Octadecanoic acid, reaction products with 2-amino-2-methyl-1-propanol

Endpoint summary

Description of key information

Based on the available weight of evidence fromin silico(OECD QSAR Tool box v.4.1) andin vitrostudies (DPRA, h-CLAT and KeratinoSens^TMassays), the test substance, C16-18 AMP, is overall concluded to be a non-sensitising to skin.​

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:

weight of evidence

Study period:

From August 04, 2017 to August 15, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

OECD TG 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), adopted 29 Jul 2016.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol No. 158 human Cell Line Activation Test (h-CLAT)

Version / remarks:

EURL ECVAM DB-ALM Protocol No. 158 human Cell Line Activation Test (h-CLAT); Skin Sensitisation and Allergic Contact Dermatitis (Issued: 01 Jul 2015)

Deviations:

no

GLP compliance:

yes

Type of study:

activation of dendritic cells

Details on the study design:

Test System Material Source
The human monocytic leukaemia cell line, THP-1, (Cat# TIB-202) were obtained from American Type Culture Collection (ATCC) via their UK subsidiary LGC standards; LGC Standards, Queens Road, Teddington, Middlesex TW11 0LY, UK. Two initial vials of cells were received in April 2016 and data to verify the referenced sources was archived.

OECD Test Guideline relating to the test method
OECD TG 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), adopted 29 Jul 2016.

XCellR8 SOP
The study was performed using the h-CLAT method as detailed in OECD TG 442E and also in EURL ECVAM DB-ALM Protocol No. 158 (Issued 01 Jul 2015). Methodology is detailed in an XCellR8 SOP (L0094) that covers the h-CLAT. Study data was collected using XCellR8 internal protocols (IPs).

Method workflow summary
Solubility was first determined for the test item using either culture medium (RPMI 1640) or DMSO. Note that for this method, test items with a Log Kow (octanol/water partition coefficient) of up to 3.5 have been tested successfully. However, test items with a Log Kow of greater than 3.5 can still be tested at lower soluble concentrations. In such a case, a negative result (non-sensitiser) should be considered inconclusive, whereas a positive result (sensitiser) could still be considered valid. THP-1 cells were pre-cultured for either 48 or 72 h. Following this, the cells were dosed with the test item over an 8 dose range and incubated for 24 ±0.5 h. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The dose of test item that yields 75% cell viability (CV75) was calculated and taken forward for the next stage of testing. This dose finding assay was carried out over two independent runs. THP-1 cells were pre-cultured again for either 48 or 72 h. Once the CV75 was determined, a narrower dilution series based around the CV75 value was produced for the test item. This dilution range was used to dose the cells again for 24 ±0.5 h. The cells were then washed and stained with propidium iodide and also with antibodies that detect CD54 and CD86 expression as well as a negative control antibody. This allowed for discrimination of live/dead cells and also changes in CD54 and CD86 marker expression by flow cytometry.

Key result

Run / experiment:

other: Average of two run

Parameter:

other: CV75 (µg/mL)

Remarks:

A concentration showing 75% of THP-1 cell survival (25% cytotoxicity)

Value:

250

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Average of two representatives

Parameter:

other: CD54 expression measured as RFI (up to 300 µg/mL, top dose concentration)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Average of two representatives

Parameter:

other: CD86 expression measured as RFI (up to 300 µg/mL, top dose concentration)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression.

Results

Solvent Selection and CV75 Determination

Prior to the CV75 determination, the test substance was assessed for solubility and was found to be soluble in Isopropanol at 25 mg/mL.

The CV75 value derived from two independent experiments was as follows:

CV75 (Rep1)	CV75 (Rep2)	Average CV75
>250	77.1	163.55 µg/mL

 

The final CV75 was taken as the top dose (250 µg/mL) even though the CV75 was lower in Rep2, as the average of the viabilities across the 2 replicates at the top dose was 64.59% and the top dose produced cytotoxicity in both replicates. In the second lowest dose cytotoxicity was not observed in Rep1. The graph for the two replicates has been included for clarity, note that the second lowest dose for Rep1 was considered to be an outlier datapoint.

Acceptance Criteria

Acceptance criteria for all controls and the test item were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression.

CV75 Determination
Criterion	Run 1	Run 2	Outcome
Cell viability must be ≥ 75% at the lowest dose.	96.89%	92.55%	PASS
The highest test substance concentration should produce cytotoxicity (< 90% cell viability) unless 5mg/mL in medium, 1mg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test substance.	86.13%	43.04%	PASS
Measurement of CD54 and CD86 Expression
Criterion	Run 1	Run 2	Outcome
Cell viabilities of medium and solvent controls should be higher than 90%	Medium: 98.02% Isopropanol: 95.75%	Medium: 97.49% Isopropanol: 94.98%	PASS
In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) compared to the medium control	CD54 = 118 CD86 = 122	CD54 = 98 CD86 = 124	PASS
For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%	Medium CD54: 140.50 CD86: 127.20 Isopropanol  CD54: 142.24 CD86: 129.13	Medium CD54: 148.44 CD86: 127.21 Isopropanol  CD54: 142.69 CD86: 130.43	PASS
In the positive control (Nickel Sulphate), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be greater than 50%.	RFI CD54: 384 CD86: 165 Viability (%) CD54: 91.02 CD86: 90.33	RFI CD54: 280 CD86: 153 Viability (%) CD54: 89.43 CD86: 91.20	PASS
For each test substance, the cell viability should be greater than 50% in at least four tested concentrations in each run	6/8	8/8	PASS
Negative results are acceptable only for test items exhibiting a cell viability of less than 90% at the highest concentration tested, unless 5mg/mL in medium, 1mg/mL in DMSO or the highest soluble concentration in solvent is used as the maximal test concentration of a test substance.	N/A as test item positive	N/A as test item positive	PASS

RFI - Relative Fluorescence Intensity, MFI - Mean Fluorescence Intensity.

Interpretation of results:

other: Category 1 (skin sensitising) based on EU CLP criteria

Conclusions:

Under study conditions, the test substance was concluded to be a skin sensitiser.

Executive summary:

A study was conducted to determine the skin sensitisation potential of test substance, C16-18 AMP, using the in vitro human Cell Line Activation Test (h-CLAT) method, according to OECD 442E, in compliance of GLP. The h-CLAT assay measures markers of dendritic cell (DC) activation in the human leukaemia cell line THP-1. The ability of a chemical to induce activation of CD86 and CD54 in THP-1 cells was used as an assessment of the chemical’s skin sensitisation potential. Activation of CD54 and CD86 protein markers was measured by flow cytometry following 24 h exposure to the test substance. The concentration of the test substance used was selected on the basis of a pre-determined CV75 value (i.e. the estimated concentration yielding 75% cell viability). For the CV75 determination, THP-1 cells (a cell line that mimics DCs) were pre-cultured for either 48 or 72 h. Following this, the cells were dosed with the test substance over an 8 dose range (300, 250, 208.33, 173.61, 144.68, 120.56, 100.47, 83.72 µg/mL) and incubated for 24 ±0.5 h. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The CV75 was found to be 250 µg/mL. The EC200 and EC150 values for CD54 and CD86 expression were found to be 12 µg/mL and 11 µg/mL respectively. As the CD54/CD86 expression crossed the sensitisation threshold the test substance was classified as a skin sensitiser. Cell viability only fell marginally below 50% at the 120.56 µg/mL test substance concentration in Run 1. All other concentrations that induced sensitisation had a cell viability of >50% and therefore the final result were deemed to be valid. An inverse relationship of dose to viability was observed in the main test, however this has not affected the end result, as all test concentrations (with the exception noted above) had cell viabilities above 50% and the RFI data were conclusive and consistent in both runs. Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression. Under study conditions, the test substance was concluded to be a skin sensitiser (XcellR8, 2017).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From August 31 to September 08, 2017

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)

Deviations:

not specified

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

Solvent: 1% Ethanol in cell culture medium
Administration method: In cell culture medium
Concentrations to be tested (µg/mL): 20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.02, 0.01

Preliminary testing: Determination of the top concentration by solubility testing
Day 1: Seeding cells (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT).
Day 2: 24 h after seeding, apply test items and controls for 48 ± 2 h
Day 4: Evaluate the luciferase activity by luminescence (3 plates) and the cell viability by MTT testing (2 plates)

Key result

Parameter:

other: Imax

Remarks:

Maximal fold induction of the luciferase activity over solvent (negative) control

Value:

1.5

Vehicle controls validity:

valid

Remarks:

1% Ethanol (≥99.5% purity) in cell culture medium

Negative controls validity:

valid

Remarks:

1% Ethanol (≥99.5% purity) in cell culture medium

Positive controls validity:

valid

Remarks:

8 µM to 128 µM Cinnamic aldehyde (≥99% purity) in 1% Ethanol cell culture medium

Remarks on result:

no indication of skin sensitisation

Table 1: Determination criteria for the skin sensitisation potential of test substance

Determination criteria for the skin sensitisation potential of test substance
	REP 1	REP 2	REP 3
Does at least one concentration of test substance induce luciferase activity≥1.5 fold	No	No	No
Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%	N/A	N/A	N/A
Does EC1.5 value occur at a concentration <1000 um (or <200 ug/mL	N/A	N/A	N/A
Does the test substance induce the luciferase in a dose dependent manner	N/A	N/A	N/A
Classification	Non-sensitiser	Non-sensitiser	Non-sensitiser

 

Table 2: Assay acceptance criteria (Mean of the 3 repetitions)

Criteria	Result	Pass or fail
1-Positive control (PC) (Cinnamic aldehyde) Induction≥1.5 fold in at least one concentration	Yes	Pass
2-Average induction of PC at 32 µm is [1.6-3]	1.318	Fail*
3-EC1.5 value is [6-39 µm]	35.3 µm	Pass
4-CV % of blank values <20 %	Yes (13.7 %)	Pass

* Acceptance criterion 2 was not met, however, all other acceptance were met and there was dose-dependent increase of induction with the positive control. Therefore the results were considered valid.

Interpretation of results:

other: Non skin sensitiser based on EU CLP criteria

Conclusions:

Under study conditions, the test substance was concluded to be non skin sensitiser.

Executive summary:

A study was conducted to determine skin sensitisation potential of the test substance, C16-18 AMP, using ARE-Nrf2 Luciferase test method, according to the OECD Guideline 442D, in compliance with GLP. Concentrations of test substance tested were: 20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.02, 0.01 µg/mL. The sensitisation potential of the test substance was quantified by calculating two parameters known as the EC1.5 and Imax values. In each repetition of the study, no concentration of test substance caused luciferase induction ≥1.5. Therefore, test substance was classified as non-sensitiser. The maximum induction was observed for repetition 1 at a test concentration of 0.039 µg/mL, which showed an Imax value of 1.284; maximum induction value for repetition 2 was observed at 0.625 µg/mL with an Imax value of 1.384 and maximum induction value for repetition 3 was observed at 0.313 µg/mL with an Imax value of 1.112. For reference, during the test validation, sensitising proficiency chemicals produced Imax values of up to 36 fold over untreated controls. All of the formal acceptance criteria were met except acceptance criterion 2. However, as all other acceptance criteria were met and there was a dose-dependent increase of induction with the positive control, results were considered valid. Under study conditions, the test substance was concluded to be non skin sensitiser (XcellR8, 2017).

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From October 11, 2017 to January 05, 2018

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))

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol No. 154 (Direct Peptide Reactivity Assay for Skin Sensitisation Testing)

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

OECD TG 442C cites the DPRA model as a validated method for skin sensitisation testing in the context of an integrated approach to testing and assessment.

Details on the study design:

See below 'Any other information for materials and methods incl. tables' for details on study design.

Test and reference substances

Test substance
Test Substance Name: C16-18 AMP
Supplier Batch/Lot Number: 0001049267
CAS Number 68951-62-2
Purity: UVCB, assumed 100% for study purpose
Expiry Date: 08 May 2019
Physical State: Solid (Off white/cream)
Storage Conditions: In original container, ambient conditions
Solubility: Insoluble in water, soluble in Isopropyl Alcohol
Solvent used: Isopropanol
Concentration tested: 100mM

Reference Substance
Supplier: Sigma-Aldrich
Reference Substance Name: Cinnamic Aldehyde
Lot Number: MKBV4784V
CAS Number: 104-55-2
Purity: >95%
Concentration Tested: 100mM
Solvent: HPLC Grade Acetonitrile (CAS No. 75-05-8)
Expiry Date: Apr 2020
Storage Conditions: Positive control is prepared fresh on Day 1 of main test

Key result

Run / experiment:

other: 1

Parameter:

other: % Cysteine Peptide Depletion

Value:

1.092

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: % Lysine Peptide Depletion

Value:

4.654

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 3

Parameter:

other: Mean % Peptide Depletion (Cys + Lys)

Value:

2.873

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Acceptance criteria for all controls and the test substance were met in both runs with the exception of RefA for Cysteine which was marginally outside the range (0.556mM, range 0.45mM to 0.55mM). This was considered acceptable as it was only slightly outside the range and did not affect any of the other samples or controls in the run.

Results

 

Solvent Selection

Prior to the main test, the test substance was assessed for solubility and was found to be soluble in Isopropanol at 100mM.

 

Acceptance criteria

Acceptance criteria for all controls and the test substance were met in both runs with the exception of RefA for Cysteine which was marginally outside the range (0.556mM, range 0.45mM to 0.55mM). This was considered acceptable as it was only slightly outside the range and did not affect any of the other samples or controls in the run.

 

Criterion		Run 1 (Cysteine)		Run 2 (Lysine)		Outcome
Std Curve r^2>0.99		0.997		0.991		PASS
PC 60.8% to 100% depletion Cys		64.698		N/A		PASS
PC 40.2% to 69.0% depletion Lys		N/A		52.256		PASS
SDCys Depletion PC<14.9%		10.909		N/A		PASS
SDLys Depletion PC<11.6%		N/A		0.991		PASS
RefA Mean Conc 0.50 ± 0.05mM		0.556*		0.533		PASS*/PASS
Peak Area CV RefB <15.0%		8.970		0.377		PASS
Peak Area CV RefC <15.0%		5.137		0.844		PASS
SDCys Depletion Test Substance <14.9%		1.797		N/A		PASS
SDLys Depletion Test Substance <11.6%		N/A		3.924		PASS
RefC Mean Conc 0.50 ± 0.05mM		0.547		0.528		PASS

Cys = Cysteine, Lys = Lysine, SD = Standard Deviation, CV = Coefficient of Variation, PC = Positive Control. *Passed as acceptable by Study Director.

 

Results Summary

The test substance produced 2.873% mean Cysteine and Lysine peptide depletion, therefore, using the Cysteine 1:10 / Lysine 1:50 prediction model, the test substance was classified as a non-sensitiser with no or minimal reactivity. A single HPLC analysis for both the Cysteine and the Lysine peptide was considered sufficient for the test substance as the result was unequivocal.

 

Name	Test Substance ID	% Cysteine Peptide Depletion	% Lysine Peptide Depletion	Mean % Peptide Depletion (Cys + Lys)	DPRA Prediction	DPRA Reactivity Class
C16-18 AMP	TA2	1.092	4.654	2.873	Non-Sensitiser	No or Minimal Reactivity

 

 

Conclusion

The final mean % peptide depletion observed in the DPRA was 2.873%. Therefore, C16-18 AMP was classified as a non-sensitiser with no or minimal reactivity as per the Cysteine 1:10 / Lysine 1:50 prediction model.

Interpretation of results:

other: Not classified based on EU CLP Criteria

Conclusions:

Under study conditions, the test substance, C16-18 AMP was determined to be non sensitiser.

Executive summary:

A study was conducted to determine the skin sensitisating potential of the test substance, C16-18 AMP, according to the OECD Guideline 442C, in chemico Direct Peptide Reactivity Assay (DPRA), in compliance with GLP. Test substance was incubated for 24 h (± 2 h) at 25 ± 2.5˚C in solution at 100mM in combination with either Cysteine or Lysine containing peptides and then run on an high performance liquid chromatography (HPLC) system (20-minute run-time) using gradient elution and UV detection at 220 nm to measure peptide concentration. Test substance was compared to vehicle controls containing the test substance solvent in combination with either Cysteine or Lysine peptide in order to determine the relative percent peptide depletion. Cinnamic aldehyde was used as positive control substance. Relative percent peptide depletion values were used in a prediction model that assigns test substances to one of four reactivity classes. The test substance produced 2.873% mean Cysteine and Lysine peptide depletion, therefore, using the Cysteine 1:10 / Lysine 1:50 prediction model, the test substance was classified as a non sensitiser with no or minimal reactivity. A single HPLC analysis for both the cysteine and the Lysine peptide was considered sufficient for the test substance as the result was unequivocal. Acceptance criteria for all controls and the test substance were met in both runs with the exception of RefA for Cysteine which was marginally outside the range (0.556mM, range 0.45mM to 0.55mM). This was considered acceptable as it was only slightly outside the range and did not affect any of the other samples or controls in the run. Under study conditions, the test substance, C16-18 AMP was determined to be non sensitiser (XCellR8, 2018).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In absence of in vivo studies with the test substance, the skin sensitisation endpoint has been addressed based on available weight of evidence fromin silicoand in vitro studies.

In silico

In chemico analysis of the tests substance was conducted using the skin sensitisation profilers and the QSAR prediction programs of the OECD QSAR Toolbox v.4.1. The general and endpoint specific (Q)SAR profiling was run to identify structural alerts related to skin sensitization and the the automated workflow (AW) was executed to identify suitable analogues with experimental LLNA or GPMT data followed by data gap filling based on a skin sensitisation prediction. As, the Toolbox profilers or the QSAR prediction can only run for discrete chemicals, SMILES notation of the 2 major constituents (representing 90% of the composition) was used as the input parameters.The ‘DPRA 13%’ profiler indicated a low potency of binding potential for both the constituents. In addition, the constituents did not to match the structural criteria specified in the boundaries of the profiler for keratinocyte gene expression. And none of the other general protein binding potency profilers revealed any structural alerts; thus supporting an absence of protein binding mode of action for skin sensitisation. Further, theOECD QSAR Toolbox automated workflow (AW) for skin sensitization,due to structural similarity of the 2 main constituents of the test substance,identifiedsame set of analogue amides with ≥50% structural similarity. Based, on the absence of evidence of skin sensitisation in anLLNA and 2 GPMTs available on the analogues, a negative skin sensitisation prediction was obtained for both the constituents of the test substance. Therefore, based on the unambiguous QSAR profiling and the skin sensitisation prediction from OECD QSAR Toolbox for the main constituents, the test substance, C16-18 AMP, is considered not likely to have a skin sensitising potential (OECD Toolbox, 2018). Refer to the CSR for further details.

In chemico/in vitro

Study 1: A study was conducted to determine the skin sensitisating potential of the test substance, C16-18 AMP, according to the OECD Guideline 442C, in chemico Direct Peptide Reactivity Assay (DPRA), in compliance with GLP. Test substance was incubated for 24 h (± 2 h) at 25 ± 2.5˚C in solution at 100mM in combination with either Cysteine or Lysine containing peptides and then run on an high performance liquid chromatography (HPLC) system (20-minute run-time) using gradient elution and UV detection at 220 nm to measure peptide concentration. Test substance was compared to vehicle controls containing the test substance solvent in combination with either Cysteine or Lysine peptide in order to determine the relative percent peptide depletion. Cinnamic aldehyde was used as positive control substance. Relative percent peptide depletion values were used in a prediction model that assigns test substances to one of four reactivity classes. The test substance produced 2.873% mean Cysteine and Lysine peptide depletion, therefore, using the Cysteine 1:10 / Lysine 1:50 prediction model, the test substance was classified as a non sensitiser with no or minimal reactivity. A single HPLC analysis for both the cysteine and the Lysine peptide was considered sufficient for the test substance as the result was unequivocal. Acceptance criteria for all controls and the test substance were met in both runs with the exception of RefA for Cysteine which was marginally outside the range (0.556mM, range 0.45mM to 0.55mM). This was considered acceptable as it was only slightly outside the range and did not affect any of the other samples or controls in the run. Under study conditions, the test substance, C16-18 AMP was determined to be non sensitiser (XCellR8, 2018).

Study 2: A study was conducted to determine skin sensitisation potential of the test substance, C16-18 AMP, using ARE-Nrf2 Luciferase test method, according to the OECD Guideline 442D, in compliance with GLP.Concentrations of test substance tested were: 20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.02, 0.01 µg/mL. The sensitisation potential of the test substance was quantified by calculating two parameters known as the EC1.5and Imaxvalues. In each repetition of the study, no concentration of test substance caused luciferase induction≥1.5. Therefore, test substance was classified as non-sensitiser. The maximum induction was observed for repetition 1 at a test concentration of 0.039 µg/mL, which showed anImaxvalue of 1.284; maximum induction value for repetition 2 was observed at 0.625µg/mL with anImaxvalue of 1.384 and maximum induction value for repetition 3 was observed at 0.313µg/mL with anImaxvalue of 1.112. For reference, during the test validation, sensitising proficiency chemicals produced Imaxvalues of up to 36 fold over untreated controls. All of the formal acceptance criteria were met except acceptance criterion 2. However, as all other acceptance criteria were met and there was a dose-dependent increase of induction with the positive control, results were considered valid.Under study conditions, the test substance was concluded to be non-skin sensitiser (XcellR8, 2017).

Study 3: A study was conducted to determine the skin sensitisation potential of test substance, C16-18 AMP, using the in vitro human Cell Line Activation Test (h-CLAT) method, according to OECD 442E, in compliance of GLP. The h-CLAT assay measures markers of dendritic cell (DC) activation in the human leukaemia cell line THP-1. The ability of a chemical to induce activation of CD86 and CD54 in THP-1 cells was used as an assessment of the chemical’s skin sensitisation potential. Activation of CD54 and CD86 protein markers was measured by flow cytometry following 24 h exposure to the test substance. The concentration of the test substance used was selected on the basis of a pre-determined CV75 value (i.e. the estimated concentration yielding 75% cell viability). For the CV75 determination, THP-1 cells (a cell line that mimics DCs) were pre-cultured for either 48 or 72 h. Following this, the cells were dosed with the test substance over an 8 dose range (300, 250, 208.33, 173.61, 144.68, 120.56, 100.47, 83.72 µg/mL) and incubated for 24 ±0.5 h. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The CV75 was found to be 250 µg/mL. The EC200 and EC150 values for CD54 and CD86 expression were found to be 12 µg/mL and 11 µg/mL respectively. As the CD54/CD86 expression crossed the sensitisation threshold the test substance was classified as a skin sensitiser. Cell viability only fell marginally below 50% at the 120.56 µg/mL test substance concentration in Run 1. All other concentrations that induced sensitisation had a cell viability of >50% and therefore the final result were deemed to be valid. An inverse relationship of dose to viability was observed in the main test, however this has not affected the end result, as all test concentrations (with the exception noted above) had cell viabilities above 50% and the RFI data were conclusive and consistent in both runs. Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression. Under study conditions, the test substance was concluded to be a skin sensitiser (XcellR8, 2017).

Based on the results from thein vitroskin sensitisation tests and considering a two out of three weight of evidence approach, the test substance, C16-18 AMP, is overall concluded to be a non-sensitising.

​Overall, based on the available evidence fromin silico(OECD QSAR Tool box v.4.1 profiling and AW prediction) together within vitrostudies (DPRA, KeratinoSens^TMassays and h-CLAT), the test substance, C16-18 AMP, is overall concluded to be a non-sensitising to skin.​ This is further supported by absence of skin sensitisation potential of structurally similar fatty acid alkanolamides, such as Cocamide MEA in a Guinea pig maximisation study and stearamide MEA in a human repeat insult patch test (CIR, 2015).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available weight of evidence from in silico (OECD QSAR Tool box v.4.1 profiling and sensitisation predictions) and in vitro studies (DPRA, KeratinoSens^TMassays and h-CLAT), the test substance, C16-18 AMP, does not warrant classification for skin sensitisation according to EU CLP criteria (Regulation 1272/2008/EC).​