Poly(oxy-1,2-ethanediyl), α,α'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized

Endpoint summary

Description of key information

Based on the OECD GUIDANCE DOCUMENT ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION Series on Testing & Assessment No. 256 (ENV/JM/MONO(2016)29 and ENV/JM/MONO(2016)29/ANN1) an Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification (BASF) was chosen. This defined approach describes an integrated testing strategy (ITS) for the identification of the skin sensitisation hazard of a substance primarily for the purposes of classification and labelling without the use of animal testing. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification. Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.[Ref. CASE STUDY I of ENV/JM/MONO(2016)29/ANN1, Dr. Robert Landsiedel, BASF SE] The test item does neither activate the Nrf2-Keap1-ARE toxicity pathway (key event 2 of skin sensitization AOP) nor induce incresed number of cell surface markers CD54 and CD86 (key 3 of the skin sensitization AOP). In line with the "2 out of 3" integrated testing strategy approach to skin hazard identification presented classification of the test item as skin sensitizer is not justified.

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In vitro study in accordance with Testing and assessment strategy for evaluating the skin sensitisation potential of substances of Chapter R.7a: Endpoint specific guidance Version 6.0 – July 2017.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Name: Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized
Product Description: C10-16-alkyletherpropylamine, ethoxylated, DES Quat
CAS No.: 70983-58-3
Physical state: yellowish to amber viscous liquid at 20 °C
Batch No.: PFS-755-173
Re-certification date of batch: 19 April 2018
Purity: 100 % (UVCB)
Color, Gardner 8.8
pH, 5% in water 5.76
Acid Value , mg KOH/g 20.1
Moisture, % 0.127
Total Amine, mg/g 11.18
Viscosity,cps, #4@60,25C 3280
Appearance @25C pass
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Details on the study design:

The KeratinoSens assay is supposed to address the second key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP), the induction of cyto-protective signalling pathways in keratinocytes in response to electrophiles and oxidative stress. The KeratinoSens assay addresses the effect on the antioxidant response element (ARE)-dependent pathway in the KeratinoSens cell line by measuring the induction of an ARE dependent gene product, the luciferase gene. The luciferase gene induction following exposure to test chemicals is measured in cell lysates by luminescence detection, allowing the discrimination between sensitisers and non-sensitisers.

Preparation of the Test Item

All test item solutions were freshly prepared immediately prior to use.
Since the test item had no defined molecular weight, the test was performed using a pro forma molecular weight of 200 g/mol. A stock solution with a concentration of 40 mg/mL was prepared by pre-weighing the test material into a glass vial and dissolving the test item in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%, AppliChem, Lot. No.: 0000978834).
Vortex mixing was used to aid solubilisation.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent.
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.
 
Controls

A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.

Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.

Negative Control
DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.

Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

Cell line

The test was carried out using the transgenic cell line KeratinoSens (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 3 experiment 1 and 2) were used.
Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 +- 1°C and 5% CO2. For test material exposure, cells were cultured in medium.

Composition of Media

Maintenance Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum
- 1% geneticin (final concentration: 500 µg/mL)

Assay Medium

Dulbecco’s Modified Eagle Medium (GlutaMAX) (Gibco Life Science, Cat. No.: 21885-025, Lot No.: 1879620, 1880290) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum
 
Test Item Exposure Medium

Dulbecco’s Modified Eagle Medium (GlutaMAX) with 1.0 g/L D-glucose and Na-Pyruvate.
The medium was supplemented with the following components:
- 1% fetal bovine calf serum

Luciferase Assay System

The luciferase activity was determined using the following products purchased from Promega. All components were used according to the instructions of the manufacture manual.
Luciferase Assay System 10-Pack.

The kit consisted of the following components relevant for this study:
- 10 vials Luciferase Assay Substrate (lyophilized)
- 10 x 10 mL Luciferase Assay Buffer

If freshly prepared, Luciferase Assay Substrate was dissolved in Luciferase Assay Buffer.If thawed from -80 °C, Luciferase Assay Reagent was allowed to equilibrate to room temperature prior to use.
Luciferase Cell Culture Lysis 5x Reagent
The kit consisted of the following components relevant for this study:
- 30 mL Luciferase Cell Culture Lysis 5x Reagent

Prior to use lysis buffer was diluted 1:5 with dist. water.
10.7. Further Reagents

MTT solution
- MTT stock solution: 5 mg/mL MTT in DPBS

SDS solution:
- 10% (w/v) sodium dodecyl sulfate (SDS) in dist. water

DPBS:
DPBS solution (without Ca2+/Mg2+)

Dose Groups
1.Negative Control: DMSO: 1% (v/v) in test item exposure medium
2.Positive Control: CA: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
3.Test Item: 12 concentrations of the test item

Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run.

Experimental Procedure

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

Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS (Gibco Life Science; Lot No.: 1866243, 1877596). Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.

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

Calculation of EC1.5, IC50 and IC30, Cell Viability and Maximal Induction of the Luciferase Activity was performed according to OECD 442 d guideline.

Prediction Model

The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is < 200 µg/mL
- an apparent overall dose-response for luciferase induction

If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <200 µg/mL is considered as inconclusive.

Acceptance Criteria

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

Positive control results:

Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.

Negative Control
DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.

Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%) was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

Key result

Run / experiment:

other: mean of 2 experiments

Parameter:

other: EC1.5

Remarks:

[µM]

Value:

6.28

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: mean of 2 experiments

Parameter:

other: Imax

Value:

1.84

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: mean of 2 experiments

Parameter:

other: IC30

Remarks:

[µM]

Value:

7.77

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: mean of 2 experiments

Parameter:

other: IC50

Remarks:

[µM]

Value:

10.05

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: positive control - experiment 1

Parameter:

other: EC1.5

Remarks:

PC

Value:

24.29

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: positive control - experiment 2

Parameter:

other: EC1.5

Remarks:

PC

Value:

8.67

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results

The in vitro KeratinoSens assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers. In the present study the test item was dissolved in DMSO. Since the test item had no defined molecular weight, the test was be performed using a pro forma molecular weight of 200 g/mol. Based on this, a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium.

The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 1.80 was determined at a test item concentration of 7.81 µM. The corresponding cell viability was 75.9%. No further significant luciferase induction was observed. The calculated EC1.5 was < 200 µg/mL (6.35 µM corresponding to 1.27 µg/mL). In the second experiment, a max luciferase activity (Imax) induction of 1.87 was determined at a test item concentration of 7.81 µM. The corresponding cell viability was 58.0%. No further significant luciferase induction was observed. The calculated EC1.5 was < 200 µg/mL (6.21 µM corresponding to 1.24 µg/mL). No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non-sensitiser. The controls confirmed the validity of the study. The calculated EC1.5 was between 7 and 34 µM (24.29 µM in experiment 1; 8.67 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (9.6% in experiment 1; 10.4% in experiment 2). The luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment the luciferase activity was 2.27 and thus the criteria was fulfilled. In the second experiment the threshold was slightly increased with a fold induction of 8.59. As in both experiments the values of the test item were very comparable and all other acceptance criteria were met, this slight increase was accepted.

Table 1: Results of the cytotoxicity measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent control	-	100	100	100	-
Positive control	4.00 8.00 16.00 32.00 64.00	100.7 104.3 106.8 118.1 122.0	99.7 101.3 109.4 119.4 132.6	100.2 102.8 108.1 118.7 127.3	0.7 2.1 1.8 0.9 7.5
Test item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	98.9 114.0 115.2 75.9 15.9 0.2 0.4 0.8 1.1 1.3 1.6 1.5	106.2 108.4 113.3 58.0 1.3 0.3 0.5 0.7 0.8 1.2 1.3 1.1	102.6 111.2 114.3 66.9 8.6 0.2 0.4 0.7 1.0 1.2 1.4 1.3	5.2 3.9 1.3 12.6 10.3 0.0 0.1 0.0 0.2 0.1 0.2 0.3

Table 2: Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent control	-	1.00	1.00	1.00	1.00	0.00
Positive control	4.00 8.00 16.00 32.00 64.00	1.08 1.18 1.34 1.58 2.39	1.13 1.14 1.30 1.61 2.60	1.21 1.25 1.40 1.73 2.66	1.14 1.19 1.35 1.64 2.55	0.07 0.06 0.05 0.08 0.14	* *
Test item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	0.99 0.78 0.98 1.53 1.45 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.87 0.81 1.08 1.92 0.97 0.00 0.00 0.00 0.00 0.00 0.02 0.00	0.88 0.75 0.92 1.96 1.25 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.91 0.78 0.99 1.80 1.22 0.00 0.00 0.00 0.00 0.00 0.01 0.00	0.07 0.03 0.08 0.24 0.24 0.00 0.00 0.00 0.00 0.00 0.01 0.00	*

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

Table 3: Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent control	-	1.00	1.00	1.00	1.00	0.00
Positive control	4.00 8.00 16.00 32.00 64.00	1.18 1.37 1.76 3.11 6.71	1.42 1.41 2.08 3.79 9.59	1.01 1.59 2.05 3.43 9.47	1.20 1.46 1.96 3.44 8.59	0.20 0.12 0.18 0.34 1.63	* * *
Test item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	0.93 0.87 0.84 1.37 0.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.84 0.86 0.91 2.01 0.17 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.89 1.01 1.14 2.24 0.09 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.89 0.91 0.96 1.87 0.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.04 0.09 0.16 0.45 0.04 0.00 0.00 0.00 0.00 0.00 0.00 0.00

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

Table 4: Induction of Luciferase Activity – Overall Induction

Overall Induction	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent control	-	1.00	1.00	1.00	0.00
Positive control	4.00 8.00 16.00 32.00 64.00	1.14 1.19 1.35 1.64 2.55	1.20 1.46 1.96 3.44 8.59	1.17 1.32 1.66 2.54 5.57	0.04 0.19 0.44 1.27 4.27
Test item	0.98 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00 2000.00	0.91 0.78 0.99 1.80 1.22 0.00 0.00 0.00 0.00 0.00 0.01 0.00	0.89 0.91 0.96 1.87 0.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.90 0.85 0.98 1.84 0.68 0.00 0.00 0.00 0.00 0.00 0.00 0.00	0.02 0.09 0.02 0.05 0.77 0.00 0.00 0.00 0.00 0.00 0.01 0.00	*

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

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In this KeratinoSenss tudy under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Conclusion:

Based on the OECD GUIDANCE DOCUMENT ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION Series on Testing & Assessment No. 256 (ENV/JM/MONO(2016)29 and ENV/JM/MONO(2016)29/ANN1) an Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification (BASF) was chosen. This defined approach describes an integrated testing strategy (ITS) for the identification of the skin sensitisation hazard of a substance primarily for the purposes of classification and labelling without the use of animal testing. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification. Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.[Ref. CASE STUDY I of ENV/JM/MONO(2016)29/ANN1, Dr. Robert Landsiedel, BASF SE] The test item does neither activate the Nrf2-Keap1-ARE toxicity pathway (key event 2 of skin sensitization AOP) nor induce incresed number of cell surface markers CD54 and CD86 (key 3 of the skin sensitization AOP). In line with the "2 out of 3" integrated testing strategy approach to skin hazard identification presented classification of the test item as skin sensitizer is not justified.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In vitro study in accordance with testing and assessment strategy for evaluating the skin sensitisation potential of substances of Chapter R.7a: Endpoint specific guidance Version 6.0 – July 2017.

Qualifier:

according to guideline

Guideline:

other: OECD 442E guideline: human cell line activation test (h-CLAT)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

Name: Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized
Product Description: C10-16-alkyletherpropylamine, ethoxylated, DES Quat
CAS No.: 70983-58-3
Physical state: yellowish to amber viscous liquid at 20 °C
Batch No.: PFS-755-173
Re-certification date of batch: 19 April 2018
Purity: 100 % (UVCB)
Color, Gardner 8.8
pH, 5% in water 5.76
Acid Value , mg KOH/g 20.1
Moisture, % 0.127
Total Amine, mg/g 11.18
Viscosity,cps, #4@60,25C 3280
Appearance @25C pass
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Details on the study design:

This in vitro method is designed to predict and classify the skin sensitising potential of a chemical or multi-constituent substances or mixtures by assessment of its potential to upregulate cell surface markers using fluorescence-activated cell sorting (FACS).

Preparation of the Test Item

The test item was freshly prepared immediately prior to use.
The test item was soluble in dimethyl sulfoxide (DMSO) at a concentration of 500 mg/mL.
Vortex mixing was used to aid solubilisation. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2.
The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium.
The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Controls
A medium control, a solvent control, and a positive control were set up in parallel in order to confirm the validity of the test.

Medium Control
A medium control was included in the test.
 
Solvent Controls
DMSO was used as solvent control as the test item and the positive control were diluted in DMSO.
The solvent controls were diluted according to the procedure described for the test item, resulting in a final concentration of 0.2% (v/v) for DMSO.

Positive Control
2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 µg/mL (alternatively at the concentration of the CV75) was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted according to the procedure described for the test item in chapter 10.7.1, resulting in a final DMSO concentration of 0.2% (v/v).

Test System

FACS
FACS: BD Canto II
Software BD FACS DIVA 6.0

Cell line
The test was carried out using THP-1 cells (ATCC TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 106 cells/mL.
Cells were cultured in 75 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% fetal bovine serum, 25 mM HEPES, 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 µg/mL streptomycin at 37  1°C and 5% CO2.

Dose Groups
Medium Control: cell culture medium
Solvent Control: 0.2% DMSO (v/v) in cell culture medium
Positive Control: 4 µg/mL DNCB
Test Item: 8 concentrations of the test item (dose finding assay/ main experiment)
dose finding assay 1: 1000, 500, 250, 125, 62.50, 31.25, 15.63 and 7.81 µg/mL
Dose finding assay 2: 200, 100, 50, 25, 12.50, 6.25, 3.13 and 1.56 µg/mL
dose finding assay 3: 20, 14.29, 10.20, 7.29, 5.21, 3.72, 2.66 and 1.90 µg/mL
main experiment 1,2 and 3: 7.09, 5.91, 4.92, 4.10, 3.42, 2.85, 2.37 and 1.98 µg/mL
 
Pre-Experiments

Reactivity Check of the Cells Stock
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. For the reactivity check of the cell batch additional negative and positive controls were included. DNCB at a final concentration of 4 µg/mL and nickel sulphate at a final concentration of 100 µg/mL served as positive control while lactic acid at a final concentration of 1000 µg/mL served as negative control. Cells were accepted when both, DNCB and nickel sulphate produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.

Solvent Finding
Solubility of the test item was determined prior to the main experiment. The test item was dissolved in 0.9% NaCl at a final concentration of 100 mg/mL. Test items not soluble in 0.9% NaCl solution were dissolved in DMSO at a concentration of 500 mg/mL. If the test item was not soluble in DMSO, other solvents (e.g. THF) were used. It was taken care that the test chemical is dissolved or stably dispersed in the chosen solvent and that it does not interfere with the test design. If the test item was not soluble in DMSO or a different organic solvent at 500 mg/mL, the highest soluble concentration was tested by diluting the solution from 500 mg/mL with a constant factor of 1:2 up to a minimal concentration of 1 mg/mL.


Experimental Procedure

Dose Finding Assay

Starting from 500 mg/mL solutions of the test chemicals, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 250-fold into culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution. For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 10^6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 10^6 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10^6 cells/well). The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer). After washing, cells were re-suspended in 600 µL FACS buffer.
200 µL of the cell suspension were transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 µg/mL.
The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration.
The CV75 value, i.e. the concentration showing 75% cell survival, was calculated by log-linear interpolation utilizing equation 10 2. The CV75 value was used to calculate the concentration range of the test item for the main experiment.

CD54 and CD86 Expression

The test item was dissolved using DMSO as determined in the pre-experiment. Based on the concentration of the pre-determined CV75 value 8 concentrations of the test item were defined for the measurement of the surface marker expression, corresponding to 1.2 x CV75; CV75; CV75/1.2; CV75/1.22; CV75/1.23; CV75/1.24; CV75/1.25; CV75/1.26. If the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item prepared with each solvent was used as starting dose.
The test item was diluted to the concentration corresponding to 500-fold of the 1.2 × CV75. Then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 250-fold into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor.
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of
0.1 – 0.2 x 106 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 10^6 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 106 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at
37 °C ± 1 °C and 5% CO2. After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 µg/mL). The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated according to equation 10 3. The cell viability was calculated.

Positive control results:

The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86. The cell batches were accepted for further testing.

Key result

Run / experiment:

other: CD54 expression experiment 1 - 5.91 µg/ml

Parameter:

other: Relative Flourescence Intensity (RFI)

Value:

162

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: CD86 expression experiment 1 - 5.91 µg/ml

Parameter:

other: Relative Flourescence Intensity (RFI)

Value:

89

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: CD54 expression experiment 2 - 5.91 µg/ml

Parameter:

other: Relative Flourescence Intensity (RFI)

Value:

169

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: CD86 expression experiment 2 - 5.91 µg/ml

Parameter:

other: Relative Flourescence Intensity (RFI)

Value:

73

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: CD54 expression experiment 3 - 5.91 µg/ml

Parameter:

other: Relative Flourescence Intensity (RFI)

Value:

85

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: CD86 expression experiment 3 - 5.91 µg/ml

Parameter:

other: Relative Flourescence Intensity (RFI)

Value:

79

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results

Dose Finding Assay

The first dose finding assay was performed using a highest final concentration of 1000 µg/mL and the second dose finding assay was performed using a highest final concentration of 5000 µg/mL.

Table 1:  Results of the Dose Finding Assay

Sample	Experiment 2		Experiment 3
	Concentration applied [µg/ml]	Cell viability [%]	Concentration applied [µg/ml]	Cell viability [%]
Medium control	0.00	95.80	0.00	95.30
Solvent control	0.00	93.80	0.00	95.30
Test item	1.56 3.13 6.25 12.50 25.00 50.00 100.00 200.00	91.90 67.80 21.40 3.30 0.80 0.90 0.70 0.40	1.90 2.66 3.72 5.21 7.29 10.20 14.29 20.00	95.90 96.60 95.80 94.90 93.40 67.70 18.60 2.30
Calculated CV75 [µg/mL]	2.54		9.27
Mean CV75 [µg/mL]	5.91
SD CV75 [µg/mL]	4.76

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers. Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing. In the present study the test item was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 3.91 mg/mL to 500 mg/mL (first experiment), 0.78 mg/mL to 100 mg/mL (second experiment) 0.95 mg/mL to 10 mg/mL (third experiment) were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. A CV75 of 5.91 ± 4.76 µg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps: 7.09, 5.91, 4.92, 4.10, 3.42, 2.85, 2.37 and 1.98 µg/mL

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining. Severe cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 11.9% (CD86), 12.7% (CD54) and 12.4% (isotype IgG1 control) in the first experiment,15.5% (CD86), 15.8% (CD54) and 14.5% (isotype IgG1 control) in the second experiment and to. 18.9% (CD86), 18.4% (CD54) and 17.4% (isotype IgG1 control) in the third experiment. In the first experiment the expression of the cell surface marker CD86 was upregulated to 168%. The upregulation above the threshold of 150% was observed at a concentration of 3.42 µg/mL. No further upregulation of the cell surface marker CD86 was observed in the tested concentration range. In the second and third experiment the expression of CD86 was not upregulated above the threshold of 150%. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Since the test item showed no upregulation in two of three independent experiments, the test item is considered to be no skin sensitiser. The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in all experiments. The threshold of 150% for CD86 (293% experiment 1; 276% experiment 2; 398 experiment 3) and 200% for CD54 (375% experiment 1; 240% experiment 2; 464 experiment 3) were clearly exceeded. The controls confirmed the validity of the study. The viability of the solvent control was > 90% (95.2 - 96.2% experiment 1; 96.0 - 96.8% experiment 2 and 96.6 – 97.2 experiment 3). The number of tested test item concentrations with cell viability > 50% was ≥ 4 (6 experiments 1 and 2 and 5 experiment 3). The RFI for CD86 and CD54 of cells treated with the solvent DMSO was ≤ 150% (113% experiment 1; 104% experiment 2; 126 experiment 3) and ≤ 200% (113% experiment 1; 112% experiment 2; 109 experiment 3). The MFI ratio of the medium control and isotype IgG1 control was ≥ 105% for CD86 (266% experiment 1; 357% experiment 2; 182 experiment 3) and CD54 (188% experiment 1; 186% experiment 2; 162 experiment 3). The MFI ratio of the solvent control (DMSO) and isotype IgG1 control was ≥ 105% for CD86 (287% experiment 1; 383% experiment 2; 209 experiment 3) and CD54 (199% experiment 1; 203% experiment 2; 172 experiment 3).

The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Table 2: CD54 and CD86 Expression Experiment 1

Sample	Conc. [µg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control		96.2	96.1	95.9	1881	1330	708	1173	622	88	89	266	188
Solvent Control	0.2 %	96.0	95.6	95.2	2018	1411	709	1329	702	100	100	287	199
DNCB (positive control)	4.0	84.6	83.6	82.4	4527	3262	633	3894	2629	293	375	715	515
Test item	7.09 5.91 4.92 4.10 3.42 2.85 2.37 1.98	11.9 37.0 52.0 73.4 90.4 92.0 92.9 93.4	12.7 35.3 53.3 73.8 91.2 92.2 93.4 93.3	12.4 34.0 53.4 74.1 89.7 91.9 93.2 92.5	2944 2012 1966 2500 2917 2551 2327 2012	2459 1961 1949 1918 1645 1527 1526 1403	1117 823 751 652 682 674 933 684	1827 1189 1215 1848 2235 1877 1394 1328	1342 1138 1198 1266 963 853 593 719	137 89 91 139 168 141 105 100	191 162 171 180 137 122 84 102	264 244 262 383 428 378 249 294	220 238 260 294 241 227 164 205

Table 3: CD54 and CD86 Expression Experiment 2

Sample	Conc. [µg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control		96.7	96.3	96.8	2509	1309	703	1806	606	97	89	357	186
Solvent Control	0.2 %	96.8	96.4	96.0	2532	1341	661	1871	680	100	100	383	203
DNCB (positive control)	4.0	86.1	85.0	85.7	5780	2240	610	5170	1630	276	240	948	367
Test item	7.09 5.91 4.92 4.10 3.42 2.85 2.37 1.98	15.5 24.8 51.8 78.3 91.0 92.3 95.0 95.7	15.8 24.1 52.6 79.6 91.7 92.5 94.8 95.9	14.5 24.6 51.4 81.7 91.7 92.1 95.0 95.1	1497 2147 3379 3257 3022 2802 2182 2135	1416 1906 1907 1552 1402 1362 1142 1112	863 754 697 654 619 587 530 530	634 1393 2682 2603 2403 2215 1652 1605	553 1152 1210 898 783 775 612 582	34 74 143 139 128 118 88 86	81 169 178 132 115 114 90 86	173 285 485 498 488 477 412 403	164 253 274 237 226 232 215 210

Table 3: CD54 and CD86 Expression Experiment 3

	Conc. [µg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control		97.2	96.7	96.8	1191	1062	656	535	406	79	91	182	162
Solvent Control	0.2 %	96.7	96.6	96.7	1295	1064	620	675	444	100	100	209	172
DNCB (positive control)	4.0	85.2	84.2	82.4	3333	2709	649	2684	2060	398	464	514	417
Test item	7.09 5.91 4.92 4.10 3.42 2.85 2.37 1.98	18.9 29.2 35.0 58.8 77.1 87.3 92.5 94.0	18.4 28.4 43.4 59.1 77.0 86.7 91.8 94.5	17.4 27.7 41.5 59.0 77.9 86.7 91.8 94.3	1306 1324 1401 1579 1460 1302 1134 1025	1150 1144 1144 1189 1197 1137 1007 971	771 767 736 683 619 563 533 512	535 557 665 896 841 739 601 513	379 377 408 506 578 574 474 459	79 83 99 133 125 109 89 76	85 85 92 114 130 129 107 103	169 173 190 231 236 231 213 200	149 149 155 174 193 202 189 190

Table 4: Acceptance Criteria

Acceptance Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail	Experiment 3	pass/fail
cell viability medium and solvent control [%] number of test dosed with viability >50% CD86 number of test dosed with viability >50% CD54 number of test dosed with viability >50% IgG1 RFI of positive control of CD86 RFI of positive control  of CD54 RFI of solvent control of CD86 RFI of  solvent control  of CD54 MFI ratio IgG1/CD86 for medium control [%] MFI ratio IgG1/CD86 for solvent control [%] MFI ratio IgG1/CD54 for medium control [%] MFI ratio IgG1/CD54 for solvent control [%]	>90 ≥4 ≥4 ≥4 ≥150 ≥200 <150 <200 >105 >105 >105 >105	95.2 - 96.2 6 6 6 293 375 113 113 266 287 188 199	pass pass pass pass pass pass pass pass pass pass pass pass	96.0 - 96.8 6 6 6 276 240 104 112 357 383 186 203	pass pass pass pass pass pass pass pass pass pass pass pass	96.6 - 97.2 5 5 5 398 464 126 109 182 209 162 172	pass pass pass pass pass pass pass pass pass pass pass pass

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item considered to be no skin sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study the test item was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 3.91 mg/mL to 500 mg/mL (first experiment), 0.78 mg/mL to 100 mg/mL (second experiment) 0.95 mg/mL to 10 mg/mL (third experiment) were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. A CV75 of 5.91 ± 4.76 µg/mL was derived in the dose finding assay Based on the CV75, the main experiment was performed covering the following concentration steps: 7.09, 5.91, 4.92, 4.10, 3.42, 2.85, 2.37 and 1.98 µg/mL

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining. Severe cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 11.9% (CD86), 12.7% (CD54) and 12.4% (isotype IgG1 control) in the first experiment,15.5% (CD86), 15.8% (CD54) and 14.5% (isotype IgG1 control) in the second experiment and to. 18.9% (CD86), 18.4% (CD54) and 17.4% (isotype IgG1 control) in the third experiment. In the first experiment the expression of the cell surface marker CD86 was upregulated to 168%. The upregulation above the threshold of 150% was observed at a concentration of 3.42 µg/mL. No further upregulation of the cell surface marker CD86 was observed in the tested concentration range. In the second and third experiment the expression of CD86 was not upregulated above the threshold of 150% in. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Since the test item showed no upregulation in two of three independent experiments, the test item is considered to be no skin sensitiser. The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in all experiments. The threshold of 150% for CD86 (293% experiment 1; 276% experiment 2; 398 experiment 3) and 200% for CD54 (375% experiment 1; 240% experiment 2; 464 experiment 3) were clearly exceeded.

Conclusion:

Based on the OECD GUIDANCE DOCUMENT ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION Series on Testing & Assessment No. 256 (ENV/JM/MONO(2016)29 and ENV/JM/MONO(2016)29/ANN1) an Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification (BASF) was chosen. This defined approach describes an integrated testing strategy (ITS) for the identification of the skin sensitisation hazard of a substance primarily for the purposes of classification and labelling without the use of animal testing. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification. Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.[Ref. CASE STUDY I of ENV/JM/MONO(2016)29/ANN1, Dr. Robert Landsiedel, BASF SE] The test item does neither activate the Nrf2-Keap1-ARE toxicity pathway (key event 2 of skin sensitization AOP) nor induce incresed number of cell surface markers CD54 and CD86 (key 3 of the skin sensitization AOP). In line with the "2 out of 3" integrated testing strategy approach to skin hazard identification presented classification of the test item as skin sensitizer is not justified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available