Hexadecyl dihydrogen phosphate

Endpoint summary

Description of key information

The conclusion from the two in-vitro tests carried out were divided. The h-CLAT test showed a negative result whereas the KeratinoSens test showed a positive result. It was not possible to carry out the third in-vitro DPRA test due to problems with solubilisation of the test substance

The conclusion of the in-vivo GPMT test was that cetyl phosphate is not a skin sensitiser.

In-vitro testing was performed by two methods and in each test, different assays / replicates were conducted. Of these two tests performed, one proved negative and one was positive.  It was also noted that the third type of test was not suitable for this substance type.

Historical data published by CIR and the absence of reported effects from use over a period of time would make this substance unlikely to be a skin sensitiser. If taking a weight of evidence from all sources available, the conclusion is that the substance is unlikely to cause skin sensitisation at concentrations not leading to corrosivity.  

The general overview and conclusion from the CIR report for the safety of alkyl phosphates in cosmetics was that these substances are not sensitising and are safe to use in cosmetics from that view point.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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.

Specific details on test material used for the study:

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

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

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.

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

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.

Reference 2

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Skin sensitisers have been reported to induce genes that are regulated by the antioxidant response element (ARE). The KeratinoSensTM test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSensTM test method was considered scientifically valid to be used as part of an IATA (Integrated Approach to Testing and Assessment), to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The method cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by the UN GHS, for authorities implementing these two optional subcategories, nor to predict potency for safety assessment decisions. However, depending on the regulatory framework a positive result may be used on its own to classify a chemical into UN GHS category 1

Specific details on test material used for the study:

Supplier Colonial Chem, Inc
Test Item Name 1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE)
Supplier batch/lot number 64252F18
CAS number 3539-43-3
Purity 100%
Expiry Date 11OCT20 (CoA)
Physical state White to off-white powder
Storage Conditions Room Temperature
Solvent 1% DMSO in cell culture medium
Administration method In cell culture medium
Concentrations tested (µg/ml) 0.195, 0.391, 0.781, 1.563, 3.125, 6.250, 12.500, 25.000, 50.000, 100.00, 200.000, 400.000
XCellR8 test item code TA2
Study test item code COL0003

Details on the study design:

The KeratinoSensTM cell line (test system) is an immortalized adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.

Positive control results:

Per plate, a single application of 5 concentrations of the positive control, Cinnamic Aldehyde, was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1% and a single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate). One well per plate was left empty (no cells).
Positive Control (PC) (Cinnamic aldehyde) induction >1.5-fold in at least one concentration, 3 out of 5 above = PASS

Key result

Parameter:

other: The sensitisation potential of the test item was quantified by calculating 2 parameters known as the EC1.5 and the IMAX value

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Interpretation of Results and Skin Sensitisation Prediction model
A test item is considered positive using the KeratinoSens prediction model if the following conditions are met in 2 of 3 repetitions:
•The IMAX is equal to or higher than 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s T-test).
•The cellular viability is higher than 70% at the lowest concentration with induction of luciferase activity equal to or above 1.5 fold (i.e. at the EC1.5 determining concentration). Test items that only induce the gene activity at cytotoxic levels are not rated positive, as in the case for some non-sensitising skin irritants.
•The EC1.5 value is < 1000 µM or < 200 µg/mL for test items with no defined MW.
•There is an apparent overall dose-response for luciferase induction (or a biphasic response).

In this study,1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE)was classified as a Positive using the KeratinoSens prediction model.

The sensitisation potential of the test item was quantified by calculating 2 parameters known as the EC_1.5and the I_MAXvalue:

The EC_1.5value is the Effective Concentration (EC) of test item that yielded an induction of luciferase activity equal to or greater than 1.5-fold over untreated controls. If at least one concentration induces statistically significant luciferase activity>1.5, then the product is classified as positive provided the cell viability measured by MTT is greater than 70%.

 

The test item induced statistically significant luciferase induction>1.5 in all 3 repetitions. The respective EC_1.5values were calculated as 6.956 µg/ml, 5.634 µg/ml and 0.920 µg/ml for repetitions 1 to 3 respectively. The statistical significance, viability, dose response and dose acceptance criteria were all met (see section 13.2) and therefore:

The test itemwas classified as positive using the KeratinoSens prediction model.

 

The I_MAXvalue is the maximum induction observed within the concentration range tested. Although the KeratinoSens^TMtest is not validated to predict potency, the I_MAXvalue can provide a useful tool for preliminary comparison of sensitisation potential between test items. The maximum induction was observed at a test concentration of 400µg/ml in rep 1 and rep 2, and at 200µg/ml in rep 3. For reps 1-3, the I_MAXvalues were 2.637, 2.746 and 3.601, respectively. For reference, during test validation, sensitising proficiency chemicals produced I_MAXvalues of up to 36-fold over untreated controls.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The human skin sensitisation potential of 1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE) was assessed using the validated in vitro method, the KeratinoSensTM assay, adapted to animal product-free conditions by XCellR8, and validated in-house to determine keratinocyte activation.
After 48h exposure of cells with 12 concentrations of 1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE), Luciferase measurements and MTT viability testing were performed.
1-Hexadecanol, 1-(dihydrogen phosphate), (ColaFax CPE) was classified as Positive according to the KeratinoSens prediction model.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

No detail specified.

Specific details on test material used for the study:

CIR review specifies 'cetyl phosphate', which is the same as hexadecyl dihydrogen phospate.
Purity not specified

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Route:

intradermal and epicutaneous

Vehicle:

water

Concentration / amount:

intradermal induction: 0.5% in water
epidermal induction: 40%

Day(s)/duration:

48 hours

No.:

#1

Route:

intradermal and epicutaneous

Vehicle:

water

Concentration / amount:

challenge 1: 20% and 40%

No.:

#2

Route:

intradermal and epicutaneous

Vehicle:

water

Concentration / amount:

challenge 2: 1% and 10%

No. of animals per dose:

20 on test and 10 on control

Details on study design:

intradermal induction on day 1; epidermal induction (48-h occlusive patch) 24 h after intradermal induction; epidermal challenge 1 (48-h occlusive patch) 2 wks after induction; challenge 2 (24-h occlusive patch) 1 wk after challenge 1

Positive control substance(s):

not specified

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

20%

No. with + reactions:

8

Total no. in group:

20

Clinical observations:

slight erythema

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

20%

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

slight erythema

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

20%

Remarks on result:

not measured/tested

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

40% challenge

No. with + reactions:

13

Total no. in group:

20

Clinical observations:

slight erythema

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

40% challenge

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

slight erythema

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

40% challenge

Remarks on result:

not measured/tested

With the exception of the 20% negative controls, reactions persisted in all of these groups at 72hrs

Interpretation of results:

GHS criteria not met

Conclusions:

The conclusion of this GPMT test was that cetyl phosphate is not a skin sensitiser.

Reference 4

Endpoint:

skin sensitisation, other

Remarks:

assessment of available data

Type of information:

other: reveiw of available data

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

no guideline followed

Principles of method if other than guideline:

This review considers the validity of the in-vitro testing and also considers weight of evidence from similar alkylphosphates. In view of the limited validity of the in-vitro methods for surface active substances that are very poorly soluble in water, the in-vitro studies alone are unable to provide a sufficient weight of evidence either way.
This review takes data from a number of sources, including the two in-vitro assays successfully performed in 2019 and comments from the testing facility that the third method was not suitable. Each are assessed on their own merit.
Secondly, a literature search has been performed on alkyl phosphates to consider if the class of material has any know sensitising potential.
The objective is to confirm a weight of evidence and to avoid any new animal testing, but to be confident in the outcome with regard to worker and consumer safety.

GLP compliance:

no

Remarks:

GLP not required for a non-testing review of data

Type of study:

other: review of available data

Justification for non-LLNA method:

A number of non-animal tests have been completed on this substance type and it has been shown to be a low irritant to skin and a strong eye irritant, but is otherwise not considered hazardous to health.
Part of the hazards assessment is to consider potential for sensitisation and the class of substance has not demonstrated sensitising potential during historical use, but of two valid in-vitro studies, one was positive and one negative. A third method was not considered to be valid, so on the basis of current guidelines, it is not possible to assess whether the substance is a potential skin sensitiser by these methods.
Historically, animal testing has not been considered on this class of substance in view of the ethical concerns over the corrosive nature and in view of potential use in personal care products. Some work is reported in a CIR review

Parameter:

other: revew of in-vitro and in-vivo data

Remarks on result:

no indication of skin sensitisation

Findings from in-vitro assays.

Two in-vitro assays have been performed, following current guidance for the assessment of potential to induce sensitisation, but the third, generally needed to provide weight of evidence was apparently not possible to validate. It should be noted that despite these tests being recommended as a replacement to animal testing, there is an absence of historical data and many substance types are proving difficult to test; this includes certain types of surfactant, mixed molecular weight UVCB or where solubility in the media is very low.

The h-CLAT assay was performed 4 times in view of mixed results and validity criteria not being met in each case and the LuSens performed 3 times in view of marginal viability.

Both of these tests ultimately passed validity checks, but in both cases, validity criteria were marginal. Those assays proving positive were significantly below the results from positive controls, suggesting only marginal effects.

It is not considered possible to make a conclusion based on these results.

Assessment from other data sources

No references were found relating to similar alkyl phosphates or phosphonates leading to sensitisation. The one in-vivo result reported here as an end point for cetyl phosphate did not find any evidence of sensitisation.

However, many of the hydrogen phosphates are typically very acidic and are classified corrosive with test waivers as animal work was not appropriate. The higher molecular weight types are typically less irritant to skin, but this depend in part on the level of excess acid.

Since the potential to cause immune response (sensitisation) is typically based on specific chemical classes, this method of looking at phosphate and phosphonates is a valid approach and although this type of grouping is not necessarily conclusive evidence, it does help contribute to the weight of evidence. 

Few substances of this class are REACH registered and the lower molecular weight materials are classified as Corrosive and have not been assessed fully for sensitisation potential. In addition, substances in this group notified on the CLI are all 'negative', but it must be accepted that there may be no reliable data to back up these classifications.

A CIR review has been conducted to assess the class of substance for use in cosmetics. This was published 2014 and covers a wide range of fatty alkyl phosphates and some potassium and sodium salts. The range includes fatty derivatives from C9 - C18 and mono, di and tri-alkyl derivatives. The authors considered that these are sufficiently related to be covered in a single report and are all of low irritancy.

There are no reports of positive skin sensitisation in animal tests or from human assessments.

Interpretation of results:

GHS criteria not met

Conclusions:

In-vitro testing was performed by two methods and in each test, different assays / replicates were conducted. Of these two tests performed, one proved negative and one was positive. It was also noted that the third type of test was not suitable for this substance type.
Historical data published by CIR and the absence of reported effects from use over a period of time would make this substance unlikely to be a skin sensitiser. If taking a weight of evidence from all sources available, the conclusion is that the substance is unlikely to cause skin sensitisation at concentrations not leading to corrosivity.
Classification as Skin Sens 1 is not recommended.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available