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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:
17 July - 21 July 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
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:
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

Constituent 1
Chemical structure
Reference substance name:
Pregnenolone
EC Number:
205-647-4
EC Name:
Pregnenolone
Cas Number:
145-13-1
Molecular formula:
C21H32O2
IUPAC Name:
3-hydroxypregn-5-en-20-one
Test material form:
solid: particulate/powder
Details on test material:
- Physical appearance: white crystalline powder
- Storage conditions: at room temperature
Specific details on test material used for the study:
- No correction for purity required

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): 26708

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 36.8-37.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: after exposure and after incubation tissues were washed with phosphate buffered saline (once)
- 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: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment with 3 independent OD570 measurements per replicate.

ACCEPTABILITY CRITERIA
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
- The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

DECISION CRITERIA
A test item is considered corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
- 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:
- The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
- In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25.1 - 34.5 mg
- The test item was applied directly on top of the skin tissues (pre-wetted with 25 μl Milli-Q water)

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL
Duration of treatment / exposure:
3 minutes and 1 hour
Duration of post-treatment incubation (if applicable):
3 hours in MTT medium
Number of replicates:
2 tissues per test item per exposure time (4 tissues in total)

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 2 replicates
Run / experiment:
3-minute exposure
Value:
97
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
mean tissue viability: 7.1%
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 2 replicates
Run / experiment:
1-hour exposure
Value:
108
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
mean tissue viability: 10%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: the results of the positive control data were within the historical data range and thereby showing the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the two tissues of the negative control was within the laboratory historical control data range.
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following 1-hour exposure to the positive control was <15 % (i.e., 10%).
- Acceptance criteria met for variability between replicate measurements: yes, the Coefficient of Variation (CV) between tissue replicates was ≤ 30% (i.e., ≤ 9.6%)

Any other information on results incl. tables

Table 1 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

 

 

1.7098

1.8784

1.6100

1.5948

1.6407

1.867

1.8243

1.5933

1.8516

1.889

1.7996

1.6380

PRECH-PURCH

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

 

1.6134

1.7937

1.7167

1.8752

1.6743

1.8691

1.7641

1.8544

1.6936

1.8323

1.7392

1.8580

Positive control

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

 

0.1563

0.1727

0.2337

0.1955

0.1563

0.1802

0.2345

0.1965

0.1593

0.1796

0.2228

0.1946

OD = Optical density

Duplicate exposures are indicated by A and B.

Table 2 Historical data

 

Negative control

Positive control

Positive control

 

3-minute treatment

(OD570)

1-hour treatment

(OD570)

3-minute treatment

(OD570)

1-hour treatment

(OD570)

3-minute treatment

(% viability)

1-hour treatment

(% viability)

Range

1.324 – 2.615

1.361 – 2.352

0.0172 – 0.56

0.046 – 0.339

6 – 25

3 – 13

Mean

1.84

1.85

0.19

0.14

11.03

7.45

SD

0.26

0.22

0.09

0.06

4.39

2.51

n

81

83

80

77

38

38

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2013 to November 2016.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) 1272/2008
Conclusions:
In the in vitro skin corrosion test, PRECH-PURCH was found to be not corrosive to the skin (mean tissue viability of 100% and 81% after a 3-minute and a 1-hour exposure period, respectively). The test item is not classified according to GHS and according to Regulation (EC) 1272/2008.
Executive summary:

An in vitro skin corrosion test was performed, according to OECD guideline 431 and GLP principles, to assess PRECH-PURCH for its ability to induce skin corrosion. An amount of at least 25 mg was applied in deuplicate on a human three dimensional epidermal model (EpiDerm (EPI-200)) for an exposure time of 3 minutes and 1 hours each. For each exposure period, two negative controls and two positive controls were included. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 97% and 108%, respectively. The results of the negative and positive controls indicated that the test system functioned properly and the study was valid.

Based on these results, PRECH-PURCH is not corrosive to the skin and is not classified according to GHS and according to Regulation (EC) 1272/2008.