Molybdenum trioxide, reaction products with bis[O,O-bis(2-ethylhexyl)] hydrogen dithiophosphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 October 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']dioxodi-.mu.-thioxodi-, (Mo-Mo)

EC Number:

615-708-0

Cas Number:

72030-25-2

Molecular formula:

N/A

IUPAC Name:

Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']dioxodi-.mu.-thioxodi-, (Mo-Mo)

Constituent 2

Reference substance name:

Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']-.mu.-oxodioxo-.mu.-thioxodi-

Cas Number:

153128-45-1

Molecular formula:

N/A

IUPAC Name:

Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']-.mu.-oxodioxo-.mu.-thioxodi-

Constituent 3

Reference substance name:

Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']oxodi-.mu.-thioxothioxodi-

Molecular formula:

N/A

IUPAC Name:

Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']oxodi-.mu.-thioxothioxodi-

Constituent 4

Reference substance name:

Unspecified 2-ethylhexyl thiophosphate esters

Molecular formula:

N/A

IUPAC Name:

Unspecified 2-ethylhexyl thiophosphate esters

Constituent 5

Reference substance name:

Unspecified 2-ethylhexyl phosphate esters

Molecular formula:

N/A

IUPAC Name:

Unspecified 2-ethylhexyl phosphate esters

Constituent 6

Reference substance name:

Unspecified 2-ethylhexyl dithiophosphate esters

Molecular formula:

N/A

IUPAC Name:

Unspecified 2-ethylhexyl dithiophosphate esters

Test material form:

liquid

Specific details on test material used for the study:

CAS Registry Number: 72030-25-2
Purity: 100%; This substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
Physical state/Appearance: Dark green liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: reconstructed human epidermis tissues

Cell source:

other: not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. Triplicate tissues treated with 10 µL of Dulbecco’s Phosphate Buffered Saline (DPBS) served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. The plates were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period, each tissue was removed from the well using forceps, rinsed using DPBS with Ca++ and Mg++, transferred to 3 wells containing maintenance medium, and incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42 hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. Maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator IL-1α determination.

MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, incubated for 3 hours at 37 °C, 5% CO2 in air and then placed onto absorbent paper to dry. A total biopsy of the epidermis was made, the epidermis was carefully separated from the collagen matrix, and both parts (epidermis and collagen matrix) placed into labeled micro tubes containing acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and then the tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate samples were transferred to the appropriate wells of a pre labeled 96 well plate. Acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the LabTech LT 4500 microplate reader.

Control samples:

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

Amount/concentration applied:

10 µL (26.3 µL/cm^2) of the test item was applied to the epidermis surface.

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:

23.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Any other information on results incl. tables

Direct MTT Reduction

The MTT solution containing the test item turned purple which indicated that the test item directly reduced MTT. 

 

Assessment of Color Interference with the MTT Endpoint

The solution containing the test item was a dark green color, therefore additional color correction tissues were incorporated into the testing procedure. The results obtained showed that negligible color interference occurred.  It was considered unnecessary to use the results of the color correction tissues for quantitative correction of results.

 

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 23.8% (≤50%) after a 15 Minute exposure period and 42 Hour post exposure incubation period.

 

Quality Criteria

The relative mean viability for the positive control was 19.7% relative to the negative control treated tissues and the standard deviation value of the viability was 6.7% (≤18%). The positive control acceptance criteria were therefore satisfied. The mean OD570 for the negative control treated tissues was 0.874, within the range of 0.6 - 1.5 and the standard deviation value of the viability was 2.1% (≤18%). The negative control acceptance criteria were therefore satisfied. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues were 2.8% (≤18%). The test item acceptance criterion was therefore satisfied.

 

Mean Viabilities of the EPISKIN™ Tissues

Treatment	OD570 (Mean ± SD)	Relative Tissue Viability (Mean ± SD)
Test Item	0.208 ± 0.024	23.8% ±2.8%
DPBS (Negative Control)	0.874± 0.019	100%±2.1%
0.5% SDS (Positive Control)	0.172 ± 0.058	19.7% ±6.7%

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Supporting evidence of non-corrosive results in supporting study and eye irritation results.

Conclusions:

The relative mean tissue viability of the test item was 23.8%, less than 50%, and therefore, the test item 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).

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 test item was also found to have the potential to cause color interference with the MTT endpoint therefore additional tissues were incorporated into the testing to correct for this. A third set of controls was included, comprising water killed tissues, to prevent a double correction from a colored test item that also reduces MTT. 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 23.8% 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.

 

Under the conditions of the study, the relative mean tissue viability of the test item was 23.8%, less than 50%, and therefore, the test item 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).