[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 December 2020 to 19 Feb 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSensTM.

Version / remarks:

Last update: 23 July 2018

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxy-3-methylbenzenesulphonic acid

Cas Number:

7134-04-5

Molecular formula:

C7H8O4S

IUPAC Name:

4-hydroxy-3-methylbenzenesulphonic acid

Test material form:

solid

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

442D

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test item was dissolved in DMSO. To a final concentration of 200 mM (a clear colourless solution)
- Preparation of the test chemical serial dilutions: From the stock solution 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solutions were diluted 25 fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 2000, 1000, 500, 230, 125, 63, 31, 1, 7.8, 3.9, 2.0, 0.98 µM (final concentration DMSO of 1%)
- Preparation of the positive controls: the positive control used was Ethylene dimethacrylate glycol for which a 2-fold dilution series ranging 7.8 to 250 µM (final concentration DMSO of 1%)
- Preparation of the vehicle controls: the vehicle control was 1% DMSO in exposure medium
- Stable dispersion obtained: No precipitation was observed

DOSE RANGE FINDING ASSAY:
- Highest concentration used: NA
- Solubility in solvents: NA
- Solubility in incubation medium: NA
- Cytotoxicity assessment performed: NA
- Final concentration range selected on basis of: NA

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: Triplicate
- Number of repetitions Two experiments were performed
- Test chemical concentrations: 2000, 1000, 500, 230, 125, 63, 31, 1, 7.8, 3.9, 2.0, 0.98 µM
- Application procedure: 50µL of the test chemical was added to cells containing fresh exposure medium
- Exposure time:48 hr ± 1hr
- Study evaluation and decision criteria used: the Imax is equal or higher than 1.5-fold and statistically significant different as compared to the vehicle control. The cellular viability is higher (>) than 70% at the lowest concentration with induction of luciferase activity greater than or equal to 1.5-fold. The EC1.5 value is less than 1000µM (or <200µg/mL for test chemicals with no defined MW). There is an overall dose-response for luciferase induction.
- Description on study acceptance criteria: The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be statistically significant equal or above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 μM). The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2- fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control. Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded. If the variability is higher, a maximum of three of the eighteen wells may be excluded based on the Dixon’s Q-test. If the variability is still higher, the results should be discarded.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): Not stated
- Incubation conditions: All cells were incubated overnight in the incubator
- Washing conditions: Not stated
- Precipitation noted: No

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: TECAN Infinite® M200 Pro Plate Reader
- Plate used: Not stated
- Lysate preparation: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 μL of the Steady- Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 minutes at room temperature.


DATA EVALUATION
- Cytotoxicity assessment: For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh DMEM Glutamax containing MTT and cells were incubated for 3 - 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

other:

Results and discussion

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 1.88 and the EC1.5 139 μM.

Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.99 and the EC1.5 37 μM.

The EC1.5 of the positive control was within two standard deviations of the historical mean in the second experiment (i.e. 37 μM). In the first experiment the EC1.5 of the positive control was with 139 μM only slightly above the acceptable value of 120 μM.

A dose response was observed and the induction at 250 μM was higher than 2-fold in the second experiment (2.99-fold). Although, the induction at 250 μM was not higher than 2-fold in the first experiment (1.88-fold), a clear dose-response was observed with increasing luciferase activity induction at increasing concentrations, and therefore the experiment is considered acceptable.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Value:

0 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC30 [442D]

Value:

0 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Value:

0 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No damage on the test system was noted

DEMONSTRATION OF TECHNICAL PROFICIENCY: Historical data for the KeratinoSens(TM) studies performed in the laboratory have indicated technical proficiency

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Correct
- Acceptance criteria met for positive control: Correct

Any other information on results incl. tables

Table 1: Overview Luminescence Induction and Cell Viability of NC01 in Experiment 1 and 2

Concentration (µM)	0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000	2000
Exp 1 Luminescence	1.02	1.04	1.04	1.15	1.09	1.09	1.03	0.97	0.92	0.81	0.79	0.61
Exp 1 viability (%)	104	99	97	97	96	96	94	93	91	90	86	83
Exp 2 luminescence	0.95	1.05	1.04	1.09	1.13	1.15	1.07	1.06	1.00	0.94	0.84	0.80
Exp 2 viability (%)	95	98	95	95	91	92	88	90	93	94	89	91

 

Table 2: Overview Lumescence Induction and Cell Viability Positive Control EDMG in Experiement 1 and 2

Concentration (µM)	7.8	16	31	63	125	250
Exp 1 Luminescence	0.95	1.03	1.15	1.33	1.45	1.88***
Exp 1 viability (%)	99	93	90	91	92	86
Exp 2 luminescence	0.99	1.18	1.45	1.72***	2.17***	2.99***
Exp 2 viability (%)	103	101	104	107	104	105

*** = p<0.001 Students t-test

 

Table 2: Overview EC_1.5, I_max and IC₅₀ Values

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Test item Experiment 1	NA	1.15	NA	NA
Test Item Experiment 2	NA	1.15	NA	NA
Pos Control Experiment 1	139	1.88	NA	NA
Pos Control Experiment 2	37	2.99	NA	NA

NA = Not applicable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Under the experimental conditions, no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) by the test chemical was reported.

Conclusions:

Under the conditions of this study, the test item is not sensitising iand showed no toxicity and no biological relevent induction of the luciferase activity when measured at any of the concentrations. The test substance is therefore not classified according to CLP Regulation (EC) No 1271/2008.

Executive summary:

The study was performed according to OECD TG 442D under GLP to assess the second key event in the skin sensitization adverse outcome pathway (AOP )in keratinocytes. The test assesses inflammatory responses as well as gene expression associated with specific cell signalling (stress) pathways such as the antioxidant/ electrophile response element (ARE)-dependent pathways.

In duplicate experiments the KeratinoSens™ cell line was dosed with the test item at a concentration range of 0.98 to 2000 µM. Concurrent positive and vehicle controls were used.   Treated plates were incubated for 48 hours ± 1 h at 37±1.0°C in the presence of 5% CO2. In experiment 1 no precipitation was observed at the start and end of the incubation period in the 96-well plates. The test item showed no toxicity with the viability of the cells greater than 70% at all test concentrations and therefore no IC₃₀ and IC₅₀ values could be calculated. In addition no luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. Therefore the Imax was 1.15 and no EC1.5 could be calculated.

In experiment 2 no precipitation was observed at the start and end of the incubation period in the 96-well plates. Comparable to experiment 1 the test item showed no toxicity with the viability of the cells greater than 70% at all test concentrations and therefore no IC₃₀ and IC₅₀ values could be calculated. No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.15 and therefore no EC1.5 could be calculated. Under the conditions of the study, the test item showed no toxicity and no biological relevant induction of luciferase activity measured at any of the test concentrations in both experiment 1 and 2 and therefore classed as negative in the KeratinoSens^TM assay.