Phosphoric acid, mono- and dimethyl esters, compds. with C10-13-(branched)-alkylaniline

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 February 2018 to 26 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

Phosphoric acid, monomethyl ester, compd. with 4-tetrapropylenebenzenamine (1:1)

Molecular formula:

C19H36NO4P

IUPAC Name:

Phosphoric acid, monomethyl ester, compd. with 4-tetrapropylenebenzenamine (1:1)

Constituent 2

Reference substance name:

Dimethyl hydrogen phosphorate, compound with 4-tetrapropyleneaniline

EC Number:

269-016-5

EC Name:

Dimethyl hydrogen phosphorate, compound with 4-tetrapropyleneaniline

Cas Number:

68170-22-9

Molecular formula:

C20H38NO4P

Test material form:

liquid: viscous

Specific details on test material used for the study:

Purity: >94% (nominal); This substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB).
Sponsor Description: Brown paste to very viscous liquid
Envigo Description: Amber colored viscous liquid
Storage Conditions: Room temperature (15-25°C) in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

The EPISKINTM Kit consisted of one EPISKIN™ plate containing 12 reconstructed human epidermis tissues (0.38 cm2), maintenance medium (used for tissue pre-conditioning and testing purposes) and assay medium (used for MTT assay).

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours.

The test item was found to directly reduce MTT in the pre-test for direct MTT reduction and therefore additional non-viable tissues were incorporated into the testing for correction purposes. The test item was found to have the possibility to cause color interference with the MTT endpoint therefore, as a precaution, additional tissues were incorporated into the testing to correct for this.

A third set of controls was included, comprising non-viable tissues, in order to prevent a double correction from a colored test item that also reduces MTT. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator IL-1α determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item or positive control treated tissues relative to negative control tissues).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 µL (26.3 µL/cm2)

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes.

Duration of post-treatment incubation (if applicable):

42 hour post-exposure incubation period.

Number of replicates:

Three.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minute exposure period and 42 hours post exposure

Value:

ca. 30

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the study, the relative mean tissue viability of tissues treated with the test material was 30.0%, less than 50%.

Executive summary:

Under the conditions of the study, the relative mean viability of tissues treated with the test material was 30.0%, less than 50%, and therefore, the test material demonstrated the ability to cause a positive response as skin irritant. 

However this study does not allow the conclusion on whether the test item is Category 1 or Category 2 of Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP) and United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).