Ethyltriphenylphosphonium acetate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 February 2018 - 09 March 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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyltriphenylphosphonium acetate

EC Number:

252-743-7

EC Name:

Ethyltriphenylphosphonium acetate

Cas Number:

35835-94-0

Molecular formula:

C20H20P.C2H3O2

IUPAC Name:

ethyltriphenylphosphanium acetate

Test material form:

solid

Specific details on test material used for the study:

The test item was ground to a fine powder before use.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

recommended by guideline

Vehicle:

unchanged (no vehicle)

Remarks:

25 µL of sterile water was added for wetting of the test item to increase tissue surface contact.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 25884
- Delivery date: 06 March 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper and the tissue surface was swabbed. The rinsing procedure was then repeated once more to ensure the tissues were completely decontaminated.
- Observable damage in the tissue due to washing: no


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader

NUMBER OF REPLICATE TISSUES: duplicates

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): freeze killed
- N. of replicates : duplicates
- Method of calculation used: True viability = mean OD tvt-(OD tkt-OD ukt)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- 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 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 (volume or weight with unit): 25 mg; 25 µL of sterile water was added for wetting of the test item to increase tissue surface contact.


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of sterile distilled water


POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of 8.0 N Potassium Hydroxide

Duration of treatment / exposure:

3 min, 60 min

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

71

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4.7% tissue viability

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

21.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4.1% tissue viability

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.
- Colour interference with MTT: The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.750 for the 3 minute exposure period and 1.877 for the 60 minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 4.1% relative to the negative control following the 60 minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied

Any other information on results incl. tables

Tissue	Exposure Period	MeanOD570of individual tissues	Mean OD570of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.672	1.750	0.110	6.3	100
		1.827
	60 Minutes	1.756	1.877	0.171	9.1
		1.998
Positive Control	3 Minutes	0.096	0.083	0.018	na	4.7
		0.070
	60 Minutes	0.066	0.078	0.016	na	4.1
		0.089
Test Item	3 Minutes	1.438	1.242	0.277	22.3	71.0
		1.046
	60 Minutes	0.375	0.412	0.052	12.7	21.9
		0.449

 

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

The purpose of this test is to evaluate the corrosivity potential of ETPPAAc using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. 

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue.  Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control.  The results are used to make a prediction of the corrosivity potential of the test item. 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes.  Negative and positive control groups were treated for each exposure period.    At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTTloading.  After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.  

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

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

 

The relative mean viabilities for each treatment group were as follows: 

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	4.7	71.0
60 minute	100*	4.1	21.9

*The mean viability of the negative control tissues is set at 100% 

 

The quality criteria required for acceptance of results in the test were satisfied. 

The test item was considered to be non-corrosive to the skin.