0.15
	125.00	1.22	1.38	0.93	1.18	0.23
	250.00	0.97	1.22	0.68	0.95	0.27
	500.00	0.95	1.26	1.15	1.12	0.15
	1000.00	0.95	0.71	1.17	0.94	0.23
	2000.00	0.91	0.81	1.02	0.91	0.10

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

Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.17	1.34	1.25	0.12
	8.00	1.26	1.16	1.21	0.07
	16.00	1.50	1.66	1.58	0.12	*
	32.00	2.09	2.16	2.13	0.05	*
	64.00	3.95	3.17	3.56	0.55	*
Test Item	0.98	0.86	0.93	0.89	0.05
	1.95	0.90	0.90	0.90	0.00
	3.91	1.00	0.81	0.90	0.13
	7.81	1.10	1.04	1.07	0.04
	15.63	1.08	1.22	1.15	0.10
	31.25	1.11	1.30	1.20	0.13
	62.50	1.31	1.42	1.36	0.08
	125.00	1.18	1.18	1.18	0.00
	250.00	1.30	0.95	1.13	0.25
	500.00	1.17	1.12	1.14	0.03
	1000.00	1.05	0.94	1.00	0.08
	2000.00	0.94	0.91	0.93	0.02

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

 

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	n/a	n/a	n/a	n/a
Imax	1.31	1.42	1.36	0.08
IC30[µM]	n/a	n/a	n/a	n/a
IC50[µM]	n/a	n/a	n/a	n/a

n/a: not applicable

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	7.8	pass	15.8	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.0	pass	2.0	pass
EC1.5 PC	7 < x < 34 µM	16.09	pass	13.42	pass
Induction PC at 64 µM	2.00 < x < 8.00	3.95	pass	3.17	pass

Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.3	3.3	41
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.3	0.6	41
EC1.5 PC	7 < x < 34 µM	20.4	6.7	41
Induction PC at 64 µM	2.00 < x < 8.00	3.3	1.1	41

 

Interpretation of results:

other: no activation of keratinocytes

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:

A study according OECD TG 442D was conducted. The performed 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 the test item was dissolved in DMSO. Based on a molecular weight of 122.12 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 (Imax) induction of 1.31 was determined at a test item concentration of 62.50μM. The corresponding cell viability was 114%. No luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 could be calculated.

In the second experiment, a max luciferase activity (Imax) induction of 1.42 was determined at a test item concentration of 62.50μM. The corresponding cell viability was 104.3%. No luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 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. 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 not be 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

For the evaluation of the skin sensitisation potential of the test substance a Weight of Evidence approach was used. 

DPRA test

A study was conducted according to OECD TG 442C in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C. The test item was dissolved at a concentration of 100 mM in acetonitrile. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (6.66%). Based on the prediction model 1 the test item might be considered as sensitiser. According to the evaluation criteria in the guideline, for test items with combined cysteine/lysine peptide depletion between 3% and 10% a second run was performed

In the second experiment 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (2.36%). 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 68.63%. The controls confirmed the validity of the study for both, the cysteine and lysine run. As this experiment shows that the test item exhibits minimal reactivity (2.36%), the slightly positive result (6.66%) in the former experiment is not regarded as having a higher impact but a lower one. Therefore, the test item can be considered as non-sensitiser.

ARE-Nrf2 luciferase test method

A study according OECD TG 442D was conducted. The performed 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 the test item was dissolved in DMSO. Based on a molecular weight of 122.12 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 (Imax) induction of 1.31 was determined at a test item concentration of 62.50μM. The corresponding cell viability was 114%. No luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 could be calculated.

In the second experiment, a max luciferase activity (Imax) induction of 1.42 was determined at a test item concentration of 62.50μM. The corresponding cell viability was 104.3%. No luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 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. 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.

Conclusion:

Based on the results from the in chemico and in vitro studies and considering the Adverse Outcome Pathway (AOP) the test item in predicted as non-sensitizer.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data the test item is not classified as skin sensitising according to Regulation (EC) No 1272/2008 (CLP), as amended for fourteenth time in Regulation (EU) No 2020/217.