Phosphoric acid, mono- and di-C16-18-alkyl esters

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Octadecyl dihydrogen phosphate

EC Number:

220-983-1

EC Name:

Octadecyl dihydrogen phosphate

Cas Number:

2958-09-0

Molecular formula:

C18H39O4P

IUPAC Name:

octadecyl dihydrogen phosphate

Constituent 2

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

Constituent 3

Reference substance name:

Dioctadecyl hydrogen phosphate

EC Number:

221-237-8

EC Name:

Dioctadecyl hydrogen phosphate

Cas Number:

3037-89-6

Molecular formula:

C36H75O4P

IUPAC Name:

dioctadecyl hydrogen phosphate

Constituent 4

Reference substance name:

Dihexadecyl hydrogen phosphate

EC Number:

218-594-7

EC Name:

Dihexadecyl hydrogen phosphate

Cas Number:

2197-63-9

Molecular formula:

C32H67O4P

IUPAC Name:

dihexadecyl hydrogen phosphate

Constituent 5

Reference substance name:

hexadecyl octadecyl hydrogen phosphate

Cas Number:

93803-11-3

Molecular formula:

C34H71O4P

IUPAC Name:

hexadecyl octadecyl hydrogen phosphate

impurity 1

Reference substance name:

Unidentified impurities, likely the C16 and C18, and mixed C16/C18 trialkyl phosphates based 31P NMR chemical shift.

Molecular formula:

N/A

IUPAC Name:

Unidentified impurities, likely the C16 and C18, and mixed C16/C18 trialkyl phosphates based 31P NMR chemical shift.

Test material form:

solid

Details on test material:

CAS Number 39471-52-8

In vitro test system

Test system:

human skin model

Source species:

human

Test system

Controls:

not required

Duration of treatment / exposure:

15 minutes

Observation period:

Post exposure incubation period of 42 hours.

Number of animals:

Not applicable

Details on study design:

Negative control:
Dulbecco's phosphate buffered saline (DPBS) with Ca++ and Mg++ was used as the negative control.

Positive control:
Sodium dodecyl sulphate (SDS) 5% w/v was used as the positive control.

Pre-test - Assessment of direct test material reduction of MTT:
MTT dye metabolism, cell viability assay; one limitation is possible interference of the test material with MTT. The test material may directly reduce MT, thus mimicking dehydrogenase activity. The property of the test material is only a problem, if at the time of the MTT there are still sufficient amounts of the test material present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and qualified.
To identify this possible interference each test material is checked for the ability to directly reduce MTT according to the following procedure:
10 mg of material added to 2 ml of a 0.3 mg/ml MTT solution prepared in assay medium. Solution is incubated in the dark at 37ºCm 5% CO2 in air for 3 hours, Untreated MTT used as a control. The test material did not directly reduce MTT, the MTT solution containing the test material did not turn blue to indicate the result.

Pre-incubation:
2ml of medium warmed to 37ºC and piped into the first three columns of a pre-labelled 12 well plate. A different 12-well plate was used for the test material and each control, incubated at 37ºC, 5% CO2 in air overnight.
Main Test:
(Day 1): Application of test material
Triplicate tissues were treated with the test material for an exposure period of 15 minutes, 10 mg of the material topically applied to the corresponding tissues ensuring a uniform covering. The epidermis surface first being moistened with 5 ul of sterile distilled water to improve contact between the solid test material and the epidermis. Triplicate tissues treated with 10 ul of negative control and 10 ul of SDS 5% w/v serving as the positive control. For the positive control care was taken to endure contact of the dilution and the epidermis surface, after 7 minutes contact time the SDS solution was re-spread with the SDS solution to maintain distribution for the remainder of the contact period, The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes. Removal of the material involved removing each tissue from the well and rinsing with a constant stream of DPBS for a period of 40 seconds. The rinsed tissues were transferred to the second column of 3 wells containing 2ml of maintenance medium, the rinsed tissues incubated at 37ºC, 5% CO2 in air for 42 hours.
(Day 3) MTT loading/Formazan Extraction:
Following 42 hours post-exposure period each 12-well plate was placed on a shaker for 15 minutes, 1.6 ml of the maintenance medium from beneath each tissue was transferred to a micro-tube and stored in a freezer at -14ºC to -30ºC to determine inflammatory mediator determination.
2ml of a 0.3 mg/ml MTT solution pipetted into the third column of three wells and tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium. Tissues were incubated for 3 hours at 37ºC, 5% CO2 in air. A total biopsy was made using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps containing 500 ul of acidified isopropanol. The tissues were then refrigerated at 1 to 10ºC until Day 6.
(Day 6) - Absorbance /optical density measurements:
Optical density measured at 540 nm using an Anthos 2001 microplate reader.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

107.6

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Classification of irritation potential is based upon relative mean tissue viability following the 15-minute exposure period followed by the 42 hour post-exposure incubation period according to the following:
Criteria for in vitro interpretation
Relative Mean Tissue Viability is =50% - classification is Irritant (I) R38
Relative Mean Tissue Viability is =50% - classification is Non-irritant (NI)

Any other information on results incl. tables

The results were evaluated according to EU labelling regulations Commission Directive 2001/59/EC for classification and labelling of dangerous items.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In a study conducted following the OECD 439 Guideline determination of in vitro skin irritation using Reconstructed Human Epidermis, the test material was determind to be non-irritating.

Executive summary:

The results were evaluated according to EU labelling regulations Commission Directive 2001/59/EC for classification and labelling of dangerous items.

The acceptance criteria was achieved for the positive and negative controls.

Positive Control

The assay establishes acceptance criterion for an acceptable test if the relative mean tissue viability for the postive control treated tissues was =40% relative to the negative control treated tissues, and the standard deviation values of the percentage viability is =18%.

Negative Control

The assay establishes the acceptance criterion for an acceptable test if the mean OD540 for the negative control treated tissues was 0.6=, and the standard deviation value of the percentage viability is =18%.