Ethyltriphenylphosphonium acetate

Endpoint summary

Description of key information

not sensitising based on "2 out of 3" integrated testing strategy

First key event: Direct Peptide Reactivity Assay: negative

Second key event: Keratinocyte Activation: negative

Third key event: Dendritic cell activation: positive

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 March 2019 - 14 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
Synthetic peptide containing Cysteine: Ac-RFAACAA-OH
Synthetic peptide containing Lysine: Ac-RFAAKAA-OH
Positive Control: Cinnamic Aldehyde

Preparation of Stability Controls and Precision Control
Stability controls (Reference Control B), precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile/buffer.

Preparation of Positive Control Solution and Test Item Stock Solutions
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of ETPPAAc was also prepared in acetonitrile.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Triplicate solutions each of the positive control and ETPPAAc stock solutions were diluted with the Cysteine peptide stock solution to prepare solutions containing 0.5 mM Cysteine and 5 mM of Cinnamic Aldehyde or 5 mM ETPPAAc. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls
Triplicate solutions each of the positive control and ETPPAAc stock solution were diluted with the Lysine peptide stock solution to prepare solutions containing 0.5 mM Lysine and 25 mM of Cinnamic Aldehyde or 25 mM ETPPAAc. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of the ETPPAAc and positive control samples in the HPLC vials was documented following preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of ETPPAAc and the associated positive controls was quantified by HPLC using UV detection.

The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:
% Peptide depletion = 100 - (Peptide peak area in replicate depletion samples (x 100))/(Mean Peptide peak area of reference control samples B)

Positive control results:

72.4% Cysteine depletion
51.8% Lysine depletion

Run / experiment:

run/experiment 1

Parameter:

cysteine depletion

Value:

-2.41 %

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

72.4% depletion

Run / experiment:

run/experiment 1

Parameter:

lysine depletion

Value:

-0.514 %

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

51.8% depletion

Other effects / acceptance of results:

Solubility of ETPPAAc was achieved at a nominal concentration of 100 mM in acetonitrile.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

The DPRA prediction and the reactivity of the test item based on the overall mean and the individual depletion valuesin the Cysteine peptide and the Lysine peptide are presented below. All analytical acceptance criteria for each peptide run were met:

 

All analytical acceptance criteria for each peptide run were met:	Peptide	Standard Linearity	Positive control depletion (%)	Reference controls	Test item
Acceptance	Cysteine	r2>0.99	60.8-100(SD<14.9%)	0.45-0.55mM(CV <15%)	SD<14.9%
criteria	Lysine	r2>0.99	40.2-69.0 (SD<11.6%)	0.45-0.55mM (CV <15%)	SD<11.6%
Achieved	Cysteine	r2>0.999	72.4(SD, 0.58%, n=3)	B: 0.504mM (CV0.64%, n=6)	SD0.48%(n=3)
results	Lysine	r2>0.999	51.8(SD, 0.60%, n=3)	B: 0.501mM (CV1.63%, n=6)	SD0.22%(n=3)

 

 

The depletion of peptide in the presence of the test item was:

Peptide	Reference Control	Mean peak area of peptide with test item(µV.sec)	Mean peptide depletion by ETPPAAc (%)
Cysteine	Control B: 921660 (n=6)	941360 (n=3)	-2.41 (n=3)
Lysine	Control B: 783900 (n=6)	787930 (n=3)	-0.514 (n=3)

Applying the following reactivity prediction depletion model (below), reactivity of ETPPAAc is classed as “no or minimal” and the DPRA prediction is therefore negative and is therefore predicted not to be a potential skin sensitizer. 

 

Mean of cysteine and lysine% depletion	Reactivity Class	DPRA Prediction
0%≤ mean% depletion ≤6.38%	No or minimal reactivity	Negative
6.38%< mean% depletion ≤22.62%	Low reactivity	Positive
22.62%< mean% depletion ≤42.47%	Moderate reactivity
42.47%< mean% depletion ≤100%	High reactivity

There was no co-elution peaks in either the Cysteine or Lysine assay.   

 

 

 

Interpretation of results:

other: expert judgement

Remarks:

negative for the first key event of the skin sensitisation Adverse Outcome Pathway

Conclusions:

negative for the first key event of the skin sensitisation Adverse Outcome Pathway

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals,In chemicoSkin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD document TG 442C) was to assess the reactivity and sensitizing potential of ETPPAAc.

Solutions of the test item were successfully analyzed by the validated DPRA analytical method in both the Cysteine and Lysine containing synthetic peptides. There was no reactivity of both peptides in the presence of the test item. With no peptide depletion, the reactivity of the test item is classified as “no or minimal” and hence the DPRA prediction is negative. ETPPAAc is likely to be a non-skin sensitizer based as based on this assay.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November 2018 - 22 January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals: OECD 442E

Version / remarks:

In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation. Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), June 2018.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

Test system
Reasons for the Choice of THP-1 Cells
THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as a surrogate for human myeloid dendritic cells, because the THP-1 cells also show enhanced CD86 and/or CD54 expression when treated with sensitisers.

THP-1 Cell Cultures
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of the laboratory.
Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly. The cell density should not exceed 1 × 106 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30).
The passage numbers of the used THP-1 cells were 17 and 18 in the cytotoxicity tests and 15 and 17 in the h-CLAT for runs 1 and 2, respectively.

Culture Medium
RPMI 1640 Medium, GlutaMAXTM Supplement including 25 mM HEPES, supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin)

Preparation and Seeding of THP-1 Cells
On the day of the cytotoxicity or main experiment (h-CLAT) directly before the treatment of the cells, a volume of 500 μL with a cell density of 1.8 - 2 × 106 THP-1 cells/mL was seeded in each corresponding well of a 24-well flat bottom plate.

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50% (for detailed results, see "Any other information on results incl. tables")).

Run / experiment:

mean

Parameter:

CV75 [442D and 442E]

Value:

55.52 µg/mL

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Run / experiment:

run/experiment 1

Parameter:

RFI CD54>150 [442E]

Value:

187.7 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

run/experiment 1

Parameter:

RFI CD86>200 [442E]

Value:

134.5 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

run/experiment 2

Parameter:

RFI CD54>150 [442E]

Value:

178 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

run/experiment 2

Parameter:

RFI CD86>200 [442E]

Value:

150.8 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: viability > 90%: yes (Medium = 92.09%, DMSO = 92.10%); MFI ratio of CD54 and CD86 to isotype control > 105%: yes (Medium CD 54: 154.9%, Medium CD 86: 237.1%,
DMSO CD 54: 166.2%, DMSO CD 86: 228.2%)
- Acceptance criteria met for positive control: RFI values of both CD54 and CD86 exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability > 50%: yes (3 μg/mL DNCB (CD 54): 240.0%, 3 μg/mL DNCB (CD 86): 283.0%, 4 μg/mL DNCB (CD 54): 290.3%, 4 μg/mL DNCB (CD 86): 304.7%)


The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of both runs. Fluctuating RFI values and no clear dose response were observed for CD54 and CD86 in both h-CLAT runs. This might be due to the low concentrations tested and the small dilution factor of 1:1.2 (as recommended by the OECD 442E guideline) with this test item.
However, the evaluated results of both h-CLAT runs are valid according to acceptance criteria of the OECD 442E guideline in this h-CLAT study. Therefore, the h-CLAT prediction is considered positive for the test item in this h-CLAT.
In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.
This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

Dose range finding assay

Results of the first Cytotoxicity Test for the Test Item ETPPAAc

Test Group	Concentration [μg/mL]	Microscopic Evaluation / Cytotoxicity	Flow Cytometric Evaluation / Cell Viability [%]
Medium control	-	No	94.76
Test item	39.1	No	84.63
	78.1	No	71.33
	156	No	52.29
	313	Yes	40.86
	625	Yes	29.76
	1250	Yes	23.53
	2500	Yes	1.29
	5000	Yes	0.40

CV75 =64.53 μg/mL

 

Results of the second Cytotoxicity Test for the Test Item ETPPAAc

Test Group	Concentration [μg/mL]	Microscopic Evaluation / Cytotoxicity	Flow Cytometric Evaluation / Cell Viability [%]
Medium control	-	No	97.98
Test item	39.1	No	81.08
	78.1	No	56.85
	156	No	24.70
	313	Yes	28.87
	625	Yes	26.31
	1250	Yes	27.15
	2500	Yes	0.81
	5000	Yes	0.39

CV75 =46.51 μg/mL

mean CV75 of both Cytotoxicity Tests: 55.52 μg/mL

Results of the h-CLAT Test

Results of the first h-CLAT run for the Test Item ETPPAAc

	Concentration (μg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	92.09
DMSO control	-	100.0	100.0	92.10
Positive Control (DNCB)	3.0	240.0*	283.0*	80.39
	4.0	290.3*	304.7*	82.01
Test Item	19	194.6	180.0*	87.66
	22	211.5*	149.5	87.65
	27	187.7	172.9*	87.29
	32	189.2	134.5	86.74
	39	194.6	149.5	86.74
	47	220.0*	156.9*	85.19
	56	211.5*	183.1*	84.64
	67	190.0	196.9*	81.64

 

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

 

Results of the second h-CLAT run for the Test Item ETPPAAc

	Concentration (μg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	95.95
DMSO control	-	100.0	100.0	95.17
Positive Control (DNCB)	3.0	231.6*	502.9*	83.60
	4.0	395.8*	441.8*	74.36
Test Item	19	240.2*	164.4*	87.99
	22	218.3*	206.0*	87.52
	27	287.8*	150.8*	86.82
	32	213.4*	225.1*	84.41
	39	178.0	216.8*	82.28
	47	282.9*	271.5*	80.03
	56	252.4*	213.8*	77.10
	67	224.4*	272.4*	73.80

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

 

Interpretation of results:

other: expert judgement

Remarks:

positive for the third key event of the skin sensitisation Adverse Outcome Pathway

Conclusions:

The test item ETPPAAc with a log Pow of -1.60 activated THP-1 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential, i.e. the third key event of a skin sensitization Adverse Outcome Pathway (AOP) of ETPPAAc dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of ETPPAAc was previously determined by two cytotoxicity tests.

In both cytotoxicity tests, following incubation with the test item cytotoxic effects were observed starting from the test item concentration of 78.1 μg/mL up to the highest tested concentration of 5000 μg/mL. Threshold for cytotoxicity is a cell viability (CV) value < 75%. The mean CV75 value of both cytotoxicity tests was calculated to be 55.52 μg/mL.

The following concentrations of the test item were tested in the main experiments (h-CLAT): 19, 22, 27, 32, 39, 47, 56 and 67 μg/mL

The test item has a log Pow of -1.60 and was tested in 2 independent runs. The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of both runs. Fluctuating RFI values and no clear dose response were observed for CD54 and CD86 in both h-CLAT runs. This might be due to the low concentrations tested and the small dilution factor of 1:1.2 (as recommended by the OECD 442E guideline) with this test item.

However, the evaluated results of both h-CLAT runs are valid according to acceptance criteria of the OECD 442E guideline in this h-CLAT study. Therefore, the h-CLAT prediction is considered positive for the test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

In conclusion, the test item ETPPAAc with a log Pow of -1.60 activated THP-1 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 April 2019 - 26 March 2020

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 Test Method)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The cells used in this assay were the transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct supplied by Givaudan (Dubendorf, Switzerland). The cells were routinely grown and subcultured in maintenance medium at 37°C ± 2°C in a humidified atmosphere containing 5% CO2 in air. Maintenance medium was 500 mL Dulbecco’s Modified Eagles Medium containing Glutamax (DMEM), supplemented with 50 mL foetal bovine serum (FBS) and 5.5 mL Geneticin.

Preparation of Test Cell Cultures
The cells from flasks of actively growing cultures were detached and disaggregated as described above. The number of viable cells in the prepared cell suspension were determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer. The cell suspension was diluted with maintenance medium without geneticin to give 1 x 105 viable cells/mL and 100 µL volumes pipetted into all wells except one well of sterile 96-well flat-bottomed microtitre plates. On each occasion four plates were prepared in parallel: three white plates for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. One well of each plate received 100 µL maintenance medium without geneticin with no cells. The plates were incubated for 24 ± 2 hours at 37 ± 2C in a humidified atmosphere of 5% CO2 in air, to allow the cells to attach.

Test Item Solubility: The test item, was found to be soluble in DMSO at 40 mg/mL, the highest final concentration as recommended by the guideline.

Treatment of Cultured Plates
Approximately 24 hours after the test cell culture plates were established, the medium was removed from the wells by careful inversion of the plates and blotting onto sterile paper towel. 150 µL of assay medium was added to every well of the 96 well plates. 50 µL from each well of the dilution plate was transferred to equivalent wells in the 96 well plates. Three white plates were dosed for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. The plates were then covered with a plate seal and placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air for 48 ± 2 hours.

Cell Viability Measurement
After incubation, the cell culture medium was removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 100 µL fresh assay medium was added to each well. 10 µL of MTT solution (5 mg/mL in PBS) was added to each well of the 96-well plate. The plate was incubated at 37 ± 2C in a humidified atmosphere of 5% CO2 in air for 4 hours ± 10 minutes. The medium was then removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 50 µL of DMSO was added to each well. The plate was then placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air, protected from light, for at least 10 minutes. The absorbance value of each well was read using a plate reader with a 540 nm filter.

Luciferase Measurement
Luciferase was measured using the Steady Glo® Luciferase Assay system kit supplied by Promega (E2550). Steady-Glo® luciferase reagent was prepared by transferring the contents of one bottle of Steady-Glo® buffer to one bottle of Steady-Glo® substrate. The reagent was mixed by inversion until the substrate had dissolved. The reconstituted reagent was used on the same day it was prepared for test 2 and test 3. Frozen reconstituted reagent was used for test 1 and was thawed to room temperature before use.
After incubation the medium was removed from the wells of the triplicate white plates by careful inversion of the plates and blotting on sterile absorbent paper. 100 µL of fresh assay medium was added to each well before 100 µL of Steady-Glo® luciferase reagent was added to each well of the plate. The plates were shaken on a plate shaker for at least 5 minutes until the cells had lysed. Luminescence (emitted light) was measured using a SpectraMax L luminometer. Each plate was read for total photon count with an integration time of 1 second. The plates were dark adapted for 1 minute prior to measurement.

Interpretation of Results and Prediction Model
A KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens™ prediction is considered negative:
• the Imax is ≥1.5 fold and statistically significantly different as compared to the solvent vehicle control (as determined by a two-tailed, unpaired Student’s T-test);
• the cellular viability is >70% at the lowest concentration with induction of luciferase activity ≥1.5 fold (i.e. at the EC1.5 determining concentration);
• the EC1.5 value is < 200 µg/mL;
• there is an apparent overall dose-response for luciferase induction (or a biphasic response).
If in a given test, all of the first three conditions listed above are met, but a clear dose-response for the luciferase induction cannot be observed, then the result of that repetition should be considered inconclusive and further testing may be required. In addition, a negative result obtained with concentrations < 200 µg/mL and that do not reach cytotoxicity (< 70% viability) at the maximal tested concentration should also be considered as inconclusive.

Test Acceptance Criteria
In order for an assay to be accepted the following criteria must be met:
• The luciferase activity induction obtained with the positive control, cinnamic aldehyde, should be statistically significant above the threshold of 1.5 (e.g. using a t test) in at least one of the tested concentrations (4 to 64 µM).
• The EC1.5 value of the positive control should be within two standard deviations of the historical mean of the testing facility. In addition, the average induction in the three replicates for cinnamic aldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamic aldehyde 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.
• The average coefficient of variation of the luminescence reading for the negative solvent control (i.e. DMSO) should be below 20% in each repetition which consists of 6 wells tested in triplicate. If the variability is higher, results should be discarded.

Run / experiment:

other: Test 1

Parameter:

other: Imax

Value:

113.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Imax >1.5 fold and statistically significant

Run / experiment:

other: Test 1

Parameter:

other: EC1.5 (µg/mL)

Value:

12.89

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cellular viability >70% at the EC1.5 determining concentration

Remarks:

KeratinoSens™ prediction: positive

Run / experiment:

other: Test 2

Parameter:

other: Imax

Value:

13.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Imax >1.5 fold and statistically significant

Run / experiment:

other: Test 2

Parameter:

other: EC1.5 (µg/mL)

Value:

17.66

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cellular viability <70% at the EC1.5 determining concentration

Remarks:

KeratinoSens™ prediction: negative

Run / experiment:

other: Test 3

Parameter:

other: Imax

Value:

35.84

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Imax >1.5 fold and statistically significant

Run / experiment:

other: Test 3

Parameter:

other: EC1.5 (µg/mL)

Value:

17.45

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cellular viability <70% at the EC1.5 determining concentration

Remarks:

KeratinoSens™ prediction: negative

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table1              Results for ETPPAAc – Test 1

Test item conc. (µg/mL)	0.20	0.39	0.78	1.56	3.13	6.25	12.5	25	50	100	200	400
Mean fold induction	0.87	0.91	0.88	1.01	0.89	0.66	1.42	3.96	7.43	5.02	9.14	113.80
Statistically significant	No	No	No	No	No	No	No	Yes	Yes	Yes	Yes	Yes
Viability (%)	117.24	112.20	119.15	120.00	120.50	96.54	84.13	86.90	85.98	82.22	70.81	22.89
Imax	113.80
EC1.5(µg/mL)	12.89
IC30(µg/mL)	203.38
IC50(µg/mL)	286.86

 

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	Yes
Is the cellular viability >70% at the EC1.5determining concentration	Yes
Is the EC1.5value < 200 µg/mL	Yes
Is there an apparent overall dose-response for luciferase induction	Yes
KeratinoSens™ prediction	Positive

 

 

 

 

Table2              Results for Cinnamic Aldehyde – Test 1

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.07	1.18	1.59	2.05	2.83
Statistically significant	No	No	Yes	Yes	Yes
Viability (%)	96.68	98.24	101.00	106.53	111.35
Imax	2.83
EC1.5(µM)	14.19
IC30(µM)	N/A
IC50(µM)	N/A

 

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	Yes (2.83)	Pass
EC1.5of positive control within two standard deviations of the historical mean (‑4.39 to 30.86)	Yes (14.19)	Pass
CV% of blank values < 20%	Yes (13.6%)	Pass

 

 

Table3              Results for ETPPAAc – Test 2

Test item conc. (µg/mL)	0.20	0.39	0.78	1.56	3.13	6.25	12.5	25	50	100	200	400
Mean fold induction	1.02	0.97	1.02	1.11	0.88	0.46	0.94	2.30	2.82	2.17	3.23	13.60
Statistically significant	No	No	No	No	No	No	No	Yes	Yes	Yes	Yes	Yes
Viability (%)	105.61	106.90	116.35	120.57	123.72	77.76	57.78	59.43	53.56	52.34	47.40	5.73
Imax	13.60
EC1.5(µg/mL)	17.66
IC30(µg/mL)	8.68
IC50(µg/mL)	147.34

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	Yes
Is the cellular viability >70% at the EC1.5determining concentration	No
Is the EC1.5value < 200 µg/mL	Yes
Is there an apparent overall dose-response for luciferase induction	Yes
KeratinoSens™ prediction	Negative

 

 

 

Table4              Results for Cinnamic Aldehyde – Test 2

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.26	1.47	1.89	2.76	4.25
Statistically significant	No	No	Yes	Yes	Yes
Viability (%)	100.24	103.32	101.31	107.47	107.68
Imax	4.25
EC1.5(µM)	8.59
IC30(µM)	N/A
IC50(µM)	N/A

 

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	Yes (4.25)	Pass
EC1.5of positive control within two standard deviations of the historical mean (-4.39 to 30.86)	Yes (8.59)	Pass
CV% of blank values < 20%	Yes (12.0%)	Pass

 

Table5              Results for ETPPAAc – Test 3

Test item conc. (µg/mL)	0.20	0.39	0.78	1.56	3.13	6.25	12.5	25	50	100	200	400
Mean fold induction	0.88	1.01	1.03	1.05	0.90	0.67	0.95	2.34	5.12	4.71	5.24	35.84
Statistically significant	No	No	No	No	No	No	No	Yes	Yes	Yes	Yes	Yes
Viability (%)	101.54	95.92	96.68	91.83	89.95	71.71	71.09	65.89	64.36	65.47	57.70	18.52
Imax	35.84
EC1.5(µg/mL)	17.45
IC30(µg/mL)	15.12
IC50(µg/mL)	239.32

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	Yes
Is the cellular viability >70% at the EC1.5determining concentration	No
Is the EC1.5value < 200 µg/mL	Yes
Is there an apparent overall dose-response for luciferase induction	Yes
KeratinoSens™ prediction	Negative

 

 

 

Table6              Results for Cinnamic Aldehyde – Test 3

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.15	1.34	1.63	2.19	4.37
Statistically significant	No	No	Yes	Yes	Yes
Viability (%)	109.44	108.82	107.43	100.50	96.96
Imax	4.37
EC1.5(µM)	12.47
IC30(µM)	N/A
IC50(µM)	N/A

 

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	Yes (4.37)	Pass
EC1.5of positive control within two standard deviations of the historical mean (-4.39 to 30.86)	Yes (12.47)	Pass
CV% of blank values < 20%	Yes (12.7%)	Pass

 

Interpretation of results:

other: expert judgement

Remarks:

negative for the second key event of the skin sensitisation Adverse Outcome Pathway

Conclusions:

It was concluded that the test item, ETPPAAc, gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is not a skin sensitizer.

Executive summary:

The purpose of this study (based on the OECD Guideline for the Testing of Chemicals, In vitro Skin Sensitisation Assays Addressing the AOP Key Event on Keratinocyte Activation, Guideline 442D, June 2018) was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, ETPPAAc, is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The Imax for the test item was 113.80 in test 1 which was >1.5 fold and statistically significant compared to the DMSO control. The EC1.5 was 12.89 µg/mL. The IC30 value was 203.38 µg/mL and the IC50 value was 286.86 µg/mL. The graph showed an overall dose-response for luciferase induction indicating a positive response. 

The Imax for the test item in test 2 was 13.60 which was >1.5 fold and statistically significant compared to the DMSO control. The EC1.5 was 17.66 µg/mL. The IC30 value was 8.68 µg/mL and the IC50 value was 147.34 µg/mL. The graph showed an overall dose-response for luciferase induction, however, at the EC1.5 determining concentration the cell viability was below 70% and therefore the prediction was negative.

The Imax for the test item in test 3 was 35.84 which was >1.5 fold and statistically significant compared to the DMSO control. The EC1.5 was 17.45 µg/mL. The IC30 value was 15.12 µg/mL and the IC50 value was 239.32 µg/mL. The graph showed an overall dose-response for luciferase induction, however, at the EC1.5 determining concentration the cell viability was below 70% and therefore the prediction was negative.

All acceptance criteria for the positive control, cinnamic aldehyde, were met.

It was concluded that the test item, ETPPAAc,gave a negative responsein the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is not a skin sensitizer.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

The skin sensitizing potential of ETPPAAc was assessed by an in chemico/in vitro testing battery, which is based on the skin sensitisation AOP (adverse outcome pathway). This AOP identifies four key events (KEs) with KE1, the covalent binding to skin proteins (termed haptenation) either of the parent substance or of its reactive derivatives following abiotic/metabolic activation, which is postulated to be the molecular initiating event (MIE), followed by KE2, the activation of epidermal keratinocytes, KE3, the activation (maturation) and mobilisation of Langerhans cell and dermal dendritic cells (DC), and KE4, the DC-mediated antigen presentation to naïve T-cells and proliferation/activation of allergen specific T-cells.

The "2 out of 3" integrated testing strategy applied for this substance covers the first three key events of the AOP leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (via the KeratinoSens assay; OECD TG 442D); and dendritic cell activation (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.

The prediction model defines that two concordant results addressing two different KEs indicate the sensitising potential, i.e. two positive results indicate a sensitiser, two negative results indicate a non-sensitiser (OECD, 2016a; OECD, 2016b).

 

First key event: Direct Peptide Reactivity Assay

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD document TG 442C) was to assess the reactivity and sensitizing potential of ETPPAAc.

Solutions of the test item were successfully analyzed by the validated DPRA analytical method in both the Cysteine and Lysine containing synthetic peptides. There was no reactivity of both peptides in the presence of the test item. With no peptide depletion, the reactivity of the test item is classified as “no or minimal” and hence the DPRA prediction is negative.

 

Second key event: Keratinocyte Activation

The purpose of this study ((based on the OECD Guideline for the Testing of Chemicals, In vitro Skin Sensitisation Assays Addressing the AOP Key Event on Keratinocyte Activation, Guideline 442D, June 2018) was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, ETPPAAc, is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The Imax for the test item was 113.80 in test 1 which was >1.5 fold and statistically significant compared to the DMSO control. The EC1.5 was 12.89 µg/mL. The IC30 value was 203.38 µg/mL and the IC50 value was 286.86 µg/mL. The graph showed an overall dose-response for luciferase induction indicating a positive response. 

The Imax for the test item in test 2 was 13.60 which was >1.5 fold and statistically significant compared to the DMSO control. The EC1.5 was 17.66 µg/mL. The IC30 value was 8.68 µg/mL and the IC50 value was 147.34 µg/mL. The graph showed an overall dose-response for luciferase induction, however, at the EC1.5 determining concentration the cell viability was below 70% and therefore the prediction was negative.

The Imax for the test item in test 3 was 35.84 which was >1.5 fold and statistically significant compared to the DMSO control. The EC1.5 was 17.45 µg/mL. The IC30 value was 15.12 µg/mL and the IC50 value was 239.32 µg/mL. The graph showed an overall dose-response for luciferase induction, however, at the EC1.5 determining concentration the cell viability was below 70% and therefore the prediction was negative.

All acceptance criteria for the positive control, cinnamic aldehyde, were met.

It was concluded that the test item, ETPPAAc,gave a negative responsein the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is not a skin sensitizer.

 

Third key event: Dendritic cell activation

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential, i.e. the third key event of a skin sensitization Adverse Outcome Pathway (AOP) of ETPPAAc dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of ETPPAAc was previously determined by two cytotoxicity tests.

In both cytotoxicity tests, following incubation with the test item cytotoxic effects were observed starting from the test item concentration of 78.1 μg/mL up to the highest tested concentration of 5000 μg/mL. Threshold for cytotoxicity is a cell viability (CV) value < 75%. The mean CV75 value of both cytotoxicity tests was calculated to be 55.52 μg/mL.

The following concentrations of the test item were tested in the main experiments (h-CLAT): 19, 22, 27, 32, 39, 47, 56 and 67 μg/mL

The test item has a log Pow of -1.60 and was tested in 2 independent runs. The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of both runs. Fluctuating RFI values and no clear dose response were observed for CD54 and CD86 in both h-CLAT runs. This might be due to the low concentrations tested and the small dilution factor of 1:1.2 (as recommended by the OECD 442E guideline) with this test item.

However, the evaluated results of both h-CLAT runs are valid according to acceptance criteria of the OECD 442E guideline in this h-CLAT study. Therefore, the h-CLAT prediction is considered positive for the test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

In conclusion, the test item ETPPAAc with a log Pow of -1.60 activated THP-1 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

 

Conclusion

Overall, following the “2 out of 3 - Sens ITS” prediction model, ETPPAAc is not considered to be skin sensitizer.

 

The purpose of this prediction model was the skin sensitisation hazard identification for classification and labelling purposes. The use of this approach for potency assessments is not yet possible. However, since the substance was identified as not sensitising, no potency assessment is required.

 

References:

OECD, 2016a. 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

 

OECD, 2016b. Annex I: case studies to the 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/ANN

 

 

Justification for classification or non-classification

Based on the available information, the substance does not require classification with respect to skin sensitisation according to regulation (EC) 1272/2008.