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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 June 2018 - 25 June 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
20 July 2012
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)
Specific details on test material used for the study:
No purity correction factor was used.

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
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model(TM) (EPISKIN-SM(TM), 0.38 cm^2)
- Tissue batch number(s): 18-EKIN-025
- Expiration date: 25 June 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 36.6 - 37.3°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

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Histology scoring: 22.4 +/- 0.4
- Satisfactory epidermis layer
- Contamination: on blood of the same donors it was verified that there was an absence of HIV 1 and 2 antibodies, hepatitis C antibodies, hepatitis B antigen HBs. On epidermal cells of the same donors it was verified that there was an absence of bacteria, fungus and mycoplasma.

NUMBER OF REPLICATE TISSUES: 3 replicates for the test item, positive control and negative control exposed skin tissues each. OD measurements on each replicate were performed in duplicate.

MTT DIRECT INTERFERENCE:
- The test item was checked for direct MTT reduction and interference in a previous study and was concluded that it did not interfere with the MTT endpoint.

PREDICTION MODEL / DECISION CRITERIA (see table 1)
A test item is considered irritant in the skin irritation test if:
- The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
- The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

ACCEPTABILITY CRITERIA:
- The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
- The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
- The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Test item: 25 µL
- Positive control: 25 µL
- Negative control: 25 µL (re-spread after 7 minutes contact time)
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours, followed by 3 hours with MTT
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 replicates
Value:
62
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (see table 3)
- Acceptance criteria met for positive control: yes, the positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 7.3%.
- Acceptance criteria met for variability between replicate measurements: yes, the standard deviation value of the percentage viability of three tissues treated identically was ≤ 11%

Any other information on results incl. tables

Table 2 Individual OD Measurements at 570 nm

 

A

(OD570)

B

(OD570)

C

(OD570)

Negative control

OD570measurement 1

OD570measurement 2

 

1.0123

0.9921

 

0.9900

0.9680

 

1.0793

1.0356

Test item

OD570measurement 1

OD570measurement 2

 

0.7848

0.7585

 

0.5607

0.5470

 

0.6308

0.6106

Positive control

OD570measurement 1

OD570measurement 2

 

0.1118

0.1180

 

0.1221

0.1175

 

0.0982

0.1070

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Table 3 Historical data

 

Negative control

(absorption; OD570)

Positive control

(absorption; OD570)

Range

0.422 – 1.547

0.023 – 0.437

Mean

0.98

0.13

SD

0.18

0.08

n

174

173

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.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) No. 1272/2008
Conclusions:
Based on the results of an in vitro skin irritation study, performed according to OECD guideline 439 and GLP principles, Tetrahydro-6propyl-2H-pyran-2-one (delta octalactone) is determined to be non-irritant to the skin (mean tissue viability of 62%). The test item is not classified according to GHS and according to Regulation (EC) No. 1272/2008.