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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:
13 March 2017 - 17 March 2017
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 guidelineopen allclose all
Qualifier:
according to
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:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
paste
Details on test material:
Name as cited in report: DDDA TEA salt
Physical appearance: beige to pale yellow paste
Storage conditions: at room temperature
Specific details on test material used for the study:
- Correction factor for purity: 1.25
- Stability at higher temperatures: no, maximum temperature 25°C

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 25714
- Surface: 0.6 cm^2
- On the day of receipt the tissues were kept on agarose and stored in the refrigerator.

- Test for color interference by the test item: 25 mg test item was added to 0.3 mL Milli-Q water and the mixture was incubated for approx. 1 hour at 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. A negative control (Milli-Q water) was tested concurrently.
- Test for reduction of MTT by the test item: 25 mg test item was added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approx. 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. A negative control (Milli-Q water) was tested concurrently.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minute exposure: room temperature, 1 hour exposure: 36.7 - 37.5°C.
- Temperature of post-treatment incubation: 37°C.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with phosphate buffered saline (one washing step)
- Observable damage in the tissue due to washing: no

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

NUMBER OF REPLICATE TISSUES: 4 (2 tissues for the 3 minute exposure period, 2 tissues for the 1 hour exposure period) + 2 tissues for the negative and the positive control for each exposure period

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (see table 1 in ''any other informatin on methods'')
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%.

ACCEPTABILITY CRITERIA:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8)
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) 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 applied: 50 μl (liquefied in a water bath at 37 ºC)

NEGATIVE CONTROL
- Amount applied: 50 μl Milli-Q water

POSITIVE CONTROL
- Amount: 50 μl 8N KNOH
Duration of treatment / exposure:
- 3 minutes
- 1 hour
Duration of post-treatment incubation (if applicable):
3 hours in MTT medium
Number of replicates:
4 in total; 2 per exposure period

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure (mean of 2 replicates)
Value:
69
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
9%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure (mean of 2 replicates)
Value:
88
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
11%
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: yes, the absolute mean OD570 was between the acceptance limits (i.e., 1.929 for the 3-minute application and 2.085 for the 1-hour application).
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following 1 hour exposure was <15% (i.e., 9%).
- Acceptance criteria met for variability between replicate measurements: yes, CV between tissue replicates was ≤30% (i.e., 10% for the 3 minute exposure period and 4.5% for the 1 hour exposure period).

- Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, it is considered to be not corrosive.

Any other information on results incl. tables

Table 2 Individual OD measurements at 570 nm

 

3-minute application (OD570)

       A               B

1-hour application (OD570)

       A               B

Negative control

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

 

2.0673

2.0059

2.1307

2.1289

1.9581

1.9140

2.1032

2.1403

1.9677

1.9110

2.0648

2.1936

DDDA TEA SALT

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

 

1.6717

1.8425

1.4712

1.4782

1.6627

1.8051

1.4199

1.4986

1.6090

1.8333

1.4316

1.5443

Positive control

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

 

0.2264

0.2996

0.2663

0.1833

0.2143

0.2804

0.2623

0.1793

0.2216

0.2854

0.2654

0.1836

OD = Optical density

Duplicate exposures are indicated by A and B.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) 1272/2008
Conclusions:
The results of an in vitro skin corrosion test, performed according to OECD guideline 431, showed that DDDA TEA salt was not corrosive to the skin. The test substance is therefore not classified according to GHS and according to Regulation (EC) 1272/2008.