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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:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3',5,5'-tetramethylbiphenyl-2,2'-diol
EC Number:
607-957-9
Cas Number:
26567-10-2
Molecular formula:
C16 H18 O2
IUPAC Name:
3,3',5,5'-tetramethylbiphenyl-2,2'-diol

Test animals

Species:
human
Strain:
other: EPISKIN

Test system

Amount / concentration applied:
- at least 10 µL distilled water was applied to the epidermal surface (in order to improve further contact between powder and epidermis).
- 20 mg of the solid test item were applied evenly to the epidermal surface.
- 50 µL of positive control (5% (w/v) SDS solution) or negative control (PBS) were added to each skin units by using a suitable pipette.
Duration of treatment / exposure:
15 minutes
Number of animals:
In this assay, 3 replicates were used for test item. 3 negative controls and 3 positive controls were also run in the assay. Furthermore, as the test item was coloured, one additional test item-treated tissue was used for the non specific OD evaluation.
Details on study design:
TEST SITE
- Area of exposure: 0.38 cm2

REMOVAL OF TEST SUBSTANCE
- Washing: Phosphate Buffered Saline
- Time after start of exposure: 15 min

PRE-INCUBATION (DAY [-1])
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate were filled with the pre-warmed medium. The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere.

APPLICATION AND RINSING (DAY 0)
As the test item was solid, first an appropriate amount (at least 10 µL) distilled water was applied to the epidermal surface (in order to improve further contact between powder and epidermis) and then 20 mg of the test item were applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a curved flat spatula (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.
50 µL of positive control (5% (w/v) SDS solution) or negative control (PBS) were added to each skin units by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.
The plates with the test item treated, negative and positive control treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (21.9-22.4°C).
After the incubation time, the EPISKIN-SM units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2.

MTT TEST (DAY 2)
After the 42 hours incubation, all EPISKIN-SM units (except of one colour control unit) were transferred into the MTT solution filled wells. Then, all transferred EPISKIN-SM units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light.

FORMAZAN EXTRACTION (DAY 2)
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation for formazan extraction.

CELL VIABILITY MEASUREMENTS (DAY 2)
Following the formazan extraction, the OD (optical density or absorbance) of the samples were measured using a plate reader at 540 nm. The mean of 6 wells of acidified isopropanol solution was used as blank.

Results and discussion

In vitro

Results
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

VALIDITY OF THE TEST

- After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

- The mean OD value of the two negative control tissues was in the recommended range (0.952). Standard deviation for negative control samples was 4.1.

- The positive control treated tissues showed 17.4 % viability demonstrating the proper performance of the assay. The standard deviation value for positive control samples was 4.1.

- The difference of viability between the three test item-treated tissue samples in the MTT assay was 9.6.

All these parameters were within acceptable limits and therefore the study was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, in this in vitro EPISKIN model test, the results indicate that the test item is Non Irritant (NI).