[No public or meaningful name is available]

Endpoint summary

Description of key information

OECD TG 442E (Gijsbrechts, 2021): not sensitizing 

OECD TG 442D (Gijsbrechts, 2021): not sensitizing 

 

The in vitro skin sensitization studies conducted were conclusive to classify the test substance as not sensitizing to the skin according to CLP REgualtion (EC) No 1272/2008. 

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:

08 December 2020 to 19 Feb 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted June 2018

Deviations:

no

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

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:

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.

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

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

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

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

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

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 December 2020 to 08 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 183: U937 Cell Line Activation Test for Skin Sensitization (U-SENSTM)

Version / remarks:

Adopted October, 2017

Deviations:

not specified

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:

25 June 2018

Deviations:

yes

Remarks:

Before acquisition 5 uL of a 0.5ug/mL Propidium Iodide (PI) solution was added, this deviated from the TG which states the use of PI solution with a final concentration of 3ug/mL. This deviation is not expected to impact the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

U937 cell line activation test (U-SENS™)

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended for skin sensitization to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the U-SENSTM assay, which is recommended by the OECD

Details of test system:

U-937 cell line [442E]

Details on the study design:

442E

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test item was dissolved in complete medium at 0.4 mg/mL
- Preparation of the test chemical serial dilutions: The stock was diluted to final test concentration of 200, 100, 50, 20, 10 and 1 ug/mL in the 96 well plate
- Preparation of the positive controls: A TNBS 10 mg/mL solution was prepared in RPMI, the solution was diluted 1:100 in order to obtain a 0.1 mg/mL stock solution where the final dose level was 50 ug/mL
- Preparation of the negative control: A lactic acid 10 mg/mL solution was prepared in RPMI medium. The solution was diluted 1:25 in order to obtain a 0.4 mg/mL stock solution where the final dose level was 200 ug/mL
- Log Kow of the test chemical: Not stated


DOSE RANGE FINDING ASSAY:
- Highest concentration used: 200 ug/mL
- Solubility in solvents: Not stated
- Solubility in incubation medium: Not stated
- Final concentration range selected on basis of individual results of previous runs

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: Two replicates of the test item with three replicates of untreated control, vehicle control, negative control and positive control
- Number of repetitions: Two valid experiments
- Test chemical concentrations: 1.0, 10, 20, 50, 100 and 200 ug/mL
- Exposure time: 45± 3 hours with the selected doses or controls
- Description on study acceptance criteria: the acceptability criteria outlined in the OECD TG


MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT and USENS
- Flow cytometry used: Just before acquisition, 5 μL of a 0.5 μg/mL propidium iodide (PI) solution was added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. For the acquisition the BD FACSCantoTM flow cytometer was used and for further analysis BD FACSDivaTM software was used. All analysis parameters were set on the RPMI wells for IgG1 and remained unchanged, for the analysis of all the other wells. The P1 region was adjusted if necessary in a SSC (X-axis) and FSC (Y-axis) plot. The P2 region was defined for the PI negative cells among P1 in a histogram with counts (Y-axis) and PI fluorescence (X-axis). The amount of cytotoxicity was analyzed as percentage of cells in P2. The P2 region was then plotted in a Dot-plot as fluorescence (X-axis) and SSC (Y-axis) and a quadrant was placed according acceptability criterion b. The percentage of cells in the UR quadrant was used to calculate the stimulation index.
- Plate used: 96 well plate

Vehicle / solvent control:

cell culture medium

Negative control:

DL-Lactic acid

Positive control:

picrylsulfonic acid/2,4,6-trinitro-benzene-sulfonic acid (TNBS) [442E]

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

CV70 [442E]

Value:

0 µg/mL

Cell viability:

No cytotoxicity was observed

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC150, CD86 [442E]

Value:

0 µg/mL

Cell viability:

No cytotoxicity observed

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC150, CD86 [442E]

Value:

0 µg/mL

Cell viability:

No toxicity observed

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

CV70 [442E]

Value:

0 µg/mL

Cell viability:

No toxicity was observed

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: Not stated

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

Table 1: Overview Stimulation index of CD86 and Cell Viability in Experiment 1 and 2 of NC01 (Test substance)

Test item	Dose	% Viability (Mean)*		CD86-IgG1 S.1. *		Colour Interference S.I.*
		Experiment		Experiment		Experiment
NC01		1	2	1	2	1	2
	1	99	100	184	111	102	104
	10	99	100	86	55	102	107
	20	99	100	129	83	99	104
	50	99	99	73	97	100	104
	100	100	100	116	125	100	107
	200	100	100	110	74	99	107

* Red vlaues are either below 70% viability or above 150 S.I

 

Table 2: Overview Stimulation index of CD86 and Cell viability in Experiment 1 and 2 of Positive, Negative and Vehicle Control

 

Controls	% Viability (Mean)*		CD86-IgG1 S.I.*
	Experiment		Experiment
	1	2	1	2
LA1	99	100	86	74
LA2	100	100	116	60
LA3	99	100	153	74
TNBS1	99	99	1114	1163
TNBS2	100	100	869	1265
TNBS3	99	100	857	1034
	IgG1 Value (%)		CD86 basal expression (%)		CD86-IgG1 expression (%)
	Experiment		Experiment		Experiment
	1	2	1	2	1	2
RPMI1	0.8	0.6	2.4	2.5	1.6	1.9
RPMI2	1.0	1.4	2.6	3.6	1.6	2.2
RPMI3	0.8	0.8	2.5	3.2	1.7	2.4
RPMI Mean Viability			99	100
RPMI Drift			6%	17%
LA Drift			52%	10%

* Red values are either below 70% viability, above 150 S.I.

 

 

Table 3: Overview EC150 and CV70 Values

 

	EC150(ug/mL)	CV70 (ug/mL)
Test item experiment 1	NA	NA
Test item Experiment 2	NA	NA

NA= Not applicable

 

Interpretation of results:

GHS criteria not met

Remarks:

Under the experimental conditions of the study, the test item was not considered to have a skin sensitising potential.

Conclusions:

Under the conditions of this study, the test item is classed as negative in the U-Sens(TM) assay since no biologically relevant inductions were observed at all test concentrations with a cell viability of <70% compared to the vehicle control.

Executive summary:

The study was performed according to OECD TG 442E under GLP to assess the skin sensitization potential of the test item to U937 human monocytes. In duplicate experiments the test material was assessed at dose of concentrations of between 1.0 and 200 ug/m. In the first experiment the test item showed no toxicity (No CV₇₀ value), an induction of the CD86 activity was measured at the lowest concentration only. Since no clear dose relation was observed, the increase (184%) was only slightly above the cut-off value of 150%.

In the second experiment, the test item showed no toxicity (No CV70 value). No induction of CD86 was not reproducible in the second experiment and therefore the increase was not considered to biologically relevant. Additionally, no biologically relevant induction of the CD86 activity (No EC150 value) was measured at any of the test concentrations. Therefore, this run is considered negative. Under the conditions of the study, the test item was not considered to increase the expression of the CD86 cell surface marker in the U937 cell line.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin Sensitization

Assessment for Skin Sensitization was conducted according to OECD TG 442E: In Vitro skin sensitization: U937 Cell Line Activiation Test (U-Sens^TM). The test item was applied to U937 human monocytes at a concentration of 1.0 and 200 ug/mL. The results indicated that there was no biologically relevant induction of CD86 activity (No Ec₁₅₀ value) with the cell viability  >70% when compared to the control. However, no prediction on the potential for skin sensitization could be made with additional testing required in order to conclude on the classification of the substance. 

In accordance with the Integrated Testing Strategy an additional In Vitro Skin Sensitization study was required in order to fullfil the acceptability criteria of 2 or 3 of 4 Key events (KE) in the OECD Integrated Approaches To Testing and Assessment Adverse Outcome Pathways. Secondary assessment was undertaken according to OECD TG 442D: In Vitro Skin Sensitization: ARE-Nrf2 Luciferase Test Method. The test item was applied to the transgenic cell line at concentrations of 0.98 to 2000 µM. The results indicated no toxicity (no IC₃₀ and no IC₅₀ value) and no biologically relevant indiction of the luciferase activity (no EC_1.5 vlaue) at any concentrations. Under the conditions of the test the test substance showed no skin seneitization. 

The results of both in vitro studies provided adequate informtion to classify the substance with no further testing required. The test item was not classified in accordance with the CLP Regulation (EC) No 1272/2008. 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The Skin Sensitization potential was established following results obtained from OECD TG 442E and OECD TG 442D. Based on the conditions of the test, the test item does not meet the criteria for classification as a Skin Sensitizer according to the CLP Regualtion (EC) No 1272/2008.