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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:
2015-02-17 to 2015-02-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
other: EpiDerm™

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μl
- Concentration (if solution): undiluted

CONTROLS
Negative control: Dulbecco’s phosphate buffered saline (DPBS; Gibco, Cat. No. 14040-091, Lot No.: 1581502).
Positive control: 5% sodium dodecyl sulfate in H2O (TC-SDS-5%; MatTek, CAS No.: 151-21-3, Lot No.: 020615TMG).
Duration of treatment / exposure:
60 ± 1 min
Observation period:
42 h
Details on study design:
TEST SYSTEM: reconstituted three-dimensional human skin model EpiDerm™ (MatTek). This skin model consists of normal (non-cancerous), human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. The EpiDerm skin model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous, granular and cornified layers analogous to those found in vivo.

PRE-EXPERIMENT To check the non-specific MTT-reducing capability of the test item 30 μl of the test item were mixed per 1 ml MTT medium and incubated for 1 h at 37 ± 1 °C in the dark. If the mixture turns blue/purple, the test item is presumed to have reduced MTT. [MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. To check the colouring potential of the test item 30 μl of the test item were mixed per 300 μl distilled water in a transparent recipient and incubated at 37 ± 1°C for 60 min.


EXPERIMENTAL PROCEDURE Tissues were pre-incubated overnight at 37 ± 1 °C, 5.0% CO2, then each group (negative control (first), test item and positive control) was treated in triplicate. After incubation for 35 ± 1 minutes, the tissues were placed under sterile flow until the 60 ± 1 min incubation time of the first dosed tissue were over. After 60 ± 1 min the tissues were washed intensively with DPBS, staggered in one-minute intervals. Excess DPBS was removed by blotting the bottom with blotting paper. The tissues were then placed in fresh assay medium and post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95% for 24 ± 2 h. Following this incubation the tissues were transferred to new medium and incubated for additional 18 ± 2 h (total postincubation time 42 h).

After incubation the tissues were transferred to a prepared 24-well plate containing 300 μl pre-warmed MTT medium and further incubated for 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%.

After the 3 h MTT incubation the tissues were rinsed three times with DPBS and afterwards dried on blotting paper. The tissues were extracted by immersion in 2 ml of isopropanol, sealed and incubated at room temperature for at least 2 hours and mixed by shaking until the solution colour became homogeneous.

2 x 200 μl aliquots of the extract of each tissue were transferred into a 96-well plate and OD was measured at 550 nm without reference wavelength in a plate spectrophotometer.

The irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with PBS.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
82.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

In vivo

Irritant / corrosive response data:
PRE-EXPERIMENT- The test item showed no reduction of MTT relative to negative control. No colouring was detected by unaided eye when 30 μl of the test item were mixed with 300 μl distilled water.

EXPERIMENT: Total mean OD550 of 3 replicate tissues (blank-corrected): negative control: 2.623*; positive control: 0.154; test item: 2.163.
Mean relative tissue viability [%]: negative control: 100.0; positive control: 12.0; test item: 82.5

QUALITY CRITERIA:
Mean absolute OD550 nm NC: pass
mean % viability PC: pass
SD viability (%): pass

* Corrected mean OD550 of the negative control corresponds to 100% absolute tissue viability.
NC negative control
PC positive control

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Tris(isopropenyloxy)(vinyl)silane has been tested in a reliable in vitro study for skin irritation conducted according to OECD 439 and in compliance with GLP using EpiDerm™ tissue. The relative tissue viability of the test-item treated tissues, assessed by mean optical density at 550 nm relative to solvent control after 60 minutes exposure and 42 h post incubation, was 82.5%, which is greater than the 50% threshold for skin irritation. The positive control produced the expected reduction in viability. Therefore it is concluded that the test substance is not irritating to the skin.