Benzamide, N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxy-

Endpoint summary

Description of key information

Skin Sensitization:

 

In vivo

OECD 406, GPMT: not sensitising

 

In vitro

Key event 2, OECD 442D, LuSens: negative

Key event 3, OECD 442E, h-CLAT: negative

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 - 14 Feb 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

non-GLP

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

no

Remarks:

The study is supplementary to the main study, which is fully reliable on its own. The study is not intended to replace the already existing in vivo study for skin sensitization, only to substantiate the data package.

Type of study:

ARE-Nrf2 luciferase LuSens test method

Details of test system:

Lusens transgenic cell line [442D]

Details on the study design:

CELL LINE
- The LuSens cell-line is an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The plasmid contains a luciferase gene (reporter gene) which is under transcriptional control of an antioxidant response element (ARE) of the rat NQO1 gene. Genes dependent on the ARE such as NQO1 are known to be upregulated by contact sensitisers.
- The HaCaT human keratinocytes (provided by RWTH, Aachen, Germany) were genetically modified at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of Wruck). The LuSens cells were obtained by BASF and in 2019 a contract services agreement was established between ICCR-Roßdorf GmbH (Licensee) and Promega.
- The passage numbers of the used LuSens cells were 9 in the cytotoxicity test and 11 in the LuSens test for the main experiments 1 and 2, respectively.

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: on the day of the experiment (immediately before treatment) the test-item was dissolved or stably dispersed/suspended in DMSO to prepare a stock solution with a concentration of 200 mM in accordance to the OECD Guideline 442D.
- Preparation of the test chemical serial dilutions: for the MTT test (dose finding assay) twelve concentrations of the test item were analysed. Therefore, dilutions were prepared by 1:2 serial dilutions from the highest soluble/dispersible concentration.
Six test item concentrations were tested in the main experiments. The highest concentration used was CV75 × 1.2. Five further test item dilutions were prepared by serial dilution with a dilution factor of 1.2.
- Preparation of the positive controls: EGDMA (final concentration 120 µM), CAS 97-90-5, purity: ≥ 97.5%. The solvent for the positive control was Treatment Medium including 1% (v/v) DMSO
- Preparation of the solvent control: DMSO (final concentration 1% (v/v) in Treatment Medium), purity: ≥ 99%.
- Preparation of the negative control: Lactic acid (final concentration 5000 µM), CAS 50-21-5, purity: ~ 90%. The solvent for the negative control was Treatment Medium including 1% (v/v) DMSO.

CULTURE MEDIUM
DMEM with supplements listed below was used to culture the cells during the assay. The culture medium was warmed to room temperature just before use.
- Cultivation Medium: DMEM culture medium with Penicillin/Streptomycin (1% v/v), FBS (10% v/v) and puromycin (0.005% v/v)
- Seeding Medium: DMEM culture medium with FBS (10% v/v)
- Treatment Medium: DMEM culture medium with FBS (1% v/v)

MTT DOSE RANGE FINDING ASSAY
- One cytotoxicity experiment (dose finding assay) was performed to obtain a CV75, if possible.
- The MTT working solution consisted of two components, the MTT stock solution (5 mg/mL MTT in Ca2+/Mg2+ free DPBS) and Treatment Medium. Both components were mixed right before application at a ratio of 1:10.
- For the treatment of the cells in the dose finding assay a stock solution of the test item and the controls were prepared. The test item was dissolved in the solvent and 1:2 serially diluted in the solvent to obtain the desired test item concentrations (twelve concentrations).
- The solvent (twelve replicates), the positive (two replicates) and the negative (three replicates) controls as well as the test item concentrations (each three replicates) were subsequently diluted 1:25 in Treatment Medium. The final test-item concentrations in the test medium were as follows:
0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µM
- 24 hours ± 30 minutes after seeding of the cells, the Seeding Medium were removed from the wells. Thereafter, 150 µL Treatment Medium were added per well and 50 µL of the test item dilutions, the solvent, negative and positive controls (1:25 dilution in Treatment Medium) and
the medium control (six replicates) were added to the wells, respectively. At the end of the incubation period of 48 ± 1 hours under standard cell culture conditions, the cell cultures were microscopically evaluated for morphological alterations, precipitation or phase separation.
- At the end of the incubation period, Treatment Medium was removed from the wells and the cells were washed at least twice with 200 µL DPBS including Ca2+/Mg2+. Thereafter, 200 µL of the MTT working solution were added to each treatment well and the cells were incubated for 3
hours ± 30 minutes under standard cell culture conditions. After rinsing the MTT working solution, the cells of each well were treated with 100 µL MTT lysis agent (Isopropanol with 0.04 N HCl) for at least 30 minutes, while gently shaking. Thereafter the microplate was transferred to a microplate reader (Multimode Reader (TriStar2 LB 942) by Berthold Technologies GmbH Co KG, Germany) equipped with a 570 nm filter to measure the absorbance (reference wavelength 690 nm).
- Cell viability of the test-item treated cells was calculated relative to the solvent control.
- The MTT assay was considered to be acceptable if it meets the following criteria: mean absorbance of the solvent control is ≥ 0.2.

MAIN EXPERIMENT (LuSens and MTT):
- Number of replicates: test item (three replicates per concentration, solvent control (twenty-four replicates), positive control (five replicates) and negative control (six replicates).
- Number of repetitions: 2 independent experiments.
- Test chemical concentrations: 321, 385, 462, 554, 665 and 798 µM
- Preparation and Seeding of Cells conditions: between 4 and 6 x 10E+5 or 6 and 8 x 10E+5 cells were seeded in 15 mL Cultivation Medium and pre-cultured at least twice a week in culture flasks (80 – 90% confluent). The cell density between approximately 80 and 90% should not be exceeded.
After microscopic assessment of the cells, the cells were washed at least twice with 10 mL Ca2+/Mg2+ free DPBS including EDTA. Thereafter, the cells were trypsinated with 1 to 2 mL 0.05% Trypsin/EDTA for approximately 5 minutes at 37 ± 1.5 °C and 5.0 ± 0.5 % CO2. The cells were resuspended in 10 mL Cultivation Medium to neutralise the trypsin.
For seeding of the cells, the Cultivation Medium was removed and the cells were transferred into Seeding Medium. Each treatment well of a 96 well microtiter plate was seeded with 100 µL cell suspension (9000 to 11000 LuSens cells per well), except the well of the blank control. The cells were incubated for 24 hours ± 30 minutes under standard cell culture conditions.
- Incubation conditions: 37 ± 1.5 °C and 5.0 ± 0.5 % CO2
- Application procedure: after incubation of the LuSens cells, Seeding Medium was removed and 150 µL of Treatment Medium was distributed in each well. Thereafter, 50 µL of the test item and control dilutions and the medium control (twelve replicates) were added into the corresponding wells.
- Exposure time: 48 ± 1 hours

LUCIFERASE ACTIVITY MEASUREMENTS
- The Steady-Glo® Mix was be prepared by adding Steady-Glo® buffer to one bottle of SteadyGlo® Substrate and mixing by inversion until the substrate was dissolved. The Steady-Glo® working solution was prepared by mixing one part of DPBS (without Ca2+/Mg2+) with one part
of Steady-Glo®-Mix.
At the end of the incubation period, the Treatment Medium was removed from the wells and the cells were washed at least twice with 200 µL DPBS including Ca2+/Mg2+. Thereafter, 200 µL of the Steady-Glo® working solution was added in each well. After slowly shaking of the microtiter
plate for at least 10 min in the dark, the plate was transferred to a microplate reader and the luminescence was measured for 2 seconds per well.
- Luminometer: Multimode Reader (TriStar2 LB 942) by Berthold Technologies GmbH Co KG, Germany.

MTT VIABILITY ASSAY
- The treatment of the cells with the MTT labelling mixture and the measurement of the cell viability for each main experiment was performed as described above in the dose range finder experiment.

PREDICTION MODEL
If the luciferase induction is ≥ 1.5 fold and statistically significant compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations (cell viability ≥ 70%) and at least 3 tested concentrations are non-cytotoxic, the main experiment of the LuSens prediction is considered positive.
If these conditions are met in 2 of 2 or in 2 of 3 main experiments, the LuSens prediction is considered positive, otherwise the LuSens prediction is considered negative.
A negative result obtained with a test item that does not form a stable dispersion and was not tested up to 2000 μM (or 2000 μg/mL for test items with no defined MW) and for which no cytotoxicity is observed in any of the tested concentration should be considered as inconclusive (OECD 442E).
If no clear dose-response curve or a biphasic dose-response curve is observed, the experiment should be repeated to verify whether this is specific to the test item or due to an experimental artefact. If the biphasic response (i.e. when the threshold of 1.5 is crossed twice) is reproducible in an independent verification experiment, the lower concentration of a ≥ 1.5 induction should be reported (i.e. when the threshold of 1.5 is crossed the first time).
Mono constituent test items with a Log Pow > 7 may be insoluble in the culture medium. However, if the test item is soluble or can be stably dispersed/suspended, the LuSens test may be performed.

ACCEPTABILITY CRITERIA
The following acceptance criteria should be met in the LuSens test method (OECD 442D):
• The average luciferase activity induction obtained with the positive control, 120 μM EGDMA should be ≥ 2.5, and the positive control should have a relative cell viability ≥ 70% as compared to the solvent control.
• The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid, as well as the basal expression of untreated cells should be < 1.5 fold as compared to the average solvent control.
• The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) should be below 20% in each main experiment.
• At least three test item concentrations should have cell viability of at least 70% relative to the solvent controls. Moreover, in case a result is to be considered negative, at least one concentration should be cytotoxic, i.e. have a cell viability < 70%, or the maximum concentration of 2000 μM (or 2000 μg/ mL for substances with no defined MW) should have been tested.

Vehicle / solvent control:

DMSO

Negative control:

DL-Lactic acid

Positive control:

other: EGDMA (120 µM) [442D]

Positive control results:

The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (Experiment 1: 5.812; Experiment 2: 5.817).
The positive control had a relative cell viability ≥ 70% as compared to the solvent control (Experiment 1: 101.842; Experiment 2: 80.545).

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Cell viability:

Cell viability was < 70% at 665 and 798 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: luciferase induction was < 1.5 fold compared to the solvent control at all the tested concentrations

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Cell viability:

Cell viability was > 70% at all tested concentrations

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: luciferase induction was < 1.5 fold compared to the solvent control at all the tested concentrations

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

CYTOTOXICITY DOSE RANGE FINDER:
- In the cytotoxicity test, cytotoxic effects were observed following incubation with the test item starting with the concentration of 1000 µM up to the highest tested concentration of 2000 µM (threshold of cytotoxicity: < 75%). The CV75 value of the cytotoxicity test was calculated as 665.1 µM.

MAIN EXPERIMENT (LuSens and MTT):
- MTT cytotoxicity assay: in Experiment 1, cytotoxicity (cell viability < 70%) was observed at 665 and 798 µM. In Experiment 2, no cytotoxicity was observed (cell viability was >70% at all tested concentrations).
- LuSens assay: after treatment with the test item for 48 ± 1 hours the luciferase induction was < 1.5 fold compared to the solvent control in at least 2 consecutive tested concentrations. Since these conditions are met in 2 out of 2 main experiments, the LuSens prediction was considered negative (tabulated results are presented in section "any other information on results incl. tables").

ACCEPTANCE OF RESULTS:
The acceptance criteria were met:
- The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 1: 5.812; ME 2: 5.817).
- The positive control had a relative cell viability ≥ 70% as compared to the solvent control (ME 1: 101.842; ME 2: 80.545).
- The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid, as well as the basal expression of untreated cells was < 1.5 fold as compared to the average solvent control (ME 1: 1.023; ME 2: 0.967).
- At least three test concentrations had a cell viability of at least 70% relative to the solvent/vehicle controls. Moreover, since the result is considered negative, at least one concentration was cytotoxic, i.e. had a cell viability < 70%, or the maximum concentration of 2000 μM have been tested.
- Historical control data is included in the attached background material.

Table 1: Results of the Dose Finding Assay (Cytotoxicity Test)

Treatment Group	Concentration			OD570			Mean OD570	SD OD570	Mean OD570 blank corr.	Cell viability [%]
				Well 1	Well 2	Well 3
Blank	-			0.015	-	-	-	-	-	-
Solvent control	-			-	-	-	0.598	0.035	0.583	100.00
Medium control	-			-	-	-	0.811	0.081	0.796	136.67
Positive Control		120	µM	0.608	0.659	-	0.634	0.036	0.619	106.15
Negative Control		5000	µM	0.630	0.647	0.574	0.617	0.038	0.602	103.32
Test Item	C1	0.98	µM	0.527	0.628	0.547	0.567	0.053	0.552	94.79
	C2	1.95	µM	0.602	0.502	0.498	0.534	0.059	0.519	89.07
	C3	3.91	µM	0.600	0.589	0.598	0.596	0.006	0.581	99.66
	C4	7.81	µM	0.498	0.523	0.503	0.508	0.013	0.493	84.61
	C5	15.6	µM	0.536	0.580	0.476	0.531	0.052	0.516	88.50
	C6	31.3	µM	0.491	0.556	0.532	0.526	0.033	0.511	87.76
	C7	62.5	µM	0.562	0.556	0.541	0.553	0.011	0.538	92.33
	C8	125	µM	0.529	0.622	0.604	0.585	0.049	0.570	97.83
	C9	250	µM	0.516	0.488	0.504	0.503	0.014	0.488	83.70
	C10	500	µM	0.483	0.460	0.448	0.464	0.018	0.449	77.00
	C11	1000	µM	0.441	0.451	0.393	0.428	0.031	0.413	70.94
	C12	2000	µM	0.337	0.297	0.335	0.323	0.023	0.308	52.86

The CV75 value of the cytotoxicity test was calculated as 665.1 µM.

 

Table 2: Results of the main experiment 1 (Cell viability)

Treatment Group	Concentration			OD570						Mean OD570	SD OD570	Mean OD570 blank corr.	Cell viability [%]
				Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank	-			0.014	-	-	-	-	-	-	-	-	-
Solvent control	-			-	-	-	-	-	-	0.350	0.056	0.336	100.00
Medium control	-			-	-	-	-	-	-	0.337	0.089	0.323	96.15
Positive Control		120	µM	0.282	0.372	0.430	0.298	0.400	-	0.356	0.064	0.342	101.84
Negative Control		5000	µM	0.457	0.373	0.393	0.362	0.269	0.369	0.371	0.061	0.357	106.04
Test Item	C1	321	µM	0.430	0.428	0.440	-	-	-	0.433	0.006	0.419	124.53
	C2	385	µM	0.399	0.370	0.377	-	-	-	0.382	0.015	0.368	109.46
	C3	462	µM	0.426	0.385	0.378	-	-	-	0.396	0.026	0.382	113.72
	C4	554	µM	0.311	0.349	0.360	-	-	-	0.340	0.026	0.326	96.96
	C5	665	µM	0.172	0.177	0.183	-	-	-	0.177	0.006	0.163	48.58
	C6	798	µM	0.130	0.197	0.175	-	-	-	0.167	0.034	0.153	45.61

 

Table 3: Results of the main experiment 1 (Fold induction)

Treatment Group	Concentration			Luminescence						Mean Luminescence	SD Luminescence	Mean Luminescence blank corr.	Fold Induction
				Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank	-			177	-	-	-	-	-	-	-	-	-
Solvent control	-			-	-	-	-	-	-	292.792	22.853	115.792	1.00
Medium control	-			-	-	-	-	-	-	324.583	18.676	147.583	1.275
Positive Control		120	µM	835	776	732	939	968		850.000	101.821	673.000	5.812
Negative Control		5000	µM	288	333	266	288	273	325	295.500	27.443	118.500	1.023
Test Item	C1	321	µM	303	296	310	-	-	-	303.000	7.000	126.000	1.088
	C2	385	µM	281	266	296	-	-	-	281.000	15.000	104.000	0.898
	C3	462	µM	296	273	318	-	-	-	295.667	22.502	118.667	1.025
	C4	554	µM	310	333	325	-	-	-	322.667	11.676	145.667	1.258
	C5	665	µM	266	273	355	-	-	-	298.000	49.487	121.000	1.045
	C6	798	µM	273	303	288	-	-	-	288.000	15.000	111.000	0.959

 

Table 4: Results of the main experiment 2 (Cell viability)

Treatment Group	Concentration			OD570						Mean OD570	SD OD570	Mean OD570 blank corr.	Cell viability [%]
				Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank	-			0.014	-	-	-	-	-	-	-	-	-
Solvent control	-			-	-	-	-	-	-	0.534	0.128	0.520	100.00
Medium control	-			-	-	-	-	-	-	0.605	0.115	0.591	113.70
Positive Control		120	µM	0.486	0.528	0.413	0.390	0.346	-	0.433	0.074	0.419	80.55
Negative Control		5000	µM	0.541	0.611	0.576	0.554	0.495	0.496	0.546	0.045	0.532	102.27
Test Item	C1	321	µM	0.496	0.524	0.463	-	-	-	0.494	0.031	0.480	92.42
	C2	385	µM	0.504	0.501	0.476	-	-	-	0.494	0.015	0.480	92.30
	C3	462	µM	0.500	0.594	0.558	-	-	-	0.551	0.047	0.537	103.26
	C4	554	µM	0.479	0.484	0.489	-	-	-	0.484	0.005	0.470	90.44
	C5	665	µM	0.367	0.484	0.438	-	-	-	0.430	0.059	0.416	79.98
	C6	798	µM	0.395	0.471	0.446	-	-	-	0.437	0.039	0.423	81.46

 

Table 5: Results of the main experiment 2 (Fold induction)

Treatment Group	Concentration			Luminescence						Mean Luminescence	SD Luminescence	Mean Luminescence blank corr.	Fold Induction
				Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank	-			155	-	-	-	-	-	-	-	-	-
Solvent control	-			-	-	-	-	-	-	263.542	25.044	108.542	1.00
Medium control	-			-	-	-	-	-	-	278.500	25.508	123.500	1.138
Positive Control		120	µM	732	909	761	791	739	-	786.400	72.290	631.400	5.817
Negative Control		5000	µM	229	259	266	244	281	281	260.000	20.669	105.000	0.967
Test Item	C1	321	µM	347	244	273	-	-	-	288.000	53.113	133.000	1.225
	C2	385	µM	303	288	222	-	-	-	271.000	43.093	116.000	1.069
	C3	462	µM	303	296	266	-	-	-	288.333	19.655	133.333	1.228
	C4	554	µM	251	236	236	-	-	-	241.000	8.660	86.000	0.792
	C5	665	µM	244	251	251	-	-	-	248.667	4.041	93.667	0.863
	C6	798	µM	266	266	273	-	-	-	268.333	4.041	113.333	1.044

 

Interpretation of results:

other: no skin sensitising potential based on the key event “inflammatory response in keratinocytes”

Conclusions:

The study was performed according to OECD guideline 442D. The test item did not activate the LuSens cells up to a concentration of 798 µg/mL under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 - 31 Jan 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

non-GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 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:

June 2018

Deviations:

no

GLP compliance:

no

Remarks:

The study is supplementary to the main study, which is fully reliable on its own. The study is not intended to replace the already existing in vivo study for skin sensitization, only to substantiate the data package.

Type of study:

human Cell Line Activation Test (h-CLAT)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

TEST CELL LINE:
- Source: ATCC (American Type Culture Collection), ATCC no.: TIB-202

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: on the day of the experiment AE0001789 was dissolved in DMSO
- Preparation of the test chemical serial dilutions: stock solutions were further diluted in culture medium to reach the desired final test concentration (final concentration of DMSO in culture medium = 0.2% (v/v))
- Preparation of the positive controls: stock solution prepared in DMSO and then further diluted in culture medium to a final concentration of 3 and 4 µ/mL (final concentration of DMSO in culture medium = 0.2% (v/v))
- Preparation of the solvent: DMSO (final concentration for cytotoxicity and for h-CLAT 0.2%)
- Log Kow of the test chemical: 2.5

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 250 µg/mL in 0.2% (v/v) DMSO in culture medium
- For the cytotoxicity test (dose finding assay) eight concentrations of the test item were analysed (1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125 and 250 µg/mL) For this, dilutions of the stock solution were prepared by 1:2 serial dilutions
- Solubility in incubation medium: as determined in a solubility pretest, 250 µg/mL was the highest soluble concentration in the incubation medium
- Results of selecting appropriate concentration and determination of cytotoxicity e.g. CV75: as no cytotoxicity was observed in the dose range finder, the highest concentration selected for the main experiment was 250 µg/mL (highest soluble concentration)

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3
- Number of repetitions: 3
- Test chemical concentrations: 69.8, 83.7, 100, 121, 145, 174, 208 and 250 µg/mL
For the test item exposure, the highest soluble test item concentration of the cytotoxicity test (without precipitations) was used instead of 1.2 × CV75, since no CV75 could be determined.
A further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. Each solution was diluted with culture medium before application of the test solution to the cells to reach a final concentration of 0.2% (v/v) DMSO in the medium.
- Application procedure: each volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells
- Exposure time: 24 ± 0.5 hours

SEEDING, INCUBATION AND STAINING
- Seeding conditions: on the day of the cytotoxicity or main experiment (h-CLAT) directly before the treatment of the cells, a volume of 500 µL with a cell density of 1.8 - 2 × 10E+6 THP-1 cells/mL was seeded in each corresponding well of a 24-well flat bottom plate.
The passage numbers of the used THP-1 cells were 10 and 12 in the cytotoxicity tests and 13, 15 and 16 in the h-CLAT for runs 1, 2 and 3, respectively.
- Culture medium: RPMI 1640 Medium, GlutaMAXTM Supplement including 25 mM HEPES, supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin) was used to culture the cells during the assay
- Incubation conditions: 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere
- Washing conditions: the triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA).
Thereafter, the cells were centrifuged, re-suspended and blocked with 600 µL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 µL FACS buffer. The cells were stained with FITC labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).
After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-amino-actinomycin D (7-AAD) solution were added.

MEASUREMENT OF CELL SURFACE EXPRESSION
- Flow cytometry used: FACSCalibur, Becton Dickinson GmbH

DATA EVALUATION
- The relative fluorescence intensity (RFI) was used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical:

RFI (%) = ((MFI of test item treated cells) - (MFI of test item treated isotype control cells) / (MFI of solvent control cells) - (MFI of solvent isotype control cells)) * 100

MFI = Geometric Mean Flourescence Intensity (GeoMean)

- The cell viability from the isotype control cells, CD54 and CD86 cells is calculated according to the following equation:

Cell Viability (%) = (Number of living cells / Number of acquired cells) * 100

Where only the isotype control cells (which are stained with mouse IgG1 (isotype) antibodies) are used for the cell viability evaluation.

The cytotoxicity test is considered to be acceptable if the cell viability of the medium and solvent control was more than 90%.

EVALUATION CRITERIA
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):

- The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
- The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).

Otherwise, the h-CLAT prediction is considered NEGATIVE

Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline).

A flow chart of the prediction model used in the h-CLAT test method is included as attached background material.

ACCEPTANCE CRITERIA
The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control and DMSO control should be more than 90%.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations.
• For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability is > 90% (OECD 442E guideline).

Vehicle / solvent control:

DMSO

Negative control:

other: culture medium

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was > 50%. Therefore, the acceptance criteria with respect to the positive control was fulfilled.

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC200, CD54 [442E]

Value:

217.6 %

Cell viability:

No cytotoxicity observed

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: RFI for CD54 exceeded 200 at 121 µg/mL

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC150, CD86 [442E]

Value:

158.7 %

Cell viability:

No cytotoxicity observed

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: RFI for CD86 exceeded 150 at 121 and 145 µg/mL

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC200, CD54 [442E]

Cell viability:

No cytotoxicity observed

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: RFI did not exceed 200 at any tested concentration

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC150, CD86 [442E]

Cell viability:

No cytotoxicity observed

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: RFI did not exceed 150 at any tested concentration

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC200, CD54 [442E]

Cell viability:

No cytotoxicity observed

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: RFI did not exceed 200 at any tested concentration

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC150, CD86 [442E]

Cell viability:

No cytotoxicity observed

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: RFI did not exceed 150 at any tested concentration

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

CYTOTOXICITY ASSAY:
Cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (250 µg/mL). Due to the lack of cytotoxicity, a CV75 value could not be calculated. Therefore, the highest soluble test item concentration (250 µg/mL) was used for the hCLAT runs.

h-CLAT ASSAY:
- In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
- The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.
- The RFI of CD86 or CD54 was not equal or greater than 150% and 200%, respectively at any dose in at least 2 out of 3 independent runs. Therefore the h-CLAT prediction is considered negative for the tested test item in this study.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (see Annex 2 in the attached study results)
- Acceptance criteria met for positive control: yes (see Annex 2 in the attached study results)
- Historical control data: see Annex 3 in the attached study results for the laboratories historical control data

Dose Range Finding Assay

 

Table 1: Results of the first Cytotoxicity Test

Test Group	Concentration (µg/mL)	Microscopic Evaluation /Cytotoxicity	Flow Cytometric Evaluation / Cell Viability [%]
Medium control	-	no	92.15
DMSO control	-	no	94.26
Test item	1.95	no	97.19
	3.91	no	96.67
	7.81	no	95.76
	15.63	no	94.72
	31.25	no	90.17
	62.50	no	93.81
	125.00	no	94.71
	250.00	no	87.97

Due to the lack of cytotoxicity in the Flow Cytometric Evaluation of the Cytotoxicity Test, a CV75 value could not be calculated.

 

Table 2: Results of the second Cytotoxicity Test

Test Group	Concentration (µg/mL)	Microscopic Evaluation /Cytotoxicity	Flow Cytometric Evaluation / Cell Viability [%]
Medium control	-	no	95.52
DMSO control	-	no	96.10
Test item	1.95	no	97.31
	3.91	no	97.84
	7.81	no	96.35
	15.63	no	95.27
	31.25	no	92.57
	62.50	no	96.04
	125.00	no	94.34
	250.00	no	89.27

Due to the lack of cytotoxicity in the Flow Cytometric Evaluation of the Cytotoxicity Test, a CV75 value could not be calculated.

Therefore the highest test item concentration of the cytotoxicity tests (250 µg/mL) is used for the h-CLAT runs.

 

Results of the h-CLAT Test

 

Table 3: Results of the first h-CLAT run

Test Group	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium control	-	100.0	100.0	98.31
DMSO control	-	100.0	100.0	98.05
Positive control (DNCB)	3.0	554.9*	410.5*	86.27
	4.0	962.7*	484.6*	84.23
Test item	69.8	100.0	83.7	97.44
	83.7	129.4	120.2	97.71
	100.0	125.5	151.5	98.08
	121.0	217.6*	152.7*	97.33
	145.0	149.0	158.7*	97.29
	174.0	141.2	134.9	95.54
	208.0	198.0	122.9	94.49
	250.0	174.5	115.4	94.33

* RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

 

Table 4: Results of the second h-CLAT run

Test Group	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium control	-	100.0	100.0	98.36
DMSO control	-	100.0	100.0	98.01
Positive control (DNCB)	3.0	309.1*	557.5*	91.06
	4.0	395.5*	515.8*	90.28
Test item	69.8	104.5	105.4	96.68
	83.7	97.0	113.5	97.72
	100.0	78.8	102.3	97.30
	121.0	71.2	103.5	97.04
	145.0	87.9	98.8	97.10
	174.0	106.1	93.1	97.05
	208.0	113.6	101.9	95.45
	250.0	115.2	99.2	94.68

* RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

 

Table 5: Results of the third h-CLAT run

Test Group	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium control	-	100.0	100.0	97.44
DMSO control	-	100.0	100.0	97.44
Positive control (DNCB)	3.0	388.6*	634.1*	88.93
	4.0	484.8*	647.3*	86.28
Test item	69.8	101.3	122.0	96.72
	83.7	94.9	110.2	96.66
	100.0	67.1	103.9	94.51
	121.0	88.6	125.9	93.92
	145.0	86.1	109.8	96.23
	174.0	89.9	111.7	95.84
	208.0	92.4	106.8	94.68
	250.0	86.1	117.1	93.43

* RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Interpretation of results:

other: no skin sensitising potential based on the key event “dendritic cell activation”

Conclusions:

The study was performed according to OECD guideline 442E. The test item did not activate THP-1 cells up to a concentration of 250 µg/mL under the test conditions of this study. Therefore, the test item is considered negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Feb - 18 Mar 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted 17 Jul 1992

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Principles of method if other than guideline:

According to OECD 406, the concentration of the test substance used for each induction exposure should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation. In the present study, after the intradermal induction the animals in the control group and the test item group showed red wheal and white wheal with red surrounding up to encrustation at the injection sites of the first induction. The topical induction led to no skin effects in the animals of the control group and of the test item group. In these circumstances, sodium lauryl sulfate should be used to induce skin irritation prior to topical induction with the test substance. However, 7 days after intradermal induction the injection sites in the test group the application sites still presented with wheals and encrustation. As topical induction was conducted at the same site as intradermal induction, and irritation was already present, then using sodium lauryl sulfate to induce further irritation can be considered unnecessary.

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A non-LLNA test is available that was performed prior to the current data requirements, stipulated in Regulation (EC) No 1907/2006. In accordance with the same Regulation, the data was included to avoid unnecessary testing.

Species:

guinea pig

Strain:

other: Crl: HA

Sex:

female

Details on test animals and environmental conditions:

Source: Charles River, 88353 KiBlegg, Germany (females were nulliparous and non-pregnant).
- Microbiological status of animals: Only healthy animals exhibiting no clinical signs were used for the study.
Age at study initiation: 4 - 5 weeks
Body weight at start of study: 250 - 358 g
Diet: "PROVIMI KLIBA 3420 - Maintenance Diet for Guinea Pigs", ad libitum.
Water: Tap water from polycarbonate bottles, ad libitum
Acclimatization period: At least 5 days.
Housing: Type IV Makrolon® cages in groups of five during the adaptation period and in groups of two or three per cage
throughout the study period. Cages were exchanged for ones with clean bedding at least two times per week. Low dust wood shaving was used as
bedding.

ENVIRONMENTAL CONDITIONS
Room temperature (°C): 22 ± 3
Air humidity (%) : 40 - 70
Ventilation (per hr): approx. 10 air changes
Light/ Dark cycle (hrs dark / hrs light): 12/12

Route:

intradermal

Vehicle:

polyethylene glycol

Concentration / amount:

5%

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Concentration / amount:

50%

Day(s)/duration:

48 hours

Adequacy of induction:

other: The topical induction led to no skin irritation effects, however, wheals and encrustation were still present at the induction site following the intradermal induction.

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Concentration / amount:

50%

Day(s)/duration:

24

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 (controls), 20 (in test groups)

Details on study design:

Preliminary:
RANGE FINDING TESTS:
Intradermal induction with 0.1 mL of 0, 1, 2.5 and 5 % produced white wheal with red surrounding after 24 and 48 h.
Topical induction with 0.5 mL of 0, 12, 25 and 50 % test substance formulation under occlusive dressing for 24 h produced no skin reaction at 24 and 48 h after patch removal.
Challenge treatment with 0, 12, 25 and 50% test formulation under semiocclusive conditions for 24 h showed no skin reactions at 24 and 48 h after patch removal.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: Single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections):
Injection 1: 1:1 mixture (v/v) FCA/sterile physiological saline
Injection 2: 5 % test substance in polyethylene glycol 400
Injection 3: 5 % test substance in a 1:1 mixture (v/v) FCA/polyethylene glycol 400

Epicutaneous: 0.5mL 50 % test substance in polyethylene glycol 400

- Control group:
Intradermal (3 pairs of injections):
Injection 1: 1:1 mixture (v/v) FCA/sterile physiological saline
Injection 2: Polyethylene glycol 400
Injection 3: 1:1 mixture (v/v) FCA/polyethylene glycol 400

Epicutaneous: 0.5 mL polyethylene glycol 400

- Site: shoulder region (intradermal + epicutaneous)
- Frequency of applications: single
- Duration: Days 1-8

B. CHALLENGE EXPOSURE
- No. of exposures: Single challenge.
- Day(s) of challenge: 21 days after the intradermal induction.
- Exposure period: 24 h
- Test groups: Test substance and vehicle only.
- Control group: Test substance and vehicle only.
- Site: Right flank (caudal) test substance and control groups; right flank (cranial) vehicle only as control.
- Concentrations: 50%
- Evaluation (hr after challenge): 24 and 48 h after patch removal.

OTHER: The animals were observed for clinical signs at least once daily throughout the entire study period. The body weights of the animals were recorded on Day 1 before the first induction and at the end of the study (Day 25 in the test item group and control group and Day 18 in the range-finding group.

Challenge controls:

The control group is actually a challenge control.

Positive control substance(s):

yes

Remarks:

12% alpha hexyl cinnamic aldehyde formulated in polyethylene glycol 400.

Positive control results:

Positive control tests from a seperate study confirmed the sensitivity of the test system. Challenge with 12 % alpha hexyl cinnamic acid produced skin reaction scores of 1-3 at 24 and 48 h after patch removal (100 % response) compared with no effects at the control patches thus confirming the sensitivity of the test system.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

5%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

5%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

5%

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

5%

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

General Examinations:
Appearance and behaviour of the test item group were not different from the control group.

At the end of the study, the mean body weight of the treatment group animals was in the same range than that of the control group animals.

After the intradermal induction (first induction) the animals in the control group and the test item group showed after 48 hours:
• red wheal
• white wheal with red surrounding
After 7 days the following effects were recorded at the injection sites in the control group and in the test item group: wheals and encrustations.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

The study was performed in accordance to OECD TG 406 under GLP conditions and is considered reliable. The test substance was determined to be non-sensitising.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In Vivo

Skin sensitising properties of the test substance were evaluated in guinea pigs according to the maximization method of Magnusson and Kligman (OECD 406) in compliance with GLP (M-249757-01-1, 2005). Concentrations of 5 and 50% were chosen for intradermal and epicutaneous induction, respectively. The test group comprised 20 and the control group 10 male guinea pigs (strain Crl: HA). On Day 1, 3 pairs of intradermal injections were performed in the interscapular region of all animals:

1) Freund's adjuvant diluted at 50% with sterile physiological saline solution (treated and control groups).

2) Test substance at a concentration of 5% in polyethylene glycol 400 (treated group) or vehicle alone (control group),

3) Test substance at a concentration of 5% in a mixture Freund's adjuvant and polyethylene glycol (treated group) or a mixture Freund's adjuvant and polyethylene glycol (control group).

On Day 8, the test substance (treated group) or the vehicle (control group) was applied topically and covered by an occlusive dressing for 48 hours. The challenge with 50% occurred on the right flank of all animals on Day 22. Test substance was held in place by an occlusive dressing for 24 hours. No mortality or signs of clinical symptoms were recorded until the end of the observation period and body weight gain was comparable among the groups. No cutaneous reactions were observed in control and test substance treated animals. Thus, the test substance did not induce skin sensitisation in guinea pigs under the experimental conditions chosen.

 

In Vitro

Two in vitro skin sensitization studies were conducted, addressing key event 2 (inflammatory response in keratinocytes, OECD 442D) and key advent 3 (activation of dendritic cells, OECD 442E) of the skin sensitization adverse outcome pathway.

Key Event 2:

The study was conducted in accordance to OECD guideline 442D, but not in compliance with GLP (M-761831-01-1, 2020). This in vitro Skin Sensitisation Test "ARE-Nrf2 Luciferase Test Method (LuSens)" was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization AOP) of the test item.

In the cytotoxicity test, cytotoxic effects were observed following incubation with the test item starting with the concentration of 1000 µM up to the highest tested concentration of 2000 µM (threshold of cytotoxicity: < 75%). The CV75 value of the cytotoxicity test was calculated as 665.1 µM.

The test item was tested in 2 independent main experiments. The following concentrations of the test item were tested in the main experiments (LuSens):

321, 385, 462, 554, 665 and 798 µM

After treatment with the test item for 48 ± 1 hours the luciferase induction is < 1.5 fold compared to the solvent control in at least 2 consecutive tested concentrations. Since these conditions are met in 2 out of 2 main experiments, the LuSens prediction is considered negative.

The acceptance criteria of the assay were met:

- The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 1: 5.812; ME 2: 5.817).

- The positive control had a relative cell viability ≥ 70% as compared to the solvent control (ME 1: 101.842; ME 2: 80.545).

- The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid, as well as the basal expression of untreated cells was < 1.5 fold as compared to the average solvent control (ME 1: 1.023; ME 2: 0.967).

- At least three test concentrations had a cell viability of at least 70% relative to the solvent/vehicle controls. Moreover, since the result is considered negative, at least one concentration was cytotoxic, i.e. had a cell viability < 70%, or the maximum concentration of 2000 μM have been tested.

In conclusion, the test item did not activate the LuSens cells up to a concentration of 798 µg/mL under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Key Event 3:

The study was conducted in accordance to OECD guideline 442E, but not in compliance with GLP (M-761830-01-1, 2020). This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of the test item.

The test item was dissolved in 0.2% (v/v) DMSO in culture medium when administered to THP-1 cells for 24 ± 0.5 hours.

The highest test item concentration for the main experiment (h-CLAT) was determined by two cytotoxicity tests. Cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (250 µg/mL). Due to the lack of cytotoxicity, a CV75 value could not be calculated. Therefore, the highest soluble test item concentration (250 µg/mL) was used for the h-CLAT runs.

The following concentrations of the test item were tested in the main experiments (h-CLAT):

69.8; 83.7; 100; 121; 145; 174; 208 and 250 µg/mL

The test item with a log Pow of 2.5 was tested in 3 independent runs. The RFI of CD86 or CD54 was not equal or greater than 150% and 200%, respectively at any dose in at least 2 out of 3 independent runs. Therefore, the h-CLAT prediction is considered negative for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (2,4-dinitrochlorobenzene) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. Therefore, the acceptability criteria for the solvent and positive control was met, demonstrating the test system functioned correctly.

In conclusion, the test item did not activate THP-1 cells up to a concentration of 250 µg/mL under the test conditions of this study. Therefore, the test item is considered negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Conclusions

There is both in vitro and in vivo data available for the registered substance in which skin sensitization potential has been investigated. In vitro data for key events 2 and 3 of the adverse outcome pathway for skin sensitization (inflammatory response in keratinocytes and activation of dendritic cells, respectively) were both negative, therefore, 2 out of 3 of in chemico/in vitro testing battery are confirmed as negative. The findings in these in vitro studies correlate with the results of the in vivo study (GPMT), in which no evidence of skin sensitization was observed. Overall the available data confirms that there is no concern for skin sensitization for the registered substance. 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.