Dibutyl [(dipropoxyphosphinothioyl)thio]succinate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 November 2017 to 16 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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:

Dibutyl 2-[(dipropoxyphosphorothioyl)sulfanyl]succinate

Cas Number:

68413-47-8

Molecular formula:

C18H35O6PS2

IUPAC Name:

Dibutyl 2-[(dipropoxyphosphorothioyl)sulfanyl]succinate

Test material form:

liquid

Specific details on test material used for the study:

- Description: Light yellow, clear liquid
- Storage: 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

Details on test system:

Test for Direct MTT Reduction:
A test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 μL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.

If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin irritation potential was performed in parallel on viable and water-killed tissues.

This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues.
Water-killed tissues were prepared prior to the study by placing untreated EPISKINTM tissues in a 12-well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5% CO2 in air for a minimum of 48 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (−14 to −30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.

In addition to the normal test procedure, the MTT reducing test item was applied to three water-killed tissues. In addition, three water-killed tissues remained untreated. The untreated water-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

Assessment of Colour Interference with the MTT endpoint:
A test item may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.

10 μL of test item was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker at room temperature, a visual assessment of the colour was made.

Control samples:

other: Negative Control: Dulbecco's Phosphate Buffered Saline (DPBS) with Ca++and Mg+ and Positive Control: Sodium Dodecyl Sulphate

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):

At the end of the exposure period, each tissue was rinsed before incubating for 42 hours.

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 incubation period.

Value:

88.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Direct MTT reduction:

The test item was shown to directly reduce MTT in the direct MTT reduction test.

 

Assessment of Colour Interference with the MTT endpoint:

The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

 

Test Item, Positive Control Item and Negative Control Item:

The relative mean viability of the test item treated tissues was 88.2% (>50%) after a 15-minute exposure period and 42-hour post-exposure incubation period.

 

Quality Criteria:

The relative mean viability for tissues treated with 5% (w/v) SDS aqueous solution (positive control) was 27.7% relative to the negative control treated tissues and the standard deviation value of the viability was 2.2% (≤18%). The positive control acceptance criteria were therefore satisfied.

 

Mean Viabilities of the EPISKIN™ Tissues:

Mean Viabilities of the EPISKIN™Tissues Treatment	OD570(Mean ± SD)	Relative Tissue Viability (Mean ± SD)
Test Item*	0.753 ± 0.064	88.2% ± 7.5%
DPBS (Negative Control)	0.854 ± 0.047	100% ±5.5%
0.5% SDS (Positive Control)	0.236 ± 0.019	27.7% ± 2.2%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant. The following classification criteria apply: EU CLP Not classified for Irritation and UN GHS Not classified for Irritation (category 3 cannot be determined).

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. Tissue viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt in the test item treated tissues relative to the negative controls.

 

Triplicate tissues were treated with the test item for 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; therefore, additional non-viable tissues were incorporated into the testing for correction purposes. The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

 

At the end of the post exposure incubation period 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. 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. Data was presented in the form of percentage viability (MTT reduction in the test item or positive control treated tissues relative to negative control tissues).

 

The relative mean viability of the test item treated tissues was 88.2%% after the 15 minute exposure period and 42 Hours post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied. The test item was classified as non-irritant. The following classification criteria apply: EU CLP Not classified for Irritation and UN GHS Not classified for Irritation (category 3

cannot be determined).