Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 - 05 Aug 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
OGYÉI, Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary

Test material

Constituent 1
Chemical structure
Reference substance name:
3,5-di-tert-butyl-4-hydroxybenzyl alcohol
EC Number:
201-815-6
EC Name:
3,5-di-tert-butyl-4-hydroxybenzyl alcohol
Cas Number:
88-26-6
Molecular formula:
C15H24O2
IUPAC Name:
2,6-di-tert-butyl-4-(hydroxymethyl)phenol
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Rahn AG, Zürich, Switzerland

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, dry conditions. Protect from heat and direct sunlight


In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: adult human (no further information provided)
Justification for test system used:
The EPISKIN model has been validated for irritation testing. It is used as a replacement for the Draize Skin Irritation test (OECD TG 404) for the purpose of distinguishing between skin irritating and non-skin irritating test substances.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France
- Tissue batch number(s): 16-EKIN-031
- Date of initiation of testing: 03 Aug 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume of washing steps: rinsed thoroughly with 25 mL PBS 1x solution
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours (± 5 min)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 22.1 ± 0.2
- Barrier function: 2.8 mg/mL
- Morphology: Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: no
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Method of calculation used:
Blank: The mean of 6 blank OD values is calculated
Negative control:
- Individual negative control OD values are corrected with the mean blank OD:
OD Negative Control (ODNC) = ODNC raw (ODNC raw value) – OD blank mean
- The corrected mean OD of the 3 negative control values is calculated, which corresponds to 100% viability
Positive control:
- Individual positive control OD values are corrected with the mean blank OD: :
OD Positive Control (ODPC) = ODPC raw – OD blank mean
- The corrected mean OD of the 3 positive control values is calculated
- The % viability for each positive control replicate is calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
- The mean value of the 3 individual viability % for positive control is calculated:
Mean PC % = (%PC1 + %PC2 + %PC3) / 3
Test item:
- Individual test item OD values are corrected with the mean blank OD: :
OD Treated Tissue (ODTT) = ODTT raw – OD blank mean
- The corrected mean OD of the 3 test item values is calculated
- The % viability for each test item replicate is calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
- The mean value of the 3 individual viability % for test item is calculated
Mean TT % = (%TT1 + %TT2 + %TT3) / 3

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive or irritant to skin if the mean relative viability after 15 minutes exposure and 42 h post incubation is less or equal to 50% of the negative control.
- The test substance is considered to be non-corrosive or non-irritant to skin if the tissue viability after exposure and post-treatment incubation is more than 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 mg (the epidermal surface was moistened with 5 µl deionised water to improve skin contact)

NEGATIVE CONTROL
- Amount(s) applied: 10 μL

POSITIVE CONTROL
- Amount(s) applied:10 μL
- Concentration (if solution): 5 %
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 ± 1 h
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of negative control (DPBS)
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of positive control (5% SDS)
Value:
16
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of test item (10 mg/ 0.38 cm²)
Value:
80
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
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: yes

ACCEPTANCE OF RESULTS:
The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be ≤ 18.
The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be ≤ 18.
For test chemicals, the standard deviation value (SD) of the % viability should be ≤ 18.

- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1: MTT assay after 3 h (± 5 min) exposure

Substance

Tissue No.

OD570

OD570 (mean)

Relative viability
[%]

Relative viability (mean)

[%]

Standard Deviation of relative viability
[%]

Negative
Control

1

0.823

0.766

107

100

8.20

2

0.776

101

3

0.699

91

Positive
Control

1

0.117

0.119

15

16

0.22

2

0.119

16

3

0.120

16

Test
Item

1

0.702

0.611

92

80

11.04

2

0.594

78

3

0.535

70

Applicant's summary and conclusion

Interpretation of results:
other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified