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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:
From 06 June 2018 to 08 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
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiSkin(TM) SOP, INVITTOX Protocol No 18
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Episkin(TM) SOP, Version 1.8
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-tert-butyl-1,4-dimethoxybenzene
EC Number:
244-216-5
EC Name:
2-tert-butyl-1,4-dimethoxybenzene
Cas Number:
21112-37-8
Molecular formula:
C12H18O2
IUPAC Name:
2-tert-butyl-1,4-dimethoxybenzene
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test Material: Vetimoss, (2-tert-butyl-1,4-dimethoxybenzene)
- CAS Number: 21112-37-8
- EC Number: 244-216-5
- Source and lot/batch No.of test material: A180118C
- Expiration date of the lot/batch: 06 June 2019
- Purity test date: 99.78 %
- Appearance: Clear, almost colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage Conditions: Room temperature (15-25 °C, ≤ 70% relative humidity (RH)),
under inert gas, protected from humidity (tightly closed container)
- Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat)
for unknown materials were applied to assure personnel health and safety.

PREPARATION OF THE TEST ITEM
- The test item was applied as supplied, no formulation was required.


In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: NA
Source strain:
other: NA
Details on animal used as source of test system:
TEST SYSTEM: Human Skin
EPISKIN(TM) (SM) (Manufacturer: SkinEthic, France, Batch No.: 18-EKIN-023, Expiry Date: 11 June 2018) is a three-dimensional human epidermis model. Adult humanderived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Justification for test system used:
The EPISKIN(TM) (SM) model has been validated for corrosivity and irritation testing in an international validation studies and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM (SM) (Manufacturer: SkinEthic, France, Batch No.: 18-EKIN-023, Expiry Date: 11 June 2018)
- Units: EPISKIN TM (SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm*2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
- Plate: 12-well assay plates
- Punch: EPISKIN TM (SM) biopsy punch for easy sampling of epidermis
- A flask of sterile “Maintenance Medium” (Batch No.: 18 MAIN3 028; Exp. Date: 13 June 2018); a flask of sterile “Assay Medium” (Batch No.: 18 ESSC 024; Exp. Date: 13 June 2018)
- Storage: The kits were kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8 °C until the initiation of the test.

MTT SOLUTION
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solutions were stored in refrigerator (2-8 °C) protected from light. These were diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

ACIDIFIED ISOPROPANOL
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

CHEMICALS USED IN THE EXPERIMENT:
- MTT
- Isopropanol
- Hydrochloric acid
- Phosphate buffered saline (PBS)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
General validity criteria
- The mean OD value of the two or three negative control tissues should be ≥ 0.6 and ≤ 1.5.
- The mean OD value of the blank samples (acidified isopropanol) should be < 0.1.
Specific criteria for corrosivity
- The difference of viability between the two tissue replicates should not exceed 30%.
- The acceptable mean percentage viability range for positive controls is ≤ 20%.


Control samples:
other: Negative controls - Corrosion test: Physiological saline (0.9% (w/v) NaCl solution) - Irritation test: Phosphate Buffered Saline Positive controls - Corrosion test: Glacial acetic acid - Irritation test: 5% (w/v) Sodium Dodecyl Sulphate solution
Amount/concentration applied:
TEST MATERIAL
The Assay Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath.
50 µL of test item was applied evenly to each of two test units and each additional control skin units.

NEGATIVE AND POSITIVE CONTROL S
50 µL of negative control (Physiological saline) or positive control (glacial acetic acid) were added to each skin unit by using a suitable pipette.

Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for the exposure time of 4 hours at room temperature (21.9-23.4°C).
Number of replicates:
TEST ITEM TEST AND CONTROLS:
- Two replicates per time point were used for test item
- Two negative controls and two positive controls
- Two additional test item-treated living tissues were used for the non-specific OD evaluation

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
97.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No indication of skin corrosion
Remarks:
Mean experiments 1,2: 112.2%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
126.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No indication of skin corrosion
Remarks:
Mean experiments 1,2: 112.2%
Other effects / acceptance of results:
General validity criteria:
- After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions.
- The mean OD value of the two negative control tissues in the corrosivity test was in the recommended range (0.804).
- The mean OD value of the blank samples (acidified isopropanol) in the corrosivity test was 0.048.

Specific criteria for corrosivity testing:
- The two positive control treated tissues showed 0.3% viability demonstrating the proper performance of the assay.
- The difference of viability between the two test item-treated tissue samples in the MTT assay was 25.4%.
- The difference of viability between the two negative control tissue samples in the MTT assay was 0.5%.

Indicator for potential false viability
- Chemical action by the test material on MTT may mimic that of cellular metabolism leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement. MTT reduction and interferences were checked and corrected.




Any other information on results incl. tables

INTERPRETATION OF THE TEST RESULTS

Corrosivity test:

The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).

For 2 disks:

- Mean viability of ≥ 35% = Non Corrosive

- Mean viability of < 35% = Corrosive

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The results of the corrosivity test indicated that the mean cell viability was 112.2% compared to the negative control. The validity criteria were acceptable.
In conclusion, the test material Vetimoss, (2-tert-butyl-1,4-dimethoxybenzene) was considered non-corrosive, according to the result of this study.
Executive summary:

The aim of the study was to predict the corrosivity potential of the test material Vetimoss, (2-tert-butyl-1,4-dimethoxybenzene) using the in vitro skin irritation corrosivity test EPISKIN TM (SM), which is designed to predict and classify the corrosivity and irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

The study was conducted according the OECD Guideline 439 and performed under GLP conditions.

The test systems EPISKIN TM (SM) were treated with the test item and incubated for for 4 hours at room temperature. At the end of the exposure time, the disks were rinsed with Phosphate Buffered Saline (PBS) and incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere.

 

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid were used as negative and positive control, respectively.

Two additional disks were used to provide in each case an estimate of colour contribution from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity test, if the mean relative viability after 4 hours of exposure is <35% the test item is considered to be corrosive to skin.

The results of the corrosivity test indicated that the mean cell viability was 112.2% compared to the negative control.

In conclusion, the test material Vetimoss, (2-tert-butyl-1,4-dimethoxybenzene) was considered non-corrosive, according to the result of this study.