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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:
14 May 2018 - 18 May 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 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:
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Appearance: Clear colourless to pale yellow liquid
Storage: In refrigerator (2-8°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:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin corrosion test system is the EpiDerm Skin Model, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Lot no.: 28356
- Surface: 0.6 cm^2
- Date of analysis: 16 May 2018

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.784 ±0.191 (within acceptance criteria)
- Barrier function: 8.42 hours (within acceptance criteria)
- Contamination: no contamination was detected

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during 3-minute treatment: room temperature
- Temperature used during 1-hour treatment: 36.3 - 37.2°C
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: once with phosphate buffered saline
- Observable damage in the tissue due to washing: no

The test item was checked for possible interference with the MTT endpoint before the study was started.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Amount of MTT used: 300 μL
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2 for the test item, the negative control and the positive control for each exposure period

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one experiment with a 3-minute exposure period and one experiment with a 1-hour exposure period.

DECISION CRITERIA (see table 1 in 'any other information on materials and 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
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
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

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL
Duration of treatment / exposure:
3-minute and 1-hour
Duration of post-treatment incubation (if applicable):
3 hours with MTT
Number of replicates:
2 for each exposure period (4 in total)

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
104
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Tissue viability: 11%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour exposure
Value:
38
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Tissue viability: 5.6%
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 (optical density at 570 nm) of the negative control tissues was within the acceptance limits (i.e. 1.769 for the 3-minute exposure and 1.841 for the 1-hour exposure)
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following the 1-hour exposure to the positive control was 5.6%.
- Acceptance criteria met for variability between replicate measurements: yes, in the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 30% (i.e. ≤17%) for the negative control, indicating that the test system functioned properly

Any other information on results incl. tables

Table 1 Historical data

 

Negative control

Positive control

 

3-minute treatment

(OD570)

1-hour treatment

(OD570)

3-minute treatment

(OD570)

1-hour treatment

(OD570)

Range

1.258 – 2.615

1.371 – 2.371

0.0172 – 0.56

0.046 – 0.339

Mean

1.80

1.82

0.19

0.14

SD

0.26

0.22

0.09

0.05

n

111

110

106

103

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 2014 to November 2017.

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

 

 

1.6578

1.9712

1.8766

1.9152

1.6490

1.9674

1.8973

1.8840

1.6464

1.9758

1.8675

1.8570

Test item

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

 

1.9951

1.7455

0.9267

0.5980

2.0076

1.7590

0.8916

0.5868

1.9966

1.7676

0.8955

0.5764

Positive control

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

 

0.2894

0.1751

0.1334

0.1601

0.2863

0.1716

0.1327

0.1559

0.2915

0.1722

0.1288

0.1567

OD = Optical density

Duplicate exposures are indicated by A and B.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of an in vitro skin corrosion study, performed according to OECD guideline 431 and GLP principles, Tetrahydro-6propyl-2H-pyran-2-one (delta octalactone) is considered not corrosive (mean tissue viability of 104% and 38% after 3 minutes and 1 hour exposure, respectively) and is therefore not classified according to GHS and Regulation (EC) 1272/2008.