3,5-di-tert-butyl-4-hydroxybenzyl alcohol

Skin irritation / corrosion

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

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

Results

Results 1

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

Results 2

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

Results 3

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