O,O-bis(2-ethylhexyl) hydrogen dithiophosphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Oct - 04 Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted Jul 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

O,O-bis(2-ethylhexyl) hydrogen dithiophosphate

EC Number:

227-376-0

EC Name:

O,O-bis(2-ethylhexyl) hydrogen dithiophosphate

Cas Number:

5810-88-8

Molecular formula:

C16H35O2PS2

IUPAC Name:

O,O-bis(2-ethylhexyl) sulfanylphosphonothioate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ tissues

Source strain:

other: Keratinocyte strain 00267

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissue (EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number(s): 23372

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a wash bottle / multipipette containing DPBS to remove any residual test material (20 times).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax®, Molecular Devices, SoftMax Pro Enterprise
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by a MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 μL of 1% Triton X-100. The ET-50 value was determined to be 6.25 h.
- Contamination: All biological components of the epidermis and the culture medium were tested by the manufacturer for viral, bacterial, fungal and mycoplasma contamination.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No control tissues were needed as the optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour and optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%. The test substance can be classified in the optional Sub-category 1A, if the viability after 3 minutes exposure is less than 25%, and in a combination of the optional Sub-categories 1B and 1C, if the viability after 3 minutes exposure is greater than or equal to 25%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8.0 N

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

Duplicate tissues; from each tissue, 3 absorbance measurements after MTT incubation were performed.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

3 min exposure

Value:

78.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

60 min exposure

Value:

9.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (range: 1.586 to 1.814).
- Acceptance criteria met for positive control: the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (5.3%).
- Acceptance criteria met for variability between replicate measurements: the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 3.6% to 7.3%).
- Range of historical values: the results of the negative and positive control fell within the historical control range except of the viability determined after 3 min exposure with the test substance that was slightly outside the range of the historical control data (see Table 1 under "Any other information on results incl. tables").

Any other information on results incl. tables

Table 1 Historical control data

Positive control		Negative control
Mean viability [%]	20.1	Mean OD	1.58
CV [%]	4.16	CV [OD]	9.32
Range of Viabilities [%]	15 - 23	Range of Ods	1.34 - 1.77

Table 2 Summary of results

Test group	Mean Absorbance of 2 tissues	CV [%]	Rel. Absorbance [% of Negative Control]**	Mean Absorbance of 2 tissues	CV [%]	Rel. Absorbance [% of Negative Control]**
	3 min			60 min
Negative control	1.619	5.7	100	1.666	3.6	100
Test substance	1.271	7.3	78.5	0.160	17.8	9.6
Positive control	0.384	4.1	23.7	0.088	16.1	5.3

** relative absorbance [rounded values]: 100 x absorbance (test item / positive control) / absorbance (negative control)

Applicant's summary and conclusion

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

Under the conditions of the in vitro test method the test substance showed corrosive properties.
CLP: 1B, H314