Hexadecyl dihydrogen phosphate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

The human Cell Line Activation Test (h-CLAT) method as detailed in OECD TG 442E (adopted 25 Jun 2018) and also in EURL ECVAM DB-ALM Protocol No. 158 human Cell Line Activation Test (h-CLAT); Skin Sensitisation and Allergic Contact Dermatitis (Issued: 23 Jul 2018).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

Skin sensitisers have been reported to induce the expression of cell membrane markers associated with Dendritic Cell (DC) activation. The h-CLAT method is an in vitro assay that quantifies these changes in cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells (a cell line that mimics DCs), following 24-hour exposure to the test item. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.

Test material

Test material information

Constituent 1

Reference substance name:

Hexadecyl dihydrogen phosphate

EC Number:

222-581-1

EC Name:

Hexadecyl dihydrogen phosphate

Cas Number:

3539-43-3

Molecular formula:

C16H35O4P

IUPAC Name:

hexadecyl dihydrogen phosphate

Test material form:

solid: particulate/powder

Details on test material:

Product commercial name is ColaFax CPE

Specific details on test material used for the study:

Supplier Batch/Lot Number: 64252F18
CAS Number: 3539-43-3
Purity: 100%

In vitro test system

Details on the study design:

Summary of the test method is as follows:
Solubility assessment
Dose range finding experiment (CV75)
• Seed Cells for CV75 (two independent runs)
• Dose cells for CV75 (two independent runs)
• Detect live/dead cells using flow cytometry (two independent runs)
• Data analysis and CV75 determination

CD54/86 expression measurement
• Seed cells for h-CLAT CD54 and CD86 expression (four independent runs)
• Dose cells for h-CLAT CD54 and CD86 expression (four independent runs)
• Detect CD54 and 86 expression using flow cytometry (four independent runs)
• Data analysis and classification of test item

Results and discussion

Positive control results:

Positive Control - CV75
Reference Item name 2,4-dinitrochlorobenzene (DNCB)
Lot number BCBP8259V
Purity 99.5%
Expiry 20FEB24
Concentration tested 8µg/ml (final)
Solvent Dimethyl sulphoxide (DMSO; Neat)

CV75 Acceptance Criteria
Cell viability must be ≥ 75% at the lowest dose.
The highest test item 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 item.

Positive Control - CD54 and CD86 Expression
Reference Item name Nickel Sulphate
Lot number SZBF2960V
Purity 99.4%
Expiry 15JUN29
Concentration tested 100µg/ml (final)
Solvent RPMI (culture medium)

CD54/CD86 Expression: Run acceptance criteria
Cell viability of medium and DMSO controls should be greater than 90%
In the positive control (Nickel Sulphate; 100µg/ml), RFI values of both CD86 and CD54 should meet the criteria for a positive result (CD86 ≥ 150 and CD54 ≥ 200) and cell viability should be > 50%
In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 should not cross the threshold that denotes a positive response (CD86 ≥ 150 and CD54 ≥ 200)
For both medium and DMSO controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%

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.

In vitro / in chemico

Results

Key result

Parameter:

other: CV75

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: For 1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE) the CV75 was unable to be determined as the test item did not reduce cell viability below 75%

Remarks:

For 1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE) the CV75 was unable to be determined as the test item did not reduce cell viability below 75% As the expression threshold for CD54 was only crossed in one repetition (at the lowest four concentrations tested) and the expression threshold for CD86 was not crossed in any repetitions performed, 1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE) was classified as Negative as per the prediction model. This result should be considered in the context of the Log Kow value being unknown for the test item.

Other effects / acceptance of results:

Prior to the CV75 determination, the test item was assessed for solubility and was found to be soluble in DMSO at 250 mg/ml.
A Log Kow value was unable to be provided by the sponsor and the result reported here should be considered in the context of this.
The CV75 value was not able to be determined from two independent runs as the cell viability did not fall below 75%.
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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study, the skin sensitisation potential of 1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE) was assessed using the In Vitro human Cell Line Activation Test (h-CLAT) method according to OECD Test Guideline 442E. After a 24h incubation with the test item the expression of cell surface markers CD54 and CD86 on THP-1 cells was measured by flow cytometry.
For 1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE) the dose that gave 75% cell viability was not determined as the viability was not decreased below 75% by the test item. CD54 crossed the expression threshold for rep 1, however the thresholds for CD54 and CD86 marker expression were not crossed in the subsequent repetitions, and therefore, 1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE) was classified as Negative as per the prediction model. A Log Kow value was not able to be provided by the Sponsor, and therefore the result should be considered in the context of this.