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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 July 2019 - 05 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Salts of phosphoric acid, 2-ethylhexyl esters with C16-18 (even numbered) and C18-unsaturated-alkylamines
EC Number:
949-859-1
Molecular formula:
not applicable
IUPAC Name:
Salts of phosphoric acid, 2-ethylhexyl esters with C16-18 (even numbered) and C18-unsaturated-alkylamines
Test material form:
liquid
Details on test material:
- Appearance: Clear yellow liquid
- Storage condition: At room temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: MatTek Corporation, Ashland MA, U.S.A.
Source strain:
other: not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissue
- Tissue batch number(s): 30903

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes exposure and 60 minute exposure: 37 ± 1.0 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.
- Observable damage in the tissue due to washing: no

TEST FOR DIRECT MTT REDUCTION AND COLOUR INTERFERENCE
The substance was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 µL of the test item or 50 µL Milli-Q water as a negative control were added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.
The substance was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the color interference, 50 µL of the test item or 50 µL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple color change was observed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Microplate reader: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 exposure times

EVALUATION
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100

PREDICTION MODEL / DECISION CRITERIA
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Text Table 1 in section any other information on materials and methods incl. tables, presents the data interpretation and optional sub-categorisation in case a test item will be corrosive.

ACCEPTABILITY CRITERIA:
1. The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
2. The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
3. In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be <= 30%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL
Duration of treatment / exposure:
3 minutes and 60 minutes
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes exposure / mean of 2 replicates
Value:
95
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
11
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure / mean of 2 replicates
Value:
78
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
6.4
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range.
- Acceptance criteria met for positive control: The mean relative tissue viability following the 1-hour exposure to the positive control was 6.4% (< 15%).
- Acceptance criteria met for variability between replicate measurements: In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤10%, indicating that the test system functioned properly.

Any other information on results incl. tables

Mean Absorption in the in vitro Skin Corrosion Test with the substance

 

3-minute application

1-hour application

A (OD570)

B (OD570)

Mean

(OD570)

SD

A (OD570)

B (OD570)

Mean

(OD570)

SD

Negative control

1.690

1.674

1.682

±

0.012

1.929

1.735

1.832

±

0.137

The substance

1.632

1.557

1.595

±

0.053

1.369

1.490

1.429

±

0.085

Positive control

0.148

0.206

0.177

±

0.041

0.141

0.096

0.118

±

0.032

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0449). Isopropanol was used to measure the background absorption.

Mean Tissue Viability in the in vitro Skin Corrosion Test with the substance

 

3-minute application

viability (percentage of control)

1-hour application

viability (percentage of control)

Negative control

100

100

The substance

95

78

Positive control

11

6.4

Coefficient of Variation between Tissue Replicates

 

3 minute

1 hour

Negative control

1.0

10

The substance

4.6

8.1

Positive control

28

32

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

Applicant's summary and conclusion

Interpretation of results:
other: Not corrosive
Remarks:
According to Regulation (EC) No. 1272/2008 and its amendments.
Conclusions:
The results of an in vitro skin corrosion test showed that the substance was not corrosive to the skin.
Executive summary:

The substance was tested in duplicate in an in vitro skin corrosion test according to OECD TG 431 test guideline and GLP principles. Tissues were exposed to the substance, a negative control (Milli-Q water) and

a positive control (8.0 N KOH) for 3 minutes and 60 minutes. The substance was tested for direct MTT reduction and colour interference and both results were negative. Acceptability criteria for the negative

control, positive control and variability between measurements were met.

The cell viability of the tissues exposed to the substance were 95% and 78% for 3 minutes and 60 minutes exposure, respectively. Both values did not exceed thereshold for corrosivity (50% after 3 minutes exposure and 15% after 60 minutes exposure), therefore the substance is considered not to be corrosive according to Regulation (EC) No. 1272/2008 and its amendments..