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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

485-320-2

EC Name:

-

Cas Number:

221667-31-8

Molecular formula:

C18H18N205S

IUPAC Name:

N-[4-(cyclopropylcarbamoyl)benzenesulfonyl]-2-methoxybenzamide

In vitro test system

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]

Results and discussion

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).

In vitro / in chemico

Results

Results 1

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

Results 2

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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).