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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

273-159-9

EC Name:

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

Cas Number:

68951-62-2

Molecular formula:

C20H41NO2 (C16 chain representative) & C22H45NO2 (C18 chain representative)

IUPAC Name:

N-(1-hydroxy-2-methylpropan-2-yl)hexadecanamide; N-(1-hydroxy-2-methylpropan-2-yl)octadecanamide

Test material form:

solid

Details on test material:

Appearance: Off white cream

In vitro test system

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.

Results and discussion

In vitro / in chemico

Results

Results 1

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

Results 2

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

Results 3

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).